TL;DR
— Advance in understanding actin sheds light on cell function — atomic level insight into proteins furthers fundamental understanding of human life;
— Rare genetic variants from birth have negative effect on lifespan — the researchers found that the combined effects of rare, damaging mutations present at birth have a negative impact on healthspan and longevity;
— Artificial Intelligence is helping biotech get real — Whether modeling the real world or making sense of real-time data, AI can enhance drug development from candidate screening to trial management;
— Researchers move closer to industrial production of heparin in cell culture;
— Using JAX NSG™ mice to test car T therapy for breast cancer;
— Super-charging drug development for COVID-19 — cell-free production method scales up yield by 5,000 times;
— COVID-19: Genetic network analysis provides ‘snapshot’ of pandemic origins;
— Switching on a key cancer gene could provide first curative treatment for heart disease;
— Artificial proteins give living cells a computational upgrade;
— Predicting the evolution of genetic mutations;
— Role of gene mutations in kidney disease described in new study;
— Study finds remdesivir effective against a key enzyme of coronavirus that causes COVID-19;
— Technique offers path for biomanufacturing medicines during space flights — Research simulating instrument aboard international space station nurtures E. coli bacteria;
— New tool to combat major wheat disease described in a fresh research;
— Novel role for dopamine that impacts gene expression related to cocaine abuse;
— Development of gene editing strategies for human β-globin (HBB) gene mutations;
..and a lot more!
Overview
Genetic technology is defined as the term which includes a range of activities concerned with the understanding of gene expression, advantages of natural genetic variation, modifying genes and transferring genes to new hosts. Genes are found in all living organisms and are transferred from one generation to the next. Gene technology encompasses several techniques including marker-assisted breeding, RNAi and genetic modification. Only some gene technologies produce genetically modified organisms.
Modern genetic technologies like genome editing would not be possible without all the previous generations of genetic technologies that have enabled scientists to discover what genes are, what they do and how DNA can be modified to add, remove or replace genes. Let’s look at the major milestones of the past:
Gene Technology Market
- The valuation of the genetic engineering market is projected to escalate to USD 6.90 MN by the end of 2027.
- Global Genetic Engineering Market is projected to grow at 12.48% CAGR during the assessment period (2017–2027).
- North America holds the largest share in the global genetic engineering market, followed by Europe and the Asia Pacific, respectively.
- Merck KGaA (Germany), Thermo Fisher Scientific Inc. (US), Horizon Discovery Group Plc. (UK), Transposagen Biopharmaceuticals Inc. (US), Genscript Biotech Corporation (US), New England Biolabs (US), Lonza Group Ltd. (Switzerland), Inc. (US), and Integrated DNA Technologies, Inc. (US), and Origene Technologies, Inc. (US), are some of the major players operating in the genetic engineering market.
- Another research provider, MarketsandMarkets, forecasts the genome editing, genome engineering market to grow from USD 3.19 billion in 2017 to USD 6.28 billion by 2022, at a compounded annual growth rate (CAGR) of 14.5% during the forecast period. The key factors propelling market growth are rising government funding and growth in the number of genomics projects, high prevalence of infectious diseases (like COVID-19) and cancer, technological advancements, increasing production of genetically modified (GM) crops, and growing application areas of genomics.
- Recent Market-Players’ Developments:
- In 2019, Merck signed a license agreement with Evotec SE. This agreement will enable Evotec access to Merck’s foundational CRISPR intellectual property.
- In 2018, Thermo Fisher Scientific, Inc. expanded its Genome Editing IP Portfolio.
- In 2018, Horizon Discovery launched Edit-R CRISPRa arrayed crRNA (CRISPR RNA) libraries, an addition to its CRISPR activation (CRISPRa) reagent platform.
Genetics News & Researches
Original research: The architecture of SARS-CoV-2 transcriptome by Dongwan Kim, Joo-Yeon Lee, Jeong-Sun Yang, Jun Won Kim, V. Narry Kim, and Hyeshik Chang
Researchers in Korea have harnessed two complementary sequencing techniques to better understand the genetic architecture of the SARS-CoV-2 genome. By combining nanopore-based direct RNA sequencing (DRS) sequencing, and DNA nanoball (DNB) sequencing, the scientists, led by Narry Kim, PhD, and Hyeshik Chang, PhD, at the Center for RNA Research within the Institute for Basic Science (IBS) in Seoul, generated new insights into subgenomic RNAs (sgRNAs) that are translated into viral proteins. Analyzing the sequence information of each RNA also revealed where the genes are located on the long viral genomic RNA.
“Now we have secured a high-resolution gene map of the new coronavirus that guides us where to find each bit of genes on all of the total SARS-CoV-2 RNAs (transcriptome) and all modifications RNAs (epitranscriptome),” Kim stated. “It is time to explore the functions of the newly discovered genes and the mechanism underlying viral gene fusion. We also have to work on the RNA modifications to see if they play a role in virus replication and immune response. We firmly believe that our study will contribute to the development of diagnostics and therapeutics to combat the virus more effectively.”
- Advance in understanding actin sheds light on cell function — Atomic-level insight into proteins furthers fundamental understanding of human life
Original paper: Mechanism of actin N-terminal acetylation by Grzegorz Rebowski, Malgorzata Boczkowska, Adrian Drazic, Rasmus Ree, Marianne Goris, Thomas Arnesen, Roberto Dominguez in Science Advances, 2020
A tiny chemical modification on one of the most abundant and important proteins in cells, actin, has long been somewhat mysterious, its function not fully understood, but scientists have now taken a big step towards clearing up the mystery. The scientists, who report their discovery on the post-translational modification of actin, believe their discovery sheds light on the foundational construction of life.
- Rare Genetic Variants from Birth Have Negative Effect on Lifespan — The researchers found that the combined effects of rare, damaging mutations present at birth have a negative impact on healthspan and longevity.
Findings from the new study — published recently in eLife through an article titled “Germline burden of rare damaging variants negatively affects human healthspan and lifespan” — suggest one additional inherited damaging mutation could carve off six months of life, and combinations of these rare mutations determine how soon someone will develop diseases such as cancer, heart disease, and dementia.
“Genetic variants identified so far can explain only a small fraction of lifespan heritability in humans,” the authors wrote. “Here, we report that the burden of rarest protein-truncating variants (PTVs) in two large cohorts is negatively associated with human healthspan and lifespan, accounting for 0.4 and 1.3 years of their variability, respectively. In addition, longer-living individuals possess both fewer rarest PTVs and less damaging PTVs. We further find that somatic accumulation of PTVs accounts for only a small fraction of mortality and morbidity acceleration and hence is unlikely to be causal in aging.”
The scientists found that people who had a high burden of ultra-rare PTVs had a shorter healthspan and lifespan. Each additional ultra-rare PTV in a person’s genome accounted for a reduction in lifespan of six months and a reduction in healthspan of two months.
Cellectis says the U.S. patent issued to the company this month will facilitate the development of future T-cell immunotherapy treatments based on CRISPR-Cas. The company’s clinical-phase pipeline consists entirely of treatments that apply its proprietary TALEN® gene-editing technology.
- Artificial Intelligence Is Helping Biotech Get Real by Catherine Shaffer in Gen Edge, April Vol.50 №4 — Whether modeling the real world or making sense of real-time data, AI can enhance drug development from candidate screening to trial management.AI may be used to extract insights from millions of experimental affinity measurements and thousands of protein structures to predict the binding of small molecules to proteins. This approach is being realized by Atomwise, the developer of AtomNet, a structure-based, deep convolutional neural network designed to predict the bioactivity of small molecules for drug discovery applications. AtomNet helped Stanford University researchers screen 6.8 million small molecules for their ability to target Miro1, a protein implicated in Parkinson’s disease. In the space-filling structure for Miro1 shown here, the area in white represents the screening site. The most promising compound, the Miro1 Reducer, appears in the inset.
- Researchers Move Closer to Industrial Production of Heparin in Cell Culture
Scientists at the University of California-San Diego say they have moved one step closer to the ability to make heparin in cultured cells. Heparin is a potent anticoagulant and the most prescribed drug in hospitals, yet cell-culture-based production of heparin is currently not possible, according to the researchers who published their study, “ZNF263 is a transcriptional regulator of heparin and heparan sulfate biosynthesis” in PNAS.
In particular, the researchers found a critical gene in heparin biosynthesis: ZNF263 (zinc-finger protein 263). The team believes this gene regulator is a key discovery on the way to industrial heparin production. The idea would be to control this regulator in industrial cell lines using genetic engineering, paving the way for safe industrial production of heparin in well-controlled cell culture.
- Using JAX NSG™ Mice to Test Car T Therapy for Breast Cancer by Maya Dubey, PhD with the support of the Jackson Laboratory.
In a recent study by Zhou and colleagues, the authors examined the impact of a modified CAR T therapy on NSG™ mice from JAX, injected with a TNBC tumor cell line in one mammary fat pad. Using a human CAR that utilized the scFv motif from the monoclonal antibody, TAB004, the authors injected a single dose of the engineered T cells to tumor-bearing mice, after the tumor had been established. They observed that, in comparison to the control group, the tumor growth was dramatically reduced and the reduction was maintained until the study endpoint at day 57. Further analysis showed that the modified CAR T cells that infiltrated the tumors expressed high levels of both CD25 and PD1, which, as noted by the authors, indicates further activation by in vivo tumor antigen stimulation from the glycosylated tumor form of MUC1 (tMUC1).
Then, to determine whether the effects of the modified CAR T cells could extend beyond 57 days, the authors conducted a similar experiment as noted above, but extended the study endpoint to 81 days. They observed that, in comparison to the control group, the therapy reduced tumor growth until the study endpoint. However, they also observed that the treated tumors started to increase their growth after approximately 60 days post-treatment. The authors note that this may be due to several possibilities including requiring more than a single treatment injection, the loss of tMUC1 in the remaining tumors that progressed, or the increased PD1 expression in the tumor-infiltrating lymphocytes may block the anti-tumor response. They tested each possibility and found that the most likely solution to remain active would require additional injections of the therapy. In addition, the combination of CAR T cells and PD1 antibody enhanced the anti-tumor immune response.
Collectively, the data collected by the authors demonstrate the effectiveness of using a modified CAR T therapy to reduce TNBC tumor growth effectively, with minimal damage to normal cells. Moreover, they are exploring the use of this type of treatment against other tumor types as well as combining this therapy with others as potential treatments for solid tumors.
- Super-charging drug development for COVID-19 — Cell-free production method scales up yield by 5,000 times.
Materials provided by Northwestern University. Originally written by Amanda Morris.
Researchers are ramping up the production of a promising drug that has proven effective in obliterating SARS-CoV in cellular cultures. The team hopes that the drug might also be effective in the fight against SARS’s close genetic cousin, the novel coronavirus (COVID-19).
Original materials: Peter Forster, Lucy Forster, Colin Renfrew, Michael Forster. Phylogenetic network analysis of SARS-CoV-2 genomes in “Proceedings of the National Academy of Sciences”, 2020
The first use of phylogenetic techniques shows the ‘ancestral’ virus genome closest to those in bats was not Wuhan’s predominant virus type. The study charts the ‘incipient supernova’ of COVID-19 through genetic mutations as it spread from China and Asia to Australia, Europe, and North America. Researchers say their methods could be used to help identify undocumented infection sources. By analysing the first 160 complete virus genomes to be sequenced from human patients, the scientists have mapped some of the original spread of the new coronavirus through its mutations, which creates different viral lineages.
“The viral network we have detailed is a snapshot of the early stages of an epidemic, before the evolutionary paths of COVID-19 become obscured by vast numbers of mutations. It’s like catching an incipient supernova in the act” they stated.
Original research paper, Reactivation of Myc transcription in the mouse heart unlocks its proliferative capacity, was presented by Megan J. Bywater, Deborah L. Burkhart, Jasmin Straube, Arianna Sabò, Vera Pendino, James E. Hudson, Gregory A. Quaife-Ryan, Enzo R. Porrello, James Rae, Robert G. Parton, Theresia R. Kress, Bruno Amati, Trevor D. Littlewood, Gerard I. Evan, Catherine H. Wilson in Nature Communications, 2020.
Researchers trying to turn off a gene that allows cancers to spread have made a surprising U-turn. By making the gene overactive and functional in the hearts of mice, they have triggered heart cell regeneration. Since adult hearts cannot usually repair themselves once damaged, harnessing the power of this gene represents major progress towards the first curative treatment for heart disease.
“This is really exciting because scientists have been trying to make heart cells proliferate for a long time. None of the current heart disease treatments are able to reverse degeneration of the heart tissue — they only slow progression of the disease. Now we’ve found a way to do it in a mouse model,” said Dr Catherine Wilson, a researcher in the University of Cambridge’s Department of Pharmacology, who led the study.
Call it Protein Logic Gate 2.0. The 1.0 version, which rewired native signaling pathways, was admirably direct, bypassing awkward rewiring at the DNA and RNA levels, but it wasn’t very scalable or extensible. It relied on a limited pool of building blocks, in this case, native proteins presenting interfaces open to fabricated protein-protein interactions. Artificial proteins can offer more flexibility. They can come in sets of modular units that interact with each other in clearly defined ways to read inputs, complete logical operations, and generate outputs.
The equivalent of Protein Logic Gate 2.0 has been launched by scientists based at the University of Washington (UW) School of Medicine. They’ve already run demonstrations of how it can be used to manipulate gene expression. These demonstrations, which involved cell-free extracts, yeast cells, and T cells, were presented in the journal Science, in an article titled, “De novo design of protein logic gates.”
This graphic table compares how electronic and protein AND logic gates respond when no input is present, when only A or B is present, and when both A and B are present. Source: UW Medicine Institute for Protein Design
Original paper: Minimum epistasis interpolation for sequence-function relationships by Juannan Zhou, David M. McCandlish in Nature Communications, 2020
Quantitative biologists have designed a new machine learning technique for predicting evolutionary pathways. It could prove a valuable tool for biologists studying rapidly evolving viruses or cancer. Described in Nature Communications, the algorithm called “minimum epistasis interpolation” results in a visualization of how a protein could evolve to either become highly effective or not effective at all. They compared the functionality of thousands of versions of the protein, finding patterns in how mutations cause the protein to evolve from one functional form to another.
Scientists from the Center for Precision Disease Modeling at the University of Maryland School of Medicine (UMSOM) say they have uncovered a mechanism that appears to explain how certain genetic mutations give rise to a rare genetic kidney disorder called nephrotic syndrome. Using a drosophila model, they found mutations in genes that code for certain proteins leads to a disruption of the recycling of the cell membrane. This disruption leads to an abnormal kidney cell structure and function, according to the study “Exocyst Genes Are Essential for Recycling Membrane Proteins and Maintaining Slit Diaphragm in Drosophila Nephrocytes” published in the Journal of the American Society of Nephrology.
Original article: Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome Coronavirus 2 with high potency by Calvin J Gordon, Egor P Tchesnokov, Emma Woolner, Jason K Perry, Joy Y. Feng, Danielle P Porter, Matthias Gotte in the Journal of Biological Chemistry, April 13, 2020
Scientists have shown that drug remdesivir is highly effective in stopping the replication mechanism of the coronavirus that causes COVID-19. The finding follows closely on research demonstrating how the drug worked against the Middle East Respiratory Syndrome (MERS) virus, a related coronavirus.
by Qiuxia Xu, Min Wang, Sujing Huang, Lin Xu, Hongqiong Guan, Hong Zhu from the Department of Obstetrics, The Second Affiliated Hospital of Hainan Medical College, Haikou City, Hainan Province, China
This paper studies the correlation between TSC1/TSC2 mutations and phenotypes of tuberous sclerosis (TSC) gene in prenatal diagnosis based on sensor measurement technology high-throughput sequencing platform. The paper mainly performs sensor measurement technology-based high-throughput sequencing of the gene coding region of TSC1/TSC2 and adjacent intron regions of 10 familial probands with the clinical diagnosis of tuberous sclerosis. High-throughput sequencing was used to validate the discovered gene mutations and prenatal diagnosis for disease-causing mutations. The results of the study found that 10 cases of probands found TSC1/TSC2 mutations, including 1 case of TSC1 mutation, 9 cases of TSC2 mutation, 9 cases of missense mutations in 6 cases, 2 cases of nonsense mutations, frameshift 1 case of mutation. Further, 9 prenatal diagnoses were made for the mother, and one mutant positive foetus was found. The research has found that the high-throughput sequencing platform based on sensor measurement technology can effectively verify and detect gene mutations, which has certain advantages and effectiveness. Therefore, it can be widely used in clinical diagnosis and treatment of gene mutations, which is effective for preventing gene mutations in postpartum infants.
The paper discovered and reported a new TSC gene mutation, namely TSC2 gene C. It not only provides direction for genetic counseling of sick families but also assists in the formulation and prognosis analysis of perinatal management strategies, directly affecting the outcome and outcome of the foetus.
- Technique offers path for biomanufacturing medicines during space flights — Research simulating instrument aboard international space station nurtures E. coli bacteria.
Original research Growth of microorganisms in an interfacially driven space bioreactor analog by Joe A. Adam, Shreyash Gulati, Amir H. Hirsa, Richard P. Bonocora in npj Microgravity, 2020
Research published in Nature Microgravity used an Earth-bound simulator of the space station instrument to grow E. coli, demonstrating that it can be nurtured with methods that promise to be more suitable for space travel than existing alternatives. An instrument currently aboard the International Space Station could grow E. coli bacteria in space, opening a new path to bio-manufacturing drugs during long term space flights.
“If we can get microorganisms to grow well in space, astronauts can use them to make pharmaceuticals on demand. This could be vital for survival on long missions where resupplying is not an option.” said Richard Bonocora, senior author and a faculty member in the Department of Biological Sciences at Rensselaer Polytechnic Institute. “Here we were asking: ‘Is there a better way to grow microorganisms that what is currently being used is space?’ And what we find is that — with shear force — yes, there likely is.”
Original paper: Horizontal gene transfer of Fhb7 from fungus underlies Fusarium head blight resistance in wheat by Hongwei Wang, Silong Sun, Wenyang Ge, Lanfei Zhao, Bingqian Hou, Kai Wang, Zhongfan Lyu, Liyang Chen, Shoushen Xu, Jun Guo, Min Li, Peisen Su, Xuefeng Li, Guiping Wang, Cunyao Bo, Xiaojian Fang, Wenwen Zhuang, Xinxin Cheng, Jianwen Wu, Luhao Dong, Wuying Chen, Wen Li, Guilian Xiao, Jinxiao Zhao, Yongchao Hao, Ying Xu, Yu Gao, Wenjing Liu, Yanhe Liu, Huayan Yin, Jiazhu Li, Xiang Li, Yan Zhao, Xiaoqian Wang, Fei Ni, Xin Ma, Anfei Li, Steven S. Xu, Guihua Bai, Eviatar Nevo, Caixia Gao, Herbert Ohm, Lingrang Kong in Science, 2020
Agricultural Research Service (ARS) scientists and their colleagues have discovered a gene that can be used to develop varieties of wheat that will be more resistant to Fusarium Head Blight (FHB), a disease that is a major threat both overseas and to the nation’s $10 billion annual wheat crop.
Scientists have discovered a new role for the brain chemical dopamine that is independent of classic neurotransmission. The new role appears to be critical to changes in gene expression related to chronic exposure to, or abuse of, cocaine. To be more precise, scientists at the Icahn School of Medicine at Mount Sinai have discovered a new role for the brain chemical dopamine that is independent of classic neurotransmission. The new role appears to be critical to changes in gene expression related to chronic exposure to, or abuse of, cocaine, according to a study published Friday, April 10, in the journal Science.
- Vascular Supply of the Human Spiral Ganglion: Novel Three-Dimensional Analysis Using Synchrotron Phase-Contrast Imaging and Histology by Xueshuang Mei, Rudolf Glueckert, Annelies Schrott-Fischer, Hao Li, Hanif M. Ladak, Sumit K. Agrawal, Helge Rask-Andersen in Scientific Reports, 2020
What does it actually look like deep inside our ears? This has been very difficult to study as the inner ear is protected by the hardest bone in the body. But with the help of synchrotron X-rays, it is now possible to depict details inside the ear three-dimensionally. Researchers have now used the method to map the blood vessels of the inner ear.
Human spiral ganglion (HSG) cell bodies located in the bony cochlea depend on a rich vascular supply to maintain excitability. These neurons are targeted by cochlear implantation (CI) to treat deafness, and their viability is critical to ensure successful clinical outcomes. The blood supply of the HSG is difficult to study due to its helical structure and encasement in hard bone. The objective of this study was to present the first three-dimensional (3D) reconstruction and analysis of the HSG blood supply using synchrotron radiation phase-contrast imaging (SR-PCI) in combination with histological analyses of archival human cochlear sections. Twenty-six human temporal bones underwent SR-PCI. Data were processed using volume-rendering software, and a representative three-dimensional (3D) model was created to allow visualization of the vascular anatomy. Histologic analysis was used to verify the segmentations. Results revealed that the HSG is supplied by radial vascular twigs which are separate from the rest of the inner ear and encased in bone. Unlike most organs, the arteries and veins in the human cochlea do not follow the same conduits. There is a dual venous outflow and a modiolar arterial supply. This organization may explain why the HSG may endure even in cases of advanced cochlear pathology.
by Batuhan Mert Kalkana, Ezgi Yagmur Kalaa, Melek Yucea, Medine Karadag Alpaslana, Fatih Kocabasa from a Regenerative Biology Research Laboratory, Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey
Recent developments in gene-editing technology have enabled scientists to modify DNA sequence by using engineered endonucleases. These gene-editing tools are promising candidates for clinical applications, especially for the treatment of inherited disorders like sickle cell disease (SCD). SCD is caused by a point mutation in the human βglobin gene (HBB). Clinical strategies have demonstrated substantial success, however, there is not any permanent cure for SCD available. CRISPR/Cas9 platform uses a single endonuclease and a single guide RNA (gRNA) to induce sequence-specific DNA double-strand break (DSB). When this accompanies a repair template, it allows repairing the mutated gene. In this study, it was aimed to target the HBB gene via the CRISPR/Cas9 genome editing tool to introduce nucleotide alterations for efficient genome editing and correction of point mutations causing SCD in a human cell line, by Homology Directed Repair (HDR). They have achieved to induce target specific nucleotide changes on HBB gene in the locus of mutation causing SCD. The effect of the on-target activity of bone fide standard gRNA and newly developed longer gRNA were examined. It is observed that longer gRNA has a higher affinity to target DNA while having the same performance for targeting and Cas9 induced DSBs. HDR mechanism was triggered by the co-delivery of donor DNA repair templates in circular plasmid form. In conclusion, they have suggested a methodological pipeline for efficient targeting with a higher affinity to target DNA and generating desired modifications on HBB gene.
Industry Important Articles
by Sheeba Naaz and Syed Naqui Kazim from the Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India
Traditional cloning is the cloning in which it is used, the restriction endonucleases to produce DNA fragments with specific complementary end sequences that can be joined together with a DNA ligase enzyme, prior to the transformation. The purpose of this study was to reconstruct the mammalian expression vector by replacing the junk DNA with its natural multiple cloning sites. The plasmid was propagated and digested with Xho1 and Kpn1 enzymes from both sides of junk DNA a 600 bp DNA was cut from the vector and then ligation of 30 bp primer which was designed similarly as its multiple sequence sites. Correct insertion was confirmed by plasmid isolation, plasmid colony PCR, PCR of cloned plasmid and comparison on gel electrophoresis with the original one and finally by sequencing. An extra DNA fragment of 600 bp was cut from the vector after restriction digestion. Ligated colonies appeared on the agar plate from which 60 bp colony PCR products was produced. Plasmid isolation after cloning and colony PCR and PCR of cloned plasmid confirmed the cloning. Cloning of multiple cloning sites in pcDNA3.0 mammalian expression vector was performed successfully. Ligation of multiple cloning sites in pcDNA vector provided various restriction enzymes recognition sites for simple and fast cloning.
As a result, the reconstruction of a mammalian expression vector pcDNA3.0 was successfully done after ligation of its proper multiple cloning sites. It was confirmed by the generation of 60 bp band in colony PCR and generation of nearly 5.4 kb band after (DNA isolation). Cloning was also confirmed by the difference produced in bands of cloned DNA with the original DNA in agarose gel electrophoresis. PCR results also showing clear difference between the cloned pcDNA with the original one. From these results, they were successful to clone pcDNA3.0 with its natural structure.
by Yihua Gao, Jian Li, Bin Chen, Qingjin Yang, Junjie Zhang, Lijian Zhang, Qionglin Huang, Xiaoxia Ye, and Chun Cai from the Analysis Center of Guangdong Medical University, Zhanjiang, Guangdong, P. R. China.
5-carboxylcytosine (5caC) is an important derivative of cytosine modification in DNA demethylation pathway. However, the accurate quantification of 5caC is a challenge, especially in mammalian tissue. This plight probably results from the trace amount of 5caC in mammalian and the inadequate sensitivity of the existing technologies. Herein, they report a novel LC-MS/MS method to precisely quantify 5caC in mammalian cells. The 5caC standard reagent was synthesized, and the genomic DNA was hydrolyzed with formic acid and the target compound 5caC was detected by HILIC LC-MS/MS. The results showed 5caC generally exist in mice organs and cancer tissues. The content of 5caC in mice brain was higher than in lungs and liver, and was obviously decreased in colorectal cancer tissues compared with the adjacent tissue. This result suggests that 5caC probably associates with tumorigenesis and plays a dominated role in epigenetic control of neuronal function.
They have confirmed and quantified the 5caC in mammalian tissues with excellent accuracy, and found that 5caC was normally presented in mammalian organs and the content was
higher in tumor-adjacent tissues than colorectal cancer congeners. In accordance with the results, 5caC was probably suggested to participate in regulation of neuronal activities and cancer development. In their method, the sample preparation was simple, and the nucleobases could be fully hydrolyzed by formic acid within a short time and directly used for LC-MS analysis. In addition, the modified cytosine instead of its derivatives was determined, which made the qualitation and quantification of target compounds more direct and accurate. This study will provide a firm foundation for the exploration of the functions of 5caC in the DNA demethylation process in mammalians.
by Deniz Kor, Berna Seker Yılmaz, Ozden Ozgur Horoz, Gulay Ceylaner, Selcuk Sızmaz, Fadli Demir, and Neslihan Onenli Mungan from the Department of Pediatric Metabolism and Nutrition, Faculty of Medicine, Cukurova University, Adana, Turkey
Mutations in the AGK gene are known to cause Sengers Syndrome, a rare recessive disorder characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance and lactic acidosis with normal mental development. Since the first report in 1975 by Sengers et al. about 50 individuals have been described as having this syndrome. In their research paper, they reported two novel mutations in the AGK gene in two patients with neonatal Sengers syndrome.
In the differential diagnosis of hypertrophic cardiomyopathy, the key point of diagnosis of Sengers Syndrome is bilateral cataracts. The confirmation of the diagnosis should be done with mutation analysis of the AGK gene. They are reporting these patients due to novel mutations in such a rare disorder. In their patients, these novel mutations caused severe clinical symptoms, including congenital cataracts, hypertrophic cardiomyopathy, lactic acidosis and early death. It also causes skeletal myopathy in one of their cases. Additive clinical data and genetic investigations are still necessary to learn more about this rare disease.
by López Del Amo, V., Bishop, A.L., Sánchez C, H.M., Bennett, J.B., Feng, X., Marshall, J.M.b, Bier, E., Gantz, V.M. from the Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093, United States
CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. In the research, they address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split–gene-drive (tGD) and demonstrate its ability to promote the super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. They employ the tGD and a small–molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.
by Maniyadath, B., Sandra, U.S., Kolthur-Seetharam, U. from the Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Eukaryotic complexity and thus their ability to respond to diverse cues are largely driven by varying expression of gene products, qualitatively and quantitatively. Protein adducts in the form of post-translational modifications, most of which are derived from metabolic intermediates, allow fine-tuning of gene expression at multiple levels. With the advent of high-throughput and high-resolution mapping technologies, there has been an explosion in terms of the kind of modifications on chromatin and other factors that govern gene expression. Moreover, even the classical notion of acetylation and methylation-dependent regulation of transcription is now known to be intrinsically coupled to biochemical pathways, which were otherwise regarded as ‘mundane’. They have not only reviewed some of the recent literature but also have highlighted the dependence of gene regulatory mechanisms on metabolic inputs, both direct and indirect. They have also tried to bring forth some of the open questions, and how the understanding of gene expression has changed dramatically over the last few years, which has largely become metabolism centric. Finally, metabolic regulation of epigenome and gene expression has gained much traction due to the increased incidence of lifestyle and age-related diseases.
by Mayumi Yamada, Shinji C. Nagasaki, Takeaki Ozawa, Itaru Imayoshi from a Research Center for Dynamic Living Systems, Graduate School of Biostudies, Kyoto University, Kyoto, 606–8501, Japan
Taking advantage of the recent development of genetically-defined photo-activatable actuator molecules, cellular functions, including gene expression, can be controlled by exposure to light. Such optogenetic strategies enable precise temporal and spatial manipulation of targeted single cells or groups of cells at a level hitherto impossible. In this review, they introduce light-controllable gene expression systems exploiting blue or red/far-red wavelengths and discuss their inherent properties potentially affecting induced downstream gene expression patterns. They also discuss recent advances in optical devices that will extend the application of optical gene expression control technologies into many different areas of biology and medicine.
by Johnston, J. from The Hastings Center, 21 Malcolm Gordon Road, Garrison, NY 10524, United States
Today’s debate about the use of gene-editing technologies to alter human DNA brings together two longstanding lines of inquiry in bioethics: the ethics of human enhancement, and the ethics of heritable genetic modification. This article traces that lineage by identifying key distinctions and ethics questions in these preexisting lines of inquiry that are also employed in four recent policy and ethics statements on human gene editing. These distinctions and ethics questions can be helpful heuristics for organizing discussion, learning from existing analysis, and highlighting what is at stake with new gene-editing technologies. Yet scientists, policymakers, and others new to the ethics of emerging technologies should also be aware of both the limitations of these distinctions and past challenges in adequately addressing the ethics questions they raise. In particular, the treatment-enhancement distinction and the somatic-germline distinction are not as clear-cut as they might initially appear. More importantly, they cannot be used to definitively differentiate right from wrong uses of the technologies in question.
by McCarty, N.S., Graham, A.E., Studená, L., Ledesma-Amaro, R. from the Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, United States and the Department of Bioengineering and Imperial College Centre for Synthetic Biology, Imperial College London, London, United Kingdom
Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing and transcriptional regulation. In this review, they discuss multiplexed CRISPR technologies and describe methods for the assembly, expression, and processing of synthetic guide RNA arrays in vivo. Applications that benefit from multiplexed CRISPR technologies, including cellular recorders, genetic circuits, biosensors, combinatorial genetic perturbations, large-scale genome engineering and the rewiring of metabolic pathways, are highlighted. They also offer a glimpse of emerging challenges and emphasize experimental considerations for future studies.
by Chang, C., Sung, C.-Y., Hsiao, H., Chen, J., Chen, I.-H., Kuo, W.-T., Cheng, L.-F, Korla, P.K.b, Chung, M.-J.a, Wu, P.-J., Yu, C.-C, Sheu, J.J.-C from the National Sun Yat-sen University, Kaohsiung, Taiwan.
Recent advances in high-throughput genomic technologies have nurtured a growing demand for statistical tools to facilitate the identification of molecular changes as potential prognostic biomarkers or drugable targets for personalized precision medicine. In this study, they developed a web-based interactive and user-friendly platform for high-dimensional analysis of molecular alterations in cancer (HDMAC). On HDMAC, several penalized regression models that are suitable for high-dimensional data analysis, Ridge, Lasso and adaptive Lasso, are offered, with Cox regression for survival and logistic regression for binary outcomes. The choice of a first-step screening is provided to address the multiple-comparison issue that often arises with large-volume genomic data. The hazard ratio or estimated coefficient is provided with each selected gene so that a multivariate regression model may be built based on the genes selected. Cross-validation is provided as the method to estimate the prediction power of each regression model. In addition, R codes are also provided to facilitate the download of whole sets of molecular variables from TCGA. In this study, the illustration of the use of HDMAC was made through a set of data on gene mutations and a set on mRNA expression from ovarian cancer patients and a set on mRNA expression from a bladder cancer patients. From the analysis of each set of data, a list of candidate genes was obtained that might be associated with mutations or abnormal expression of genes in ovarian and bladder cancers. HDMAC offers a solution for rigorous and validation analysis of high-dimensional genomic data.
