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Utilization of Modified Induced Pluripotent Stem Cells as the Advance | OPTH – Dove Medical Press

Introduction

Glaucoma is one of the optic neuropathy disorders characterized by the progressive degeneration of retinal ganglion cells (RGC), which eventually lead to cupping of the optic disc and decreased visual field.1 Glaucoma is also closely related to an increase in intraocular pressure caused by the damage of trabecular meshwork (TBM), which results in optic nerve damage, characterized by the loss of retinal ganglion cells.2,3 Globally, in 2020, more than 76 million people are suffering from glaucoma, and it is expected to increase to 111.8 million people by 2040.4,5 Glaucoma is also a severe and complex medical problem because it often causes blindness. According to the World Health Organization (WHO), the most common causes of blindness are cataracts (51%), followed by glaucoma (8%), and age-related macular degeneration (5%).6 This data shows that glaucoma is the worlds second most common cause of blindness after cataracts. Symptoms that are often asymptomatic at an early stage and the low public awareness have contributed to the disorders seriousness.

Handling and treating glaucoma cases is difficult, especially because no therapy can cure glaucoma. Current treatment, both medical and surgical, is focused solely on lowering intraocular pressure. Treatment of glaucoma cases should also be carried out for life to maintain normal intraocular pressure and prevent the progression of intraocular damage due to glaucoma.7 Based on these problems, innovation is needed to handle glaucoma effectively. Besides, solutions are also required to repair the damage to retinal ganglion cells in glaucoma. One of the therapies that researchers are trying to take advantage of is stem cell therapy, a technology where cells can develop into many specific cells desired.8 In cases of glaucoma or optic neuropathies, damaged RGCs can be replaced with new ones grown from stem cells.9 Another option for RGC regeneration is to use retinal stem cells to regenerate RGCs. Indeed, stem cell therapy relies on exogenous stem cell sources due to their limited availability. Currently, many stem cell therapies for eye diseases that are created and studied are limited to treating the damage of photoreceptors and retinal pigment epithelium. iPSC-derived RGCs can serve as an excellent model for formulating approaches to promote de novo-generated RGCs to connect with their targets. Therefore, researchers have been looking into the potential use of modified stem cell therapy to treat the intraocular injury in glaucoma cases.10

This review aims to synthesize and prove the efficacy and further modification of this method so that it can be eligible for treatment and can also give data collection for the scientific community. This systematic review is expected to provide detailed information regarding the possible applications of modified stem cell therapy in treating intraocular damage in glaucoma patients.

In the present literature review, literature regarding the potential utilization of stem cells as an advanced therapy for intraocular glaucomatous damage was searched. The stages of this literature review include five steps: i) identifying the research question, ii) identifying relevant studies, iii) study selection, iv) charting the data, and v) summarizing and reporting the results.

This literature review was conducted to answer the following research questions:

The literature search was carried out from January to February 2021. Keywords and synonyms used to conduct literature searches related to the research question are attached in Table 1. Boolean operators (OR, AND, NOT) combine keywords when searching for literature. The search was conducted on seven online databases, namely PubMed, ScienceDirect, ProQuest, EBSCOhost, SAGE, Clinicalkey, and Scopus.

Table 1 Keywords That Were Used in the Database Search

The inclusion criteria for the literature search consisted of journals published in English and journals published in the last ten years. The exclusion criteria for selected studies consisted of journals that were not fully accessible due to the limited facilities owned as supporting access. We thoroughly screened the titles and abstracts of the studies obtained to suit the purpose of this literature review. Abstracts that were not relevant to the research objectives were excluded. Then a full article screening was carried out from the selected abstracts to identify whether the full article was suitable for the research objectives and whether the full article could be used to answer research questions.

Information obtained from all selected study articles is then displayed in the charting table The information displayed includes the author, year of publication, study objectives, location, study design, inclusion and exclusion criteria, results, and conclusions.

The researcher did not assess the quality of the selected articles because this study was only a literature review. The data from selected studies are reported to produce recommendations for further research regarding the use of stem cell therapy in glaucoma cases.

Based on the literature search that has been conducted, a total of 2262 studies and abstracts were included in the journal screening process at an early stage. From this screening process, 362 duplicate articles were excluded from the selection. The remaining 1900 articles then entered the abstract eligibility screening stage. Only 53 articles were selected, while 1879 other articles were excluded. Of the 53 articles, 18 articles appeared relevant to the study and met the inclusion criteria for review throughout the study. Meanwhile, 35 other studies were excluded because the focus in these studies did not match the objectives of this literature review. After assessing the full articles, six studies met the inclusion criteria in this literature review (Figure 1).

Figure 1 Flow diagram of the literature review process.

In Table 2, a summary of the characteristics of the selected studies is presented. The data used from selected studies include research objectivity, study design, results, outputs, and conclusions from the study. Of all the selected studies, there were six studies that had experimental methods. Almost all studies have the aim of evaluating and proving the potential of using stem cells to replace damaged tissue and restore and restore the function of damaged eye tissue, particularly due to degenerative processes such as disease of the retina or glaucoma.

Table 2 Results Summary of the Characteristics of the Selected Studies

Glaucoma is characterized by the degeneration of retinal ganglion cells. Based on the pathophysiology, glaucoma can be divided into two categories, namely open-angle glaucoma and closed-angle glaucoma. In patients with open-angle glaucoma, there is increased resistance to the aqueous humors outflow through the trabecular meshwork. This increased resistance is often caused by apoptosis and senescence of trabecular meshwork cells with increasing age.15 Degradation and abnormalities of the cytoskeleton arrangement of trabecular meshwork cells resulting in thickening of the drainage pathways and abnormal extracellular matrix deposition also worsen trabecular meshwork function in open-angle glaucoma.16 In closed-angle glaucoma, the aqueous humor cannot reach the trabecular meshwork due to obstruction.17 Examples of obstructions that often cause closed-angle glaucoma are anterior synechiae, the attachment of the iris to the trabecular meshwork, and posterior synechiae, where the iris is attached to the lens. This adhesion causes the aqueous humor to fail to reach the drainage system and the trabecular meshwork.18

Glaucoma is closely related to increased intraocular pressure, which is determined by the balance between the production of aqueous humor by the ciliary body and the drainage of the aqueous humor through the trabecular meshwork. The disturbance of the balance between production and drainage increases the humor Aquos, which at a later stage can increase the intraocular pressure.19 Studies have shown a link between increased intraocular pressure and retinal ganglion cell death. This study has also proven that the longer the intraocular pressure increases, the higher the degree of retinal ganglion cell damage.20 However, data show as many as 3040% of patients with glaucoma have normal intraocular pressure. One of the causes of glaucoma at normal intraocular pressure is a decrease in neurotrophic factors needed in the maintenance of neurons in the optic nerve. Neurotrophic factors are required to maintain retinal ganglion cells, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and cell line-derived neurotrophic factor.21 Furthermore, microcirculation disorders, changes in immune system conditions, and increased levels of oxidative stress can also cause glaucoma at normal intraocular pressure.21

Stem cells are cells with the ability to differentiate and form all tissues in the human body. They are one of the potential therapies used in cases that require tissue repair and regeneration, one of which is glaucoma. For a cell to be called a stem cell, it must have two essential characteristics. The first one is the stem cell must produce offspring with the exact features the cell originates from, and the second one, the stem cell must be able to differentiate into the specific cell desired.22 There are two types of stem cells found in multicellular organisms, including humans. The first stem cells are embryonic stem cells or multipotent cells found in blastocysts, while the second stem cells are adult stem cells or pluripotent cells that can be found in a wide variety of adult tissues.23

Research has also succeeded in inducing adult cells to return to the pluripotent stage using molecular manipulation. The cells produced by this molecular manipulation are then called induced pluripotent stem cells (iPS).24 Most iPS manufacturing uses viruses such as retroviruses and lentiviruses to carry genes encoding transcription factors to adult cells to be modified. This gene will then undergo transcription and translation into a protein capable of inducing the adult cell nucleus to return to an embryonic state.25

An important concept that needs attention in stem cell therapy is how to induce stem cells to become the desired differentiated cells.26 It is necessary so that the cells can be used to treat various diseases, including glaucoma. We can further achieve differentiation of stem cells into specific desired cells by adding various growth factors and signaling pathways to resemble the conditions of their original development.27

The research conducted successfully isolates cultures and confirms that the trabecular meshwork stem cells around the Schwalbe line are multipotent with the ability to differentiate into a wide variety of cells, including trabecular meshwork cells adipocytes osteocytes, and chondrocytes.28 Other studies have also been able to induce stem cells on the Schwalbe line trabecular meshwork to proliferate and differentiate into photoreceptors under certain conditions.29 Apart from trabecular meshwork stem cells, other stem cells that can differentiate into functional meshwork trabecular cells are adipose-derived stem cells (ADSC), mesenchymal stem cells (MSC), and iPS. iPS cells can also differentiate into trabecular meshwork cells after culturing the extracellular matrix with cell-derived trabecular meshwork. The success of a wide variety of stem cells to differentiate into functional meshwork trabecular cells provides a more effective alternative to cutting-edge therapy in treating glaucoma, especially open-angle glaucoma.3

One of the stem cell therapies successfully applied and able to regenerate damaged retinal ganglion cells is iPS cell therapy. This therapy uses induced adult fibroblasts to return to pluripotent cells using four transcription factors, namely Oct3/4, Sox2, Klf4, and c-Myc. The results of the iPS are pluripotent cell colonies that are morphologically similar to ESCs, which are able to differentiate into the three germ cell layers.30

Because iPS can be programmed from the patients somatic cells, this therapy can maintain the unique genome of each individual. Currently, various modifications to the iPS therapy have been made to increase its acceptability and effectiveness of iPS therapy. One of them is the use of plasmid vectors and miRNA instead of retroviruses to avoid mutagenesis of the adult cells used.31,32

One of the significant challenges in stem cell therapy is to achieve the differentiation of stem cells into the desired cells, in this case, the differentiation of stem cells to retinal ganglion cells. Usually, in vivo, the differentiation of stem cells into retinal ganglion cells is regulated by several transcription factors such as Ath5, Brn3, and Notch. The transcription factors Ath5 and Brn3 play a vital role in the differentiation of retinal ganglion cells, and their levels are increased in the process of eye development.33 Meanwhile, Notch is a negative regulator of retinal ganglion cell differentiation, and its levels are decreased in normal eye development. Therefore, the addition of the transcription factors Ath5 and Brn3 and the Notch antagonist is a strategy to differentiate retinal ganglion cells from stem cells.34 Apart from transcription factors, various neurotrophic pathways and factors have been identified in the differentiation of stem cells into retinal ganglion cells. These pathways consist of fibroblast growth factor (FGF), insulin-like growth factor (IGF), bone morphogenetic protein (BMP), nodal, and Wnt signaling pathways. All of these pathways regulate retinal development, whereas FGF and IGF provide positive regulation. Meanwhile, BMP, nodal, and Wnt signaling pathways provide negative regulation.35

Another major challenge in the clinical application of stem cell therapy in glaucoma sufferers is that not only do the stem cells successfully differentiate into retinal ganglion cells, but they must also be able to reach the central nervous system.36 Modifications must be made so that new retinal ganglion cells can reach the visual cortex of the cerebrum. Recent research has found that a combination of genetic modification and stimulation of the signaling pathway stimulates regeneration of the optic nerve until it reaches the central nervous system. The addition of ephrin molecules, proteoglycans, cell-adhesion molecules, and semaphorin is able to guide the axons of the developing retinal ganglion cells to reach the optic chiasm.13 Meanwhile, the addition of cadherin, ephrin, and the Wnt signaling pathway can guide and stimulate synapse formation in the superior colliculus and the visual cortex.12,37

In addition, because of the adverse intraocular environment in glaucoma, stem cell therapy needs to be combined with neuroprotective compounds. It is also associated with a decrease in neurotrophic factors required to maintain neurons and causes progression of retinal ganglion cell damage in glaucoma sufferers. Therefore, the addition of BDNF and other neurotrophic factors such as glial cell-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) should be considered for combined stem cell therapy.38

The stem cells are used in cases of glaucoma, which require repair and regeneration of trabecular meshwork cells and retinal ganglion cells. iPS has been shown the ability to differentiate to replace damaged trabecular meshwork cells and retinal ganglion cells in glaucoma. Some modifications are required so that stem cells that have differentiated into trabecular meshwork cells and retinal ganglion cells can reach the central nervous system. These modifications include the addition of ephrin molecules, proteoglycans, cell-adhesion molecules, semaphorin, cadherin, and the Wnt signaling pathway. The combination of stem cells with neuroprotective factors such as BDNF, GDNF, and CNTF also needs to be considered to maintain neuronal maintenance and inhibit the progression of cell damage.

The development of new stem cell technologies not only paves the way for us to gain a better understanding of the biology associated with glaucoma and create models for the development of new drugs, but it also opens the door to the prospect of cell-based therapies that can help patients regain their vision. More specifically in relation to the field of glaucoma, there have been recent developments in the process of developing protocols for the differentiation of stem cells into trabecular meshwork and retinal ganglion cells. Further research on the effectiveness of using modified stem cells as a therapy for glaucoma and in vivo research can be carried out immediately so that clinical trials can be carried out, which in turn can be used by the community to control symptoms and reduce blindness due to glaucoma.

The authors report no conflicts of interest in this work.

1. Choudhari NS, Neog A, Fudnawala V, George R. Cupped disc with normal intraocular pressure: the long road to avoid misdiagnosis. Indian J Ophthalmol. 2011;59(6):491. doi:10.4103/0301-4738.86320

2. Braunger BM, Ademoglu B, Koschade SE, et al. Identification of adult stem cells in Schwalbes line region of the primate eye. Invest Ophthalmol Vis Sci. 2014;55(11):7499. doi:10.1167/iovs.14-14872

3. Abu-Hassan DW, Li X, Ryan EI, Acott TS, Kelley MJ. Induced pluripotent stem cells restore function in a human cell loss model of open-angle glaucoma. Stem Cells. 2015;33(3):751761. doi:10.1002/stem.1885

4. Cook C, Foster P. Epidemiology of glaucoma: whats new? Can J Ophthalmol. 2012;47(3):223226. doi:10.1016/j.jcjo.2012.02.003

5. Hapsari DM. The Relationship Between Family Support and Adherence in Care in Glaucoma Clients in the Working Area of the Balung Health Center. Jember Regency. Uni; 2017.

6. World Health Organization. Global data on visual impairments 2010; 2012. https://www.iapb.org/wp-content/uploads/GLOBALDATAFINALforweb.pdf. Accessed August 24, 2022.

7. Harasymowycz P, Birt C, Gooi P, et al. Medical management of glaucoma in the 21st century from a canadian perspective. J Ophthalmol. 2016;2016:122. doi:10.1155/2016/6509809

8. Romito A, Cobellis G. Pluripotent stem cells: current understanding and future directions. Stem Cells Int. 2016;2016:120. doi:10.1155/2016/9451492

9. Cen L-P, Ng TK. Stem cell therapy for retinal ganglion cell degeneration. Neural Regen Res. 2018;13(8):1352. doi:10.4103/1673-5374.235237

10. ner A. Stem cell treatment in retinal diseases: recent developments. Turk J Ophthalmol. 2018;48(1). doi:10.4274/tjo.89972

11. Manuguerra-Gagn R, Boulos PR, Ammar A, et al. Transplantation of mesenchymal stem cells promotes tissue regeneration in a glaucoma model through laser-induced paracrine factor secretion and progenitor cell recruitment. Stem Cells. 2013;31(6):11361148. doi:10.1002/stem.1364

12. Osterhout JA, Josten N, Yamada J, et al. Cadherin-6 mediates axon-target matching in a non-image-forming visual circuit. Neuron. 2011;71(4):632639. doi:10.1016/j.neuron.2011.07.006

13. De lima S, Koriyama Y, Kurimoto T, et al. Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors. Proc Natl Acad Sci U S A. 2012;109(23):91499154. doi:10.1073/pnas.1119449109

14. Deng F, Chen M, Liu Y, et al. Stage-specific differentiation of iPSCs toward retinal ganglion cell lineage. Mol Vis. 2016;22:536.

15. Pulliero A, Seydel A, Camoirano A, Sacc SC, Sandri M, Izzotti A. Oxidative damage and autophagy in the human trabecular meshwork as related with ageing. PLoS One. 2014;9(6):e98106. doi:10.1371/journal.pone.0098106

16. Porter KM, Epstein DL, Liton PB. Up-regulated expression of extracellular matrix remodeling genes in phagocytically challenged trabecular meshwork cells. PLoS One. 2012;7(4):e34792. doi:10.1371/journal.pone.0034792

17. Lai J, Choy BNK, Shum JWH. Management of primary angle-closure glaucoma. Asia Pac J Ophthalmol. 2016;5(1):5962. doi:10.1097/APO.0000000000000180

18. Sun X, Dai Y, Chen Y, et al. Primary angle closure glaucoma: what we know and what we dont know. Prog Retin Eye Res. 2017;57:2645. doi:10.1016/j.preteyeres.2016.12.003

19. Gemenetzi M, Yang Y, Lotery AJ. Current concepts on primary open-angle glaucoma genetics: a contribution to disease pathophysiology and future treatment. Eye. 2012;26(3):355369. doi:10.1038/eye.2011.309

20. Khan Kareem A, Tse DY, van der Heijden ME, et al. Prolonged elevation of intraocular pressure results in retinal ganglion cell loss and abnormal retinal function in mice. Exp Eye Res. 2015;130. doi:10.1016/j.exer.2014.11.007

21. Almasieh M, Wilson AM, Morquette B, Cueva Vargas JL, Di Polo A. The molecular basis of retinal ganglion cell death in glaucoma. Prog Retin Eye Res. 2012;31(2):152181. doi:10.1016/j.preteyeres.2011.11.002

22. Cao J, Ng ES, Mcnaughton D, et al. The characterisation of pluripotent and multipotent stem cells using Fourier transform infrared microspectroscopy. Int J Mol Sci. 2013;14(9):1745317476. doi:10.3390/ijms140917453

23. Makhani K, Ali SM, Yousuf S, Siddiqui S. Therapeutic potential of totipotent, pluripotent and multipotent stem cells. MOJ Cell Sci Rep. 2015;2(5):00041. doi:10.15406/mojcsr.2015.02.00041

24. Zaveri L, Dhawan J. Cycling to meet fate: connecting pluripotency to the cell cycle. Front Cell Dev Biol. 2018;6. doi:10.3389/fcell.2018.00057

25. Wattanapanitch M. Recent updates on induced pluripotent stem cells in hematological disorders. Stem Cells Int. 2019;2019:115. doi:10.1155/2019/5171032

26. Kolios G, Moodley Y. Introduction to stem cells and regenerative medicine. Respiration. 2012;85(1):310. doi:10.1159/000345615

27. Biehl JK, Russell B. Introduction to stem cell therapy. J Cardiovasc Nurs. 2009;24(2):98103; quiz 1045. doi:10.1097/JCN.0b013e318197a6a5

28. Tay CY, Sathiyanathan P, Chu SWL, Stanton LW, Wong TT. Identification and characterization of mesenchymal stem cells derived from the trabecular meshwork of the human eye. Stem Cells Dev. 2012;21(9):13811390. doi:10.1089/scd.2011.0655

29. Nadri S, Yazdani S, Arefian E, et al. Mesenchymal stem cells from trabecular meshwork become photoreceptor-like cells on amniotic membrane. Neurosci Lett. 2013;541:4348. doi:10.1016/j.neulet.2012.12.055

30. Omole AE, Fakoya AOJ. Ten years of progress and promise of induced pluripotent stem cells: historical origins, characteristics, mechanisms, limitations, and potential applications. PeerJ. 2018;2018. doi:10.7717/peerj.4370

31. Miyoshi N, Ishii H, Nagano H, et al. Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell. 2011;8(6):633638. doi:10.1016/j.stem.2011.05.001

32. Ikushima S, Ono R, Fukuda K, Sakayori M, Awano N, Kondo K. Trousseaus syndrome: cancer-associated thrombosis. Jpn J Clin Oncol. 2016;46(3):204208. doi:10.1093/jjco/hyv165

33. Ji SL, Tang SB. Differentiation of retinal ganglion cells from induced pluripotent stem cells: a review. Int J Ophthalmol. 2019;12(1):152160. doi:10.18240/ijo.2019.01.22

34. Li L, Chen LP, Liu QH. Effect of the notch signaling pathway on retinal ganglion cells and its neuroprotection in rats with acute ocular hypertension. Int J Ophthalmol. 2018;11(2). doi:10.18240/ijo.2018.02.05

35. Ritchey ER, Zelinka CP, Tang J, Liu J, Fischer AJ. The combination of IGF1 and FGF2 and the induction of excessive ocular growth and extreme myopia. Exp Eye Res. 2012;99(1):116. doi:10.1016/j.exer.2012.03.019

36. Georgiev D. Photons do collapse in the retina not in the brain cortex: evidence from visual illusions. NeuroQuantology. 2011;9(2). doi:10.14704/nq.2011.9.2.403

37. He CW, Liao CP, Pan CL. Wnt signalling in the development of axon, dendrites and synapses. Open Biol. 2018;8(10):180116. doi:10.1098/rsob.180116

38. Mysona BA, Zhao J, Bollinger KE. Role of BDNF/TrkB pathway in the visual system: therapeutic implications for glaucoma. Expert Rev Ophthalmol. 2017;12(1):6981. doi:10.1080/17469899.2017.1259566

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Utilization of Modified Induced Pluripotent Stem Cells as the Advance | OPTH - Dove Medical Press

COVID-19 Can Make the Brain Age by 2 Decades; Heres 1 … – Stem Cell

A new British study found that the original SARS-CoV-2 virus (i.e. Wuhan strain) can impair cognitive ability in a way equivalent to making the brain age by two decades. Currently, 67 percent of the worlds population has been vaccinated against the COVID-19 pandemic. Is it possible that the vaccines themselves can also cause aging damage?

Nevertheless, theres no need to worry, aging is proven to be a process, and there is a way to help reverse the aging process.

The study was published in the journal EClinicalMedicine. Experts from the University of Cambridge and Imperial College London Medical School evaluated the cognitive effects of the COVID-19 infection in humans.

The research subjects consisted of COVID-19 patients who were hospitalized for severe illness between March and July 2020.

After these patients recovery from acute infection, the researchers conducted follow-up visits for an average of six months to analyze and evaluate their anxiety, depression, and post-traumatic stress. The researchers discovered a significant decline in the patients attention, complex problem solving skills, and memory, along with reduced accuracy, and prolonged reaction time.

These cognitive deficits are similar to the cognitive decline a person would experience between the ages of 50 to 70, which is equivalent to aging by two decades and losing 10 IQ points.

In addition, the recovery of cognitive ability is very slow in patients with COVID-19 infections.

Why does COVID-19 infection cause abnormal aging? Lets first take a look at one concept.

The relationship between genes and epigenetics are like seeds and soil. Genes are like seeds, while epigenetics is like soil. Genes in the human body do not usually change after birth. They are like seeds that lie dormant in the soil, and some will grow, and some will not. What determines whether these seeds will grow or not are the genetic switches, or epigenetics.

Epigenetics is the study of these factors that affect genes being turned on or off. Specifically, one common type of gene switchDNA methylationcan change the expression of genes, shutting them down and making them non-functional. Methylation is a somewhat complex biochemical process in the body, the important thing to remember is that it is one of the ways epigenetic takes place and a process by which genes get turned on and off.

As a result of DNA methylation, there are variations in whether the same genes can be expressed in different organs and at different ages, and the amount of expression.

Cells become senescent as we age. That means they stop dividing and enter a kind of stasis. Instead of dying off as they normally would, they persist, but change shape and size andsecrete inflammatory molecules that cause other nearby cells to become senescent.

In an article published in Nature Reviews Genetics, Steve Horvath, a professor of human genetics and biostatistician at the University of California Los Angeles, concluded that as people age and have more senescent cells, there are characteristic changes in the methylation status of human DNA.

Human beings experience birth, aging, illness, and death, which is now discovered by our scientists to be controlled by our internal epigenetic clock. This is similar to the observations that everything in our universe has its cycle of formation, stasis, degeneration, and destruction.

Professor Horvath summarized the DNA methylation profiles associated with aging in an epigenetic clock of aging. While our years on Earth are are chronological age, how we live and inherent factors affect how long we will actually live, which is our biological age, or the age of our body. Biological ages can be estimated by using the methylation profiles of aging-related genes. In other words, scientists can focus in on genes linked to aging and then look at how those genes are methylating and from this gauge how far along the person is in that process of degeneration and death. These genetic focal points were carefully selected by scientists, independent of gender, body part, comorbidities, and other factors. And the results have been highly accurate, with a precision of over 95 percent in gauging a persons biological age.

Humans have a normal aging methylation curve. If a persons DNA methylation is above the curve, he or she will age faster than his or her peers; if it is below the curve, he or she will appear younger than his or her peers.

So, what factors can accelerate aging, i.e. epigenetic clock of aging?

A study conducted in Belgium was published in 2018 in the journal Aging. It found that the following factors accelerate epigenetic aging in humans:

This makes us wonder if COVID-19 infection can accelerate the epigenetic aging clock.

A study published in the journal Nature Communications answers this question. The study collected blood samples from 232 healthy individuals, 194 non-severe COVID-19 patients and 213 severe COVID-19 patients for DNA methylation analysis and found that the epigenetic age of COVID-19 patients was significantly accelerated.

In addition, the epigenetic age acceleration in COVID-19 patients is related to the stage of the disease. The age acceleration is fastest during the acute inflammatory phase, when the body and the virus are in intense combat; and it is slightly reversed during the recovery phase.

Even after the infection is over, many people still have symptoms of long COVID. Is this related to the aging caused by COVID-19?

Epigenetic aging is seen in the graying of hair and loosening of teeth. However, on a cellular level, cells in the human body also gradually age.

Cellular senescence refers to a state of cell cycle arrest when cells are stressed, as well as the secretion of various inflammatory cytokines at the same time. In a paper published in Nature Aging, a Japanese research team stated that senescent cells do not die immediately, but instead, they spread inflammatory cytokines to nearby uninfected cells, causing more cells to senesce as well.

So, what are the effects of cellular senescence on our health?

Cellular senescence plays an important role in many age-related diseases, such as degenerative diseases of the nerves, eyes, lungs, and heart.

The aforementioned study was conducted prior to the Omicron variant outbreak, and the Omicron variant is clearly less pathogenic than the old strain. In fact, some of the mutation sites of the Omicron variant counteracted the factors that caused cellular senescence. It is estimated that Omicron causes significantly less accelerated senescence or sequelae than the old strain.

However, the vaccines we are currently administering are still developed using the spike proteins of the old strain of early 2020, so is there a risk of accelerated aging?

The COVID-19 vaccines mainly express spike proteins in the human body. In a study published in the Journal of Virology in 2021, researchers from Saint Louis University in Missouri transfected spike proteins of the old strain into cells in vitro. It was later discovered that a large number of cellular senescence markers (including specific cytokines, interleukins, and specific enzymes, etc) were found in the spike-transfected cells, compared to the control group.

In addition, the spike proteins increase inflammatory factors, cause mitochondrial damage, produce misfolded proteins, and cause genomic instability, all of which accelerate cellular aging.

Reversing aging sounds like a dream come true for everyone. We have already understood so many mechanisms related to aging, so is it possible to find a way to slow down or even reverse aging?

In fact, our daily diet, work habits, and lifestyle all affect the epigenetic aging clock. For instance, during high-temperature cooking, red meat produces glycosylated end products, which are associated with cellular aging; poultry and fish are relatively healthy; and the vitamins in fruits and vegetables help keep cells young, which can help slow down or reverse the aging clock.

In addition, an article published in 2017 in the journal Psychoneuroendocrinology, American and French scholars investigated whether or not sitting in meditation affects the epigenetic aging clock.

The studys subjects were 18 individuals who had been meditating for at least 10 years and meditated for at least 30 minutes a day, and 20 non-meditators. They were divided into two groups: under and over 52 years of age, respectively. The researchers measured the DNA methylation in their blood cells for estimation of their epigenetic aging acceleration.

The results showed that the epigenetic aging acceleration increased in elderly non-meditators, while the acceleration in elderly meditators was more similar to that of younger people and was not affected by the epigenetic aging effect.

Gene expression is also associated with changes in our appearance, so meditators appear younger than their actual age. Furthermore, meditators also have younger brains.

The University of California Los Angeles and the Australian National University jointly published a study in 2016 in the journal NeuroImage. The studys subjects were 250 meditators and 50 non-meditators, both groups with an average age of 51.4 years.

The researchers analyzed and compared the brain ages of the two groups and found that the brain age of the meditators was younger than their actual age. For instance, 50-year-old meditators had the same brain age as a 42.5-year-old non-meditator, while 60-year-old meditators had the same brain age as the 51-year-old non-meditators in the control group.

Interestingly, for the meditators over 50, each additional year of their actual age would make their brain age one month and 22 days younger than their actual age.

In summary, damage caused by SARS-CoV-2 to the human body speeds up the human epigenetic clock of aging and dumbs down the brain. Vaccines based on the old strains in 2020 may also harm the human body in this regard.

At least 67 percent of the worlds people have now been vaccinated; will people in the future get older more quickly? No. It is too early to conclude anything based on cellular data alone rather than human studies. However, the cellular data is a clear reminder for us to take precautions to prevent this.

Meanwhile, it is quite worrisome that when we discussed this topic during a Health 1+1 live broadcast on July 12 2022, quite a few audience members had commented that they themselves or their friends or relatives had become obviously older after receiving the jabs.

The good news is that aging is a long process. During this process, for example, we can develop the habits of healthy living, including eating well, exercising,and daily meditation, which can help slow down or even reverse the aging process, and furthermore bring us various positive health benefits, which will be detailed in future articles.

Views expressed in this article are the opinions of the author and do not necessarily reflect the views of The Epoch Times. Epoch Health welcomes professional discussion and friendly debate. To submit an opinion piece, please follow these guidelines and submit through our form here.

View post: COVID-19 Can Make the Brain Age by 2 Decades; Here's 1 Way to Reverse Aging - The Epoch Times

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COVID-19 Can Make the Brain Age by 2 Decades; Heres 1 ... - Stem Cell

Green job – Wikipedia

Green jobs (green-collar jobs, sustainability jobs, eco jobs or environmental jobs[1]) are, according to theUnited Nations Environment Program, "work in agricultural, manufacturing, research and development(R&D), administrative, and service activities that contribute(s) substantially to preserving or restoring environmental quality. Specifically, but not exclusively, this includes jobs that help to protect ecosystems and biodiversity; reduce energy, materials, and water consumption through high efficiency strategies; de-carbonize the economy; and minimize or altogether avoid generation of all forms of waste and pollution." The environmental sector has the dual benefit of mitigating environmental challenges as well as helping economic growth.

Green jobs, according to theU.S. Bureau of Labor Statistics,are classified as, "jobs in business that produce goods or services that benefit the environment or conserve natural resources"[2] or "jobs in which workers' duties involve making their establishment's production processes more environmentally friendly or use fewer natural resources".[3] The Bureau of Labor Statistics categorizes green jobs into the following: water conservation, sustainable forestry, biofuels, geothermal energy, environmental remediation, sustainability, energy auditors, recycling, electric vehicles, solar power, and wind energy.[4]

These definitions include jobs which seek to use or develop renewable forms of energy (i.e. wind, hydropower, geothermal, wind, landfill gas and municipal solid waste) as well as increase their efficiency. Under the green jobs domain education, training, and public awareness are also included. These jobs seek to enforce regulations, support education, and increase public influence for the benefit of the environment.

This list is not exhaustive, it lists some of the more common environmental jobs and also some of the jobs which have see the fastest recent growth.[5]

Specialise in agricultural productivity. They study commercial plants, animals and cultivation techniques in order to improve the productivity and sustainability of farms and agricultural industries[6] and[7] Agricultural scientists have a higher-than-average proportion of full-time jobs and earnings are above average.[8]

Research and present data on the structure and dynamics of our climate system.[9] Currently there is scientific consensus from a number of American scientific societies that the earth's temperature is warming.[10]

Advance and ensure protection of the natural environment and resources via educating communities and encouraging involvement, and awareness.[11] The growth of these jobs along with forester jobs in the US is predicted to be around 6 per cent (in line with average occupation growth) from 2016 to 2026[12]

Investigate ecosystems as a whole i.e. they investigate both the living and non-living components of the environment. They study the various animals and plants that live within an ecosystem and the relationship between the two.[13][14]

Use science and maths to design and develop electric automobile technology. They then undertake evaluations with respect to measure the safety, efficiency, cost, reliability, and safety of these aforementioned designs.[15] An Electric Car Engineer is just one of a number of possible jobs in the electric vehicle industry and this type of Engineer will work in teams with other types of Engineers to produce electric automobiles.[16]

Examine and mitigate the effects of human and other activities on both the natural and built environment. This could include reducing pollution and protecting the air, water, soil, and humans from actions which may harm either them or the environment.[17] According to CNBC, in 2018 across the world, this was one of the fastest growing environmental jobs.[18] The University of Portland recently reported that they will add a clean energy technology minor to enable their environmental engineering graduates compete in an expanding environmental job market.[19]

They examine the environment (for example by sampling the land, water, air or other natural resources) and develop policies and plans designed to prevent, control or reduce the harmful effects of human activity on the environment.[20][21] Also one of the fastest growing environmental jobs in the world in 2018 according to CNBC.[22]

Analyse and provide advice on policies and processes which guide the design, implementation and modification of either commercial or government environmental operations and programs.[23] Environmental consultants are often employed to ensure environmental legislation is being adhered to during construction projects.[24] Listed as one of the top ten fastest growing green jobs in Australia in 2018.[25]

Measure risk and develop, oversee, implement and monitor legislation which governs public health for both the built and natural environment. Environmental health officers carry out these aforementioned duties to promote good human health and best practices environmentally.[26] and.[27] Also one of the fastest growing environmental jobs in Australia in 2018.[28]

Supervise the environmental performance of private companies and public institutions. They also formulate, execute and oversee environmental strategies that encourage sustainable development.[29] An environmental manager can be employed by a single company to ensure any negative environmental impacts caused by their operation are minimised.[30]

In a nutshell they are responsible for the cultivation of forests.[31] Map out and lead the planting, growth, harvesting and conservation of forests for wood production. To ensure balance and sustainable development Foresters may become involved the production of multipurpose forests, sustainable forest management and the reforestation of native woodlands.[32][33] The growth of these jobs along with conservation officer jobs in the US is predicted to be around 6 per cent (in line with average occupation growth) from 2016 to 2026[34]

According to the Food and Agriculture Organization of the United Nations, forests provide more than 86 million green jobs and support the livelihoods of many more people.[35] An estimated 880 million people worldwide spend part of their time collecting fuelwood or producing charcoal, many of them women.[35] Human populations tend to be low in areas of low-income countries with high forest cover and high forest biodiversity, but poverty rates in these areas tend to be high.[35] Some 252 million people living in forests and savannahs have incomes of less than USD 1.25 per day.[35]

Design buildings (they can be homes, offices, schools, hospitals, or any other type of building) that in their design, construction or operation, reduce or eliminate negative impacts, and can create positive impacts, on our climate and natural environment.[36] They also try to reduce negative environmental impacts in terms of reducing the contributions to landfill.[37]

Analyse the interplay of marine life (animals and plants) with coastal areas and the atmosphere.[38] and [39] Crucial today is their role in measuring the impact of climate change on our oceans and how much ocean acidification is present and potentially damaging our coral reef ecosystems.[40]

In a typical recycling plant (MRF), the workers are sort labourers, equipment operators, managers (various levels) and maintenance mechanics. Also included are drivers who collect the recyclate from residential, commercial, and non-hazardous industrial sources. Sort labourers 'clean' the stream at various stages of the mechanized sortation process to prevent equipment stoppages and produce bales acceptable to the remanufacturing sector depending on the commodity (metal, paper, cardboard, plastics, etc.). Equipment operators operate heavy equipment designed to help with processes around the plant. Typical equipment includes skid-steers (For cleaning up and moving large amounts of material to bale), forklifts (to move bales and containers of material to places within the plant collected from the sortation process). Managers typically manage the operations/sales and human resources of the MRF so the sortation process requiring sorters and equipment operators runs properly day to day. Maintenance mechanics work to ensure all equipment works properly (such as unjamming a conveyor or fixing electronic equipment associated with the sortation process).[41]

Study how to best supply energy from renewable or sustainable sources of energy, such as wind energy, solar power wave energy and biofuels.[42] They focus on ways of producing energy that are deemed to be safer for the environment.[43] An energy engineer was listed as one of the fastest growing jobs in Australia in 2018.[28]

Assemble and carry out the installation of solar panels on rooftops or other areas such as ground mounted solar panels.[44] A growing industry for example, has seen job creation and on the job training by a non-profit called GRID Alternatives.[45]

Responsible for growing food in a city or heavily populated town.[46] Green roof tops can provide locally sourced foods that help protect the environment by reducing the use of pesticides, fossil fuels, and other resources which are often used to grow and transport food to market from larger commercial farms.[47]

Ensure that minimum standards of water quality are met and that these standards ensure human safety and minimise harm to the natural environment. Water quality scientists ensure that these global standards and other compliance requirements are met in three areas - groundwater, surface water (lakes, rivers, ponds, etc.), and drinking water.[48] "The fact that water is the lifeblood of our planet means that there are thousands of opportunities for environmental workers in this area".[49]

Wind technicians install, inspect, maintain, operate and repair wind turbines. Wind technicians have the knowledge to identify and fix issues that could cause the turbine to be break or fail to operate as it should.[50] Globally one of the fastest growing environmental jobs in 2017.[51] The U.S. Department of Energy is working with six leading wind turbine manufacturers towards achieving 20 per cent wind power in the United States by 2030. However, the dropping number of students in power engineering programs in recent years means that, the labour requirements needed to facilitate this aim won't be met, unless this trend is reversed.[52]

Eco-innovation drives the creation of environmental jobs worldwide.[53] It simultaneously increases labour productivity and wages while increasing energy and environmental production efficiency.[53]

In 2018 Australia generated 21 per cent of its total power from renewables and this sector accounted for more than 20,000 jobs.[54]

According to the International Renewable Energy Agency (IRENA) in 2016, Brazil has 934,000 renewable energy jobs, the second highest in the world. While Brazil is the global leader in liquid biofuels with a total of 845,000 jobs, it also has 41,000 jobs in solar, 36,000 jobs in wind, and 12,000 jobs in small hydro power.[55] A report produced by IRENA in 2018 showed Brazil to have the largest liquid biofuel workforce,[56] an 893,000 workers in the overall renewable energy industry.[56] In 2011, green employment accounted for 3.1 million jobs or 2.4 per cent of total employment in 2010 and 3.4 million jobs or 2.6 per cent of total US employment.[57]

China currently produces the most Photovoltaic equipment worldwide and is the world's largest installation market. With respect to employment China accounted for about two thirds of PV employment worldwide, or some 2.2 million jobs in 2018.[56] With respect to total jobs in the renewable energy sector as a whole the number for China was 3.8 million in 2017.[56]

Was the leading installer of PV Capacity Installations.[58] until overtaken by China, The United States, India and Japan.[59] In 2018 Germany had 332,000 workers in the renewable energy sector overall.[56]

The Thought Leadership Series by the Copenhagen Climate Council published a report in 2009, stating that Japanese solar PV manufacturers represent 26 per cent of the global market and that the solar industry is able to operate without dependence on subsidies.[60] According to a report by the International Renewable Energy Agency, Japanese solar PV jobs increased by 28 per cent in 2014.[34] In 2016, Japan was listed as the third largest employer of solar PV jobs with 377,100 workers, based on direct and indirect labour.[34] In terms of renewable energy, Japan employs 3,000 jobs in liquid biofuels, 5,000 jobs in wind power, 700 jobs in solar cooling and heating, and 2,000 jobs in geothermal energy.[34] In 2018 Japan's slowing economy meant that employment in the solar PV industry fell from 302,000 in 2016 to an estimated 272,000 jobs in 2017.[56]

In 2010 Green Goods and Services survey found there are 3.1 million Green Goods and Services (GGS) jobs in the United States which accounts for 2.4 per cent of all United States salary and wage employment.[61][62] The private sector had 2.3 million GGS jobs, and the public sector had 860,000 GGS jobs.[62] From 2010 the data indicates that green jobs are continuing to grow rapidly in the United States. The US is currently undergoing an energy revolution from coal fire power plants to renewable energy. The majority of these additions are coming from three main resources: solar (9.5 GW), natural gas (8 GW), and wind (6.8 GW).[63] Together, these three sources make up 93 per cent of total additions.[63] The shift from fossil fuels to renewables will be mirrored by US employment as workers turn away from jobs like coal mining and towards green jobs.[63] This is made evident by a report published by the Bureau of Land Management published 17 April 2017 that states wind turbine service technicians are currently and projected to continue to be the fastest growing profession in the United States between 2017 and 2024 with projected growth of 108.0 per cent[64]

President Reagan said, "Trees cause more pollution than automobiles do."[65] As governor of California, Reagan advocated on behalf of the environment; a large portion of Californian constituents were pro-environment.[66] It states in the book The Enduring Wilderness, "President Ronald Reagan signed more wilderness laws than any other president - forty-three laws designating 10.6 million acres of wilderness in thirty-one states."[67] President Reagan also set a new precedent as president by leasing over twenty million acres of national land for coal, oil, and gas development.[66]

The Business Energy Investment Tax credit is a federal policy introduced in 2005 under the Bush administration to promote implementation of green energy sources through a 30 per cent federal tax return in both residential and commercial projects. Individuals and companies were able to apply credit for investments in green energy technologies including solar, fuel cell and wind energy technology[68] The ITC has been extended multiple times, most recently in 2015 through a multi-year extension that will maintain the 30 per cent return up until 2019, afterward decreasing to 26 per cent until 2020 and 22 per cent until 2021. After 2021, commercial credits would reduce to 10 per cent and 0 per cent for residential projects. The Solar Energy Industries Association has attributed stability in the growth of solar energy industries in the U.S. to the implementation of the ITC since 2006 [69] Since the implementation of the ITC, the U.S. solar industry has experienced growth in implementation of solar technology, mainly due to the rapidly decreasing overhead costs as the solar industry was spurred to production and development through the ITC.[70] The solar industry is projected to employ over 420,000 individuals by 2020- nearly double of the 260,000 solar workers in 2016- and contribute $30 billion to the United States economy annually.[71]

President Obama campaigned under the promise of creating 5 million new green jobs in the United States.[72] President Obamas plan included the American Clean Energy and Security Act of 2009 (ACES) proposed a cap and trade system which would bring in revenue that would be used to invest in clean energy technology creating 5 million new jobs[73] The bill was passed through the house but never made it to the senate floor and therefore was never written into law. Secondly, due to the 2013 Balanced Budget and Emergency Deficit Control Act the federal government "discontinued measuring all green jobs" which makes tracking job growth extremely difficult.[73]

Although it is unclear if President Obama met his 5 million jobs goal, there was significant growth under his administration. As of March 2016 according to a nonpartisan group, Environmental Entrepreneurs, there were 2.5 million jobs in clean energy with 77,088 jobs solely in the wind industry.[73] During this period of time employment in the solar field was also on the rise. According to the 2015 National Solar Census 2015 marked the third consecutive year in which solar growth was at 20 per cent.[74]

Additionally, the American Recovery and Reinvestment Act (ARRA), passed in early 2009, includes provisions for new jobs in industries such as energy, utilities, construction, and manufacturing with a focus toward energy efficiency and more environmentally-friendly practices.[75][76]

In March 2009, U.S. President Barack Obama appointed Van Jones as Special Advisor for Green Jobs, Enterprise and Innovation at the White House Council on Environmental Quality (CEQ). Following Jones' resignation in September 2009, no further candidates appear to have been appointed to this position.

On 23 January 2017 President Trump signed an executive order, "Presidential Memorandum Regarding the Hiring Freeze", regarding a hiring freeze on government positions across the executive branch.[77] Trump placed a hold on grants distributed through the EPA that could amount to $4 billion per year. The measure was recanted days later, but Trump has proclaimed his intent to "drastically cut the EPA". Myron Ebell, a former member of the Trump transition team, when asked about United States Environmental Protection Agency cuts in an interview with Associated Press, responded "Let's aim [to cut] for half and see how it works out, and then maybe we'll want to go further."

In the 2018 "Make America Great Again Blueprint", the Trump administration projected EPA funding cuts of 31 per cent and discontinued funding for the Clean Power Plan, international climate change programs, and climate change research and partnership programs.[28]

In 2008 the United Nations Environment Programme (UNEP), the International Labour Organization (ILO), the International Trade Union Confederation (ITUC), and the International Employers Organization (IEO) jointly launched the Green Jobs Initiative. The purpose is to bring a just transition to a green economy by providing space for workers, employers, and governments to negotiate on policy effective in providing equitable opportunity to green jobs.[78]

$8 million was invested to produce and measure data on green-collar Jobs and green economic activity through the Department of Labor, the Bureau of Labor Statistics, and Federal agencies (Environmental Protection Agency, Department of Energy and Commerce, Employment and Training Administration). Methods on the approach target business that produce green goods and services and include special employer surveys, aggregate data gathering on employment and wages, and tabulations that distinguish between occupation and industry.[81]

Data collection and upkeep on Green Goods and Services (GGS) jobs has been discontinued due to the Balanced Budget and Emergency Deficit Control Act in 2013. All "measuring green jobs" programs in the US government were eliminated by this Act.[82]

The Green Jobs Act of 2007 (H.R. 2847), introduced by Reps. Hilda Solis (D-CA) and John Tierney (D-MA), "authorized up to $125 million in funding to establish national and state job training programs, administered by the U.S. Department of Labor, to help address job shortages that are impairing growth in green industries, such as energy efficient buildings and construction, renewable electric power, energy efficient vehicles, and biofuels development."[83] The Energy Independence and Security Act passed in December 2007 incorporates the Green Jobs Act of 2007.

Pathways out of Poverty (POP) is a national workforce training program that was established on 14 August 2009 by the Obama administration and funded by the American Recovery and Reinvestment Act (ARRA) of 2009. POP targets individuals living below or near the poverty level to provide them with skills needed to enter the green job market, focusing on the energy efficiency and renewable energy industries. The training programs focus on teaching basic literacy and job readiness skills. Some of the programs also provide supportive assistance with childcare and transportation to overcome barriers to employment.[84]

(MTTP) provides job training opportunities specifically to low-income, job insecure 18-24 year-olds who have a high school degree or GED. In 2009, secure full-time salaries of twice the New York State minimum wage of $7.25 were provided to graduates of MTTP by a grant from the US Forest Service. Out of the 16 employed graduates that were interviewed for a study by USDA Forest Service Northern Research Station, 75 per cent were male, 25 per cent were female, 81 per cent were black, 19 per cent were brown, 75 per cent had a high school diploma, 19 per cent had a GED, and 6 per cent went to some high school. Most employees with personal support who graduate from MTTP stay in their green job; not all employees have personal support networks.[85]

According to the Green Equity Toolkit by Race Forward, green jobs are disproportionately occupied by white men.[86] Historically, the environmental movement has been white, middle- and upper-class.[87] In 1990, minorities consisted of 1.9 per cent (14 out of the 745) of workers for four of the largest environmental organizations (Natural Resources Defense Council, Friends of the Earth, Audubon Society, and Sierra Club); out of sixty-three mainstream environmental organizations, 32 per cent had no minorities staffed, 22 per cent had no board members of colour, 19 per cent had no volunteers of colour, 16 per cent had no members of colour.[47] According to a journal in the Ecology Law Quarterly published in 1992, white people disproportionately occupy green jobs since said jobs address environmental concerns not confronted by low-income people and people of colour.[22]Environmental lawyers (who are disproportionately white, middle- and upper-class) focus on environmental issues based on aesthetics, recreation, and protecting natural lands outside of their communities; they often do not face environmental problems in their communities.[22]Low-income communities and people of colour who face environmental problems, such as pollution, do not often have access or will to seek green jobs due to the immediate health hazards in their communities. Instead of green jobs, they often engage in grassroots environmental activism to prevent mortality in their communities from toxicities, such as superfund sites, landfills, incinerators and other health hazards.[22]

A report published in 2014 titled, The State of Diversity in Environmental Organizations, states there has been increasing racial diversity over the past 50 years, but at a disproportionately slow rate. People of colour consist of 38 per cent of the US population and do not exceed 16 per cent of the staff of the environmental organizations studied (191 conservation and preservation organizations, 74 government environmental agencies, 28 environmental grant-making foundations). Employed ethnic minorities disproportionately occupy lower-ranked positions in environmental organizations and fewer than 13 per cent occupy leadership positions. A small number of environmental organizations have a diversity manager, diversity committee, or collaborate with low-income or ethnic organizations. Environmental organizations rarely recruit from minority-serving institutions, minority professional gatherings, and other pipelines with talented minorities. Minority interns to environmental organizations are hired less often than their white counterparts. Promotions often go to white females in environmental organizations.[88]

The National Council for Workforce Education and AED published a report, "Going Green: The Vital Role of Community Colleges in Building a Sustainable Future and Green Workforce" that examines how workforce education and community colleges contribute to the overall efforts in the move toward renewable and clean energy. The report gives examples of initiatives currently in effect nationally as well as offering information as to how to implement programs.

The nuclear power industry is also growing and contributing to the green job sector. The World Nuclear Association released a report in July 2020 titled "Employment in the Nuclear and Wind Electricity Generating Sectors". Overall, the report found that a 100 GWe nuclear fleet will employ perhaps more than three times as many workers as a wind fleet of the same capacity.[89] These statistics highlight growing opportunities for green jobs in the nuclear industry. Green jobs are attractive options as they directly address the climate crisis while also providing competitive pay and good benefits. Nuclear power specifically has the potential of creating thousands of high skilled, long term, local jobs.[90]

In response to high unemployment and a distressed economy workers need skills that are relevant to their specific geographical locations. "Instead of making green jobs we need to make jobs green" says Ken Warden, an administrator in workforce education.[citation needed]

There are many different solar industry jobs.[91] The SEIA maintains a resource for those looking for solar jobs.[92] A 2016 study indicates that the declining coal industry could protect their workers by retraining them for the solar industry.[93] There are also some indications that the solar industry "welcomes coal workers with open arms".[94]

For the forest sector, the Team of Specialists (ToS) from the Food and Agriculture Organizations of the United Nations (FAO) and the United Nations Economic Commission for Europe (ECE) mapped out potential green jobs in the forest sector.[95] The ToS identified 19 fields of activities with 30 examples of forestry jobs listed.

See also

This article incorporates text from a free content work. Licensed under CC BY-SA 3.0 IGO License statement/permission. Text taken from The State of the World's Forests 2020. Forests, biodiversity and people In brief, FAO & UNEP, FAO & UNEP. To learn how to add open license text to Wikipedia articles, please see this how-to page. For information on reusing text from Wikipedia, please see the terms of use.

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Green job - Wikipedia

Annealing Temperature of 55C and Specificity of Primer Binding in PCR …

1. Introduction

Since polymerase chain reaction (PCR) was invented in the mid-1980s, it has made its way into all molecular biology, genetic, microbiology or biochemistry laboratories, where it is, due to its simplicity and efficiency, used in a very wide range of (PCR)-based techniques and applications [1, 2]. In just a few hours with a certain amount of cycles consisting of three simple stepsDNA denaturation, annealing of primers and extension [2]the desired DNA sequence is multiplied about a million fold [3]. The crucial step in PCR is the annealing of primers, where the annealing temperature determines the specificity of primer annealing.The annealing temperature of a standard PCR protocol is either 55C [2, 3] or 60C [4]. The chosen temperature depends on the strand-melting temperature ofthe primers and the desired specificity. For greater stringency higher temperatures are recommended [2].

PCR is very often used to amplify specific DNA fragments that are later cloned as inserts in plasmid vectors and used then in subsequent experiments. Examples of such subsequent experiments are nucleotide sequencing, in order to determine the nucleotide sequence of the insert or invitro transcription, and translation, in order to obtain a certain protein.

In our experiments, the aim was to determine the nucleotide sequence of several fimbrial genes from different Escherichia coli(E. coli) strains isolated from faecal samples of dogs with diarrhoea. The genes of interest were papA, papG, papEFof the P-fimbriae and F17Gof the F17-fimbriae. Therefore, from a collection of 24 clinical haemolytic E. colistrains from faecal samples of dogs with diarrhoea [5], genomic DNA was isolated and used as the matrix DNA to amplify these genes of interest with gene-specific primers with PCR.Further, the obtained PCR products were cloned into a TA cloning vector, and the nucleotide sequence was determined.

Escherichia coliis one of the best studied organisms. It belongs to the family of Enterobacteriaceae. It is a Gram-negative rod-shaped bacterium, non-sporulating, nonmotile or motile by peritrichous flagella, chemoorganotrophic, facultative anaerobic, producing acid from glucose, catalase positive, oxidase negative and mesophilic [6].

It is a well-known commensal bacterium that is part of the gut microbiota of humans and other warm-blooded organisms. However, also pathogenic strains of E. colido exist and can cause a variety of intestinal and extraintestinal infections in humans and many animal hosts. E. coliis considered to be one of the most important pathogens; it is the most frequently isolated species in clinical microbiology laboratories [7]. Intestinal pathogenic E. coli(IPEC) strains, also called diarrhoeagenic E. coli(DEC) strains, are divided into six different well-described categories, i.e. pathotypes: enteropathogenic E. coli(EPEC), enterohaemorrhagic E. coli(EHEC), enterotoxigenic E. coli(ETEC), enteroaggregative E. coli(EAEC), enteroinvasive E. coli(EIEC) and diffusely adherent E. coli(DAEC) [8]. DEC causes diarrhoea syndromes that vary in clinical presentation and pathogenesis depending on the strains pathotype [7]. E. colistrains involved in diarrhoeal diseases are one of the most important among the various etiological agents of diarrhoea [9]. The extraintestinal pathogenic E. coli(ExPEC) strain group is comprised of different E. coliassociated with infections of extraintestinal anatomic sites [10]. Traditionally, the ExPEC isolates are separated into groups determined by disease association, i.e. uropathogenic E. coli(UPEC), neonatal meningitis-associated E. coli(NMEC) and sepsis-causing E. coli(SEPEC), naming the most important ExPEC groups. But ExPEC strains are also implicated in infections originating from abdominal and pelvic sources (e.g. biliary infections, infective peritonitis and pelvic inflammatory disease) and also associated with skin and soft tissue infections and hospital-acquired pneumonia [11]. Due to its genotypic and phenotypic diversity, E. coliis known as the paradigm for a versatile bacterial species [12].

The pathogenic strains possess specialised virulence factors such as adhesins, toxins, iron acquisition systems, polysaccharide coats and invasins that are not present in commensal strains [7].

Adhesins play a very important role in the host-microbe interactions, as they convey the adherence to the epithelial hosts cells, surface structures or molecules. Adhesion is the essential first step for most commensal and pathogenic bacteria in order to colonise and persist within the host [13]. While adhesion to abiotic surfacesis usually mediated by non-specific interactions, adhesion to biotic surfaces typically involves specific receptor-ligand interaction [14]. Adhesins are structures on the bacterial surface that help the bacteria to bind to receptors on hosts cells (Figure 1).

Scanning electron microscopy ofEscherichia colistrain 963 adhering to 19-day-old Caco-2 cells [5]. The fimbrial structures on the bacterial surfaces are promoting the bacterial adherence to receptors on host cells.

Adhesins are not just involved in adherence but also in bacterial invasion, survival, biofilm formation, serum resistance and cytotoxicity [15]. Moreover, they are also involved in bacterial motility and DNA transfer [13]. They differ in their architecture and receptor specificities. Types of adhesin vary depending on the Gram nature of bacteria [15].

Adhesins are among the most important virulence-associated properties of E. coli, as they are the main virulence factors of bacteria needed in bacterial colonisation. There are two types of bacterial adhesins: fimbrial and afimbrial [16].

Fimbrial adhesins, i.e. fimbriae, are rodlike structures with a diameter of 57nm. Each fimbria consists out of several hundred copies of a protein, whose generic name is major subunit, and other proteins, present in one or a very few copy number and called minor subunits that are positioned either at the basis or at the top of the fimbriae or intercalated between the major subunits [16]. Fimbriae can be even longer than 1m [13]. On the bacterial surface of wild-type E. colistrains, there are around 500 fimbriae [17]. P-fimbriae and F-17 fimbriae belong to the fimbrial adhesins.

Non-fimbrial adhesins are monomeric or trimeric structures that decorate the surface of bacteria. These adhesins are anchored to the surface of the outer membrane and due to their small size, the size of non-fimbrial adhesins is approximately 15nm, allow an intimate contact between the bacterial cell surface and specific substrates. One of the major classes of non-fimbrial adhesins is autotransporter adhesins [13].

P-fimbriae are the most extensively studied adhesins. They are also the first virulence-associated factor found among uropathogenic E. coli. These fimbriae bind to Gal(14)Gal moieties of the membrane glycolipids on human erythrocytes of the P blood group and on uroepithelial cell fimbriae [18]. Further receptors for P-fimbriae are present on erythrocytes from pigs, pigeon, fowl, goats and dogs but not on those from cows, guinea pigs or horses [19]. These fimbriae are encoded in the papoperon, consisting of 11 different genes (see Figure 2A): papA(558bp), papB(315bp), papC(2511bp), papD(720bp), papE(522bp), papF(504bp), papG(1008bp), papH(588bp), papI(234bp), papJ(582bp) and papK(537bp) [20].

Scheme ofpapandF17operon and annealing sites of the used primers. Genes in the operon are presented as boxes. The positions of used primers to amplify the studied genes are marked with arrows. (A) Scheme ofpapoperon. The scheme ofpapoperon was drawn based on the GenBank deposited nucleotide sequence X61239.1 [20] and (B) scheme ofF17operon. The scheme ofF17operon was drawn based on the GenBank deposited nucleotide sequence L77091.1 [26].

The product of the papAgene is the major subunit protein A (19.5kDa) [19]. In papBa regulatory protein (13kDa) is encoded. PapB is necessary for the activation of the papAexpression [21]. PapC (80kDa) is located in the outer membrane and forms the assembly platform for fimbrial growth. PapD (27.5kDa) is present in the periplasmic space and is involved in the translocation of fimbrial subunits across the periplasmic space to the outer membrane prior to assembly. PapE (16.5kDa), PapF (15kDa) and PapG (35kDa) are minor fimbrial components. PapG is the adhesin molecule conferring the binding specificity [19]. PapH (20kDa) terminates fimbrial assembly and helps anchor the fully grown fimbriae to the cell surface [22]. PapI (12kDa) is another regulatory protein involved in papAexpression due to activation of papBpromoter [21]. PapJ (18kDa) is a periplasmic protein required to maintain the integrity of P-fimbriae [23]. PapK (20kDa) regulates the length of the tip fibrillum and joins it to the rod [24].

Many variants of P-fimbriae exist. PapA molecules from different P-fimbrial serovariants have a high degree of similarity at the N and C termini, while the central portions of PapA exhibit a great variation in the primary structure. This central part of PapA is hydrophilic and exposed and hence under selective pressure from the host immune system. Substantial heterogeneity is also between different minor fimbrial subunits (PapE, PapF and PapG) [19]. In addition also P-fimbria-related fimbriae, the so-called Prs-fimbriae, exist. Prs-fimbriae are encoded in the prs(pap-related sequence) operon [18].

F17-fimbriae are found on pathogenic E. colistrains, isolated from infections in domestic animals. They are mainly detected on bovine and ovine E. coliassociated with diarrhoea or septicaemia but also on E. colifrom other hosts, including humans. The F17 adhesin binds to N-acetyl-d-glucosamin receptors of bovine intestinal cells; however, F17 subtypes were also found to bind to N-acetyl-d-glucosamin receptors of human uroepithelial and intestinal cells [25]. The F17-fimbriae are encoded in the F17operon, consisting of four genes: F17A(546bp), F17D(723bp), F17C(2469bp) and F17G(1035bp) (see Figure 2B) [26].

F17A protein (20kDa [25]) is the structural component of the F17-fimbriae (major subunit protein). The F17A protein is homologous to PapA protein of the P-fimbriae [27]. F17C protein (90kDa) probably functions as a base protein on which the fimbrial subunits are polymerised. F17D protein (28kDa) has a close homology to the PapD protein of the P-fimbriae [28]. It functions as the periplasmic transport protein [29]. F17G protein (36kDa [25]) is the minor fimbrial component required for the binding of the F17-fimbriae to its receptor on the host cell [30].

Several variants of F17-fimbriae exist. The diversity is based on differences in F17A and F17G genes. The variant of F17-fimbriae found in humans is designated as G-fimbriae, encoded in the gafoperon [25].

The analysed 24 clinical haemolytic E. colistrains [5] originated from dogs with diarrhoea and were isolated at the Veterinary Microbiological Diagnostics Centre of Utrecht University, the Netherlands. Some more details about the strains are given in Table 1. As positive control strains, a dog uropathogenic E. colistrain (strain 1473) and a cattle mastitis E. colistrain (strain E5) from Wim Gaastras E. colicollection were used [31].

Characteristics of the 24 studied E. colistrains [5, 31].

MSHA is an abbreviation of mannose-sensitive haemagglutination, and MRHA is an abbreviation of mannose-resistant haemagglutination. The erythrocytes are abbreviated as follows: B, bovine erythrocytes; E, equine erythrocytes; C, canine; O, ovine; P, porcine. NT, non-typable.

All used bacterial strains were stored at 80C as a suspension in a 1:1 mixture of L-broth and glycerol as published by Garcia etal. [32]. The strains were grown overnight on LB plates and in liquid LB medium at 37C.When grown in liquid LB medium, the flasks with the bacterial culture were incubated with aeration.

Chromosomal DNA was isolated from all 24 clinical haemolytic E. colistrains [5] and strains used for positive controls [31] using a slightly modified protocol based on the protocol of miniprep of bacterial genomic DNA published by Ausubel etal. [33]. To summarise, 2ml of an overnight bacterial culture was centrifuged for 2min at 14,000rpm at room temperature. The obtained bacterial pellet was resuspended in 567l of buffer TE and 6l of 0.5M EDTA.The suspension was incubated for 15min at 80C.Following the incubation at 80C, the suspension was thawed, and 10l of 25mg/ml proteinase K solution was added. The suspension was mixed thoroughly, and 30l of 10% SDS was added to the suspension and mixed thoroughly again. A 2-hour incubation at 37C followed, and then 100l of 5M NaCl was added to the suspension and mixed thoroughly. Next 80l of CTAB/NaCl was added, mixed thoroughly again and incubated at 65C for 10min. After the incubation the suspension was treated with 200l of chloroform/isoamyl alcohol and centrifuged for 5min at 14,000rpm at room temperature. The aqueous supernatant was transferred to a fresh microcentrifuge tube and treated with 100l of phenol/chloroform/isoamyl alcohol and centrifuged for 5min at 14,000rpm at room temperature. The aqueous supernatant was transferred to a fresh microcentrifuge tube, and the DNA in the aqueous supernatant was precipitated with addition of 0.6 volume of isopropanol. The precipitated chromosomal DNA was transferred to a fresh microcentrifuge tube containing 100l of 70% ethanol. The precipitated DNA in 70% ethanol was pelleted with centrifugation (10min at 14,000rpm at room temperature). The 70% ethanol was then removed and the chromosomal DNA pellet air-dried at 37C.Finally the chromosomal DNA pellet was dissolved in 100l of sterile distilled water.

One l of the isolated chromosomal DNA was used in 50l PCR mixtures consisting of 20pmol of each primer, 0.2mM dNTP mixture and 0.625U of Taq-polymerase in PCR buffer [5]. In PCRs for P-fimbrial genes for positive control samples, the isolated chromosomal DNA of the dog uropathogenic E. colistrain (strain 1473) was used. In PCRs for F17-fimbrial gene for positive control samples, the isolated chromosomal DNA of the cattle mastitis E. colistrain (strain E5) was used. In all PCRs for the negative control, sterile distilled water was used [31].

Primers used in the PCRs to amplify the studied genes are listed in Table 2.

Primers and their melting temperatures (Tm) used to amplify the studied genes.

Predicted primer annealing sites of the used primers on the target operons are shown in Figure 2.

The PCR amplification in all the reactions for all studied genes was carried out in the following steps: heating at 94C for 4min, followed by 35cycles of denaturation at 94C for 1min, annealing at 55C for 1min, extension at 72C for 1min and the final extension for 10min at 72C.

The expected sizes of PCR products were determined with the Primer-BLAST online tool (data set nr organism Escherichia coli) on the Internet page of the National Center for Biotechnology Information, US National Library of Medicine (http://www.ncbi.nlm.nih.gov) as follows: papA552bp (GenBank deposited nucleotide sequence LR134092.1), 555bp (GenBank deposited nucleotide sequence CP025703.1), 534bp (GenBank deposited nucleotide sequence CP018957.1), 564bp (GenBank deposited nucleotide sequence CP029579.1) and 561bp (GenBank deposited nucleotide sequence CP024886.1); papEF1372bp (GenBank deposited nucleotide sequence CP027701.1), 1373bp (GenBank deposited nucleotide sequence CP028304.1), 1367bp (GenBank deposited nucleotide sequence CP026853.1) and 1371bp (GenBank deposited nucleotide sequence LR134238.1); papG1000bp (GenBank deposited nucleotide sequence CP026853.1) and 1003bp (GenBank deposited nucleotide sequence M20181.1); and F17G888bp (GenBank deposited nucleotide sequence AF055313.1) and 885bp (GenBank deposited nucleotide sequence CP001162.1).

Samples of isolated chromosomal DNA (5l of isolated chromosomal DNA and 1l of 6loading dye) were subjected to analysis with agarose gel electrophoresis using 1% of agarose gels with 0.5g/ml ethidium bromide, run in 0.5TBE electrophoresis buffer. Samples of obtained PCR products (25l of PCR products, 5l of 6loading dye) were subjected to analysis with agarose gel electrophoresis using 1% of agarose gels with 0.5g/ml ethidium bromide, run in 1TAE electrophoresis buffer. Used protocols for agarose gel electrophoresis were based on Sambrook etal. [34]. For DNA ladder the lambda bacteriophage DNA digested with the restriction endonuclease PstI was used.

Cloning of PCR products and DNA sequencing of cloned PCR products obtained in the PCRs for P- and F17-fimbrial genes was done as described by Stari etal. [5]. In short, obtained PCR products were cut out of the agarose gel, cleaned with the GeneClean II Kit, inserted into the TA cloning vector pMOSBlue and then transformed to electrocompetent E. colipMOSBlue cells. Subsequently, the plasmid DNA was isolated from pMOSBlue cells using the FlexiPrep Kit, and the nucleotide sequence was determined with the dideoxynucleotide chain termination method using an automated laser fluorescence sequencer. All procedures were performed according to the manufacturers protocols.

Sequence analysis of the cloned fragments, originated from PCR products obtained in PCRs for P- and F17-fimbrial genes, was performed with the computer program BLAST on the Internet page of the National Center for Biotechnology Information, US National Library of Medicine (http://www.ncbi.nlm.nih.gov) searching for homology in the GenBank nr database.

An annealing temperature of 55C was used in the PCRs for the amplification of the papAgene with primers 22 and 23. The obtained PCR products were all of the expected size (around 600bp). However, the nucleotide sequence analysis of the eight obtained cloned PCR products revealed that six clones harboured false, non-papAinserts. Four of these false clones derived from amplification of part of methylisocitrate lyase gene, and two clones derived from amplification of part of the RNA-binding protein Hfq gene and part of the GTPase HflX gene, as revealed by BLAST analysis. In both cases even though both primers, forward primer 22 and reverse primer 23, were added to the PCR mixture, the primer 22 was used as the forward but also the reverse primer. Further nucleotide analysis revealed that in the case of the amplification of part of the methylisocitrate lyase gene, the forward primer annealed downstream from the c348059 position of the 3 5 strand and reverse primer upstream of the 348539 position on the 5 3 DNA strand of the E. coliK-12 MG1655 sequence as deposited in the CP025268.1 nucleotide sequence [35]. The anticipated annealing sites for non-specific papA-primer binding in this case are presented in Figure 3.

Anticipated annealing sites for non-specificpapA-primer binding in the methylisocitrate lyase gene. The shown sequences are enumerated according to the CP025268.1 GenBank deposited sequence [35]. The sequence and the complement chromosomal sequence are given. For the forward primer annealing site, the sequence from 348033 to 348059nt is shown, and for the reverse primer annealing site, the sequence from 348539 to 348565nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR.The methylisocitrate lyase gene is positioned in the deposited sequence from 347733 to 348623nt.

In the case of the amplification of part of the RNA-binding protein Hfq gene and part of the GTPase HflX gene, the forward primer annealed downstream from the c4402446 position on the 3 5 DNA strand, and the reverse primer annealed upstream from the 4402903 position on the 5 3 DNA strand of the E. coliK-12 MG1655 sequence as deposited in the CP025268.1 nucleotide sequence [35]. The anticipated annealing sites for non-specific papA-primer binding in this case are presented in Figure 4.

Anticipated annealing sites for non-specificpapA-primer binding in the RNA-binding protein Hfq gene (forward primer) and GTPase HflX gene (reverse primer). The shown sequences are enumerated according the CP025268.1 GenBank deposited sequence [35]. The sequence and the complement chromosomal sequence are given. For the forward primer annealing site, the sequence from 4402419 to 4402446nt is shown, and for the reverse primer annealing site, the sequence from 4402903 to 4402934nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR.The RNA-binding protein Hfq gene is positioned in the deposited sequence from 4402214 to 4402522nt, and the GTPase HflX gene is positioned from 4402598 to 4403878nt.

In the PCRs for the papEFamplification, also the annealing temperature of 55C was used. Seven PCR products, all of the expected size, of around 1400bp, were cloned, and the obtained insert sequences were analysed. All seven clones harboured the amplified papEF-related sequence, the prsEFsequence of the Prs-fimbriae (GenBank X61238.1 [36]); however, in all seven cases, only the forward POP primer annealed to the correct complementary sequence from c27 to c48 nt on the 3 5 DNA strand of the X61238.1, while the reverse primer was not as expected the BAD primer but again the POP primer, which annealed at another partially complementary sequence of the prsEFgene from 1357 upstream on the 5 3 DNA strand. Further BLAST analysis showed that the BAD primer has only a partial complementary region of nine nucleotides at the position 3229 to 3237in the 5 3 DNA strand and at the position c3238 to c3230in the 3 5 strand of the X61238.1 sequence. The anticipated annealing sites of POP primer on the analysed X61238.1 nucleotide sequences are presented in Figure 5.

Anticipated annealing sites of primer POP in theprsEFsequence. The shown sequences are enumerated according the X61238.1 GenBank deposited sequence [36]. The sequence and the complement chromosomal sequence are given. For the forward primer annealing site, the sequence from 27 to 48nt is shown, and for the reverse primer annealing site, the sequence from 1357 to 1375nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR.TheprsEgene is positioned in the deposited sequence from 79 to 600nt, and theprsFgene is positioned from 676 to 1179nt.

In the PCRs for the papGamplification, also the annealing temperature of 55C was used. Three PCR products, all of the expected size, of around 1000bp, were obtained and cloned, and the obtained insert sequences were analysed. All three clones harboured the expected papGsequence. In all three PCR amplifications, both primers, the forward GOD1 and reverse GOD2 primer, annealed at the expected positions. The anticipated annealing sites for specific papG-primer binding of GOD1 and GOD2 as revealed by analysis of the nucleotide E. colisequence CP027701.1 [37] are presented in Figure 6.

Anticipated annealing sites of primer GOD1 and primer GOD2in thepapGsequence. The shown sequences are enumerated according the CP027701.1 GenBank deposited sequence [37]. The sequence and the complement chromosomal sequence are given. For the forward primer annealing site, the sequence from 507724 to 507748nt is shown, and for the reverse primer annealing site, the sequence from 508702 to 508723nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR.ThepapGgene is positioned in the deposited sequence from 507716 to 508726nt.

At the annealing temperature of 55C with primers specific for the F17Ggene, eight PCR products, again all of the expected size of approximately 900bp, were obtained and cloned. Nucleotide sequence analysis of all eight clones showed that four harboured correct and four harboured false inserts. All four false inserts were, as BLAST revealed, sequences of the protein rtngene of the E. coliK-12 MG1655 chromosome, as deposited in the CP025268.1 nucleotide sequence [35]. Further nucleotide analysis revealed that in the case of the rtngene amplification, the forward primer F17G-1 annealed downstream from the c2275331 position on the 3 5 DNA strand and reverse primer F17G-2 upstream of the 2276103 position on the 5 3 DNA strand of the E. coliK-12 MG1655 CP025268.1 nucleotide sequence. The anticipated annealing sites for non-specific F17G-primer binding in analysed nucleotide sequences are presented in Figure 7.

Anticipated annealing sites for non-specificF17G-primer binding in thertngene. The shown sequences are enumerated according the CP025268.1 GenBank deposited sequence [35]. The sequence and the complement chromosomal sequence are given. For the forward primer, F17G-1, annealing site, the sequence from 2275306 to 2275331nt is shown, and for the reverse primer, F17G-2, annealing site, the sequence from 2276103 to 2276129nt is shown. The primer sequence is in the grey box. The arrows mark the direction of DNA elongation in the PCR.Thertngene is positioned in the deposited sequence from 2274762 to 2276318nt.

The main aim of our research was to determine the sequences of chosen P- and F17-fimbriae genes among E. coliisolated from faecal samples of diarrhoeic dogs. As we assumed that the fimbriae of such E. colistrains, due to already known variations of P- and F17-fimbriae, might have nucleotide differences, the annealing temperature of 55C in the PCRs was used. To our surprise, even though only PCR products of expected sizes were cloned, many of the obtained PCR clones, in the case of PCR products obtained with papAprimers 75% and in the case of PCR product obtained with F17Gprimers 50%, carried false inserts. Nucleotide sequence analysis revealed that also in the case of papEFclones, even though the cloned inserts were as hoped for fimbrial inserts, even if they were Prs-fimbrial genes, the binding site of the reverse primer was not the expected one. The high percentages of false PCR products were obtained when PCR primers with a high melting temperature (Tm) were used at the annealing temperature of 55Cprimer 22 has the Tm of 84.1C, and 75% of false PCR products were obtained with this primer; primers F17G-1 and F17G-2 have the Tm of 72.4C and 76.4C, respectively, and 50% of false PCR products were obtained with them. In the consecutive PCR amplifications with the primers 22 and 23, the annealing temperature was raised to 60C, and from these PCRs more PCR products were obtained, namely, 16. All 16 were cloned and analysed, and all clones were with correct inserts (data not shown).

To conclude, we all know that with PCR, we can obtain false unspecific products, and we believe that such PCR products will be distinguished from right PCR products, because the false PCR products will not be of the correct expected size; however, our results showed that also PCR products of the expected size can be false PCR products. In order to avoid false positive PCR results, it is therefore essential to use the right annealing temperature that should not be too different from the primers melting temperature.

The author is very thankful to Wim Gaastra for the primer nucleotide sequences. This analysis was supported by the Slovenian Research Agency (P1-0198).

The author has no conflict of interest.

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Annealing Temperature of 55C and Specificity of Primer Binding in PCR ...

Servers – Mindustry Wiki – GitHub Pages

Servers are a large part of Mindustry in that they offer the ability to play the game with other people. There are two main types of servers; dedicated servers and local LAN servers.

Dedicated servers are standalone, headless versions of the game that are focused only on providing a means for people to play Multiplayer. They are usually run on a computer as a separate program rather than in-game, and are operated from the terminal. These are usually stronger than a local LAN server as they have more resources available to them to support more than two or three players, and can be run 24/7. They are also more versatile and powerful in that they have many commands to provide the administrator with more control over it, and they can easily be modded to fit the administrator's needs.

You can connect to one using the "Join Game" button under the "Play" menu. Unlike local LAN servers, you will have to enter the host's IP address and port. Also unlike local LAN servers, once you add a server, it will automatically show up on your server list when you open it, and the game will automatically check the server's status.

To establish a dedicated server, a dedicated Linux or Windows machine is highly recommended.

Unless you have already enabled port forwarding, your dedicated server can only be connected to by clients within your local network. If you want to make your server globally available, read below.

In simplified terms, an IP address is a number that identifies your computer on the internet. You can connect to someone's Mindustry server if you know their IP address. There are two types; a public and a local address.

Most of the time, this is what you should remember; never share your public IP with the public if you're hosting from your home, unless you acknowledge the implications of doing so! Your public IP is tied to your household, and if it falls into the wrong hands, and when put into the wrong hands, can open up your network to vulnerabilities and dangers. Exercise caution, do your research, and use a VPN or webhost if possible.

It is also recommended and that you use a domain name or DNS service to mask your IP for public servers for ease of use, or even better, use a cloud service e.g. Amazon AWS or a dedicated server/VM from a hosting provider such as Linode or DigitalOcean, which is much safer. Do your research, and determine which option best fits your needs.

A local LAN or Steam server is a server that is built into the game, and can be started using the "Host Multiplayer Game" button in the in-game menu. It is meant to be simple and straightforward, for sessions between a few players under a LAN network (aka in your household's WiFi network). It is not really meant for several players, as it takes more and more resources from your device to be able to use it that way; for that you will need a dedicated server mentioned above. It can only run when the game is open, and is immediately terminated when it is closed.

You can connect to one using the "Join Game" button under the "Play" menu. Unlike dedicated servers, your device will automatically find the host device and it will ususally appear in the server list without you having to enter the host's IP address in.

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Stem Cell Therapy Market worth $558 million by 2027 – Exclusive Report by MarketsandMarkets – PR Newswire

CHICAGO, Sept. 2, 2022 /PRNewswire/ --Stem Cell Therapy Marketis projected to reach USD 558 million by 2027 from USD 257 million in 2022, at a CAGR of 16.8% during the forecast period, according to a new report by MarketsandMarkets.Key drivers of the stem cell therapy market include increase in stem cell research funding, expanding number of clinical trials related to stem cell therapies, and growing number of GMP-certified cell therapy production facilities. However, high costs associated with the development of stem cell therapy along with the ethical concerns related to embryonic stem cells are likely to hamper the market growth to a certain extent.

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Browse in-depth TOC on "Stem Cell Therapy Market"155 Tables43 Figures166 Pages

The adipose tissue-derived MSCs segment dominates the cell source market in the stem cell therapy through 2020-2027.

The global stem cell therapy market is segmented into adipose tissue-derived MSCs (mesenchymal stem cells), bone marrow-derived MSCs, placenta/umbilical cord-derived MSCs, and other cell sources. Adipose-derived stem cell tissues can be obtained easily and also possess a variety of the regenerative properties similar to other mesenchymal stem cells/tissues. These cells are multipotent and are easy to isolate & harvest; these qualities have collectively rendered the adipose tissue-derived MSCs segment highest revenue in 2021.

In 2021, the musculoskeletal disorders ranked first in terms of revenue in the stem cell therapy market.

Based on therapeutic application, the global stem cell therapy market is segmented into musculoskeletal disorders, wounds & injuries, cardiovascular diseases, surgeries, inflammatory & autoimmune diseases, neurological disorders, and other therapeutic applications. In 2021, the musculoskeletal disorders application segment accounted for the largest share of the stem cell therapy market. Increasing market availability of stem cell-based therapeutic products across major markets and the growing patient preference for effective & early treatment strategies are driving the growth of this segment.

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The Asia Pacific region is the fastest-growing region of the stem cell therapy market in 2021.

The Asia Pacific region is estimated to grow at the highest CAGR in the stem cell therapy market during the forecast period. Japan and South Korea are the key revenue contributors of the Asia Pacific stem cell therapy market. Favorable government support for product approvals and the presence of major players in these countries are anticipated to drive the regional market growth.

The stem cell therapy market is consolidated in nature with prominent players in the stem cell therapy market include Smith+Nephew (UK), MEDIPOST Co., Ltd. (South Korea), Anterogen Co., Ltd. (South Korea), CORESTEM (South Korea), Pharmicell Co., Ltd. (South Korea), NuVasive, Inc. (US), RTI Surgical (US), AlloSource (US), JCR Pharmaceuticals Co., Ltd. (Japan), Takeda Pharmaceutical Company Limited (Japan), Holostem Terapie Avanzate Srl (Italy), Orthofix (US), Regrow Biosciences Pvt Ltd. (India), and STEMPEUTICS RESEARCH PVT LTD. (India).

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Stem Cell Manufacturing Market by Product (Consumables, Instrument, HSCs, MSCs, iPSCs, ESCs), Application (Research, Clinical (Autologous, Allogenic), Cell & Tissue Banking), End User (Pharma & Biotech, Hospitals, Tissue Bank) - Global Forecast to 2026

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Stem Cell Therapy Market worth $558 million by 2027 - Exclusive Report by MarketsandMarkets - PR Newswire

From optimized stem cell transplants to CAR T cell therapy: Advancing options for cancer, HIV and more – City of Hope

City of Hope recently shared significant news at the 24th Annual AIDS Conference about a patient treated in 2019 whose HIV has been in remission. The man had been living with HIV for 31 years before coming to City of Hope with another grave diagnosisacute myeloid leukemia.One of the best hopes for long-term remission of acute myeloid leukemia (AML) is a stem cell transplant, and City of Hope has one of the nations leading transplant programs, having performed more than 17,000 transplants since 1976. In addition, the institution is at the forefront of using transplants to treat older adults with blood cancers, including increasing efficacy and safety in those over 60 and those with comorbidities, like the then 63-year-old City of Hope patient with HIV. The research was presented by Jana K. Dickter, M.D., City of Hope associate clinical professor in the Division of Infectious Diseases.

City of Hope hematologist Ahmed Aribi, M.D., assistant professor in the Division of Leukemia, prepared the patient for an allogeneic blood stem cell transplant with a chemotherapy-based, reduced-intensity regimen developed for treatment of older patients with blood cancers. Reduced-intensity chemotherapy makes the transplant more tolerable for older patients and reduces the potential for transplant-related complications from the procedure.

Aribi and his team worked with City of Hopes Unrelated Donor BMT Program directed by Monzr M. Al Malki, M.D. to find a donor who was a perfect match for the patient and had the rare genetic mutation, homozygous CCR5 Delta 32, which is found in just 1 to 2% of the general population.

People who have this mutation have a resistance to acquiring HIV. CCR5 is a receptor on CD4+ immune cells, and most strains of HIV use that receptor to enter and attack the immune system. But the CCR5 mutation blocks that pathway, which stops HIV from replicating.

After this successful transplant for both AML and HIV, the patient has been in remission for HIV since stopping ART in March 2021. While this outcome has happened in three other patients, the City of Hope patient was both the oldest to undergo a transplant with HIV and leukemia and go into remission for both. He had also lived with HIV the longest 31 years.

The City of Hope patient is another major advancement. It demonstrates that research and clinical care developed and led at City of Hope are changing the meaning of an HIV diagnosis for patients across the United States and the world, said John Zaia, M.D., director of City of Hopes Center for Gene Therapy, Aaron D. Miller and Edith Miller Chair for Gene Therapy and a leader in HIV research. City of Hope remains at the forefront of clinical research that changes peoples lives for the better.

When I was diagnosed with HIV in 1988, like many others, I thought it was a death sentence. I never thought I would live to see the day that I no longer have HIV. City of Hope made that possible, and I am beyond grateful. The City of Hope patient

The story above is one significant example of several important advances being made at City of Hope in the care of people with HIV. When many centers still treated patients with low-intensity, noncurative treatment approaches for HIV-related lymphoma, City of Hope challenged that paradigm by demonstrating that autologous transplantation could be used to cure patients who would otherwise die.

More recently, City of Hope is leveraging its leadership in CAR T cell therapya groundbreaking treatment currently used to rally the bodys natural defenses against cancer and exploring its potential in tandem with another advance, City of Hopes vaccine for cytomegalovirus (CMV).

In a proof-of-concept study, funded by theCalifornia Institute for Regenerative Medicine, lab models demonstrated that the combination therapy could recognize and eliminate HIV without serious toxicity to cells in the virus host. In cultured human cells, the CAR T cells killed cells tagged with the gp120 protein, and kept killing them, without significant signs of risking damage to healthy cells. In a mouse model for HIV/AIDS, high doses of the dual-action CAR T cells followed by the CMV vaccine were successful in controlling HIV, and even nestled into the bone marrow, indicating potential for treatment to keep working over the long term.

In addition to achieving breakthrough outcomes in cancer and HIV, City of Hope has been recognized as the seventh "Best Hospital" for cancer in the nation according to U.S. News & World Report's 2022-23 Best Hospitals: Specialty Ranking. This marks the first time the cancer treatment center has cracked the top 10 of the U.S. News & World Report annual rankings and the 16th consecutive year it has been distinguished as one of the nation's elite cancer hospitals. It was also rated as high performing in four cancer surgery specialties: lung, colon, prostate and ovarian cancers.

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From optimized stem cell transplants to CAR T cell therapy: Advancing options for cancer, HIV and more - City of Hope

UBC researchers are accelerating the immune cell production pipeline – UBC Faculty of Medicine – UBC Faculty of Medicine

Breakthrough research led by postdoctoral fellow Dr. Yale Michaels (above) reveals how to produce cancer fighting immune cells faster and more efficiently.

Great gains have been made in the field of stem cell research, especially in the last five years with immune cells emerging as a promising treatment for cancer. Despite the progress, the bottleneck of costly and laborious means of production for personalized medicine has remained a barrier. Now, researchers at the Michael Smith Laboratories and the School of Biomedical Engineering have published a breakthrough paper in Science Advances that just might change the game.

The research team, based out of the Zandstra Laboratory, has refined a process to produce cancer fighting immune cells from stem cells faster and more efficiently than previously known methods. With a patent application in the works, this exciting finding could help transform the field of stem cell medicine from an expensive, niche endeavour to something easily scalable and broadly applicable in the clinic.

CAR T therapy, a well-known and successful cancer treatment, involves obtaining immune cells from a patient and genetically modifying them to fight against the patients cancer. Although this type of therapy has an efficacy rate of close to 50 per cent for some cancers, a new batch of medicine needs to be created for each treatment, costing roughly half a million dollars each round.

Because the main cost associated with these treatments is the fact that theyre made individually, a more cost-effective strategy could be figuring out how to manufacture those immune cells in the lab using stem cells, instead of taking them directly from a patient, explains Dr. Yale Michaels, a UBC postdoctoral fellow and first author of the study.

Dr. Yale Michaels

Building on a large body of previous work in the area, Dr. Michaels and team have discovered an efficient way to do exactly that.

Weve figured out the minimal necessary steps to efficiently guide pluripotent stem cells to develop in the dish into immune cells, in particular, T cells, he adds.

T cells are important infection fighters in our body that originate from pluripotent stem cells (PSCs), and until now, their developmental pipeline has been difficult to replicate effectively in the lab. Dr. Michaels and his team discovered that by providing two proteins to the cells during a key window of development improved the efficiency of immune cell production by eighty times.

Their novel method is the fastest known means of producing immune cells in the lab and could mean that hundreds of doses of medicine could be derived from a single cell.

One of the next steps were working on is to scale this up and make it work more efficiently so that we can make enough cells to treat patients. I hope that this publication and the ongoing work in the lab will contribute to future clinical pipelines, says Dr. Michaels.

The improvement of this production pipeline is one step towards solving a variety of human health challenges from cancer to other immune diseases. The success of the research project is the culmination of effort from a diverse team of people within the Zandstra lab and beyond, including engineers, molecular biologists, computer scientist and more.

One of the next steps were working on is to scale this up and make it work more efficiently so that we can make enough cells to treat patients. I hope this ongoing work will contribute to future clinical pipelines. Dr. Yale Michaels

Its been quite rewarding seeing all the fruits of those different knowledge bases coming together, Dr. Michaels reflects. Its the collective work of hundreds or thousands of people, each making important contributions that collectively enable this to succeed. People have made tremendous progress over the last 20 years; this breakthrough is an exciting continuum.

Seeing the potential for findings like these to work towards treating human problems like cancer, immune diseases and transplant rejection is hugely motivating for Dr. Michaels.

Its fascinating to think that one single cell can produce all the cells, with their diverse functions, required to make up an adult human body. And how that happens is an interesting fundamental question about what makes us. The more we learn about how this process unfolds, the better we are able to apply it to treating human disease.

A version of this story originally appeared on the Michael Smith Laboratories website.

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UBC researchers are accelerating the immune cell production pipeline - UBC Faculty of Medicine - UBC Faculty of Medicine