Analysis Of Genomic DNA Research Articles

SPARC: Development of a Transgenic Pig Expressing mCherry and CRE Recombinase Fusion Proteins Under the Control of the Murine Choline Acetyltransferase Promoter

Pigs can serve as useful models for advancing the biomedical field due to anatomy and physiology that more closely resembles human anatomy and physiology compared to traditional rodent models. However, there is a paucity of porcine CRE-driver lines available, limiting their utility for refined neural mapping and neural modulation studies. Here we describe the development of pigs with mCherry and CRE recombinase proteins expressed in cholinergic neurons. A 6,419 bp DNA fragment containing the Mus musculus choline acetyltransferase (ChAT) promoter was used to drive production of a mCherry–P2A–CRE recombinase fusion protein (mmChAT–mCherry–P2A–CRE). Linearized mmChAT–mCherry–P2A–CRE plasmid DNA was introduced into male porcine fetal fibroblasts by electroporation. Positive colonies were selected with 400µg/mL G-418 and screened for transgene insertion by PCR. Two colonies showing targeted insertion were used for somatic cell nuclear transfer and the resulting blastocysts were transferred to surrogate gilts. Two early pregnancies were established, and one of them went to term. Six Duroc-Landrace male piglets were born by natural delivery. PCR analysis of genomic DNA showed that one piglet carried the transgene whereas the others were determined to be wild-type. The transgenic piglet was phenotypically normal with no obvious difference in appearance compared to the wild-type littermates. The piglet was euthanized at 7-weeks of age for further characterization. Major tissues known to have cholinergic cell bodies and cholinergic innervation (hippocampus, cerebellum, spinal cord, basal ganglion, dorsal root ganglion, nodose ganglion, vagus nerve, trachea, bronchus, heart, bladder, duodenum and pancreas) were evaluated regarding mCherry and CRE recombinase mRNA presence. End-point PCR revealed CRE recombinase mRNA in basal ganglion, nodose ganglion, dorsal root ganglion and heart. mCherry mRNA was found in bronchus homogenates. Studies are ongoing to confirm integration sites into the genome, and mCherry and CRE recombinase expression at the protein level in cholinergic neurons. If successful, this model will allow for simultaneous dissection of cholinergic circuit connectivity and control of neural activity through CRE-mediated recombination.

Electrophysiological and pharmacological analysis with LQT2 patient (453delC‐KNCH2)‐derived induced pluripotent stem cell‐derived cardiomyocytes

Objective Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are promising options for cell-based cardiac repair and in vitro models of human cardiomyocytes. The hiPSC-CMs are used as a diagnostic disease modeling and drug toxicity screening platform. Because hiPSCs retain the genetic information of the cells from which they are derived, cells differentiated from iPSCs can potentially recapitulate the disease phenotype. Methods In this study, we investigated the electrophysiological properties of hiPSC-CMs derived from a LQT2 patient with mutation in the KNCH2 gene (453delC-KNCH2) for hERG channel (LQT2 453delC-iPSC-CM). Results The genomic DNA analysis of LQT2 453delC-iPSC-CM confirmed the heterozygous mutation of 453delC KCNH2. The mRNA expression of cardiac ion channels and ventricular markers were analyzed to verify cardiomyocyte maturity. In the whole-cell patch clamp study, LQT2 453delC-iPSC-CMs showed smaller amplitude of hERG-type K+ current (IKr) and prolonged action potential duration compare to the control hiPSC-CMs. The results were consistent with the loss-of-function mutation of KCNH2 and LQT2 phenotype. In a multi-electrode array (MEA) study, TdP risk was compared between the control hiPSC-CMs and LQT2 453delC-iPSC-CMs. High, intermediate, and low risk TdP drugs were applied, and the drug-induced changes of field potential were analyzed to compare TdP risk. Conclusion The data set might be helpful for the drug investigation in the LQT syndrome and the development of patient-specific antiarrhythmic strategies.

OriC-ENS: A sequence-based ensemble classifier for predicting origin of replication in S. cerevisiae

DNA Replication plays the most crucial part in biological inheritance, ensuring an even flow of genetic information from parent to offspring. The beginning site of DNA Replication which is called the Origin of Replication (ORI), plays a significant role in understanding the molecular mechanisms and genomic analysis of DNA. Hence, it is paramount to accurately identify the origin of replication to gain a more accurate understanding of the biochemical and genomic properties of DNA. In this paper, We have proposed a new approach named OriC-ENS that uses sequence-based feature extraction techniques, K-mer, K-gapped Mono-Di, and Di Mono, and an ensemble classification technique that uses majority voting for the identification of Origin of Replication. We have used three SVM classifiers, one for the K-mer features and two more for K-Gapped Mono-Di and K-Gapped Di-mono features. Finally, we used majority voting to combine the prediction by each predictor. Experimental results on the S. Cerevisiae dataset have shown that our method achieves an accuracy of 91.62 % which outperforms other state-of-the-art methods by a significant margin. We have also tested our method using other evaluation metrics such as Matthews Correlation Coefficient (MCC), Area Under Curve(AUC), Sensitivity, and Specificity, where it has achieved a score of 0.83, 0.98, 0.90, and 0.92 respectively. We have further evaluated our model on an independent test set collected from OriDB, consisting of the sequences of Schizosaccharomyces pombe where we have seen that our model can predict the origin of replication efficiently and with great precision. We have made our python-based source code available at https://github.com/MehediAzim/OriC-ENS.

Solid-state nanopore analysis of human genomic DNA shows unaltered global 5-hydroxymethylcytosine content associated with early-stage breast cancer

5-Hydroxymethylcytosine (5hmC), the first oxidized form of the well-known epigenetic modification 5-methylcytosine, is an independent regulator of gene expression and therefore a potential marker for disease. Here, we report on methods developed for a selective solid-state nanopore assay that enable direct analysis of global 5hmC content in human tissue. We first describe protocols for preparing genomic DNA derived from both healthy breast tissue and stage 1 breast tumor tissue and then use our approach to probe the net abundance of the modified base in each cohort. Then, we employ empirical data to adjust for the impact of nanopore diameter on the quantification. Correcting for variations in nanopore diameter among the devices used for analysis reveals no detectable difference in global 5hmC content between healthy and tumor tissue. These results suggest that 5hmC changes may not be associated with early-stage breast cancer and instead are a downstream consequence of the disease.

Reclassification of Haloactinobacterium glacieicola as Occultella glacieicola gen. nov., comb. nov., of Haloactinobacterium album as Ruania alba comb. nov, with an emended description of the genus Ruania, recognition that the genus names Haloactinobacterium and Ruania are heterotypic synonyms and description of Occultella aeris sp. nov., a halotolerant isolate from surface soil sampled at an ancient copper smelter.

In the course of screening the surface soils of ancient copper mines and smelters (East Harz, Germany) an aerobic, non-motile and halotolerant actinobacterium forming small rods or cocci was isolated. The strain designated F300T developed creamy to yellow colonies on tryptone soy agar and grew optimally at 28 °C, pH 7-8 and with 0.5-2 % (m/v) NaCl. Its peptidoglycan was of type A4α l-Lys-l-Glu (A11.54). The menaquinone profile was dominated by MK-8(II, III-H4) and contained minor amounts of MK-8(H2), MK-8(H6) and MK-9(H4). The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, mono and diacylated phosphatidylinositol dimannosides, and components that were not fully characterized, including two phospholipids, two glycolipids and an uncharacterized lipid. Major whole-cell sugars were rhamnose and ribose. The fatty acid profile contained mainly iso and anteiso branched fatty acids (anteiso-C15 : 0, iso-C14 : 0) and aldehydes/dimethylacetals (i.e. not fatty acids). Sequence analysis of its genomic DNA and subsequent analysis of the data placed the isolate in the group currently defined by members of the genera Ruania and Haloactinobacterium (family Ruaniaceae, order Micrococcales) as a sister taxon to the previously described species Haloactinobacterium glacieicola, sharing an average nucleotide identity and average amino acid identity values of 85.3 and 85.7 %, respectively. Genotypic and chemotaxonomic analyses support the view that strain F300T (=DSM 108350T=CIP 111667T) is the type strain of a new genus and new species for which the name Occultella aeris gen. nov., sp. nov. is proposed. Based on revised chemotaxonomic and additional genome based data, it is necessary to discuss and evaluate the results in the light of the classification and nomenclature of members of the family Ruaniaceae, i.e. the genera Haloactinobacterium and Ruania. Consequently, the reclassification of Haloactinobacterium glacieicola as Occultella glacieicola comb. nov. and Haloactinobacterium album as Ruania alba comb. nov., with an emended description of the genus Ruania are proposed.

Preventive Effects of Bacillus licheniformis on Heat Stroke in Rats by Sustaining Intestinal Barrier Function and Modulating Gut Microbiota.

Heat stroke (HS) models in rats are associated with severe intestinal injury, which is often considered as the key event at the onset of HS. Probiotics can regulate the gut microbiota by inhibiting the colonization of harmful bacteria and promoting the proliferation of beneficial bacteria. Here, we investigated the preventive effects of a probiotic Bacillus licheniformis strain (BL, CMCC 63516) on HS rats as well as its effects on intestinal barrier function and gut microbiota. All rats were randomly divided into four groups: control (Con) + PBS (pre-administration with 1 ml PBS twice a day for 7 days, without HS induction), Con + BL group (pre-administration with 1 ml 1 × 108 CFU/ml BL twice a day for 7 days, without HS induction), HS + PBS (PBS, with HS induction), and HS + BL (BL, with HS induction). Before the study, the BL strain was identified by genomic DNA analysis. Experimental HS was induced by placing rats in a hot and humid chamber for 60 min until meeting the diagnostic criterion of HS onset. Body weight, core body temperature, survival rate, biochemical markers, inflammatory cytokines, and histopathology were investigated to evaluate the preventive effects of BL on HS. D-Lactate, I-FABP, endotoxin, and tight-junction proteins were investigated, and the fluorescein isothiocyanate-dextran (FD-4) test administered, to assess the degree of intestinal injury and integrity. Gut microbiota of rats in each group were analyzed by 16S rRNA sequencing. The results showed that pre-administration with BL significantly attenuated hyperthermia, reduced HS-induced death, alleviated multiple-organ injury, and decreased the levels of serum inflammatory cytokines. Furthermore, BL sustained the intestinal barrier integrity of HS rats by alleviating intestinal injury and improving tight junctions. We also found that BL significantly increased the ratios of two probiotic bacteria, Lactobacillus and Lactococcus. In addition, Romboutsia, a candidate biomarker for HS diagnosis, was unexpectedly detected. In summary, BL pre-administration for 7 days has preventative effects on HS that may be mediated by sustaining intestinal barrier function and modulating gut microbiota.

Metagenomic Data Assembly - The Way of Decoding Unknown Microorganisms.

Metagenomics is a segment of conventional microbial genomics dedicated to the sequencing and analysis of combined genomic DNA of entire environmental samples. The most critical step of the metagenomic data analysis is the reconstruction of individual genes and genomes of the microorganisms in the communities using metagenomic assemblers – computational programs that put together small fragments of sequenced DNA generated by sequencing instruments. Here, we describe the challenges of metagenomic assembly, a wide spectrum of applications in which metagenomic assemblies were used to better understand the ecology and evolution of microbial ecosystems, and present one of the most efficient microbial assemblers, SPAdes that was upgraded to become applicable for metagenomics.

Molecular cloning, characterisation and expression of a gene encoding cellobiose dehydrogenase from Termitomyces clypeatus

Cellobiose dehydrogenase (CDH) (E.C. 1.1.99.18) is a biomass degrading enzyme secreted by various cellulolytic fungi. It has applications in renewable biofuel production, biosensor, bioremediation, lactose determination in food, bleaching of pulp, biomedical applications etc. This paper describes cloning of a previously characterised gene from the published transcriptome data of a fungus Termitomyces clypeatus, its expression in Saccharomyces cerevisiae and its molecular and biochemical characterisation etc. which ultimately led us to conclude that the gene in fact codes for a CDH at biochemical level. The full length cDNA exhibited 2523 nucleotides and the deduced protein sequence consists of 840 amino acids. Genomic DNA analysis has revealed presence of four introns within this gene. The protein contains 4 putative N-glycosylation and 14 putative O-glycosylation sites. Cloning and molecular characterisation of this gene opens yet another new source of CDH enzyme for commercial exploitation by industry in future.

Improvement of the attenuated mutant strain of Brucella melitensis Rev1 as a potential vaccine candidate

Vaccination is the best approach to prevent brucellosis. The existing vaccines such as Rev1 are associated with disadvantages such as abortion in pregnant animals. They also induce antibodies to the O-polysaccharide which may complicate diagnosis. Therefore, it is necessary to generate an appropriate vaccine. The aim of this study is the improvement of the available mutant strain of Brucella melitensis Rev1, in which the prosamine synthetize coding sequence from the O-PS region has been replaced with kanR coding sequence, via the replacement of kanR with GFP reporter encoding sequence by using homologous recombination. The syntactic deletion cassette, containing the GFP encoding sequence flanked with the upstream and downstream sequences of kanR encoding sequence from Rev1 mutant strain, was cloned into pBluescriptIISK(-) vector by SacI and KpnI, and the suicide vector pBluescriptSK-∆kanR/gfp was electroporated into mutant strain. The constructs were confirmed by using PCR, restriction mapping and DNA sequencing. The loss of kanR function in transfected cells was confirmed by using the GFP phenotyping with flow cytometry and fluorescence microscopy, and following the phenotypic characterization of the mutant strain on selective media and the genomic DNA analysis with specific primers. This kanR-free Rev1 mutant strain lack the O-polysaccharide and have a reduced ability to enter and survive in the host cell. Therefore, it could be evaluated as a potential vaccine candidate if eliciting protective immunity.

Abstract PD13-03: Genomic profiling of breast cancer leptomeningeal metastasis (BCLM) reveals a divergent evolution and therapeutic targets

Abstract Background: Leptomeningeal metastasis remains a devastating development in breast cancer, with a median survival of 3-4 months, no widely accepted standard treatment, and limited access to trials of novel therapies. In addition, lack of access to leptomeningeal metastatic material hampers the pre-clinical investigation of the disease process and molecular drivers. This project uses CSF as a liquid biopsy to characterise BCLM, through genomic analysis of the cell-free DNA (cfDNA), and the development of pre-clinical BCLM models by the expansion of CSF disseminated tumour cells. Methods: CSF (surplus to clinical requirements) and blood were collected from patients undergoing evaluation of leptomeningeal metastasis. cfDNA was extracted from CSF and plasma, and subjected to ultra-low pass whole genome sequencing (ulpWGS) to assess tumour-derived cfDNA fraction. Samples with >10% tumour fraction underwent whole exome sequencing, along with matched archival primary tumour, archival extra-cranial metastatic site(s) and germline DNA. CSF cells were expanded in vitro (to establish 3D patient-derived organoids (PDOs). Results: Cohort demographics are shown in Table 1. Whole exome sequencing (WES) in 21 patients reveals that 65.2% of variants found in CSF cfDNA were not shared with the primary tumour or other matched samples. Phylogenetic analysis shows a divergent evolution from extra-cranial metastatic sites, represented by plasma cfDNA (n = 12) and/or metastatic site tissue DNA (n = 7). The most frequently mutated cancer-associated genes in CSF were MUC16 (12/21), TP53 (11/21), CDH1 (10/21), and KMT2D (7/21). The common occurrence of CDH1 loss-of-function mutations was in keeping with the large number of lobular cases, however were also discovered in CSF of two cases with E-cadherin positive ductal primary tumours. Furthermore, mutations (including frameshift indels) in JAK family proteins (JAK1, JAK3 and TYK2) were present in 5/21 cases, and were private to CSF in 4/21. Potential actionable gene alterations private to CSF include; IDH2 (3/21), GLI1 (3/21), PIK3CA (2/21) and PTCH1 (2/21). Further, there was an enrichment for somatic BRCA1/2 mutations (5/21, 2 private to CSF) indicating potential for platinum and/or PARP inhibitor therapy in these individuals. Patient-derived organoids (PDOs) were established using CSF tumour cells from 3 ER+/HER2- and 2 TNBC BCLM cases. WES of PDOs revealed high concordance with genomic variants identified in the matched CSF cfDNA. Therapeutic compound testing revealed 3/5 PDOs did not display sensitivity to methotrexate, the most commonly used BCLM intrathecal treatment. Patient-derived xenograft (PDX) models have been established by mammary fat pad, intraductal, intracardiac and intracerebroventricular injection routes. Conclusion/future plans: WES of CSF cfDNA provides insight into genomic changes in BCLM, including the divergent evolution, and BCLM specific alterations, some of which are potentially targetable. Parallel PDO and PDX models are being used to validate potential drivers and therapeutic targets. Treatment options beyond intrathecal methotrexate are urgently needed and in future might be molecularly tailored based on alterations discovered by CSF cfDNA sequencing. Table 1. Clinical demographicsDemographicsMedian, years (range)Age at BC diagnosis45 (24 – 66)Time from primary BC to BCLM4.7 (0.7 – 14.7)Time from first metastasis to BCLM1.2 (0.0 – 6.5)Histological Typen (%)Lobular10 (48)Ductal8 (38)Mixed ductal/lobular3 (14)Immuno-histochemical phenotypen (%)ER+ HER2-13 (62)TNBC5 (24)ER+ HER2+2 (9)ER- HER2+1 (5)Metastatic sitesn (%)No other metastatic sites (BCLM only)3 (14)Bone12 (57)Serosal8 (38)Brain6 (29)Liver4 (19)Ovary4 (19) Citation Format: Amanda Fitzpatrick, Marjan Iravani, Adam Mills, Eleanor Knight, Thanussuyah Alaguthurai, Vandna Shah, Alicia Okines, Nick Turner, Mark Harries, Syed Haider, Andrew Tutt, Clare Isacke. Genomic profiling of breast cancer leptomeningeal metastasis (BCLM) reveals a divergent evolution and therapeutic targets [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-03.

SPACEWALK: A Remote Participation Study of ALK Resistance Leveraging Plasma Cell-Free DNA Genotyping

IntroductionRemote consent and enrollment offer a unique opportunity to provide rare cancer populations with access to clinical research. The genomic analysis of plasma cell-free DNA (cfDNA) permits remote characterization of the cancer genome. We hypothesized we could leverage these approaches to remotely study drug resistance in patients with metastatic ALK-positive NSCLC. MethodsThe SPACEWALK study (Study of Plasma Next-Generation Sequencing for Remote Assessment, Characterization, Evaluation of Patients With ALK Drug Resistance) enrolled patients with ALK-positive NSCLC and progression on a next-generation ALK inhibitor who could participate remotely. Plasma was collected for next-generation sequencing (NGS) of cfDNA before initiating subsequent therapy, with results returned and subsequent therapy studied. ResultsOf the 62 patients enrolled, an ALK fusion was detected in 27 (44%) with a median allelic fraction of 2.6%. Among these 27 patients, a potential resistance mechanism was identified in 17 patients (63%): eight cases (30%) had secondary ALK kinase domain resistance mutations, three cases (11%) had bypass track resistance, and six cases (22%) had both ALK resistance mutations and bypass resistance. The most frequently detected mechanism of bypass resistance was MET amplification. Repeat plasma NGS was performed in 14 patients after subsequent treatment was initiated, with seven (50%) patients exhibiting greater than 50% reductions in ALK fusion allelic fraction. ConclusionsThrough the leveraging of remote participation, plasma NGS offers an optimal mechanism for characterizing resistance to emerging targeted therapies in rare cancer populations, though sensitivity depends on adequate tumor DNA samples. Repeat cfDNA analysis on therapy may offer an objective monitoring approach to remotely study treatment response.

Novel-iridoviral kinase induces mortality and reduces performance of green peach aphids (Myzus persicae) in transgenic Arabidopsis plants

Insect pests are a serious threat to agricultural productivity. Genetically-modified crops offer a promising alternative, and Bacillus thuringiensis endotoxin genes have played a major role in this respect. However, to overcome insect tolerance issues and to broaden the target range, it is critical to identify alternative insecticidal toxins working through novel mechanisms. In our previous study, a kinase from Chilo iridescent virus (CIV) that has insecticidal activity was identified and designated as Iridovirus Serine/Threonine Kinase (ISTK). A 35 kDa truncated form of ISTK, designated iridoptin, was obtained during expression and purification of ISTK in the yeast system. This yeast-expressed CIV toxin induced 50% mortality in cotton aphids and 100% mortality in green peach aphids (GPA). In this study, codon optimized ISTK gene and iridoptin fragment (designated oISTK and oIRI, respectively) were designed by altering the codon usage features that are seldom present in plant exons. Codon-optimized gene(s) cloned into plant expression vectors were used to stably transform Arabidopsis plants. PCR analysis of genomic DNA of transformed plants confirmed the presence of the DNA insert (oISTK/oIRI) in selected transgenic lines. Further screening was performed by selecting the PCR positive lines, which showed expression of respective toxins at the polypeptide level, using Western blot analyses. Codon-optimized gene constructs resulted in significant improvement in levels of expression of ISTK and iridoptin polypeptide and confirmed its stability in planta. The stable lines expressing either of the two toxin forms induced moderate to very high mortality in GPAs and significantly affected GPA development and fecundity.

Microbiome as Mediator of Diet on Colorectal Cancer Risk: The Role of Vitamin D, Markers of Inflammation and Adipokines

Obesity and diet are associated with colorectal cancer (CRC) risk, and microbiome could mediate this risk factor. To investigate this interaction, we performed a case–control study (34 CRC cases and 32 controls) and analyzed fecal microbiota composition using 16S rRNA metabarcoding and sub-sequential shotgun analyses of genomic bacterial DNA to evaluate the role of microbiome and diet in CRC etiology, taking into account vitamin D and other risk biomarkers. Dietary habits were evaluated using a short questionnaire. Multivariate methods for data integration and mediation analysis models were used to investigate causal relationships. CRC cases were significantly more often deficient in vitamin D than controls (p = 0.04); FokI and CYP24A1 polymorphism frequency were different between cases and controls (p = 0.03 and p = 0.02, respectively). A diet poor in fatty fish and rich in carbohydrates was found to be significantly associated with CRC risk (p = 0.011). The mediation analysis confirmed the significant role of the microbiome in mediating CRC risk—increasing levels of Bifidobacteria/Escherichia genera ratio, an indicator of “healthy” intestinal microbiome, can overcome the effect of diet on CRC risk (p = 0.03). This study suggests that microbiome mediates the diet effect on CRC risk, and that vitamin D, markers of inflammation, and adipokines are other factors to consider in order to achieve a better knowledge of the whole carcinogenic process.

Identifying genome-wide off-target sites of CRISPR RNA-guided nucleases and deaminases with Digenome-seq.

Digested genome sequencing (Digenome-seq) is a highly sensitive, easy-to-carry-out, cell-free method for experimentally identifying genome-wide off-target sites of programmable nucleases and deaminases (also known as base editors). Genomic DNA is digested in vitro using clustered regularly interspaced short palindromic repeats ribonucleoproteins (RNPs; plus DNA-modifying enzymes to cleave both strands of DNA at sites containing deaminated base products, in the case of base editors) and subjected to whole-genome sequencing (WGS) with a typical sequencing depth of 30×. A web-based program is available to map in vitro cleavage sites corresponding to on- and off-target sites. Chromatin DNA, in parallel with histone-free genomic DNA, can also be used to account for the effects of chromatin structure on off-target nuclease activity. Digenome-seq is more sensitive and comprehensive than cell-based methods for identifying off-target sites. Unlike other cell-free methods, Digenome-seq does not involve enrichment of DNA ends through PCR amplification. The entire process other than WGS, which takes ~1-2 weeks, including purification and preparation of RNPs, digestion of genomic DNA and bioinformatic analysis after WGS, takes about several weeks.

Citramalate synthase yields a biosynthetic pathway for isoleucine and straight- and branched-chain ester formation in ripening apple fruit

A plant pathway that initiates with the formation of citramalate from pyruvate and acetyl-CoA by citramalate synthase (CMS) is shown to contribute to the synthesis of α-ketoacids and important odor-active esters in apple (Malus × domestica) fruit. Microarray screening led to the discovery of a gene with high amino acid similarity to 2-isopropylmalate synthase (IPMS). However, functional analysis of recombinant protein revealed its substrate preference differed substantially from IPMS and was more typical of CMS. MdCMS also lacked the regulatory region present in MdIPMS and was not sensitive to feedback inhibition. 13C-acetate feeding of apple tissue labeled citramalate and α-ketoacids in a manner consistent with the presence of the citramalate pathway, labeling both straight- and branched-chain esters. Analysis of genomic DNA (gDNA) revealed the presence of two nearly identical alleles in "Jonagold" fruit (MdCMS_1 and MdCMS_2), differing by two nonsynonymous single-nucleotide polymorphisms (SNPs). The mature proteins differed only at amino acid 387, possessing either glutamine387 (MdCMS_1) or glutamate387 (MdCMS_2). Glutamate387 was associated with near complete loss of activity. MdCMS expression was fruit-specific, increasing severalfold during ripening. The translated protein product was detected in ripe fruit. Transient expression of MdCMS_1 in Nicotiana benthamiana induced the accumulation of high levels of citramalate, whereas MdCMS_2 did not. Domesticated apple lines with MdCMS isozymes containing only glutamate387 produced a very low proportion of 2-methylbutanol- and 2-methylbutanoate (2MB) and 1-propanol and propanoate (PROP) esters. The citramalate pathway, previously only described in microorganisms, is shown to function in ripening apple and contribute to isoleucine and 2MB and PROP ester biosynthesis without feedback regulation.

Molecular genetic characteristics of hemostasis in hemorrhagic fever with renal syndrome

Aim. To assess the predictive value of single-nucleotide polymorphisms of hemostasis and folate cycle genes in hemorrhagic fever with renal syndrome (HFRS).
 Methods. 43 patients undergoing HFRS were examined based on the Republican clinical infectious diseases hospital in Izhevsk. Toxic shock syndrome (TSS) in the decompensated phase, pulmonary edema in the alveolar phase, and acute kidney injury (AKI) at stage F [RIFLE criteria (risk, injury, failure, loss, end-stage renal disease)] were registered as complications. Molecular analysis of patients genomic DNA was performed after its isolation from peripheral blood cells. Genotyping was performed by using multiplex real-time PCR with conformationally restricted probes. Statistical analysis was performed by the licensed program SPSS 22.0; the significance level of difference between groups was determined using the nonparametric MannWhitney test (for quantitative variables) and the Fishers exact test (for qualitative variables).
 Results. The C/C genotype of the ITGB3:1565T/C gene (p=0.0278), and the C/C genotype of the MTHFR1298 A/C gene (p=0.0407) was less common in severe cases, while the G allele of FGB:455G/A gene (p=0.046) and the T allele of the ITGB3:1565T/C gene (p=0.0166) was more frequent. More frequent detection of the 5G/4G genotype of the PAI-1:675 5G/4G gene was found in the case of TSS (p=0.0433). Genotype C/C of the ITGB3:1565T/C gene (p=0.0145) and a combination of pathological genotypes A/C and C/C of the MTHFR1298A/C gene (p=0.0004) are less common in the development of AKI at stage F.
 Conclusion. The molecular genetic analysis makes it possible to identify patients with genotypes predisposing to a severe and complicated course of hemorrhagic fever with renal syndrome.

Survival Implications of the Relationship between Tissue versus Circulating Tumor DNA TP53 Mutations-A Perspective from a Real-World Precision Medicine Cohort.

Interrogating the genomics of circulating tumor DNA (ctDNA; the liquid biopsy) has advantages in patients in whom tissue biopsy is difficult. However, the reported concordance between genomic analysis of tissue DNA and ctDNA is variable among studies. Herein, we characterized the clinical implications of the relationship between mutations in TP53 genes in tissue DNA versus ctDNA. The molecular profiles of both liquid (Guardant Health) and tissue (Foundation Medicine) biopsies from 433 patients were analyzed (pan-cancer setting). In 71/433 (16%) cases, all same TP53 mutations were detected in both tissue DNA and ctDNA; in 18/433 (4%), same mutation plus additional mutation/mutations; and in 27/433 (6%), different TP53 mutations were detected. In 99/433 (23%) cases, TP53 mutations were detected only in tissue DNA; in 43/433 (10%), only in ctDNA; and in 175/433 (40%), no TP53 mutations were detected in either test. When TP53 mutations were identical in tissue and ctDNA, the alterations were enriched for nonsense mutations, and survival was significantly shorter in multivariate analysis (as compared with different mutations in ctDNA vs. tissue or no mutations); this finding was independent of tumor type, time interval between tests, and the %ctDNA for TP53 mutations. In summary, in 16% of 433 patients with diverse cancers, TP53 mutations were identical in tissue DNA and ctDNA. In these individuals, the alterations were enriched for stop-gain (nonsense) mutations (results in a premature termination codon). Though unknown confounders cannot be ruled out, these patients fared significantly worse than those whose ctDNA and tissue DNA harbored different TP53 mutation portfolios or no TP53 mutations.

Molecular identification and optimization of cultural conditions for mycelial biomass production of wild strain of Chlorophyllum molybdites (G.Mey) Massee from the Philippines

Comparative analyses of genomic DNA extracted from freshwater fish tissues preserved in formaldehyde and alcohol in different periods of timeR. K. Garg, Khushboo Sengar, R. K. Singh

Antimicrobial peptides in the seedling transcriptome of the tree legume Peltophorum dubium

Antimicrobial peptides (AMPs) play an essential role in plant defense against invading pathogens. Due to their biological properties, these molecules have been considered useful for drug development, as novel agents in disease therapeutics, applicable to both agriculture and medicine. New technologies of massive sequencing open opportunities to discover novel AMP encoding genes in wild plant species. This work aimed to identify cysteine-rich AMPs from Peltophorum dubium, a legume tree from South America. We performed whole-transcriptome sequencing of P. dubium seedlings followed by de novo transcriptome assembly, uncovering 78 AMP transcripts classified into five families: hevein-like, lipid-transfer proteins (LTPs), alpha hairpinins, defensins, and snakin/GASA (Giberellic Acid Stimulated in Arabidopsis) peptides. No transcripts with similarity to cyclotide or thionin genes were identified. Genomic DNA analysis by PCR confirmed the presence of 18 genes encoding six putative defensins and 12 snakin/GASA peptides and allowed the characterization of their exon-intron structure. The present work demonstrates that AMP prediction from a wild species is possible using RNA sequencing and de novo transcriptome assembly, regarding a starting point for studies focused on AMP gene evolution and expression. Moreover, this study allowed the detection of strong AMP candidates for drug development and novel biotechnological products.

Concomitant Occurrence of Polyclonal Hematopoiesis and Cell-Autonomous Megakaryopoiesis in Triple-Negative Essential Thrombocythemia

Somatic mutations in JAK2, MPL, and CALR are found in approximately 80% of patients with essential thrombocythemia (ET), whereas the remaining patients are negative for disease-defining mutations and are defined as triple-negative (TN). Studies have shown that some patients with TN-ET harbor non-canonical mutations in JAK2 and MPL; however, the failure to identify recurrent mutations in most patients has made the pathogenesis of TN-ET ambiguous (Milosevic Feenstra et al. Blood 2016, Cabagnols et al. Blood 2016). In this study, we screened 483 patients suspected as having ET in a single center, performed mutation analysis for JAK2 V617F, CALR exon 9, and MPL exon 10, and centrally reviewed bone marrow specimens. We identified 23 patients with TN-ET based on the WHO 2016 criteria. Sequencing analysis of these patients revealed non-canonical mutations in JAK2 and MPL in 4 cases. Whole exome-sequencing analysis of genomic DNA from peripheral blood and CD3-positive cells from 9 patients revealed that 2 patients harbored somatic mutations in other genes; 7 patients showed no detectable somatic mutation. A STAT5 reporter assay revealed that unlike JAK2 V617F and MPL W515L, all non-canonical mutants of JAK2 or MPL activated STAT5 similar to wild-type proteins, suggesting that these mutations did not drive the disease. Statistical analysis of clinical records revealed that patients with TN-ET were mostly young (median age of 36.0 years), female (18/23, 78.3%), and had neither a history of thrombosis nor progression to secondary myelofibrosis and leukemia, demonstrating the unique characteristics of TN-ET. The presence of clonal hematopoiesis, analyzed using genomic DNA purified from granulocytes of peripheral blood from female patients in a human androgen receptor assay, revealed that only 1 out of 15 patients was clonal. Hypothesizing that TN-ET was reactive thrombocytosis, the concentrations of cytokines promoting platelet production such as thrombopoietin (TPO) and interleukin-6 (IL-6) in the serum were analyzed. However, no significant differences in concentrations were observed among ET with driver mutations, TN-ET, and healthy individuals. We next examined the capacity of hematopoietic stem cells from patients with TN-ET to form megakaryocytic colonies. CD34-positive cells purified from cryopreserved bone marrow cells were cultured in the absence or presence of TPO. CD34-positive cells derived from patients with TN-ET exhibited an equivalent capacity to form megakaryocytic colonies compared to those from patients with ET harboring a driver mutation, even in the absence of TPO (Figure 1). Thus, in TN-ET, megakaryopoiesis may have been induced in a cell-autonomous manner. In 10 patients with TN-ET with available blood count data, no sign of thrombocytosis was observed before ET development, indicating that thrombocytosis was not hereditary but rather occurred via an alternate mechanism, such as aberrations in epigenomic regulation that induced cellular transformation (Ohnishi et al, Cell 2015). Taken together, TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis. Figure 1 Disclosures Komatsu: Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy.