Region Primers Research Articles

Child abuse: knowledge, awareness, and experience among dentists in India

BackgroundMedical child abuse and neglect are part of a spectrum of conditions that can lead to significant morbidity and mortality in children. Recognition of these forms of child maltreatment is crucial to prevent harm. Dentists should have familiarity with the framework for diagnosing and reporting child abuse as the orofacial complex is the prime anatomical region to show any signs of abuse. AIM: The purpose of this cross-sectional study was to assess the knowledge, awareness, and experience of dentists in India on child abuse and their role as primary responders. METHODS: A self-administered questionnaire comprising 16 questions was distributed among dentists in India and responses were collected. The questionnaire included questions on the knowledge, awareness, and experience of dentists in child abuse identification, reporting, legal proceedings, and proposals for the betterment of the current scenario. The data collected were statistically analyzed.ResultsA total of 203 dentists responded to the questionnaires through online platforms. Among them, 78.3% were females, and 65.3% had an institutional practice. While the majority (72.4%) were confident in identifying the indicators of child abuse, the knowledge on reporting was alarming. 98% of participants expressed their need for comprehensive education and training.ConclusionThe gap between the knowledge about indicators and the confidence to report suspicious cases are notable in this study. Adequate interventions in the hour of need require the involvement of support agencies and funding at both government and local levels.

Promotion of hepatic stellate cell activation and liver fibrosis by microRNA-33a-5p through targeting the Dickkopf-1-mediated wingless-related integration site/beta-catenin pathway.

Liver fibrosis occurs in response to chronic liver injury and is characterized by the production of excess extracellular matrix (ECM) proteins, largely by activated hepatic stellate cells (HSCs). Numerous studies have implicated micro-ribonucleic acids (miRNAs) in liver fibrosis, but the mechanisms remain unclear. Herein, HSC activation by miR-33a-5p during hepatic fibrosis was investigated. The miR-33a-5p was increased in the fibrotic mice and activated HSCs. AntagomiR-33a-5p inhibited HSC activation, proliferation, and migration in vitro, while simultaneously inducing apoptosis. The luciferase reporter assays indicated that the miR-33a-5p bound to the three prime untranslated region (3'UTR) of Dickkopf-1 (DKK1). Further investigation revealed that the miR-33a-5p targeted DKK1-modulated wingless-related integration site (Wnt)/β-catenin signaling directly to control hepatic fibrosis. Notably, the mice treated with antgomiR-33a-5p exhibited increased expression of DKK1 and reduced expression of fibrosis markers, along with reduced fibrosis. The RNA was isolated from activated and quiescent LX-2 cells and subsequently sequenced. Transcriptomic and bioinformatic analyses indicated strong downregulation of DKK1 during LX-2 cell activation. This paper presents the first demonstration of the miR-33a-5p-mediated modulation of liver fibrosis, with miR-33a-5p found to interact with DKK1, leading to regulation of Wnt/β-catenin signaling. The transcriptomic changes occurring during HSC activation were also defined. Overall, the findings suggest that both miR-33a-5p and DKK1 may be useful targets for treating liver fibrosis.

Structure-based pharmacophore modeling and DFT studies of Indian Ocean-derived red algal compounds as PI3Kα inhibitors.

Phosphoinositide kinases (PIKs) are a type of lipid kinase that acts as an upstream activator of oncogenic signaling. Presently accessible therapeutic compounds have downsides, such as toxicity and dubious efficacy, as well as lengthy treatment durations, which have bred resistance. Here we attempt to screen the Indian Ocean-derived red algal compounds to be used as a promising lead for PI3Kα inhibitor development. Experimental structure of the PI3K alpha Isoform-Specific Inhibitor alpelisib complex-based pharmacophore model was constructed and used as key to mark off the suitable lead compounds from the pool of marine-derived red algal compounds of Indian Ocean. Besides, the study encompasses pharmacophore scaffold screening as well as physicochemical and pharmacokinetic parameter assessment. We employed molecular docking and molecular dynamics simulation to assess the binding type and stability of 21 red algal derivatives. Twelve compounds demonstrated a sustained binding mode within the PI3Kα binding pocket with an optimal protein backbone root-mean-square deviation, also prompted hydrogen bonding throughout the simulations, and also implies that these MNPs have firmly mediated the interaction with prime hinge region residues in the PI3Kα ATP binding pocket. DFT studies revealed that proposed compounds had the greatest occupied molecular orbital electrophilicity index, basicity, and dipole moment, all of which attributed their stability as well as binding affinity at the PI3Kα active site. Our study's findings revealed that CMNPD31054, CMNPD4798, CMNPD27861, CMNPD4799, CMNPD27860, CMNPD9533, CMNPD3732, CMNPD4221, CMNPD31058, CMNPD31052, CMNPD29281, and CMNPD31055 can be used as lead compounds for PI3KΑ isoform inhibitors design.

Ginkgo biloba extract (GbE) restores gut microbiota dysbiosis in a rat model of lard-rich diet-induced obesity

BackgroundGut microbiota (GM) modulation has been considered a nutritional approach to manage obesity. Reduced Firmicutes/Bacteroidetes ratio (F/B) is associated with reduced energy harvesting capacity from the diet, ameliorates endotoxemia and inflammation, and restores gut hormone signaling related to hypothalamic control of energy homeostasis. As anti-obesogenic and anti-inflammatory properties have been attributed to Ginkgo biloba extract (GbE), the present study investigated whether GbE supplementation for two weeks modulates the GM composition of obese rats. MethodFifty-six 2-month-old male Wistar rats were submitted to a lard-rich diet-induced obesity protocol for 60 days (high-fat diet, HFD). Following the obesity-inducing period, rats were gavaged daily with GbE at 500 mg/kg (HFD+G group), or saline (HFD group), for 14 days. A 3rd group (pair-fed group, PF) was performed by mimicking the HFD group (saline administration) but with its food intake matched to the HFD+G group. Rats were euthanized on the 14th supplementation day. Stool DNA was extracted and amplified with V3–V4 region primers of the 16S rRNA gene. ResultsIn comparison to both HFD and PF groups, GbE supplementation increased the number of Bacteroidetes colon community and concomitantly reduced the abundance of Firmicutes, reducing the F/B ratio. Hierarchical clustering showed that communities of the HFD+G group were less likely related to HFD and PF groups. ConclusionAs GbE modulated the GM structure and diversity in GbE-supplemented obese rats, our results show that GbE possesses phytotherapeutic potential to modulate obesity by improving GM and lessening the consequences of obesity-related GM dysbiosis.

Identifying Growth Pattern as Regional Classification in Central Java and Yogyakarta

This study is the preliminary research of spatial inequality in Central Java. The regional differences are substantively irrepressible. The perception of north-south regions of Java exists and persist. As many of north regions have specialized in non-agriculture, the northern had been assumed as dynamic regions. This assumption has led to the differentiation of regional policy focused on north-south unbalance development. This study aims to evaluate the spatial pattern of Central Java and Yogyakarta based on economic growth from 1990-2010. The pattern indicates the relation of urban and its surrounding and no longer the contest of north and south region. Some current facts define that spread effects from the prime regions is beneficial for its surroundings. In this study, Semarang, Solo, and Yogyakarta, has been identified as the predominant cities toward their surroundings creating a cluster of growth pattern. The nearby surroundings obtain economic growth spill-over from those cities. On the other side, the regions locate away from prime cities have experienced lower growth. Consequently, the spatial inequality may occur across the regions.

Nematicidal effects of silver nanoparticles (AG-NPs) on the root-knot nematode, Meloidogyne javanica associated with Swiss chard (Beta vulgaris L.).

Root-knot nematodes (RKNs) are important nematode pests, causing huge economic losses on vegetable crops worldwide. A decline in the yield of Swiss chard (Beta vulgaris L.) which was associated with RKNs was observed on an organic vegetable farm in the Western Cape Province of South Africa. Nematodes were extracted from galled plant roots and identified using molecular tools. PCR-based Sequence Characterised Amplified Region (SCAR) primers was used to confirm the specie of the RKN associated with the infected plants. Thereafter, a pot assay was conducted to determine the response of artificially infected Swiss chard plants to varying concentrations of bio-synthesized silver nanoparticle Ag-NP (1 μg/mL, 2 μg/mL, and 3 μg/mL) under controlled conditions. The results of the study showed that Swiss chard is highly susceptible to M. javanica with an egg-laying-female index of >5 in all infected plants. Significantly lower values (at P=0.05) in egg masses (EM), juveniles (J2s), and reproduction factor (RF) of nematodes were recorded on plants treated with 3 μg/mL, indicating a potential for nematode control. A negative correlation was also observed in the number of egg masses, J2s, and RF of the nematodes with increasing concentrations of the Ag-NP. This study confirms that Swiss chard is highly susceptible to M. javanica and demonstrates the potential nematicidal property of Ag-NP in controlling the nematode pest of Swiss chard.

Next-Generation Sequencing of the Microbial Community Profile In Free-Range Chicken (Gallus gallus domesticus) Cecum from East Nusa Tenggara Province

Free-range chicken is livestock reared to support the humans' need for protein alongside its ritualistic use in traditional medicine to treat diseases. This study investigates the diversity of bacterial communities in the free-range chicken cecum reared in different East Nusa Tenggara Province localities comprising Sambi 1, Sambi 2 villages, Labuan Bajo, and Kupang City. The extracted chromosomal DNA was subjected to next-generation sequencing using the V3-V4 region primers. Results revealed that the Kupang chicken cecum had the highest total tags, while the Sambi 2 village recorded the lowest. Similarly, Sambi 2 chicken cecum exhibited the highest unique tags (6662) and OTUs number (1261), while the Kupang samples gave the lowest at 2550 and 745, respectively. The Shannon diversity index for bacterial diversity demonstrated that cecum samples from Labuan Bajo (5.679) were more diverse than Sambi 1 (5.378), Sambi 2 (5.653), and Kupang samples (3.77). The bacteria with the highest dominance index (0.935) was found in Sambi 2, while the lowest was observed in the Kupang samples (0.082). The three bacterial phyla showing the highest relative abundance were those from Sambi 1, Sambi 2, and Labuan Bajo cecum samples, comprising Firmicutes, Bacteroidota, and Actinobacteriota.Conversely, the Kupang samples showed an abundance of Firmicutes, Bacteroidota, and Campilobacterota, compared to the Lactobacillus-dominated Kupang, Sambi 1, and Sambi 2 chicken cecum samples. The highest relative abundance for Bifidobacterium occurred in Sambi 1 and Sambi 2 chicken cecum samples, the Kupang samples were Campylobacter dominated, and Olsenella was abundant in the Labuan Bajo samples. Intriguingly, the bacterial composition in the tested chicken cecum samples largely comprised beneficial bacteria such as the lactic acid bacteria group. This bacterial group can be further characterized for obtaining probiotic cultures that could improve the health of free-range chickens.

Ecoinformatic Analysis of the Gut Ecological Diversity of Wild and Captive Long-Tailed Gorals Using Improved ITS2 Region Primers to Support Their Conservation.

Ex situ conservation is used to protect endangered wildlife. As captive and wild long-tailed gorals are known to be similar, individuals under ex situ conservation can be reintroduced into nature. However, there is no appropriate indicator to evaluate them. Here, we amplified the internal transcribed spacer 2 (ITS2) region and compared the gut ecological information (eco-information) of captive and wild long-tailed gorals. We validated the existing ITS86F and ITS4 universal primers using reference sequences of the National Center for Biotechnology Information (NCBI) and improved their matching rates. We compared the gut eco-information of captive and wild long-tailed gorals obtained through experiments using the improved primer pair and found that the gut ecological diversity of captive gorals was low. Based on this, we suggested that the gut eco-information can be used as an evaluation index before reintroducing captive long-tailed gorals. Furthermore, we identified four plant types from the gut eco-information of wild long-tailed gorals, which can be the additional food sources to enhance the reduced intestinal ecological diversity of the captive animals.

Identification of single nucleotide variants in SLC26A9 gene in patients with cystic fibrosis (p.Phe508del homozygous) and its association to Orkambi® (Lumacaftor and Ivacaftor) response in vitro

BackgroundSince patients with cystic fibrosis with different Cystic Fibrosis Transmembrane Regulator (CFTR) genotypes present a wide response variability for modulator drugs such as Orkambi®, it is important to screen variants in candidate genes with an impact on precision and personalized medicine, such as Solute Carrier Family 26, member 9 (SLC26A9) gene. MethodsSanger sequencing for the exons and intron–exon boundary junctions of the SLC26A9 gene was employed in nine individuals with p.Phe508del homozygous genotype for the CFTR gene who were not under CFTR modulators therapy. The sequencing variants were evaluated by in silico prediction tools. The CFTR function was measured by cAMP–stimulated current (ΔIsc-eq-FSK) in polarized CFTR of human nasal epithelial cells cultured in micro–Ussing chambers with Orkambi®. ResultsWe found 24 intronic variants, three in the coding region (missense variants – rs74146719 and rs16856462 and synonymous – rs33943971), and three in the three prime untranslated region (3′ UTR) region in the SLC26A9 gene. Twenty variants were considered benign according to American College of Medical Genetics and Genomics guidelines, and ten were classified as uncertain significance. Although some variants had deleterious predictions or possible alterations in splicing, the majority of predictions were benign or neutral. When we analyzed the ΔIsc-eq-FSK response to Orkambi®, there were no significant differences within the genotypes and alleles for all 30 variants in the SLC26A9 gene. ConclusionsAmong the nine individuals with p.Phe508del homozygous genotype for the CFTR gene, no pathogenic SLC26A9 variants were found, and we did not detect associations from the 30 SLC26A9 variants and the response to the Orkambi® in vitro.

458 Assessment of MGMT Methylation Status Using High-Performance Liquid Chromatography in Newly Diagnosed Glioblastoma

INTRODUCTION: The utility of O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significant consensus exists on the optimal method of assessing MGMT methylation. METHODS: W analyzed the MGMT methylation status of 28 isocitrate dehydrogenase–wild-type GBMs treated with temozolomide using ion-exchange HPLC and bisulfite sequencing using frozen samples. We designed three primers for separate regions (regions 1–3) that had 21 to 38 CpGs for PCR and validated the MGMT promoter methylation status. RESULTS: There was a strong correlation between HPLC and bisulfite sequencing results (R = 0.794). The optimal cutoff values for MGMT methylation in HPLC were determined to allow differentiation of patient prognosis by receiver operating characteristic curve analysis. The cutoff values were 34.15% for region 1, 8.84% for region 2, and 36.72% for region 3. Kaplan–Meyer curve analysis estimated that the most differentiated prognosis was enabled in the setting of 8.84% methylation of MGMT in region 2. Progression-free survival and overall survival were significantly longer for patients in this setting of region 2 methylation (p = 0.00365 and p = 0.00258, respectively). CONCLUSIONS: The combination of our HPLC method and the original primer setting provides a new standard method for determination of MGMT methylation status in patients with GBM and is useful for refining MGMT-based drug selection.

The first study on clinicopathological changes in cats with feline infectious peritonitis with and without retrovirus coinfection.

Feline infectious peritonitis (FIP) is an infectious, immune-mediated, and fatal disease in cats caused by a mutant feline coronavirus (FCoV) infection. Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are two common retroviruses that play a role in reducing feline immune function with opportunistic retrovirus infection being a predisposing factor for the development of FIP. This study aimed to evaluate the clinicopathological parameters of FIP in cats with and without retrovirus coinfection. In total, 62 cats presenting with pleural and/or peritoneal effusion at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand, were selected for the study. Effusion samples were collected and a reverse transcriptase-polymerase chain reaction (RT-PCR) assay was performed on all samples using the 3' untranslated region primer. All FCoV-positive cats were tested for retrovirus infection using a commercial kit (Witness FeLV-FIV [Zoetis]; United States). Clinical signs, hematological, and biochemical parameters of these cats were investigated and grouped. Of the 62 cats with pleural and/or peritoneal effusion, FCoV was detected in 32, of which 21 were highly suspicious for FIP. The cats suspected of FIP were divided into three subgroups following viral detection. A total of 14 had only FCoV infection (Group A), four had FCoV and FeLV infection (Group B), and three had FCoV, FeLV, and FIV infection (Group C). Of the rest, 11 had definitive diagnoses, which included three being FCoV and FeLV-positive (Group D), and eight were retrovirus-negative (Group E). Mild anemia and lymphopenia were found in cats infected with these three viruses. An albumin-to-globulin ratio lower than 0.5 was found in FIP cats with only FCoV infection. Typically, cats with clinical effusion and FIP, with and without retrovirus coinfection, had similar hematological findings. Clinical signs, blood parameters, fluid analysis with cytological assessment, and RT-PCR assays could identify better criteria to diagnose FIP with and without retrovirus coinfection.

DbCNV: deleteriousness-based model to predict pathogenicity of copy number variations

BackgroundCopy number variation (CNV) is a type of structural variation, which is a gain or loss event with abnormal changes in copy number. Methods to predict the pathogenicity of CNVs are required to realize the relationship between these variants and clinical phenotypes. ClassifyCNV, X-CNV, StrVCTVRE, etc. have been trained to predict the pathogenicity of CNVs, but few studies have been reported based on the deleterious significance of features.ResultsFrom single nucleotide polymorphism (SNP), gene and region dimensions, we collected 79 informative features that quantitatively describe the characteristics of CNV, such as CNV length, the number of protein genes, the number of three prime untranslated region. Then, according to the deleterious significance, we formulated quantitative methods for features, which fall into two categories: the first is variable type, including maximum, minimum and mean; the second is attribute type, which is measured by numerical sum. We used Gradient Boosted Trees (GBT) algorithm to construct dbCNV, which can be used to predict pathogenicity for five-tier classification and binary classification of CNVs. We demonstrated that the distribution of most feature values was consistent with the deleterious significance. The five-tier classification model accuracy for 0.85 and 0.79 in loss and gain CNVs, which proved that it has high discrimination power in predicting the pathogenicity of five-tier classification CNVs. The binary model achieved area under curve (AUC) values of 0.96 and 0.81 in the validation set, respectively, in gain and loss CNVs.ConclusionThe performance of the dbCNV suggest that functional deleteriousness-based model of CNV is a promising approach to support the classification prediction and to further understand the pathogenic mechanism.

Phylogenetic analysis of Prunus genus using nuclear and chloroplast gene markers as a bioorganic structure profiling

Prunus species are known as model species for the family Rosaceae and other woody plants with their small genome size and relatively small life cycle as woody plants. Also Prunus species are economically important plants in the subfamily Amygdaloideae of the Rosaceae have a controversial taxonomic history due to the lack of a phylogenetic framework. Therefore, mapping, classification, determination the genetic structure and characterization of genes as a spesific chemical and fuctional units of nucleic acids and making phylogenetic analysis in these species have important implications. The aim of this study to find any gene markers as a bioorganic – chemical structure profiling to determine genetic diversity of different Prunus species from Iraq by using nuclear and chloroplast DNA markers to evaluate the phylogeny of Prunus. A total of 12 species Prunus subgenus amygdalus were collected from Iraq. Ten gene region markers on both nuclear DNA and chloroplast DNA have been tested to evaluate the genetic variation at their molecular levels by using multiplex PCR with 10 gene region primers and sequence analysis for matK. The name of the gene regions using in this study are ITS (ITS1+ITS2+5.8S coding region), rbcL, matK, rpl16, trnL-trnF, atpB-rbcL, rp136-infA-rps8, trnK-rps16, pbsM-trnD, psbA-trnH. As a result, five different phylogenetic trees were obtained from the gene sequences of the samples and the base sequences were close to each other. The estimated value of the shape parameter for the discrete Gamma Distribution is 0.05. The nucleotide frequencies are A = 29.41%, T/U = 37.37%, C = 17.52%, and G = 15.67%. The nucleotide diversity of the sequence was low with the 0.004113 value. There were 14 different informative base site between the samples. The polymorphism observed was less in agreement with higher level of conservation of the gene regions. The Prunus species from Iraq region were grouped on two closed clusters on the phylogenetic trees.

Isolation, molecular characterization of indigenous Metarhizium anisopliae (Metchnikoff) isolate, using ITS-5.8s rDNA region, and its efficacy against the Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae)

BackgroundAs a biological control agent, entomopathogenic fungus (EPF) can give an alternative to high-risk pesticides. Metarhizium anisopliae is one of the most promising pest controls EPF in the worldwide.ResultOn fungi-specific selective media, the EPF isolate SVPUAT was isolated from Western Uttar Pradesh, India. The isolate was identified as M. anisopliae (GenBank accession no. OP962431) after molecular screening utilizing PCR amplification with the internal transcribed spacer (ITS) region primer. In a contact toxicity experiment, the isolate SVPUAT was tested against the 4th instar larvae of Helicoverpa armigera (Hubner) at concentrations ranging from 103 to 1010 spores ml−1. The lethal time LT50 and LT90 values for the 4th instar larvae of H. armigera injected with 1 × 1010 spores ml−1 were 3.46 and 5.54 days, respectively.ConclusionsThe present M. anisopliae isolate SVPUAT was identified using the ITS-5.8s rDNA region from GenBank and has showed significant pathogenicity to H. armigera. More research is needed to prove the efficacy against various pests of economic important as a legitimate choice for an integrated pest management program.

The lncRNA XIST/miR-29b-3p/COL3A1 axis regulates central carbon metabolism in head and neck squamous cell carcinoma and is associated with poor tumor prognosis.

Recent evidence shows that COL3A1 promotes the progression of many types of cancer. The purpose of our study is to explore the correlation between COL3A1 and the prognosis of patients with head and neck squamous cell carcinoma (HNSCC) and its potential mechanism. We initially screened the differentially expressed gene COL3A1 in The Cancer Genome Atlas (TCGA) database, and the association between the expression level of COL3A1, prognosis, and the clinical parameters of HNSCC patients was verified. A nomogram was constructed according to the multivariate analysis results. Next, a heatmap of COL3A1 co-expressed genes was constructed in TCGA database. The TargetScan database is used to explore the microRNAs (miRNA) related to COL3A1. The starBase database was used to explore and predict the long non-coding RNAs (lncRNAs) that the candidate miRNAs might bind to. Finally, the potential mechanism of action was investigated using Gene Set Enrichment Analysis (GSEA). COL3A1 expression is elevated in HNSCC tumor tissues, and HNSCC patients with high COL3A1 expression have worse prognostic factors. COL3A1 was positively correlated with the central carbon metabolism-related proteins: epidermal growth factor receptor (EGFR), phosphoglycerate mutase 1 (PGAM1), hexokinase 3 (HK3), and phosphofructokinase, platelet (PFKP). The TargetScan database showed that the best candidate miRNA for binding to the three prime untranslated region (3'UTR) end of COL3A1 mRNA was hsa-miR-29b-3p, which was negatively correlated with COL3A1. The starBase database showed that the lncRNA X Inactive Specific Transcript (lncRNA XIST) was the best candidate upstream non-coding RNA for regulating hsa-miR-29b-3p. GSEA showed that COL3A1 may be involved in the poor prognosis of HNSCC by participating in carbon metabolism, glucose metabolism, oxidative stress, and the Wingless-Type MMTV Integration Site Family (Wnt) and vascular endothelial growth factor A-vascular endothelial growth factor receptor 2 (VEGFA-VEGFR2) pathways. Low COL3A1 expression can be employed as a new HNSCC predictive biomarker, and the prognosis of HNSCC patients with lower COL3A1 expression can be greatly improved. At the same time, we found that the lncRNA XIST/miR-29b-3p/COL3A1 axis may regulate the central carbon metabolism of HNSCC and is associated with poor prognosis. These findings point to a potential target for developing HNSCC anticancer therapies.

Racial disparities in tumor microbiome in young-onset and average-onset colorectal cancer.

13 Background: The occurrence of young-onset colorectal cancer (yoCRC) is rising alarmingly, with a disproportionate incidence in Black patients as compared to White. We and others have previously shown differences in tumor microbiomes between yoCRC and average-onset colorectal cancer (aoCRC), but it is unknown if these differences contribute to racial disparities. We therefore analyzed racial differences in the intra-tumoral microbiome of CRC in young adults (<50 years). Methods: We analyzed 277 samples of histologically confirmed cases of stage I-IV CRC patients identified as either non-Hispanic Black or non-Hispanic White, who underwent surgical resection at Cleveland Clinic from year 2000-2020, who consented to a prospective biorepository. Fresh frozen specimens from the primary tumor with paired adjacent nonmalignant tissue were analyzed. The 16s rRNA gene was amplified and sequenced using V4 region primers and Illumina’s MiSeq system. Using DADA2, the quality filtered 16S rRNA amplicon reads were clustered and annotated as amplicon sequence variants (ASVs). The abundance count matrix was analyzed for alpha and beta diversity and differential abundance analysis (DAA) using Phyloseq package. Statistical tests included analysis of variance (ANOVA), permutational multivariate analysis (PERMANOVA), linear regression and Wilcoxon test. DAA and correlation analysis were controlled for false discovery rate using Benjamini Hochberg correction. Results: Differential abundance analysis highlighted key differences in the microbiome composition of Black and White patients with yoCRC. Amongst Black patients, the most prevalent taxa were Limosilactobacillus, Bacillus, Staphylococcus, Listeria and Akkermansia, whereas amongst White patients the most prevalent taxa were Enterococcus and Escherichia-Shigella (Table). Microbial profiles were also significantly different between Black patients with yoCRC and aoCRC and between White patients with yoCRC and aoCRC. At ASV level, the microbiome of Black yoCRC is more similar (R2 =0.87) to Black aoCRC, in comparison to white yoCRC (R2=0.57). Conclusions: We found significant differences between the intra-tumoral microbiome of yoCRC in the United States by race. Future epidemiologic studies and public health interventions need to account for these differences to reduce risk of yoCRC across various US populations. [Table: see text]

Elevation of hsa-miR-7-5p level mediated by CtBP1-p300-AP1 complex targets ATXN1 to trigger NF-κB-dependent inflammation response.

Nuclear factor-κB (NF-κB)-mediated inflammation is a major cause of acute respiratory distress syndrome (ARDS). However, the regulatory mechanisms by which NF-κB transactivates proinflammatory cytokines remain unclear in the pathogenesis of ARDS. Herein, we report that the activating protein 1 (AP1) transcription factor recruits a histone acetyltransferase p300 and a transcriptional regulator C-terminal binding protein 1 (CtBP1) to assemble the CtBP1-p300-AP1 complex, which transactivates the expression of hsa-miR-7-5p in ARDS biopsies. Overexpressed hsa-miR-7-5p binds to the three prime untranslated regions (3'-UTRs) of ataxin 1 (ATXN1), suppressing its expression. Decreased ATXN1 expression relieves its repression of NF-κB, causing the induction of proinflammatory cytokine genes and triggering an inflammatory response. Depletion of CtBP1 or treatments with two CtBP1 inhibitors (NSC95397 and 4-methylthio-2-oxobutanoate (MTOB)) in human macrophages impairs the assembly of the CtBP2-p300-AP1 complex, resulting in decreased hsa-miR-7-5p levels, upregulation of ATXN1, and attenuation of proinflammatory cytokines. A similar regulatory mechanism was observed in lipopolysaccharide-treated mice. Our results reveal that increased hsa-miR-7-5p level mediated by the CtBP1-p300-AP1 complex targets ATXN1 to trigger an NF-κB-dependent inflammatory response. Interfering with this signaling pathway to block the inflammatory response may be a strategy for treating ARDS. KEY MESSAGES : The transcription factor AP1 recruits p300 and CtBP1 to form a transcriptional complex, which transactivates the expression of hsa-miR-7-5p in ARDS biopsies. Overexpressed hsa-miR-7-5p binds to the 3'-UTR of ATXN1, suppressing its expression. The decreased ATXN1 impaired its suppression of NF-κB, causing the induction of proinflammatory cytokine genes and triggering inflammation response. Disruption of the assembly of CtBP2-p300-AP1 complex upregulates ATXN1 and attenuates inflammation.

Isolation and identification of Pithomyces sacchari as a leaf spot pathogen of Helianthus annuus from Pakistan

Sunflower (Helianthus annuus L.) is an important annual crop known for its edible oil. Sunflower is susceptible to many fungal diseases including rusts, rotting, mildews, and leaf spots that result in low crop yield. Presently, infected leaves with leaf spot disease symptoms were collected from Jallo Park, Botanical Garden; University of the Punjab, Canal road, and Johar Town, Lahore for pathogen/s isolation and identification. The identification was executed morphologically as well as genetically by nucleotide sequencing of rDNA using Internal spacer region (ITS) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers. Morphological characters demonstrated a rapidly growing colony on MEA reaching 5.0–5.5 cm in diameter without zonation. The mycelial growth was rough and cottony white from the front and light pink from the reverse side. Conidia were brown, verruculose, and ellipsoidal with three to five transverse septations and one longitudinal septum ranging from 15 to 30 ± 2 µm in the broadest part. Conidiophores were long, branched, septate, 70–80 × 3–4 µm in size. Based on morphological characteristics, the pathogen was identified as Pithomyces sacchari. In genetic characterization BLAST analysis of the rDNA-ITS region of the pathogen exhibited maximum (100%) homology with other P. sacchari GenBank strains. Similarly, 99% homology was found with partial glyceraldehyde 3-phosphate dehydrogenase (GAPDH). To confirm the pathogenicity, Koch’s pathogenicity test was performed by inoculating artificial fungal suspension in pots and plate assays. The emergence of similar disease symptoms and re-isolation of the same pathogens verified Koch’s pathogenicity postulates. Conclusively this study confirms the identification of this novel pathogen of sunflowers and necessitates the quick development of management tools.

Single-Cell RNA Sequencing for the Detection of Clonotypic V(D)J Rearrangements in Multiple Myeloma.

Multiple myeloma (MM) has a highly heterogeneous genetic background, which complicates its molecular tracking over time. Nevertheless, each MM patient's malignant plasma cells (PCs) share unique V(D)J rearranged sequences at immunoglobulin loci, which represent ideal disease biomarkers. Because the tumor-specific V(D)J sequence is highly expressed in bulk RNA in MM patients, we wondered whether it can be identified by single-cell RNA sequencing (scRNA-seq). To this end we analyzed CD138+ cells purified from bone marrow aspirates of 19 samples with PC dyscrasias by both a standard method based on bulk DNA and by an implementation of the standard 10x Genomics protocol to detect expressed V(D)J sequences. A dominant clonotype was easily identified in each sample, accounting on average for 83.65% of V(D)J-rearranged cells. Compared with standard methods, scRNA-seq analysis proved highly concordant and even more effective in identifying clonal productive rearrangements, by-passing limitations related to the misannealing of consensus primers in hypermutated regions. We next validated its accuracy to track 5 clonal cells with absolute sensitivity in a virtual sample containing 3180 polyclonal cells. This shows that single-cell V(D)J analysis may be used to find rare clonal cells, laying the foundations for functional single-cell dissection of minimal residual disease.

Development of strain-specific SCAR primers to beneficial rhizobacteria Bacillus subtilis OSU-142

Background and purpose: Synthetic fertilizers damage the environment. Biofertilizers that consist of microorganisms emerge as an environmentally friendly alternative. Biofertilizers improve plant growth by mobilizing soil nutrients, triggering plant hormone synthesis, or competing with pathogenic bacteria. However, biofertilizers often fail due to insufficient colonization of the plant roots.Materials and methods: To explore the colonization dynamics of a bacterial strain commonly used in biofertilizers, Bacillus subtilis OSU-142 (OSU-142), developing a set of primers specific to OSU-142 was aimed. Since its genome is unknown, to identify genomic regions unique to OSU-142 strain, DNAs of more than 40 bacterial strains were fingerprinted, most of which belong to Bacillus subtilis using the Randomly Amplified Polymorphic DNA (RAPD) method.Results: This approach identified a polymorphic band at 880 bp, which was then cloned and sequenced. The sequence showed no perfect match to any known sequences in the tested genomic databases indicating that the region of OSU-142 DNA is highly unique. The primer was converted to Sequence-Characterized Amplified Region (SCAR) primers, and its functionality in detecting OSU-142 genome was confirmed. Newly designed primer set can specifically detect OSU-142.Conclusions: These primers can be useful for basic science or commercial applications on tracking OSU-142 in various environments, thus contributing to biofertilizers’ adoption in the long run.