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Genetic association study revealed three novel loci were ass | PGPM – Dove Medical Press

Introduction

Myopia, the most common refractive error, results in a significant threat on global public health worldwide.1 As the myopic population increases globally, the severity of its impact is predicted.2 Children with early onset are particularly susceptible to myopia-related complications, like high myopia (HM) and myopic macular degeneration.3 According to a recent school-based epidemiology study of myopia in China with 14,551 participants (ages ranging from 5 to 16 years), the overall prevalence of myopia is 78.2%.4 Myopia is a complex disease which is contributed by various environmental and genetic factors. Environmental factors includes low outdoor time and near work, dim light exposure, the use of LED lamps for homework, low sleeping hours, and short reading distance.3,5 Meanwhile, there is growing evidence that susceptibility genes play a crucial role in the risk of myopia and single nucleotide polymorphisms (SNPs) may contribute to the risk of myopia.610

Recently, a genome-wide association study (GWAS) identified six novel loci (rs2246661, rs74633073, rs76903431, rs698047, rs17029206, and rs72748160) in Asian adults, and revealed the important role of genes in the nervous system in the pathogenesis of myopia.11 The findings highlighted a nervous system role in pathogenesis of myopia. Minors are better suited to studying the genetic factors of myopia. Whether these genetic loci works for myopia in minors remains unknown and worthy to be explored. Thus, here we aimed to evaluate the potential role of these GWAS identified loci in occurrence of myopia in this case-control study including 600 myopia minors, 110 HM minors and 800 non-myopia minors.

A total of 600 consecutive myopia minors, 110 HM minors, and 800 non-myopia minors, which were frequency-matched by age and gender, were recruited in this case-control study. All subjects were Chinese Han population. Myopia was defined as mean spherical equivalent (MSE) of both eyes 0.5 diopters (D), while HM was defined as MSE less than or equal to 6.0 D.12 Patients with a predisposition to myopic eye disease, other known ocular or systemic diseases were excluded. Controls were selected from subjects coming for routine vision screening. The criteria for the control group were as follows: minors with MSE between 0.5 D and +1.0 D, best unaided visual acuity 0.8, and no other known ocular or systemic diseases.13 Patients are tested for refractive errors using an automated refractometer (Topcon RM-8000B, Topcon Co., Tokyo, Japan). The refraction was taken under cycloplegia. Information of all participants, including age, gender, body mass index (BMI, calculated using weight/height2), self-reported outdoors time, self-reported time using electronic equipment, and parental myopia, was collected through questionnaire responses, and all subjects donated 5 mL peripheral venous blood. The study protocol was approved by the ethics committee of Nanjing Tongren Hospital. All subjects gave their written informed consent, and the study complied with the Declaration of Helsinki.

Genomic DNA was extracted by blood DNA extraction kit (Promega, Madison, Wisconsin, USA) and stored in TE buffer. Genotyping was performed by TaqMan assay in 384-well ABI 7900HT Real-Time PCR system (Applied Biosystems [ABI], Foster City, CA). The qPCR reactions proceeded in a final volume of 10 L mix including 5 L TaqMan Genotyping Master Mix (Thermo Fisher Scientific), 0.5 L pre-designed TaqMan probe (Thermo Fisher Scientific), 20 ng genomic DNA and ultrapure water. Each plate included blank samples as negative controls to verify genotyping quality. Genotype data were analyzed using their System SDS Allelic Discrimination Software version 2.3 (Applied Biosystems). For quality control, about 5% of the samples were genotyped repeatedly with Sanger sequencing and the results of both methods were in good agreement.

SPSS 22.0 (SPSS, Chicago, IL) was used for statistical analysis, and a two-sided P-value of less than 0.05 was used as statistical significance. Chi -square goodness-of-fit test was adopted to derive the Hardy-Weinberg equilibrium (HWE). In the case-control study, Student t-test and/or Chi -square test were used to demonstrate how demographic and clinical characteristics and frequency of genotypes differ between case and control groups. Using unconditional logistic regression model, adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were adopted (only significant variables in Table 1 were included for adjustment) to evaluate the effects of SNPs and to quantify the association between the SNPs and myopia in minors.

Table 1 Characteristics of Participating Minors

Table 1 presented the 600 myopia minors, 110 HM minors, and 800 non- myopia minors in this case-control study. The groups were comparable in age, and gender (P>0.05). While the highly myopic spent more time using electronic devices (P<0.001), less time outdoors (P<0.001) and had more myopic parents than non-myopic ones (P=0.015). The median ages in controls, myopia, and HM were 15.1, 15.0, and 15.1, respectively, while the means standard deviation for them were 0.320.41, 3.2 1.6, and 9.82.2, respectively.

All six SNPs analyzed were in HWE in non-myopia controls, indicating that the sampled subjects were representative of the population and did not show any bias in genotype frequency (p>0.05). Subsequently, we evaluated the associations between the selected SNPs and the risk of myopia adjusting for BMI, self-reported outdoors time, and self-reported time using electronic equipment. Table 2 showed the results of genotypic frequency analysis for selected loci. SNP rs2246661 (allelic OR: 1.29; 95% CI: 1.091.52; P =0.003), rs74633073 (allelic OR: 1.41; 95% CI: 1.121.78; P =0.004), and rs76903431 (allelic OR: 1.42; 95% CI: 1.111.81; P =0.005) significantly contributed to elevated susceptibility of myopia. Under additive genetic model, all of three SNPs showed statistically significant associations. For rs2246661, the CT genotype was associated with a 1.42-fold increased risk (95% CI= 1.121.81; P=0.004), while the TT genotype conferred 1.64-fold increased risk of myopia (95% CI= 1.12.43; P=0.014), compared with the TT genotype. For rs74633073, the CT genotype was associated with a 1.39-fold increased risk (95% CI= 1.051.85; P=0.022), while the TT genotype conferred 2.61-fold increased risk of myopia (95% CI= 1.126.08; P=0.026). For rs76903431, genotype GG was associated with a 1.48-fold increased risk (95% CI= 1.111.97; P=0.007), while the GG genotype conferred 2.57-fold increased risk of myopia (95% CI= 1.046.37; P=0.041), compared with the CC genotype.

Table 2 Associations Between Candidate Loci and Myopia in Minors

We further evaluated the associations of these six candidate SNPs with HM adjusting for self-reported outdoors time, self-reported time using electronic equipment, and parental myopia. We only found rs2246661 (OR: 1.37; 95% CI: 1.021.84; P =0.035), significantly contributed to elevated susceptibility of HM (Table 3). Under additive genetic model, the CT genotype was associated with a 1.55-fold increased risk (95% CI= 1.012.37; P=0.044), while the TT genotype conferred 1.88-fold increased risk of myopia (95% CI=1.033.43; P=0.040), compared with the TT genotype.

Table 3 Associations Between Candidate Loci and High Myopia in Minors

The current study investigated the potential function of six GWAS identified loci in occurrence of minors' myopia in a case-control study in Chinese population. We found three loci, including rs2246661, rs74633073, and rs76903431, significantly contributed to elevated risk of myopia. Besides, we also found rs2246661 significantly contributed to HM in minors. Our results confirm the GWAS findings in Asian adults and further provide a causal explanation for the occurrence of myopia at the molecular level.

The prevalence of myopia grew rapidly in minors.3,5,14 Finding the causes of the disease and taking effective preventive measures are vital to controlling the damage caused by myopia in young people. To date, a series of GWASs have been conducted to characterize the molecular mechanism responsible for myopia worldwide.11, 1520 However, not all could be replicated. For example, Wang et al21 replicated findings of two Japanese GWAS in a Chinese population, and got null results. This was because myopia in adults was a genetically heterogeneous disease, which was influenced by inborn genetic factors and acquired environmental factors. On the contrary, minors are better suited to exploring the genetic factors of myopia. Thus, we attempted to classify the occurrence of myopia in minors was affected by GWAS loci identified in adults in this case-control study.

In the current study, rs2246661, rs74633073, and rs76903431 were identified to be associated risk of myopia in minors. Through searching Pubmed, we did not find any other genetic associations. According to RegulomeDB 2.0, rs2246661 and rs74633073 were located at the transcription factor (TF) binding site, which could affect the combination of TFs and their targets.22 HaploReg v4.1 revealed rs2246661 could cause Ets Motifs change, and rs74633073 could cause AP-2, RFX5 Motifs change, while rs76903431 could cause CDP, Pbx-1, RXRA Motifs change.23 This evidence supported the important role of these genetic loci.

Our study had several limitations. First, the selection bias of a case-control study design cannot be avoided. Second, early-onset myopia, which refers to myopia occurring before the age of 11 years, was not evaluated in current study, due to the limitations of sample size.24 Third, based on existing sample size, the associations might not have the strength to achieve real results, especially for HM. Fourth, the biological function of these SNPs and its detailed effect on occurrence of myopia need to be deep investigated by further biological studies. There are also several strengths in our research, including the detailed inspection and accurate diagnosis of cases, structured questionnaire by well-trained interviewers, and strict quality control of genotyping.

Conclusively, this study provides the evidence of the promotional role of rs2246661, rs74633073, and rs76903431 loci on the susceptibility of myopia. Replicated researches in independent ethnic samples and functional investigation are needed to confirm our findings.

The authors declare that they have no conflict of interest.

1. Foster PJ, Jiang Y. Epidemiology of myopia. Eye (Lond). 2014;28(2):202208. doi:10.1038/eye.2013.280

2. Angle J, Wissmann DA. The epidemiology of myopia. Am J Epidemiol. 1980;111(2):220228. doi:10.1093/oxfordjournals.aje.a112889

3. Grzybowski A, Kanclerz P, Tsubota K, Lanca C, Saw SM. A review on the epidemiology of myopia in school children worldwide. BMC Ophthalmol. 2020;20(1):27. doi:10.1186/s12886-019-1220-0

4. Wang J, Li Y, Zhao Z, et al. School-based epidemiology study of myopia in Tianjin, China. Int Ophthalmol. 2020;40(9):22132222. doi:10.1007/s10792-020-01400-w

5. Mak CY, Yam JC, Chen LJ, Lee SM, Young AL. Epidemiology of myopia and prevention of myopia progression in children in East Asia: a review. Hong Kong Med J. 2018;24(6):602609.

6. Kunceviciene E, Liutkeviciene R, Budiene B, Sriubiene M, Smalinskiene A. Independent association of whole blood miR-328 expression and polymorphism at 3UTR of the PAX6 gene with myopia. Gene. 2019;687:151155. doi:10.1016/j.gene.2018.11.030

7. Zhang D, Zeng G, Hu J, McCormick K, Shi Y, Gong B. Association of IGF1 polymorphism rs6214 with high myopia: a systematic review and meta-analysis. Ophthalmic Genet. 2017;38(5):434439. doi:10.1080/13816810.2016.1253105

8. Jin GM, Zhao XJ, Chen AM, Chen YX, Li Q. Association of COL1A1 polymorphism with high myopia: a Meta-analysis. Int J Ophthalmol. 2016;9(4):604609.

9. Liang Y, Song Y, Zhang F, Sun M, Wang N. Effect of a single nucleotide polymorphism in the LAMA1 promoter region on Transcriptional activity: implication for pathological myopia. Curr Eye Res. 2016;41(10):13791386. doi:10.3109/02713683.2015.1118129

10. Chen T, Shan G, Ma J, Zhong Y. Polymorphism in the RASGRF1 gene with high myopia: a meta-analysis. Mol Vis. 2015;21:12721280.

11. Meguro A, Yamane T, Takeuchi M, et al. Genome-wide association study in asians identifies novel loci for high myopia and highlights a nervous system role in its pathogenesis. Ophthalmology. 2020;127(12):16121624. doi:10.1016/j.ophtha.2020.05.014

12. Luo HD, Gazzard G, Liang Y, Shankar A, Tan DT, Saw SM. Defining myopia using refractive error and uncorrected logMAR visual acuity >0.3 from 1334 Singapore school children ages 79 years. Br J Ophthalmol. 2006;90(3):362366. doi:10.1136/bjo.2005.079657

13. Tideman JW, Polling JR, Voortman T, et al. Low serum vitamin D is associated with axial length and risk of myopia in young children. Eur J Epidemiol. 2016;31(5):491499. doi:10.1007/s10654-016-0128-8

14. Recko M, Stahl ED. Childhood myopia: epidemiology, risk factors, and prevention. Mo Med. 2015;112(2):116121.

15. Huang Y, Kee CS, Hocking PM, et al. A genome-wide association study for susceptibility to visual experience-induced myopia. Invest Ophthalmol Vis Sci. 2019;60(2):559569. doi:10.1167/iovs.18-25597

16. Khor CC, Miyake M, Chen LJ, et al. Genome-wide association study identifies ZFHX1B as a susceptibility locus for severe myopia. Hum Mol Genet. 2013;22(25):52885294. doi:10.1093/hmg/ddt385

17. Meng W, Butterworth J, Bradley DT, et al. A genome-wide association study provides evidence for association of chromosome 8p23 (MYP10) and 10q21.1 (MYP15) with high myopia in the French Population. Invest Ophthalmol Vis Sci. 2012;53(13):79837988. doi:10.1167/iovs.12-10409

18. Li Z, Qu J, Xu X, et al. A genome-wide association study reveals association between common variants in an intergenic region of 4q25 and high-grade myopia in the Chinese Han population. Hum Mol Genet. 2011;20(14):28612868. doi:10.1093/hmg/ddr169

19. Solouki AM, Verhoeven VJ, van Duijn CM, et al. A genome-wide association study identifies a susceptibility locus for refractive errors and myopia at 15q14. Nat Genet. 2010;42(10):897901. doi:10.1038/ng.663

20. Hysi PG, Young TL, Mackey DA, et al. A genome-wide association study for myopia and refractive error identifies a susceptibility locus at 15q25. Nat Genet. 2010;42(10):902905. doi:10.1038/ng.664

21. Wang Q, Gao Y, Wang P, et al. Replication study of significant single nucleotide polymorphisms associated with myopia from two genome-wide association studies. Mol Vis. 2011;17:32903299.

22. Boyle AP, Hong EL, Hariharan M, et al. Annotation of functional variation in personal genomes using Regulome DB. Genome Res. 2012;22(9):17901797. doi:10.1101/gr.137323.112

23. Ward LD, Kellis M. HaploReg v4: systematic mining of putative causal variants, cell types, regulators and target genes for human complex traits and disease. Nucleic Acids Res. 2016;44(D1):D877881. doi:10.1093/nar/gkv1340

24. Baird PN, Saw SM, Lanca C, et al. Myopia. Nat Rev Dis Primers. 2020;6(1):99.

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UCLA researchers make DNA detection portable, affordable using cellphones – University of California

Researchers at UCLA have developed an improved method to detect the presence of DNA biomarkers of disease that is compatible with use outside of a hospital or lab setting. The new technique leverages the sensors and optics of cellphones to read light produced by a new detector dye mixture that reports the presence of DNA molecules with a signal that is more than 10-times brighter.

Nucleic acids, such as DNA or RNA, are used in tests for infectious diseases, genetic disorders, cancer mutations that can be targeted by specific drugs, and fetal abnormality tests. The samples used in standard diagnostic tests typically contain only tiny amounts of a diseases related nucleic acids. To assist optical detection, clinicians amplify the number of nucleic acids making them easier to find with the fluorescent dyes.

Both the amplification and the optical detection steps have in the past required costly and bulky equipment, largely limiting their use to laboratories.

In a studypublished onlinein the journal ACS Nano, researchers from three UCLA entities the Henry Samueli School of Engineering and Applied Science, the California NanoSystems Institute, and the David Geffen School of Medicine showed how to take detection out of the lab and for a fraction of the cost.

The collaborative team of researchers included lead author Janay Kong, a UCLA Ph.D. student in bioengineering; Qingshan Wei, a post-doctoral researcher in electrical engineering; Aydogan Ozcan, Chancellors Professor of Electrical Engineering and Bioengineering; Dino Di Carlo, professor of bioengineering and mechanical and aerospace engineering; andOmai Garner, assistant professor of pathology and medicine at the David Geffen School of Medicine at UCLA.

The UCLA researchers focused on the challenges with low-cost optical detection. Small changes in light emitted from molecules that associate with DNA, called intercalator dyes, are used to identify DNA amplification, but these dyes are unstable and their changes are too dim for standard cellphone camera sensors.

But the team discovered an additive that stabilized the intercalator dyes and generated a large increase in fluorescent signal above the background light level, enabling the test to be integrated with inexpensive cellphone based detection methods. The combined novel dye/cellphone reader system achieved comparable results to equipment costing tens of thousands of dollars more.

To adapt a cellphone to detect the light produced from dyes associated with amplified DNA while those samples are in standard laboratory containers, such as well plates, the team developed a cost-effective, field-portable fiber optic bundle. The fibers in the bundle routed the signal from each well in the plate to a unique location of the camera sensor area. This handheld reader is able to provide comparable results to standard benchtop readers, but at a fraction of the cost, which the authors suggest is a promising sign that the reader could be applied to other fluorescence-based diagnostic tests.

Currently nucleic acid amplification tests have issues generating a stable and high signal, which often necessitates the use of calibration dyes and samples which can be limiting for point-of-care use, Di Carlo said. The unique dye combination overcomes these issues and is able to generate a thermally stable signal, with a much higher signal to noise ratio. The DNA amplification curves we see look beautiful without any of the normalization and calibration, which is usually performed, to get to the point that we start at.

Additionally, the authors emphasized that the dye combinations discovered should be able to be used universally to detect any nucleic acid amplification, allowing for their use in a multitude of other amplification approaches and tests.

The team demonstrated the approach using a process called loop-mediated isothermal amplification, or LAMP, with DNA from lambda phage as the target molecule, as a proof of concept, and now plan to adapt the assay to complex clinical samples and nucleic acids associated with pathogens such as influenza.

The newest demonstration is part of a suite of technologies aimed at democratizing disease diagnosis developed by the UCLA team. Includinglow-cost optical readout and diagnostics based on consumer-electronic devices,microfluidic-based automationandmolecular assays leveraging DNA nanotechnology.

This interdisciplinary work was supported through a team science grant from the National Science Foundation Emerging Frontiers in Research and Innovation program.

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UCLA researchers make DNA detection portable, affordable using cellphones - University of California

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UCLA researchers make DNA detection portable, affordable using cellphones – UCLA Newsroom

Researchers at UCLA have developed an improved method to detect the presence of DNA biomarkers of disease that is compatible with use outside of a hospital or lab setting. The new technique leverages the sensors and optics of cellphones to read light produced by a new detector dye mixture that reports the presence of DNA molecules with a signal that is more than 10-times brighter.

Nucleic acids, such as DNA or RNA, are used in tests for infectious diseases, genetic disorders, cancer mutations that can be targeted by specific drugs, and fetal abnormality tests. The samples used in standard diagnostic tests typically contain only tiny amounts of a diseases related nucleic acids. To assist optical detection, clinicians amplify the number of nucleic acids making them easier to find with the fluorescent dyes.

Both the amplification and the optical detection steps have in the past required costly and bulky equipment, largely limiting their use to laboratories.

In a study published onlinein the journal ACS Nano, researchers from three UCLA entities the Henry Samueli School of Engineering and Applied Science, the California NanoSystems Institute, and the David Geffen School of Medicine showed how to take detection out of the lab and for a fraction of the cost.

The collaborative team of researchers included lead author Janay Kong, a UCLA Ph.D. student in bioengineering; Qingshan Wei, a post-doctoral researcher in electrical engineering; Aydogan Ozcan, Chancellors Professor of Electrical Engineering and Bioengineering; Dino Di Carlo, professor of bioengineering and mechanical and aerospace engineering; andOmai Garner, assistant professor of pathology and medicine at the David Geffen School of Medicine at UCLA.

The UCLA researchers focused on the challenges with low-cost optical detection. Small changes in light emitted from molecules that associate with DNA, called intercalator dyes, are used to identify DNA amplification, but these dyes are unstable and their changes are too dim for standard cellphone camera sensors.

But the team discovered an additive that stabilized the intercalator dyes and generated a large increase in fluorescent signal above the background light level, enabling the test to be integrated with inexpensive cellphone based detection methods. The combined novel dye/cellphone reader system achieved comparable results to equipment costing tens of thousands of dollars more.

To adapt a cellphone to detect the light produced from dyes associated with amplified DNA while those samples are in standard laboratory containers, such as well plates, the team developed a cost-effective, field-portable fiber optic bundle. The fibers in the bundle routed the signal from each well in the plate to a unique location of the camera sensor area. This handheld reader is able to provide comparable results to standard benchtop readers, but at a fraction of the cost, which the authors suggest is a promising sign that the reader could be applied to other fluorescence-based diagnostic tests.

Currently nucleic acid amplification tests have issues generating a stable and high signal, which often necessitates the use of calibration dyes and samples which can be limiting for point-of-care use, Di Carlo said. The unique dye combination overcomes these issues and is able to generate a thermally stable signal, with a much higher signal to noise ratio. The DNA amplification curves we see look beautiful without any of the normalization and calibration, which is usually performed, to get to the point that we start at.

Additionally, the authors emphasized that the dye combinations discovered should be able to be used universally to detect any nucleic acid amplification, allowing for their use in a multitude of other amplification approaches and tests.

The team demonstrated the approach using a process called loop-mediated isothermal amplification, or LAMP, with DNA from lambda phage as the target molecule, as a proof of concept, and now plan to adapt the assay to complex clinical samples and nucleic acids associated with pathogens such as influenza.

The newest demonstration is part of a suite of technologies aimed at democratizing disease diagnosis developed by the UCLA team. Including low-cost optical readout and diagnostics based on consumer-electronic devices,microfluidic-based automation andmolecular assays leveraging DNA nanotechnology.

This interdisciplinary work was supported through a team science grant from the National Science Foundation Emerging Frontiers in Research and Innovation program.

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New bedside genetic tests pick the right drug, right away

It is being billed as the worlds first bedside genetic test for heart patients. A nurse merely swabs the inside of a patients mouth and places the saliva sample into a compact machine thats the size of a toaster. In less than an hour, the device analyzes the DNA and determines the best drug option for the patient.

Normally, this type of test would be sent to a lab that could take up to a week to return the results.

In cardiology, especially with patients coming into the hospital with heart attacks, we often have to make decisions very quickly said Derek So, an assistant professor at the University of Ottawa Heart Institute. This is essentially the first step to making fast decisions based on genetics.

The genetic test, developed by researchers at the heart institute and Ottawa-based Spartan Bioscience, helps predict how patients will respond to Plavix, a widely-prescribed blood thinner.

Plavix is often given to people with advanced cardiovascular disease to prevent blood clots that cause heart attacks and strokes. It is also used to keep blood flowing freely through stents devices that prop open narrowed blood vessels.

Plavix, or its genetic equivalent clopidogrel, is considered to be standard therapy. But not everyone does well on the treatment. In fact, research has revealed some people carry a genetic variant that can impede Plavix from working to its full potential.

And thats where the new rapid genetic test could come in handy, said Dr. So. It can pinpoint those individuals who should be getting an alternative anti-clotting agent such as Effient or Brilinta, he said.

Dr. So, along with his co-investigator Jason Roberts, recently completed a proof-of-concept study to see if the genetic test worked in a real hospital setting. As part of the study, nurses were given 30 minutes of training on how to collect the saliva samples and use the DNA analyzing machine.

The findings, published last week in The Lancet medical journal, showed the system can produce the desired results. Accurate bedside tests were performed on almost 200 patients.

The same technology could be applied to other areas of medicine where there are genetic associations to either a diagnosis or a treatment, said Dr. So.

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New bedside genetic tests pick the right drug, right away

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An Introduction to PCR – Technology Networks

Polymerase chain reaction (PCR) is a technique that has revolutionized the world of molecular biology and beyond. In this article, we will discuss a brief history of PCR and its principles, highlighting the different types of PCR and the specific purposes to which they are being applied.

In 1983, American biochemist Kary Mullis was driving home late at night when a flash of inspiration struck him. He wrote on the back of a receipt the idea that would eventually grant him the Nobel Prize for Chemistry in 1993. The concept was straightforward: reproducing in a laboratory tube the DNA replication process that takes place in cells. The outcome is the same: the generation of new complementary DNA (cDNA) strands based upon the existing ones.

Mullis used the basis of Sanger's DNA sequencing as a starting point for his new technique. He realized that the repeated use of DNA polymerase triggered a chain reaction resulting in a specific DNA segment's amplification.

The foundations for his idea were laid by a discovery in 1976 of a thermostable DNA polymerase, Taq, isolated from the bacterium Thermus aquaticus found in hot springs.1 Taq DNA polymerase has a temperature optimum of 72 C and survives prolonged exposure to temperatures as high as 96 C, meaning that it can tolerate several denaturation cycles.

Before the discovery of Taq polymerase, molecular biologists were already trying to optimize cyclic DNA amplification protocols, but they needed to add fresh polymerase at each cycle because the enzyme could not withstand the high temperatures needed for DNA denaturation. Having a thermostable enzyme meant that they could repeat the amplification process many times over without the need for fresh polymerase at every cycle, making the whole process scalable, more efficient and less time-consuming.

The first description of this polymerase chain reaction (PCR) using Taq polymerase was published in Science in 1985.2

In 1993, the first FDA-approved PCR kit came to market. Since then, PCR has been steadily and systematically improved. It has become a game-changer in everything from forensic evidence analysis and diagnostics, to disease monitoring and genetic engineering. It is undoubtedly considered one of the most important scientific advances of the 20th century.

The PCR is used to amplify a specific DNA fragment from a complex mixture of starting material called template DNA. The sample preparation and purification protocols depend on the starting material, including the sample matrix and accessibility of target DNA. Often, minimal DNA purification is needed. However, PCR does require knowledge of the DNA sequence information that flanks the DNA fragment to be amplified (called target DNA).

From a practical point of view, a PCR experiment is relatively straightforward and can be completed in a few hours. In general, a PCR reaction needs five key reagents:

DNA to be amplified: also called PCR template or template DNA. This DNA can be of any source, such as genomic DNA (gDNA), cDNA, and plasmid DNA.DNA polymerase: all PCR reactions require a DNA polymerase that can work at high temperatures. Taq polymerase is a commonly used one, which can incorporate nucleotides at a rate of 60 bases/second at 70 C and can amplify templates of up to 5 kb, making it suitable for standard PCR without special requirements. New generations of polymerases are being engineered to improve reaction performance. For example, some are engineered to be only activated at high temperatures to reduce non-specific amplification at the beginning of the reaction. Others incorporate a proofreading function, important, for example, when it is critical that the amplified sequence matches the template sequence exactly, such as during cloning.Primers: DNA polymerases require a short sequence of nucleotides to indicate where they need to initiate amplification. In a PCR, these sequences are called primers and are short pieces of single-stranded DNA (approximately 15-30 bases). When designing a PCR experiment, the researcher determines the region of DNA to be amplified and designs a pair of primers, one on the forward strand and one on the reverse, that specifically flanks the target region. Primer design is a key component of a PCR experiment and should be done carefully. Primer sequences must be chosen to target the unique DNA of interest, avoiding the possibility of binding to a similar sequence. They should have similar melting temperatures because the annealing step occurs simultaneously for both strands. The melting temperature of a primer can be impacted by the percentage of bases that are guanine (G) or cytosine (C) compared to adenine (A) or thymine (T), with higher GC contents increasing melting temperatures. Adjusting primer lengths can help to compensate for this in matching a primer pair. It is also important to avoid sequences that will tend to form secondary structures or primer dimers, as this will reduce PCR efficiency. Many free online tools are available to aid in primer design.Deoxynucleotide triphosphates (dNTPs): these serve as the building blocks to synthesize the new strands of DNA and include the four basic DNA nucleotides (dATP, dCTP, dGTP, and dTTP). dNTPs are usually added to the PCR reaction in equimolar amounts for optimal base incorporation.PCR buffer: the PCR buffer ensures that optimal conditions are maintained throughout the PCR reaction. The major components of PCR buffers include magnesium chloride (MgCl2), tris-HCl and potassium chloride (KCl). MgCl2 serves as a cofactor for the DNA polymerase, while tris-HCl and KCl maintain a stable pH during the reaction.The PCR reaction is carried out in a single tube by mixing the reagents mentioned above and placing the tube in a thermal cycler.The PCR amplification consists of three defined sets of times and temperatures termed steps: denaturation, annealing, and extension (Figure 1).

Figure 1: Steps of a single PCR cycle.

Each of these steps, termed cycles, is repeated 30-40 times, doubling the amount of DNA at each cycle and obtaining amplification (Figure 2).

Figure 2: The different stages and cycles of DNA molecule amplification by PCR.

Let's take a closer look at each step.

The first step of PCR, called denaturation, heats the template DNA up to 95 C for a few seconds, separating the two DNA strands as the hydrogen bonds between them are rapidly broken.

The reaction mixture is then cooled for 30 seconds to 1 minute. Annealing temperatures are usually 50 - 65 C however, the exact optimal temperature depends on the primers' length and sequence. It must be carefully optimized with every new set of primers.

The two DNA strands could rejoin at this temperature, but most do not because the mixture contains a large excess of primers that bind, or anneal, to the template DNA at specific, complementary positions. Once the annealing step is completed, hydrogen bonds will form between the template DNA and the primers. At this point, the polymerase is ready to extend the DNA sequence.

The temperature is then raised to the ideal working temperature for the DNA polymerase present in the mixture, typically around 72 C, 74 C in the case of Taq.

The DNA polymerase attaches to one end of each primer and synthesizes new strands of DNA, complementary to the template DNA. Now we have four strands of DNA instead of the two that were present to start with.

The temperature is raised back to 94 C and the double-stranded DNA molecules both the "original" molecules and the newly synthesized ones denature again into single strands. This begins the second cycle of denaturation-annealing-extension. At the end of this second cycle, there are eight molecules of single-stranded DNA. By repeating the cycle 30 times, the double-stranded DNA molecules present at the beginning are converted into over 130 million new double-stranded molecules, each one a copy of the region of the starting molecule delineated by the annealing sites of the two primers.

To determine if amplification has been successful, PCR products may be visualized using gel electrophoresis, indicating amplicon presence/absence, size and approximate abundance. Depending on the application and the research question, this may be the endpoint of an experiment, for example, if determining whether a gene is present or not. Otherwise, the PCR product may just be the starting point for more complex downstream investigations such as sequencing and cloning.

Thanks to their versatility, PCR techniques have evolved over recent years leading to the development or several different types of PCR technology.

Some of the most widely used ones are:

One of the most useful developments has been quantitative real-time PCR or qPCR. As the name suggests, qPCR is a quantitative technique that allows real-time monitoring of the amplification process and detection of PCR products as they are made.2 It can be used to determine the starting concentration of the target DNA, negating the need for gel electrophoresis in many cases. This is achieved thanks to the inclusion of non-specific fluorescent intercalating dyes, such as SYBR Green, that fluoresce when bound to double-stranded DNA, or DNA oligonucleotide sequence-specific fluorescent probes, such as hydrolysis (TaqMan) probes and molecular beacons. Probes bind specifically to DNA target sequences within the amplicon and use the principle of Frster Resonance Energy Transfer (FRET) to generate fluorescence via the coupling of a fluorescent molecule on one end and a quencher at the other end. For both fluorescent dyes and probes, as the number of copies of the target DNA increases, the fluorescence level increases proportionally, allowing real-time quantification of the amplification with reference to standards containing known copy numbers (Figure 3).

qPCR uses specialized thermal cyclers equipped with fluorescent detection systems that monitor the fluorescent signal as the amplification occurs.

Figure 3: Example qPCR amplification plot and standard curve used to enable quantification of copy number in unknown samples.

Reverse transcription (RT) -PCR and RT-qPCR are two commonly used PCR variants enabling gene transcription analysis and quantification of viral RNA, both in clinical and research settings.

RT is the process of making cDNA from single-stranded template RNA3 and is consequently also called first-strand cDNA synthesis. The first step of RT-PCR is to synthesize a DNA/RNA hybrid between the RNA template and a DNA oligonucleotide primer. The reverse transcriptase enzyme that catalyzes this reaction has RNase activity that then degrades the RNA portion of the hybrid. Subsequently, a single-stranded DNA molecule is synthesized by the DNA polymerase activity of the reverse transcriptase. High purity and quality starting RNA are essential for a successful RT-PCR.

RT-PCR can be performed following two approaches: one-step RT-PCR and two-step RT-PCR. In the first case, the RT reaction and the PCR reaction occur in the same tube, while in the two-step RT-PCR, the two reactions are separate and performed sequentially.

The reverse transcription described above often serves as the first step in qPCR too, quantifying RNA in biological samples (either RNA transcripts or derived from viral RNA genomes).

As with RT-PCR, there are two approaches for quantifying RNA by RT-qPCR: one-step RT-qPCR and two-step RT-qPCR. In both cases, RNA is first reverse-transcribed into cDNA, which is used as the template for qPCR amplification. In the two-step method, the reverse transcription and the qPCR amplification occur sequentially as two separate experiments. In the one-step method, RT and qPCR are performed in the same tube.

Digital PCR (dPCR) is another adaptation of the original PCR protocol.4 Like qPCR, dPCR technology uses DNA polymerase to amplify target DNA from a complex sample using a primer set and probes. The main difference, though, lies in the partitioning of the PCR reactions and data acquisition at the end.

dPCR and ddPCR are based on the concept of limiting dilutions. The PCR reaction is split into large numbers of nanoliter-sized sub-reactions (partitions). The PCR amplification is carried out within each droplet. Following PCR, each droplet is analyzed with Poisson statistics to determine the percentage of PCR-positive droplets in the original sample. Some partitions may contain one or more copies of the target, while others may contain no target sequences. Therefore, partitions classify either as positive (target detected) or negative (target not detected), providing the basis for a digital output format.

ddPCR is a recent technology that became available in 2011.5 ddPCR utilizes a water-oil emulsion to form the partitions that separate the template DNA molecules. The droplets essentially serve as individual test tubes in which the PCR reaction takes place.

The recent development of microfluidic handling systems with microchannels and microchambers has paved the way for a range of practical applications, including the amplification of DNA via PCR on microfluidic chips.

PCR performed on a chip benefits from microfluidics advantages in speed, sensitivity and low consumption of reagents. These features make microfluidic PCR particularly appealing for point-of-care testing, for example, for diagnostics applications. From a practical point of view, the sample flows through a microfluidic channel, repeatedly passing the three temperature zones reflecting the different steps of PCR. It takes just 90 seconds for a 10 L sample to perform 20 PCR cycles.6 The subsequent analysis can then be easily carried out off-chip.

The different PCR approaches all have advantages and disadvantages that impact the applications to which they are suited 7. These are summarized in Table 1.

Approach

Advantages

Limitations

PCR

Easiest PCR to perform

Low cost of equipment and reagents

Several downstream applications (e.g., cloning)

Results are only qualitative

Requires post-amplification analyses that increase time and risk of error

Products may need to be confirmed by sequencing

qPCR

Produces quantitative results

Probe use can ensure high specificity

High analytical sensitivity

Low turnaround time

Eliminates requirements for post-amplification analysis

Requires more expensive reagents and equipment

Less flexibility in primer and probe selection

Less amenable to other downstream product confirmation analyses (such as sequencing) due to the small length of the amplicon

Not suitable for some downstream applications such as cloning

RT-PCR and RT-qPCR

Can be used with all RNA types

RNA is prone to degradation

The RT step may increase the time and potential for contamination

dPCR and ddPCR

Fast

No DNA purification step

Provides absolute quantification

Increased sensitivity for detecting the target in limited clinical samples

Highly scalable

Costly

Based on several statistical assumptions

Microfluidic PCR

Accelerated PCR process

Reduced reagent consumption

Can be adapted for high throughput

Portable device for point-of-care applications

Allows single-cell analysis

Still very new technology

Requires extensive sample preparation to remove debris and unwanted compounds

Restricted choice of materials for the microfluidic device due to high temperatures

Table 1: Key advantages and disadvantages of different PCR approaches.

PCR has become an indispensable tool in modern molecular biology and has completely transformed scientific research. The technique has also opened up the investigation of cellular and molecular processes to those outside the field of molecular biology and consequently also finds utility by scientists in many disciplines.

Whilst PCR is itself a powerful standalone technique, it has also been incorporated into wider techniques, such as cloning and sequencing, as one small but important part of these workflows.

Research applications of PCR include:

Gene transcription -PCR can examine variations in gene transcription among cell types, tissues and organisms at a specific time point. In this process, RNA is isolated from samples of interest, and reverse-transcribed into cDNA. The original levels of RNA for a specific gene can then be quantified from the amount of cDNA amplified in PCR.Genotyping -PCR can detect sequence variations in alleles of specific cells or organisms. A common example is the genotyping of transgenic organisms, such as knock-out and knock-in mice. In this application, primers are designed to amplify either a transgene portion (in a transgenic animal) or the mutation (in a mutant animal).Cloning and mutagenesis- PCR cloning is a widely used technique where double-stranded DNA fragments amplified by PCR are inserted into vectors (e.g., gDNA, cDNA, plasmid DNA). This for example, enables the creation of bacterial strains from which genetic material has been deleted or inserted. Site-directed mutagenesis can also be used to introduce point mutations via cloning. This often employs a technique known as recombinant PCR, in which overlapping primers are specifically designed to incorporate base substitutions (Figure 4). This technique can also be used to create novel gene fusions.

Figure 4: Diagram depicting an example of recombinant PCR.Sequencing- PCR can be used to enrich template DNA for sequencing. The type of PCR recommended for the preparation of sequencing templates is called high-fidelity PCR and is able to maintain DNA sequence accuracy. In Sanger sequencing, PCR-amplified fragments are then purified and run in a sequencing reaction. In next-generation sequencing (NGS), PCR is used at the library preparation stage, where DNA samples are enriched by PCR to increase the starting quantity and tagged with sequencing adaptors to allow multiplexing. Bridge PCR is also an important part of the second-generation NGS sequencing process.Both as an independent technique and as a workhorse within other methods, PCR has transformed a range of disciplines. These include:

Genetic research- PCR is used in most laboratories worldwide. One of the most common applications is gene transcription analysis9, aimed at evaluating the presence or abundance of particular gene transcripts. It is a powerful technique in manipulating the genetic sequence of organisms animal, plant and microbe - through cloning. This enables genes or sections of genes to be inserted, deleted or mutated to engineer in genetic markers alter phenotypes, elucidate gene functions and develop vaccines to name but a few. In genotyping, PCR can be used to detect sequence variations in alleles in specific cells or organisms. Its use isnt restricted to humans either. Genotyping plants in agriculture assists plant breeders in selecting, refining, and improving their breeding stock. PCR is also the first step to enrich sequencing samples, as discussed above. For example, most mapping techniques in the Human Genome Project (HGP) relied on PCR.Medicine and biomedical research- PCR is used in a host of medical applications, from diagnostic testing for disease-associated genetic mutations, to the identification of infectious agents. Another great example of PCR use in the medical realm is prenatal genetic testing. Prenatal genetic testing through PCR can identify chromosome abnormalities and genetic mutations in the fetus, giving parents-to-be important information about whether their baby has certain genetic disorders. PCR can also be used as a preimplantation genetic diagnosis tool to screen embryos for in vitro fertilization (IVF) procedures.Forensic science- Our unique genetic fingerprints mean that PCR can be instrumental in both paternity testing and forensic investigations to pinpoint samples' sources. Small DNA samples isolated from a crime scene can be compared with a DNA database or with suspects' DNA, for example. These procedures have really changed the way police investigations are carried out. Authenticity testing also makes use of PCR genetic markers, for example, to determine the species from which meat is derived. Molecular archaeology too utilizes PCR to amplify DNA from archaeological remains.Environmental microbiology and food safety- Detection of pathogens by PCR, not only in patients' samples but also in matrices like food or water, can be vital in diagnosing and preventing infectious disease.PCR is the benchmark technology for detecting nucleic acids in every area, from biomedical research to forensic applications. Kary Mullis's idea, written on the back of a receipt on the side of the road, turned out to be a revolutionary one.

References1. Chien A, Edgar DB, Trela JM. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 1976;127(3):1550-57 doi: 10.1128/JB.127.3.1550-1557.1976

2. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985;230(4732):1350 doi: 10.1126/science.2999980

3. Arya M, Shergill IS, Williamson M, Gommersall L, Arya N, Patel HRH. Basic principles of real-time quantitative PCR. Expert Review of Molecular Diagnostics 2005;5(2):209-19 doi: 10.1586/14737159.5.2.209

4. Bachman J. Chapter Two - Reverse-Transcription PCR (RT-PCR). In: Lorsch J, ed. Methods in Enzymology: Academic Press, 2013:67-74. doi : 10.1016/B978-0-12-420037-1.00002-6

5. Morley AA. Digital PCR: A brief history. Biomol Detect Quantif 2014;1(1):1-2 doi: 10.1016/j.bdq.2014.06.001

6. Taylor SC, Laperriere G, Germain H. Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Scientific Reports 2017;7(1):2409 doi: 10.1038/s41598-017-02217-x

7. Ahrberg CD, Manz A, Chung BG. Polymerase chain reaction in microfluidic devices. Lab on a Chip 2016;16(20):3866-84 doi: 10.1039/C6LC00984K

8. Garibyan L, Avashia N. Polymerase chain reaction. J Invest Dermatol 2013;133(3):1-4 doi: 10.1038/jid.2013.1

9. VanGuilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for gene expression analysis. BioTechniques 2008;44(5):619-26 doi: 10.2144/000112776

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Genetherapy | Cell Therapy | Conferences | Events | 2019 …

Cell Therapy

CellTherapy or Cytotherapy is the transfer of cells into a patient with a goal ofimproving the disease. From beginning blood transfusions were consideredto be the first type of celltherapy to be practiced as routine. Later, Bone marrow transplantation hasalso become a well-established concept which involves treatment of much kind ofblood disorders including anemia, leukemia, lymphoma and rareimmunodeficiencydiseases. Alternativemedical practitioners perform celltherapy in the form of several different names including xeno-transplanttherapy, glandular therapy, and fresh celltherapy. It has been claimed by the proponents of celltherapy that it has been used successfully to repair spinal cord injuries,strengthen weaken immune system, treatsautoimmunediseaseslike AIDS, helppatients with neurological disorders like Alzheimers disease, Parkinsons diseaseand epilepsy.

GeneTherapy

GeneTherapy basically involves the introduction or alteration of geneticmaterial within a cell or organism with an intention of curing the disease.Both celltherapy and gene therapy are overlapping fields of biomedical research withthe goals of repairing the direct cause ofGeneticdiseasesin DNA orcellular population respectively, the discovery of recombinant DNA technologyin the 1970s provided tools to efficiently develop genetherapy. Scientists use these techniques to readily manipulate viralgenomes, isolate genes and identify mutations involved in human disease,characterize and regulategeneexpressions, and engineer variousviral and non-viral vectors. Various long-term treatments for anemia,hemophilia, cystic fibrosis, muscular dystrophy, Gauschers disease, lysosomalstorage diseases, cardiovascular diseases, diabetes and diseases of bones andjoints are resolved through successful gene therapy and are elusivetoday.

StemCell Therapies

CellTherapy is defined as the therapy in which cellular material is injectedinto a patient in order to recover the healthy tissue. Celltherapy is targeted at many clinical indications in multiple organs bymeans of several modes of cell delivery.Stem-CellTherapyis the use ofstem cells to treat or prevent a disease or condition. Stemcells are a class of undifferentiated cells which are able to differentiateinto required or specialized cell types. Adult or somatic stem cells exist throughout the body after embryonic development and arefound available inside the different types of tissue. The stem cell methodologyincludes the phases of Stem cell or progenitor cell engraftment,differentiation followed by long term replacement of damaged tissue.

CellCulture and Bioprocessing

A Stem-Cell line is a group of undifferentiated stem cells which is culturedinvitro and can be propagated indefinitely. While stem cells can propagateindefinitely in culture due to their inherent cellular properties, immortalizedcells would not normally divide indefinitely but have gained this ability tosustain due to mutation. The Immortalized cell lines can be generated from cells by means of isolating cells fromtumors or induce mutations to make the cells immortal. An immortalizedcell line is a population of multicellular organism cells which has notproliferates indefinitely. Due to mutation, the cells evaded normal cellularsenescence and instead undergoing continuous celldivision. A key factor in reducing the production costs ofbiopharmaceuticals is the development of cell lines which in turn produce ahigh yield of product.

TissueScience & Regenerative Medicine

Regenerative Medicine is the branch of translational research deals with the processof replacing, engineering or regenerating human cells, tissues or organs inorder to restore or establish normal functionality of cell. Regenerativemedicine is the combination oftissueengineeringand Molecular Biology. CellTherapy mediate cell repair via five primary mechanisms: providing ananti-inflammatory effect, homing to damaged tissues and recruiting other cellssuch as endothelial progenitor cells for necessary tissue growth, supportingtissue remodeling over scar formation and inhibiting apoptosis programmablecell death and differentiating tissues into bone, cartilage, tendon andligament tissue.

ClinicalTrials on Cell & Gene Therapy

Clinical Trials of Celland Gene Therapy products often varying from the clinical trials design for other types of pharmaceutical products. Thesedifferences in trial design are necessitated by the distinctive features ofthese products. The clinical trials also reflect previous clinical experienceand evidence of medicine. Early experiences with Cell and Gene Therapy products indicate that some CGT products may posesubstantial risks to subjects due to effect at cellular and genetic level. Thedesign of early-phase clinical trials of Cell and Gene Therapy products ofteninvolves the following consideration of clinical safety issues, preclinicalissues and chemistry, manufacturing and controls (CMC) issues that areencountered.

NanoTherapy

Diseases can betreated using viruses as vector to deliver genes inGeneTherapy. Viruses as genevector however, can themselves cause problems in that they may initiateinflammation and the genes may be expressed at too high a level or for too longperiod of exposure. The goal of Nano Technology in genetherapy is delivery of therapeutic genes without a virus, usingnanoparticles as non-viral vector to deliver the genes. The particles canbe made with multiple layers so the outer layer with covering of peptide thatcan target the particles to cells of interest at specific site. The emergent Nanotechnologyin gene therapy is used to develop unique approaches in treating theretinopathies and the development of micro and Nano dimensional artificialantigen presenting cells forcancerimmunotherapy. These antigenpresenting cells mimic the natural signals in immunity that killer T-cellsreceive when there is an invader (bacteria, virus, cancer cell, etc.) in thebody.

AdvancedGene Therapeutics

Functionality ofbiomaterials for these forms is depends upon the chemical reaction such aslocalized or systemic response at the surface tethered moieties or encapsulatedtherapeutic factors such as drugs, genes, cells, growth factors, hormones andother active agents to specific target sites. The application of functional biomaterials is rehabilitation, reconstruction, regeneration, repair,ophthalmic applications and act as therapeutic solutions. It has the propertyof biocompatibility and produce inertness response to the tissue.Thebiomaterial-mediated gene therapy aim to use polymeric gene therapy systems tohalt the progression of neuron loss throughneuroprotectiveroutesand it combine stemcell therapy and biomaterial delivery system in order to enhance regeneration or repairafter ischemic injury.

Geneand Cell Therapy for Rare & Common Diseases

Gene therapy is a superior method to treat uncommon hereditary maladies; fixa solitary quality deformity by presenting a 'right' quality. The main qualitytreatment preliminaries were directed utilizing patients with uncommonmonogenetic issue, however these are presently dwarfed by the clinical testingof quality therapeutics for more typical conditions, for example, malignancy,AIDS and cardiovascular illness. This is halfway because of an inability toaccomplish long haul quality articulation with early vector frameworks, a basicprerequisite for amending numerous innate hereditary deformities. Presently, with the appearance ofadeno-relatedviral(AAV) and lent viral vectors, which show steady qualityarticulation in creature thinks about, this mechanical obstruction, may havebeen survived. These vectors are foreseen to shape the premise of numerous genetherapy protocols for acquired hereditary illnesses.

CellScience and Stem Cell Research

The extract derivedfrom the plant cell culture technology is being harnessed and utilized as anactive ingredient in anti-aging skincare products. In recent years, researchershave identified naturally occurring botanicals with substantial antioxidantactivity proven to protect skin stem cells from UV-induced oxidative stress,inhibit inflammation, neutralize free radicals and reverse the effects ofphoto-aging by means ofanti-oxidantactivity. Consequently,cosmeceutical products containing plant stem cell derived extracts have theability to promote healthy cell proliferation and protect against UV-induced dermatological cellular damage in humans. In contrast to epidermal stem cells,plant stem cells are totipotent that they are capable of regenerating anentirely new, whole plant. Through innovative plant stem cell technology,scientists are able to extract tissue from botanicals and regenerate stem cells can be harnessed for use in humans. The use of stem cellsderived from botanicals plant, rather than human stem cells, avoids thecontroversy surrounding the source or methods of extraction of human stem cellswhile still harnessing the potential of these intriguing cells and its effectin anti-photo aging.

MolecularBasis of Epigenetics

Epigenetics refers to changes in a chromosomewhich has influence on gene activity and expression. It is also used todescribe any heritable phenotypic change that doesn't derive from amodification of the chromosome such as prions.Epigeneticsis the mechanism for storing andperpetuating or continuing indefinitely a memory at the cellular level. Thebasic molecular epigenetic mechanisms that are widely studied at present regulation of chromatin structure of cell through histone post-translationalmodifications and covalent modification of DNA principally through the methodof DNA methylation. Chromatin is a dynamic structure that integrates potentially hundreds ofsignals from the cell surface and has effects of coordinated and appropriate transcriptional response in cell. It is increasingly clear that epigenetic marking ofchromatin and DNA itself is an important component of the cell signalintegration of entire function that is performed by the genome. Moreover, thechanges in the epigenetic state of chromatin in cell can have lasting effectson behavioral changes.

Geneticsand Stem Cell Biology

An undifferentiated mass of cell in amulticellular animal which is prepared for offering rise to uncertain number ofcells of a comparable sort, and from which certain diverse sorts of cell riseby detachment. Undifferentiated life forms can isolate into specific cell creates. Thetwo describing characteristics of an undifferentiated cell are endlessself-restoration and the ability to isolate into a specific adult. There aretwo critical classes of youthful microorganisms: pluripotent that can end upbeing any cell in the adult body, and multipotent that is kept to transforminginto more limited masses of cells.

Regulatoryand Safety Aspects of Cell and Gene Therapy

Celltreatment things require a combination of prosperity examinations.Comparable living being and quality things are heterogeneous substances. Thereare a few zones that particularly ought to be tended to as it is extremely notthe same as that of pharmaceuticals. These range from making group consistency,thing soundness to thing prosperity, quality and sufficiency throughpre-clinical, clinical examinations and displaying endorsement. This reviewplots the present headings/administers in US, EU, India and the relatedchallenges in making SCBP with highlight on clinical point.

Markets& Future Prospects for Cell & Gene Therapy

The immense number of associations related withcell treatment has extended development incredibly in the midst of the pastcouple of years. More than 500 associations have been recognized to be lockedin with cell treatment and 305 of these are profiled 291 co-tasks. Of theseassociations, 170 are related with fundamental microorganisms. The Profiles of72 academic establishments in the US related with cell treatment close by theirbusiness facilitated efforts. Allogeneicdevelopment with in excess of 350 clinical preliminaries is prepared toorder the commercialization of cell medicines in publicize. Advance R&D incell and quality treatment is depended upon to bloom given the normally basedpurposes of intrigue.

CellBiology

Cell biology is the investigation of cell andhow the cell capacities. Cell consist of numerous organelles that performparticular capacities and assume an imperative part in the development andgrowth of an organism. Cells are of 2 composes Prokaryotic Cell and Eukaryotic Cell. Case of aProkaryoticCellincorporates,Bacteria, then again Animal Cell and Plant Cell are portrayed as EukaryoticCells

Geneediting and CRISPR based technologies

CRISPR (Clustered Regularly Interspaced ShortPalindromic Repeats) Technology is a champion among the most fit yet clearmechanical assembly for genome changing. It urges and empowers investigators toeasily change DNA groupings and modify quality limits. It has various potentialapplications that join helping innate disseminates, treating and keeping thespread of diseases and improving yields. CRISPR broadly used as CRISPR-Cas9whereCRISPRsare particular stretches out of DNA andCas9 is the protein which is an aggravate that exhibitions like a few nuclearscissors, fit for cutting DNA strands. The assurance of CRISPR advancementanyway raises moral stresses as it isn't 100% compelling. Regardless, the progressionof CRISPR-Cas9 has disturbed the designed science industry these days, being aclear and great quality changing device.

Genetics& Genomic Medicine

Genetics in Health and Disease in whichtherapy utilizesgenetics, imaging and biological indicators tounderstand predisposition to disease, what constitutes health during childhoodand throughout the life course. Gene and Protein Function are used to develop tools, skills and resources to elucidategene function and to inform development of new therapies using state-of the-arttechnologies. Personalized Medicine and Patient benefit is considered to ensurebasic science discoveries of disease mechanisms and patients genomes are usedto produce best effect to improve patients lives which include betterdiagnostics, identification of biomarkersand targeting of therapies.

BioengineeringTherapeutics

Tissue Engineering or Bioengineering is the combinational usage of cells,Engineering, materials methods, suitable biochemical and physicochemicalfactors in order to improve or replace the infected biological tissues. Thefield includes the development of materials, devices,techniques to detect and differentiate disease states, the treatmentresponse, aid tissue healing, precisely deliver treatments to tissues or cells,signal early changes in health status, and provide implantable bio-artificialreplacement organs for recover or establish of healthy tissue. Techniquesdeveloped here identify and detect biomarkers of disease sub-types,progression, and treatment response, from tissue imaging to genetic testing andSingle cell analysis, that aid the more rapid development of new treatments andguide their clinical applications in treating the disorder. It includes theusage of computational modeling, bioinformatics, andquantitativepharmacologyto integratedata from diverse experimental and clinical sources to discover new drugs andspecific drug targets, as well as to design more efficient and informativepreclinical, clinical safety and efficacy studies.

Immunogenetics& Transplantation

Immunogenetics and Transplantation providesspecialized diagnostic services for allogeneic transplantation and related research. It provides support for blood, bonemarrow, kidney, pancreas, liver, heart, lung, small bowel andcorneatransplantation. Currently Immunogenetics& Transplantation is hot topic of discussion.

Biomarkers

Biomarkersare evolving rapidly in the advance of personalized medicine and individualhealth. The identification & validation of biomarkers in drug discovery,development and in disease prognosis, diagnosis, prevention & treatmentplay an essential role in the genomic era.

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