Enzyme Fragment Research Articles

One-pot universal NicE-seq: all enzymatic downstream processing of 4% formaldehyde crosslinked cells for chromatin accessibility genomics

BackgroundAccessible chromatin landscape allows binding of transcription factors, and remodeling of promoter and enhancer elements during development. Chromatin accessibility along with integrated multiomics approaches have been used for determining molecular subtypes of cancer in patient samples.ResultsOne-pot Universal NicE-seq (One-pot UniNicE-seq) is an improved accessible chromatin profiling method that negate DNA purification and incorporate sonication free enzymatic fragmentation before library preparation and is suited to a variety of mammalian cells. One-pot UniNicE-seq is versatile, capable of profiling 4% formaldehyde fixed chromatin in as low as 25 fixed cells. Accessible chromatin profile is more efficient on formaldehyde-fixed cells using one-pot UniNicE-seq compared to Tn5 transposon mediated methods, demonstrating its versatility.ConclusionOne-pot UniNicE-seq allows the entire process of accessible chromatin labeling and enrichment in one pot at 4% formaldehyde cross-linking conditions. It doesn’t require enzyme titration, compared to other technologies, since accessible chromatin is labelled with 5mC incorporation and deter degradation by nicking enzyme, thus opening the possibility for automation.

Directed homoenzymatic fragmentation of the plant protopectin complex: Assessment criteria

Introduction. The functional basis of protopectin complex can be represented as a network of regions that consist of homogalacturonan sequences and a base of rhamnogalacturonans-I, i.e. rhamnosyl-containing branching sites. Enzymatic isolation of these regions is possible only at a certain minimal native degree of polymerization. The research objective was to develop a system of criteria for assessing the potential applicability of the enzymatic transformation of plant protopectin complex.
 Study objects and methods. The research featured the polymerization degree of the homogalacturonan regions of the protopectin complex and produced a system of assessment criteria for the enzymatic fragmentation potential of the protopectin complex. The theoretical calculations were based on the values of the mass fractions of rhamnosyl and galacturonide residues in plant cell walls. The result was a new polymerization degree analytical function.
 Results and discussion. The ratio of the mass fractions of rhamnosyl and galacturonide residues in the water-insoluble plant tissue served as a dimensionless criterion of applicability. The rational condition for the dimensionless criterion of applicability was based on the fundamental constraint for homogalacturonan regions in the protopectin complex. It was expressed by a fundamental inequation. The rational area for determining the numerical values of the applicability criterion was presented as . The functional dependence was reduced to a two-dimensional criteria space as “width of rhamnosyl branches vs. the criterion of applicability”, where each pectin-containing raw material was given a single uniquely defined position. The boundary conditions for the criteria space were determined analytically. 
 Conclusion. The new approach offers an assessment of the enzymatic fragmentation potential of the plant protopectin complex by homoenzyme preparations. The approach is in fact the second stage of the decision tree in the science-based technology for pectin and its products.

A Genome-Edited ERα-HiBiT Fusion Reporter Cell Line for the Identification of ERα Modulators Via High-Throughput Screening and CETSA.

The estrogen receptor α (ERα) is a target of intense pharmacological intervention and toxicological biomonitoring. Current methods to directly quantify cellular levels of ERα involve antibody-based assays, which are labor-intensive and of limited throughput. In this study, we generated a post-translational reporter cell line, referred to as MCF7-ERα-HiBiT, by fusing a small pro-luminescent nanoluciferase (NLuc) tag (HiBiT) to the C-terminus of endogenous ERα in MCF7 cells. The tag allows the luminescent detection and quantification of endogenous ERα protein by addition of the complementary NLuc enzyme fragment. This MCF7-ERα-HiBiT cell line was optimized for quantitative high-throughput screening (qHTS) to identify compounds that reduce ERα levels. In addition, the same cell line was optimized for a qHTS cellular thermal shift assay to identify compounds that bind and thermally stabilize ERα. Here, we interrogated the MCF7-ERα-HiBiT assay against the NCATS Pharmacological Collection (NPC) of 2,678 approved drugs and identified compounds that potently reduce and thermally stabilize ERα. Our novel post-translational reporter cell line provides a unique opportunity for profiling large pharmacological and toxicological compound libraries for their effect on ERα levels as well as for assessing direct compound binding to the receptor, thus facilitating mechanistic studies by which compounds exert their biological effects on ERα.

Genomic structure and diversity of oak populations in British parklands

Societal Impact StatementThe largest populations of veteran oak trees in Europe are found in British parklands: managed wood pastures up to 1000 years old. Here, we present genomic evidence that parkland oak populations harbour considerable diversity and grew from local seed sources. We found some evidence for natural regeneration of offspring and for hybridization between pedunculate and sessile oak. We detected signatures of past gene flow between these two species and few regions of high differentiation within their genomes. Future expansion of this dataset may allow us to test for a genomic basis of acute oak decline, a syndrome of particular concern in parkland environments.Summary The two predominant oak species in Europe, Quercus robur (English or pedunculate oak) and Quercus petraea (sessile oak), have a long history of human intervention. In Britain, a common management regime is parklands, often surrounding country houses. Little is known about how seeds were sourced for parkland oak populations nor the genetic diversity that they contain. We sequenced the whole genomes of 386 trees from four British parkland sites to characterize the nuclear and chloroplast genetic structure and diversity of oak parkland populations. We assembled the chloroplast genomes and matched these to restriction enzyme fragment chloroplast haplotypes found in previous studies of ancient woodlands. We found over two million high‐quality nuclear single‐nucleotide polymorphisms (SNPs), allowing us to identify 360 Q. robur, 10 Q. petraea and 16 hybrid individuals with confidence. We identified 81 coding regions exhibiting strong differentiation between the two species. We found evidence for selective sweeps in Q. robur near some regions containing genes with putative involvement in stress tolerance. We detected a few very close relatives within some sites, suggesting natural regeneration or local seed planting. There was little differentiation among the Q. robur populations at the four sites. Chloroplast genomes found in each parkland tended to be similar to those of local ancient woodlands, suggesting that they were derived from local seed sources.

RESCOT: Restriction enzyme set and combination optimization tools for rNMP capture techniques

The incorporation of ribonucleoside monophosphates (rNMPs) in genomic DNA is a frequent phenomenon in many species, often associated with genome instability and disease. The ribose-seq technique is one of a few techniques designed to capture and map rNMPs embedded in genomic DNA. The first step of ribose-seq is restriction enzyme (RE) fragmentation, which cuts the genome into smaller fragments for subsequent rNMP capture. The RE selection chosen for genomic DNA fragmentation in the first step of the rNMP-capture techniques determines the genomic regions in which the rNMPs can be captured. Here, we designed a computational method, Restriction Enzyme Set and Combination Optimization Tools (RESCOT), to calculate the genomic coverage of rNMP-captured regions for a given RE set and to optimize the RE set to significantly increase the rNMP-captured-region coverage. Analyses of ribose-seq libraries for which the RESCOT tools were applied reveal that many rNMPs were captured in the expected genomic regions. Since different rNMP-mapping techniques utilize RE fragmentation and purification steps based on size-selection of the DNA fragments in the protocol, we discuss the possible usage of RESCOT for other rNMP-mapping techniques. In summary, RESCOT generates optimized RE sets for the fragmentation step of many rNMP capture techniques to maximize rNMP capture rate and thus enable researchers to better study characteristics of rNMP incorporation.

Genetic characterization and phylogenetic of Anaplasma capra in Persian onagers (Equus hemionus onager)

Anaplasma spp. are among the most recognized arthropod-borne infectious agents. Although the novel A. capra has been isolated from wildlife, livestock, and hard ticks from many parts of the world, there is no report regarding the identification of this pathogen from equines and little is known about the epidemiology of A. capra in Equidae. In this study, A. capra was identified in two out of ten blood specimens of wild onagers (Equus hemionus onager) during a routine health check-up in Semnan, Iran by light microscopy and molecular analyses while other pathogens were not detected. First, inclusions on RBC's were observed in two blood smears by light microscopy. Then, the blood specimens of both animals were analyzed by realtime-PCR for Anaplasma, Ehrlichia, and Theileria infections. A 1400 bp sequence of 16S rRNA belonging to Anaplasmataceae and 874 bp fragment for groEL gene for A. capra were amplified in Anaplasma positive samples and sequenced. Preliminary BLAST analysis of sequenced fragments showed high homology to A. capra strains in GenBank database. Finally, nested PCR and restriction enzyme fragment length polymorphism techniques confirmed the pathogen as A. capra. To the best of our knowledge, this study has reported the occurrence of A. capra in wild onagers for the first time and suggests that equines could be infected with this pathogen and act as reservoirs for A. capra.

The association between PON1 gene polymorphisms (Q192R and L55M) and nephrotic syndrome in Iraqi children

Background: The role of paraoxonase 1 enzyme (PON1) and its single nucleotide polymorphisms (SNPs) in children with nephrotic syndrome (NS) has been reported previously in different ethnic and racial groups with divergent results. The human PON1 gene contains two coding region polymorphisms leading to two different PON1 isoforms. Objectives: The aim of the present study was to find out the association between the PON1 (Q192R and L55M) polymorphisms and their relation with serum PON1 activity as well as lipid profile tests (total cholesterol, TC; triglycerides, TG; high-density lipoprotein cholesterol, HDL-c; and low-density lipoprotein cholesterol, LDL-c) in children with NS. Methods: This study included a total of 80 participants (40 with NS in the age group of 2-14 years and 40 age and sex-matched healthy controls). The PON1 enzyme activity and lipid profile tests were measured in serum samples of all included participants. The PON1 genotype was determined by PCR-restriction enzyme fragment length polymorphism (PCR-RFLP) for both PON1 alleles (192 and 55) SNPs. Results: Our findings showed that the mean levels of lipid profile tests (TC, TG, LDL-c) were significantly increased in patients when compared with healthy controls (p<0.05), while the HDL-c concentration was significantly decreased in patients than that of controls. Also, the patients had significantly lower concentrations of PON1 when compared with the controls regardless of the genotype Q192R and L55M polymorphisms. Moreover, the homozygous RR genotype for PON1 SNP 192 and MM homozygous genotype for PON1 SNP 55 were significantly frequent in patients when compared with the controls. Conclusions: Our results support that the presence of the homozygous RR genotype for PON1 SNP 192 and MM homozygous genotype for PON1 SNP 55 were significantly higher in patients compared with the controls.

Cytotoxicity of Gymnopilus purpureosquamulosus extracts on hematologic malignant cells through activation of the SAPK/JNK signaling pathway.

Treatment of hematologic malignancies is a formidable challenge for hematologists and there is an urgent need to identify safe and efficacious agents either via synthesis in the laboratory or isolation from natural products. Here, we report the cytotoxicity of extracts from mushroom Gymnopilus purpureosquamulosus Høil (G. pps) and describe its molecular mechanisms. Using leukemia, lymphoma and multiple myeloma cell lines, 28–35 ppm G. pps extract inhibited cell proliferation by ~46–79%, which correlates with activation of apoptosis as indicated by increase in annexin V-positive cells (~5–8-fold), production of reactive oxygen species (~2–3-fold), cells in sub G0/G1 phase (~3–13-fold), caspase 3 enzymatic activity (~1.6–2.9-fold), DNA fragmentation, PARP1 cleavage and down-regulation of prosurvival proteins. Mitochondrial membrane potential decreased and leakage of pro-apoptotic factors to cytoplasm was observed, consistent with the activation of intrinsic apoptosis. Western blot analysis showed activation of the ASK1-MEK-SAPK/JNK and ASK1-P38 MAPK pathways possibly due to changes in the cellular redox status as suggested by decreased protein levels of peroxiredoxin, thioredoxin and thioredoxin reductase. Moreover, antioxidant N-acetylcysteine alleviated the cytotoxicity of G. pps. Pharmacological inhibition of SAPK/JNK and P38 alleviated the G. pps-mediated cytotoxicity. The extract activated apoptosis in leukemia and lymphoma patient cell samples but not in mononuclear cells from healthy donors further supporting the therapeutic values of G. pps for hematologic malignancies.

Expanding the SiMPl Plasmid Toolbox for Use with Spectinomycin/Streptomycin.

We recently developed the SiMPl plasmid toolbox, which is constituted by pairs of plasmids, generically indicated as pSiMPlx_N and pSiMPlx_C, which can be stably maintained in Escherichia coli with a single antibiotic x. The method exploits the split intein gp41–1 to reconstitute the enzyme conferring resistance toward the antibiotic x, whereby each enzyme fragment is expressed from one of the plasmids in the pair. pSiMPl plasmids are currently available for use with ampicillin, kanamycin, chloramphenicol, hygromycin, and puromycin. Here, we introduce another pair for use with spectinomycin/streptomycin, broadening the application spectrum of the SiMPl toolbox. To find functional splice sites in aminoglycoside adenylyltransferase, we apply a streamlined strategy looking exclusively at the flexibility of native cysteine and serine residues, which we first validated splitting the enzymes conferring resistance toward ampicillin, kanamycin, chloramphenicol, and hygromycin. This strategy could be used in the future to split other enzymes conferring resistance toward antibiotics.

Enzyme aggregation and fragmentation induced by catalysis relevant species.

It is usually assumed that enzymes retain their native structure during catalysis. However, the aggregation and fragmentation of proteins can be difficult to detect and sometimes conclusions are drawn based on the assumption that the protein is in its native form. We have examined three model enzymes, alkaline phosphatase (AkP), hexokinase (HK) and glucose oxidase (GOx). We find that these enzymes aggregate or fragment after addition of chemical species directly related to their catalysis. We used several independent techniques to study this behavior. Specifically, we found that glucose oxidase and hexokinase fragment in the presence of D-glucose but not L-glucose, while hexokinase aggregates in the presence of Mg2+ ion and either ATP or ADP at low pH. Alkaline phosphatase aggregates in the presence of Zn2+ ion and inorganic phosphate. The aggregation of hexokinase and alkaline phosphatase does not appear to attenuate their catalytic activity. Our study indicates that specific multimeric structures of native enzymes may not be retained during catalysis and suggests pathways for different enzymes to associate or separate over the course of substrate turnover.

Pharmacological Activation of Nrf2 by Rosolic Acid Attenuates Endoplasmic Reticulum Stress in Endothelial Cells.

Endoplasmic reticulum (ER) plays a key role in the folding, modification, and trafficking of proteins. When the homeostasis of the ER is disturbed, un/misfolded proteins accumulate in the ER which leads to ER stress. Sustained ER stress results in apoptosis, which is associated with various diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major transcription factor in redox homeostasis by regulating various genes associated with detoxification and cell-protective mechanisms. We found that Rosolic acid (RA) treatment dose-dependently activates Nrf2 in endothelial cells using the enzyme fragment complementation assay. The cytoprotective role of RA against ER stress-induced endothelial apoptosis and its molecular mechanism was explored in the present study. The Nrf2 and its target genes, as well as ER stress marker expressions, were measured by qPCR in ER stress-exposed endothelial cells. The contribution of Nrf2 in RA-mediated defense mechanism in endothelial cells was established by knockout studies using Nrf2-CRISPR/Cas9. The treatment with RA to ER stress-induced endothelial cells exhibited activation of Nrf2, as demonstrated by Nrf2 translocation and reduction of ER stress markers. We found that the Nrf2 knockout sensitized the endothelial cells against ER stress, and further, RA failed to mediate its cytoprotective effect. Proteomic studies using LC-MS/MS revealed that among the 1370 proteins detected, we found 296 differentially regulated proteins in ER stress-induced endothelial cells, and RA administration ameliorated 71 proteins towards the control levels. Of note, the ER stress in endothelial cells was attenuated by the treatment with the RA, suggesting the role of the Nrf2 activator in the pathological conditions of ER stress-associated diseases.

423. SARS-CoV-2 NGS Assay Powered by Biotia COVID-DX Software

BackgroundCOVID-19 had spread quickly, causing an international public health emergency with an alarming global shortage of COVID-19 diagnostic tests. We developed and clinically validated a next-generation sequencing (NGS)-based target enrichment assay with the COVID-DX Software tailored for the detection, characterization, and surveillance of the SARS-CoV-2 viral genome.MethodsThe SARS-CoV-2 NGS assay consists of components including library preparation, target enrichment, sequencing, and a COVID-DX Software analysis tool. The NGS library preparation starts with extracted RNA from nasopharyngeal (NP) swabs followed by cDNA synthesis and conversion to Illumina TruSeq-compatible libraries using the Twist Library Preparation Kit via Enzymatic Fragmentation and Unique Dual Indices (UDI). The library is then enriched for SARS-CoV-2 sequences using a panel of dsDNA biotin-labeled probes, specifically designed to target the SARS-CoV-2 genome, then sequenced on an Illumina NextSeq 550 platform. The COVID-DX Software analyzes sequence results and provides a clinically oriented report, including the presence/absence of SARS-CoV-2 for diagnostic use. An additional research use only report describes the assay performance, estimated viral titer, coverage across the viral genome, genetic variants, and phylogenetic analysis.ResultsThe SARS-CoV-2 NGS Assay was validated on 30 positive and 30 negative clinical samples. To measure the sensitivity and specificity of the assay, the positive and negative percent agreement (PPA, NPA) was defined in comparison to an orthogonal EUA RT-PCR assay (PPA [95% CI]: 96.77% [90.56%-100%] and NPA [95% CI]: 100% [100%-100%]). Data reported using our assay defined the limit of detection to be 40 copies/ml using heat-inactivated SARS-CoV-2 viral genome in clinical matrices. In-silico analysis provided >99.9% coverage across the SARS-CoV-2 viral genome and no cross-reactivity with evolutionarily similar respiratory pathogens.ConclusionThe SARS-CoV-2 NGS Assay powered by the COVID-DX Software can be used to detect the SARS-CoV-2 virus and provide additional insight into viral titer and genetic variants to track transmission, stratify risk, predict outcome and therapeutic response, and control the spread of infectious disease.DisclosuresDorottya Nagy-Szakal, MD PhD, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Joseph Barrows, MS, Biotia, Inc. (Employee, Shareholder) Heather L. Wells, MPH, Biotia (Consultant) Marilyne Debieu, PhD, Biotia (Employee) Courteny Hager, BS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee) Siyuan Chen, PhD, Twist Bioscience (Employee) Christopher Mason, PhD, Biotia (Board Member, Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)Twist (Other Financial or Material Support, I am CEO of Biotia and Biotia has business partnership with Twist)

Emulsifying and emulsion stabilizing properties of soy protein hydrolysates, covalently bonded to polysaccharides: The impact of enzyme choice and the degree of hydrolysis

Emulsifying and emulsion stabilizing properties of fragments, derived from vegetable proteins (soy protein isolate) and covalently linked to maltodextrin have been studied. The dual role of the degree of hydrolysis (DH) in improving the solubility of soy protein isolate (SPI) on one hand and difficulties of linking hydrolysates to polysaccharide on the other, have been highlighted. The findings have been compared to results obtained for whey protein isolate (WPI) fragments, undergoing the same enzymatic fragmentation and subsequent Maillard reaction. All experiments were conducted with two very different enzymes. Trypsin is rather selective of peptide bonds it cleaves, while alcalase is less specific. At the same DH, the actions of these two enzymes on the behaviour of the resulting conjugates were found to be broadly similar for the whey protein hydrolysates. In contrast, the trypsin generated soy protein fragments showed distinctly superior emulsifying properties than those produced by alcalase. These differences were related to the physical form of SPI existing as small colloidal aggregates in solution at the time of hydrolysis, whereas WPI was present as a molecularly well-dissolved protein. It is shown that, in realizing the best emulsion stabiliser, the improved solubility as a result of a higher level of hydrolysis is offset by the deterioration of the surface functionality of polypeptides due to further fragmentation. This leads to an optimum value for the DH of vegetable proteins for synthesising the most suitable Maillard based emulsifiers. For commercial SPI used in this study this was found to be around 8%.

A Functional Single Nucleotide Polymorphism in the Aγ Globin Gene Promoter Affects Globin Chain Synthesis

Beta Thalassemia and related hemoglobinopathies (such as sickle cell disease), are usually characterized by high levels of HbF since this compensates for the lack of, or abnormal HbA that is expressed in vivo. Further insight on how the hemoglobin switch is controlled and regulated can become crucial in the treatment of hemoglobin disorders. The present treatment options currently include routine blood transfusions with the occasional complications attributed to iron overload in major organs. Human stem cell therapy and bone marrow transplantation are some other options that are very risky and not without complications. Previous studies have shown that HbF levels differ in adults and that at least three loci independent of the β globin locus are responsible including XMN1, the HBS1L-MYB intergenic interval, and BCL11A. In another study conducted on an extended Maltese family, revealed that KLF1 also controls HbF levels in vivo. This study also suggested that other genes in conjunction with KLF1, play a part in activating the HbF switch upon birth. To date, the complete mechanism of how globin gene switching works is still elusive. A total of 355 DNA samples were tested. The majority of the DNA samples were obtained from an existing collection of normal to borderline HbA2 and HbF cases, while other samples were collected along the course of the study. Data from anonymous and randomized cord blood DNA samples were also used and obtained from the Laboratory of Molecular Genetics, University of Malta. These were tested for the rs368698783 polymorphism under study and were used for reference purposes. The SNP rs368698783 was amplified by PCR and checked by agarose gel electrophoresis using a 1.5% agarose gel. Restriction enzyme fragment length polymorphism analysis was then performed using Mnl1 restriction enzyme. Agarose gel electrophoresis was performed again in order to separate the Mnl1- incubated products according to size and thus determine the frequency of the mutation identified. Some samples were chosen at random and were genotyped by DNA sequencing. Amongst the total study population (~355 individuals), the frequency of the mutant allele was 0.14 and close to 0.25 for the random newborn cord blood DNA analyzed in a previous study. The total study population was divided into 125 healthy adults and 193 into borderline HbF/HbA2 The mean fetal hemoglobin and HbA2 for the normal sub-population were 0.39± 0.034 and 2.68± 0.02 respectively. The mean levels in the borderline population were slightly higher with a mean of 0.6± 0.04 for fetal hemoglobin and 3.7±0.04 for HbA2 The effects of rs368698783 A allele on HbF levels examined in the total population and furthermore in the two sub populations of (i) normal healthy adults and (ii) adults with borderline levels of HbF/HbA2 showed that the mutant A allele of HBG1-rs368698783 exhibited a significantly elevated level of HbF (p < 0.05) compared with the wildtype G allele in each of the cohorts from Malta. Intriguingly no association was determined between this point mutation and HbA2 levels. The data is suggestive that the variant HBG1-rs368698783 could act as a modifier associated with elevated levels of HbF in human adults. The SNP is located within the LYAR binding motif. The LYAR is a transcriptive factor which is said to silence the expression of fetal hemoglobin. thus, the G to A polymorphism reduces the DNA binding affinity increasing HbF levels invivo. Figure Disclosures No relevant conflicts of interest to declare.

RAD51 135G>C Single Nucleotide Polymorphism and Risk of Breast Cancer in Selected Filipino Cases

The RAD51 gene encodes the protein that plays a central role in the repair of DNA double-strand breaks (DSBs) through the homologous recombination pathway. Association of RAD51 single nucleotide genetic polymorphism (SNP) with the development of cancer has been observed to be tumor site- and race-specific. Thus, this study aimed to determine the potential association between RAD51 135G>C SNP and breast cancer among selected Filipinos. Patients with histologically confirmed breast cancer (n = 60) seen at the University of Santo Tomas Hospital (USTH Manila) were age- and sex-matched with clinically healthy controls (n = 60). Genomic DNA was extracted from the blood of participants and analyzed for RAD51 genotype by polymerase chain reaction – restriction enzyme fragment length polymorphism (PCR-RFLP). A significantly higher incidence of RAD51 C/C genotype was seen among the cases than the controls (p < 0.05). The more common G/C genotype was not associated with breast cancer development, while the recessive less common C/C genotype was observed to potentially increase the risk. However, passive smokers carrying the RAD51 G/C genotype had a significantly increased chance of developing breast cancer. RAD51 G/C genotype – even when combined with other established risk factors like alcohol use, active smoking, and family history – were not associated with breast cancer.

Identification of the core active structure of a Dendrobium officinale polysaccharide and its protective effect against dextran sulfate sodium-induced colitis via alleviating gut microbiota dysbiosis

It has been claimed that Dendrobium officinale applied as a functional food in China for centuries derived from the excellent anti-inflammatory activities. Herein, we aim to investigate the core structure of a Dendrobium officinale polysaccharide (DOP) based on the linear structural features by a specific endo-β-1,4-mannanase which was required for the protective effect against dextran sulfate sodium (DSS)-induced colitis in mice. Structure characterization revealed that enzymatic fragment contained the core domain (EDOP) which was composed of glucose and mannose in the molar ratio of 1.00:4.76, and consisted of (1 → 4)-β-D-Glcp and (1 → 4)-β-D-Manp with some attached 2-O-acetylated groups. In colitis mice, both DOP and EDOP could dramatically attenuate the clinical signs via blocking pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and their related mRNA), restoring the levels of short-chain fatty acids (SCFAs), activating the G-protein coupled receptors (GPRs) and modulating the gut microbiota. Gut microbiota dysbiosis is currently considered to be an important factor affecting colitis. The treatment of DOP and EDOP could recall the diversity of gut microbiota and modulate the abundance of the gut microbiota, including increasing the abundance of Bacteroides, Lactobacillus and Ruminococcaceae and reducing the abundance of Proteobacteria. Our findings have suggested that EDOP, as a core domain of DOP, retained similar structural features together with anti-inflammatory activity with DOP, and they could be potentially applied as natural candidates in the treatment of inflammatory bowel disease (IBD).

Association between platelet-derived growth factor-C single nucleotide polymorphisms and nonsyndromic cleft lip with or without cleft palate in Western Chinese population

To explore the association between two single nucleotide polymorphisms (SNPs), namely, rs4691383 and rs7667857, in the platelet-derived growth factor-C (PDGF-C) gene, the genotypes, environmental exposure factors, and nonsyndromic cleft lip with or without cleft palate (NSCL/P) in Western Chinese population. A total of 268 case-parent trios were selected, and two SNPs (rs4691383 andrs7667857) were genotyped by using polymerase chain reaction and restriction enzyme fragment length polymorphic method and direct sequencing method. Hardy-Weinberg equilibrium, linkage disequilibrium test, transmission disequilibrium test, and haplotype analysis were conducted to analyze the data. Meanwhile, the questionnaires on the epidemiology of cleft lip and palate filled by the included samples were collected, and the interaction between the genotypes of the two SNPs and environmental exposure factors was assessed by conditional logistic regression. The A allele at rs4691383 and the G allele at rs7667857 of PDGF-C gene were over-transmitted for NSCL/P (P<0.05). No interaction effect was observed between the three environmental exposure factors (history of smoking/passive smoking, folic acid supplementation, and long-term inhalation of harmful environmental gases) and the PDGF-C genotypes among NSCL/P (P>0.05). The rs4691383 and rs7667857 at PDGF-C gene are closely related to the occurrence of NSCL/P in Western Chinese population. However, the interaction between environmental exposure factors and PDGF-C genotypes is not obvious in the occurrence of NSCL/P.

Recombinant Mycobacterium bovis BCG is a promising platform to develop vaccines against Trypansoma cruzi infection

Chagas disease, caused by the hemoflagelate parasite Trypanosoma cruzi, is one of the most prevalent endemic parasitoses, affecting 7-8million people. Due to the complexity of the infection, no vaccines are available at present. The extraordinary adjuvant capacity of bacille Calmette-Guérin (BCG) was explored in this work to develop a vaccine candidate to protect against T. cruzi infection using the recombinant BCG (rBCG) vaccine platform. Three antigens of the parasite corresponding to the N and C terminal fragments of the enzyme trans-sialidase (NT-TS and CT-TS, respectively) and a fragment of the cruzipain enzyme (CZf) were cloned into the vectors pUS997 and pUS2000 and transformed into the BCG Pasteur strain. In vaccinated mice, rBCG expressing NT-TS in pUS2000 plasmid provided the highest protection and the lowest parasitemia after challenging BALB/c mice with a 50% lethal dose of parasites. When mice vaccinated with pUS2000-NT-TS were challenged with a 100% lethal dose of parasite, high levels of protection were also obtained, together with a low degree of cardiac lesions 120days after infection. In immunized mice with pUS2000-NT-TS/rBCG clone, the proliferation of CD4+ cells from splenocytes stimulated with the TS antigen was significant; this stimulation increased interferon (IFN)-γ and interleukin (IL)-17 within CD4⁺ T lymphocytes (LTCD4+ ) cells and IFN-γ and CD107 expression within LTCD8+ cells. Therefore, pUS2000-NT-TS/rBCG conferred high levels of protection, which correlated with an immune response orientated towards a T helper type 1 (Th1)/Th17 profile, together with an LTC-specific response, indicating that rBCG is a promising platform to develop vaccines against T. cruzi.

Impact of Apo Lipoprotein E Gene Polymorphism on Hepatitis C Virus Disease Progression and Response to Direct Acting Antivirals in the Egyptian Population

Background: Hepatitis C virus infection is a major health problem in Egypt. Apo lipoprotein E and lipid metabolism are closely associated with the life cycle of the virus and play an important role in facilitating this infection. Objectives: To identify the role of apolipoprotein E gene polymorphism on hepatitis C virus disease progression and response to direct-acting antivirals (DAAs). Methods: We included twenty patients with chronic hepatitis C virus infection (CHC) from those who failed to respond to DAAs treatment and 76 subjects from responders. Apo lipoprotein E genotypes were determined using PCR restriction enzyme fragment length polymorphism (RFLP). Results: Apolipoprotein genotype E3/E3 was prevalent in 75.9% of patients. The distribution for the rest of genotypes was 16.9%, 3.6%, 2.4% and 1.2 % for Apo E3/E4, E2/E3, E4/E4, and E2/E2 respectively. No statistically significant difference was found regarding any of the Apo E genotypes with liver cirrhosis or response to therapy. Conclusions: No significant impact for any Apo E genotypes on CHC disease severity or response to therapy with direct-acting antivirals was detected among Egyptian patients.

NIPT Technique Based on the Use of Long Chimeric DNA Reads.

Non-invasive prenatal testing (NIPT) for aneuploidy on Chromosomes 21 (T21), 18 (T18) and 13 (T13) is actively used in clinical practice around the world. One of the limitations of the wider implementation of this test is the high cost of the analysis itself, as high-throughput sequencing is still relatively expensive. At the same time, there is an increasing trend in the length of reads yielded by sequencers. Since extracellular DNA is short, in the order of 140–160 bp, it is not possible to effectively use long reads. The authors used high-performance sequencing of cell-free DNA (cfDNA) libraries that went through additional stages of enzymatic fragmentation and random ligation of the resulting products to create long chimeric reads. The authors used a controlled set of samples to analyze a set of cfDNA samples from pregnant women with a high risk of fetus aneuploidy according to the results of the first trimester screening and confirmed by invasive karyotyping of the fetus using laboratory and analytical approaches developed by the authors. They evaluated the sensitivity, specificity, PPV (positive predictive value), and NPV (negative predictive value) of the results. The authors developed a technique for constructing long chimeric reads from short cfDNA fragments and validated the test using a control set of extracellular DNA samples obtained from pregnant women. The obtained sensitivity and specificity parameters of the NIPT developed by the authors corresponded to the approaches proposed earlier (99.93% and 99.14% for T21; 100% and 98.34% for T18; 100% and 99.17% for T13, respectively).