Selected Target Genes Research Articles

Evaluation of Controlled Ovarian Stimulation Protocols in Patients with Normal and Low Ovarian Reserve: Analyses of miRNAs and Selected Target Genes Involved in the Proliferation of Human Cumulus Cells and Oocyte Quality.

The oocyte and the surrounding cumulus cells (CCs) are deeply linked by a complex bidirectional cross-talk. In this light, the molecular analysis of the CCs is nowadays considered to be precious in providing information on oocyte quality. It is now clear that miRNAs play a key role in several ovarian functions, such as folliculogenesis, steroidogenesis, and ovulation. Thus, in this study, specific miRNAs, together with their target genes, were selected and investigated in CCs to assess the response of patients with normal (NR) and low (LR) ovarian reserve to two different controlled ovarian stimulation (COS) protocols, based on rFSH and hMG. Moreover, a Fourier transform infrared microspectroscopy (FTIRM) analysis was performed to evaluate DNA conformational changes in CCs and to relate them with the two COS protocols. The results evidenced a modulation of the expression of miRNAs and related target genes involved in CCs’ proliferation, in vasculogenesis, angiogenesis, genomic integrity, and oocyte quality, with different effects according to the ovarian reserve of patients. Moreover, the COS protocols determined differences in DNA conformation and the methylation state. In particular, the results clearly showed that treatment with rFSH is the most appropriate in NR patients with normal ovarian reserve, while treatment with hMG appears to be the most suitable in LR patients with low ovarian reserve.

Adaptive strategies of uropathogenic Escherichia coli CFT073: from growth in lab media to virulence during host cell adhesion.

Urinary tract infections (UTIs) are a major concern in public health. The prevalent uropathogenic bacterium in healthcare settings is Escherichia coli. The increasing rate of antibiotic-resistant strains demands studies to understand E. coli pathogenesis to drive the development of new therapeutic approaches. This study compared the gene expression profile of selected target genes in the prototype uropathogenic E. coli (UPEC) strain CFT073 grown in Luria Bertani (LB), artificial urine (AU), and during adhesion to host bladder cells by semi-quantitative real-time PCR (RT-PCR) assays. AU effectively supported the growth of strain CFT073 as well as other E. coli strains with different lifestyles, thereby confirming the appropriateness of this medium for in vitro models. Unexpectedly, gene expression of strain CFT073 in LB and AU was quite similar; conversely, during the adhesion assay, adhesins and porins were upregulated, while key global regulators were downregulated with respect to lab media. Interestingly, fimH and papGII genes were significantly expressed in all tested conditions. Taken together, these results provide for the first time insights of the metabolic and pathogenic profile of strain CFT073 during the essential phase of host cell adhesion.

Expression of let-7a, miR-106b and miR-29b is changed in human gastric cancer.

MicroRNA (miRNA) are small, noncoding RNA sequences that post-transcriptionally regulate the proliferation, activity and apoptosis of human gastric cancer cells by controlling various signaling cascades. Processes that involve miRNA molecules can create a specific network of interactions in a cell, and disruption of its functioning may contribute to the transformation of normal cells into cancerous cells. Aims of our survey were: 1) study the relationship between the expression of selected miRNA types (let-7a, miR-106b, miR-29b, miR-21, miR-155, miR-222) in the gastric mucosa and pathomorphological changes determined by classical histopathological methods, 2) perform in silico analysis to select target genes for selected miRNAs and to perform functional analysis of these genes. Eighty-three subjects (45 women, 38 men; mean age 39±14 years, range: 21-80 years, were examined). Among them were 18 (21.5%) patients with chronic active gastritis, 42 (50.6%) people with chronic inactive gastritis, 9 (10.8%) patients with gastric cancer and 14 (16.9%) patients without histopathological changes. The study demonstrated that mainly the expression of 3 (miR-29b, let-7a miR-106b) out of 6 selected miRNAs are significantly different depending on the site of biopsy (body of the stomach or antrum) and the group of patients. Expression of miR-106b in the antrum and body was the highest in the group of cancer patients and the lowest in patients with chronic active gastritis. The expression of let-7a differed depending on group of patients and location. The highest expression was in the body in the group with inactive gastritis and the lowest in gastric cancer. Patients with cancer had the lowest expression of miR-29b in stomach body and it was the highest in the patients with inactive gastritis in this location. Expression of miR-21 and miR-155 determinations were not statistically significant in comparison to groups or locations, and of miR-222 was not different between the groups, but only in the control group was higher in the antrum than in the body. We conclude that identification of miRNAs may represent a promising modern complementary method in the diagnosis of gastric diseases, especially cancer.

MicroRNA regulatory networks associated with abnormal muscle repair in survivors of critical illness.

BackgroundIntensive care unit (ICU)‐acquired weakness is characterized by muscle atrophy and impaired contractility that may persist after ICU discharge. Dysregulated muscle repair and regeneration gene co‐expression networks are present in critical illness survivors with persistent muscle wasting and weakness. We aimed to identify microRNAs (miRs) regulating the gene networks and determine their role in the self‐renewal of muscle in ICU survivors.MethodsMuscle whole‐transcriptome expression was assessed with microarrays in banked quadriceps biopsies obtained at 7 days and 6 months post‐ICU discharge from critically ill patients (n = 15) in the RECOVER programme and healthy individuals (n = 8). We conducted an integrated miR–messenger RNA analysis to identify miR/gene pairs associated with muscle recovery post‐critical illness and evaluated their impact on myoblast proliferation and differentiation in human AB1167 and murine C2C12 cell lines in vitro. Select target genes were validated with quantitative PCR.ResultsTwenty‐two miRs were predicted to regulate the Day 7 post‐ICU muscle transcriptome vs. controls. Thirty per cent of all differentially expressed genes shared a 3'UTR regulatory sequence for miR‐424‐3p/5p, which was 10‐fold down‐regulated in patients (P < 0.001) and correlated with quadriceps size (R = 0.86, P < 0.001), strength (R = 0.75, P = 0.007), and physical function (Functional Independence Measures motor subscore, R = 0.92, P < 0.001) suggesting its potential role as a master regulator of early recovery of muscle mass and strength following ICU discharge. Network analysis demonstrated enrichment for cellular respiration and muscle fate commitment/development related genes. At 6 months post‐ICU discharge, a 14‐miR expression signature, including miRs‐490‐3p and ‐744‐5p, identified patients with muscle mass recovery vs. those with sustained atrophy. Constitutive overexpression of the novel miR‐490‐3p significantly inhibited AB1167 and C2C12 myoblast proliferation (cell count AB1167 miR‐490‐3p mimic or scrambled‐miR transfected myoblasts 7926 ± 4060 vs. 14 159 ± 3515 respectively, P = 0.006; proportion Ki67‐positive nuclei AB1167 miR‐490‐3p mimic or scrambled‐miR transfected myoblasts 0.38 ± 0.07 vs. 0.54 ± 0.06 respectively, P < 0.001; proliferating cell nuclear antigen expression AB1167 miR‐490‐3p mimic or scrambled‐miR transfected myoblasts 11.48 ± 1.97 vs. 16.75 ± 1.19 respectively, P = 0.040). Constitutive overexpression of miR‐744‐5p, a known regulator of myogenesis, significantly inhibited AB1167 and C2C12 myoblast differentiation (fusion index AB1167 miR‐744‐5p mimic or scrambled‐miR transfected myoblasts 8.31 ± 7.00% vs. 40.29 ± 9.37% respectively, P < 0.001; myosin heavy chain expression miR‐744‐5p mimic or scrambled‐miR transfected myoblasts 0.92 ± 0.39 vs. 13.53 ± 5.5 respectively, P = 0.01).ConclusionsCombined functional transcriptomics identified 36 miRs including miRs‐424‐3p/5p, ‐490‐3p, and ‐744‐5p as potential regulators of gene networks associated with recovery of muscle mass and strength following critical illness. MiR‐490‐3p is identified as a novel regulator of myogenesis.

Reconstruction of dynamic regulatory networks reveals signaling-induced topology changes associated with germ layer specification.

SummaryElucidating regulatory relationships between transcription factors (TFs) and target genes is fundamental to understanding how cells control their identity and behavior. Unfortunately, existing computational gene regulatory network (GRN) reconstruction methods are imprecise, computationally burdensome, and fail to reveal dynamic regulatory topologies. Here, we present Epoch, a reconstruction tool that uses single-cell transcriptomics to accurately infer dynamic networks. We apply Epoch to identify the dynamic networks underpinning directed differentiation of mouse embryonic stem cells (ESCs) guided by multiple signaling pathways, and we demonstrate that modulating these pathways drives topological changes that bias cell fate potential. We also find that Peg3 rewires the pluripotency network to favor mesoderm specification. By integrating signaling pathways with GRNs, we trace how Wnt activation and PI3K suppression govern mesoderm and endoderm specification, respectively. Finally, we identify regulatory circuits of patterning and axis formation that distinguish in vitro and in vivo mesoderm specification.

P095 Anti-inflammatory effect of high acetate concentration on organoid-derived epithelial monolayer from patients with Ulcerative Colitis

Abstract Background Short-chain fatty acids (SCFA), such as acetate and butyrate, have gained increasing interest for their potential to suppress IBD inflammation1,4. Most data have been generated for butyrate, showing beneficial effects on the gut microbiome composition, intestinal barrier function and immune system1. The effects of acetate are less known, despite its lower toxicity on epithelial cells and its ability to support growth of butyrate-producing bacteria by metabolic cross-feeding2. Furthermore, the probiotic effect of the biotherapeutic yeast Saccharomyces cerevisiae var. boulardii might be linked to its high acetic acid production3. Here, we studied the effect of acetate on inflammatory markers and barrier integrity using an organoid-derived epithelial monolayer system obtained from Ulcerative Colitis (UC) patients. Methods Confluent Transwell® organoid-derived monolayers evaluated by Transepithelial electrical resistance (TEER) of 8 UC patients were basolaterally stimulated with control medium (CTRL) or with an inflammatory mix (INFL) [100 ng/ml TNF-α, 20 ng/ml IL-1β, 1 µg/ml Flagellin] (Fig. 1). After 24h, the monolayer was stimulated apically with control medium or with high acetate (HA) [100mM] and TEER was measured at 0, 24, and 48h post-stimulation. After 48h, RNA extraction of the epithelial cell monolayers and qPCR for selected target genes (IL8, TNFα, CLDN1, CLDN2, HIF1A, ZO1, OCLDN, MUC2 and MKI67) was performed. Cytokine measurement was performed using Mesoscale Discovery (Proinflammatory panel 1) on apical and basolateral media. Data were analyzed using GraphPad Prism 9 (D’Agostino &amp; Pearson test for normality followed by Wilcoxon tests). Results Transcriptomic analysis of the monolayer cells showed a decrease in expression of IL8 (p=0.03), TNFα (p=0.08) and increase of HIF1A (p=0.03) upon HA administration in inflamed conditions (Fig. 2) whereas the proliferation marker MKI-67 was upregulated (p=0,03). Moreover, these findings were supported by significant decreases in pro-inflammatory cytokines measured apically after HA stimulation (Fig. 3A) such as IFNγ, TNFα, IL6, IL8 and IL1β (all p=&amp;lt;0.01). Basolaterally, IL8 (p=0.02) and IL13 (p=&amp;lt;0.01) increased significantly upon HA stimulation (Fig.3B). Administration of HA was associated with a numerical though non-significant increase in TEER-values (p=0.15) (Fig. 4) suggesting a potential for improved barrier integrity. Conclusion In this UC patient-derived epithelial cell culture model, high acetate administration positively affected barrier integrity gene expression and TEER-values and suppressed expression and production of pro-inflammatory cytokines.

Identification of microRNAs involved in sucrose accumulation in sugarcane (Saccharum species hybrid)

The genetic improvement efforts in sugarcane are largely centered around increasing its sucrose accumulation potential. Sugarcane genotypes show variable regulation of genes involved in synthesis, accumulation, transport, storage and subsequent retention of sucrose. MicroRNAs (miRNAs), which are a class of small non-coding RNAs play a very important role in post-transcriptional regulation of genes. Very little has been reported on the miRNA-mediated regulation of sucrose accumulation in sugarcane. In this study, two sugarcane genotypes contrasting for sucrose content along with two extreme bulks from their F1 population were used to identify differentially expressed miRNAs which might play a regulatory role in sucrose accumulation. A total of 161 miRNAs were identified, of them, 23 were significantly differential for contrasting sucrose content. The target genes of the identified miRNAs were predicted and some of them were validated using transcriptome library-based gene expression levels. Nineteen differential miRNAs and six selected target genes were validated using quantitative PCR. The study revealed that miRNA might be involved in sucrose accumulation in sugarcane, not by directly targeting the sucrose metabolism genes but by targeted regulation of transcription factors (PCF8, GRFs, ARF, etc.), and genes involved in carbohydrate metabolism, vegetative to mature phase transition, stress response, ion transport, growth and developmental pathways in sugarcane like RAV6, calcium-dependent protein kinase, HSP20, diacylglycerol kinase, squamosa promoter binding protein, etc. This study identifies miRNA candidates involved in sucrose accumulation and enriches the small RNA inventory of sugarcane.

Contamination of hay and haylage with enteric bacteria and selected antibiotic resistance genes following fertilization with dairy manure or biosolids.

The present study evaluated if enteric bacteria or antibiotic resistance genes carried in fecal amendments contaminate the hay at harvest, representing a potential route of exposure to ruminants that consume the hay. In the field experiments, dairy manure was applied to a hay field for three successive growing seasons, and biosolids were applied to a hay field for one growing season. Various enteric bacteria in the amendments were enumerated by viable plate count, and selected gene targets were quantified by qPCR. Key findings include the following: at harvest, hay receiving dairy manure or biosolids did not carry more viable enteric bacteria than hay from unamended control plots. The fermentation of hay did not result in a detectable increase in viable enteric bacteria. The application of dairy manure or biosolids resulted in a few gene targets being more abundant in hay during the first harvest. Fermentation of hay resulted in an increase in the abundance of gene targets, but this occurred with hay from both the amended and control plots. Overall, the application of fecal amendments resulted in an increase in the abundance of some gene targets associated with antibiotic resistance in the first cut hay.

A Versatile Clustered Regularly Interspaced Palindromic Repeats Toolbox to Study Neurological CaV3.2 Channelopathies by Promoter-Mediated Transcription Control.

Precise genome editing in combination with viral delivery systems provides a valuable tool for neuroscience research. Traditionally, the role of genes in neuronal circuits has been addressed by overexpression or knock-out/knock-down systems. However, those techniques do not manipulate the endogenous loci and therefore have limitations. Those constraints include that many genes exhibit extensive alternative splicing, which can be regulated by neuronal activity. This complexity cannot be easily reproduced by overexpression of one protein variant. The CRISPR activation and interference/inhibition systems (CRISPRa/i) directed to promoter sequences can modulate the expression of selected target genes in a highly specific manner. This strategy could be particularly useful for the overexpression of large proteins and for alternatively spliced genes, e.g., for studying large ion channels known to be affected in ion channelopathies in a variety of neurological diseases. Here, we demonstrate the feasibility of a newly developed CRISPRa/i toolbox to manipulate the promoter activity of the Cacna1h gene. Impaired, function of the low-voltage-activated T-Type calcium channel CaV3.2 is involved in genetic/mutational as well as acquired/transcriptional channelopathies that emerge with epileptic seizures. We show CRISPR-induced activation and inhibition of the Cacna1h locus in NS20Y cells and primary cortical neurons, as well as activation in mouse organotypic slice cultures. In future applications, the system offers the intriguing perspective to study functional effects of gain-of-function or loss-of-function variations in the Cacna1h gene in more detail. A better understanding of CaV3.2 channelopathies might result in a major advancement in the pharmacotherapy of CaV3.2 channelopathy diseases.

PSMA-specific degradable dextran for multiplexed immunotargeted siRNA therapeutics against prostate cancer.

Small interfering RNA (siRNA) is ideal for gene silencing through a sequence-specific RNA interference process. The redundancy and complexity of molecular pathways in cancer create a need for multiplexed targeting that can be achieved with multiplexed siRNA delivery. Here, we delivered multiplexed siRNA with a PSMA-targeted biocompatible dextran nanocarrier to downregulate CD46 and PD-L1 in PSMA expressing prostate cancer cells. The selected gene targets, PD-L1 and CD46, play important roles in the escape of cancer cells from immune surveillance. PSMA, abundantly expressed by prostate cancer cells, allowed the prostate cancer-specific delivery of the nanocarrier. The nanocarrier was modified with acid cleavable acetal bonds for a rapid release of siRNA. Cell imaging and flow cytometry studies confirmed the PSMA-specific delivery of CD46 and PD-L1 siRNA to high PSMA expressing PC-3 PIP cells. Immunoblot, qRT-PCR and flow cytometry methods confirmed the downregulation of CD46 and PD-L1 following treatment with multiplexed siRNA.

Screening for genes involved in cellulase regulation by expression under the control of a novel constitutive promoter in Trichoderma reesei

• We characterized the upstream region of cfe1 as promising promoter without upstream open reading frames (uORFs) or introns. • The cfe1 promoter enabled up to 400 fold overexpression. • We analyzed transcriptomic data with support from machine learning to select target genes. • We report three genes affecting cellulase regulation. The filamentous ascomycete Trichoderma reesei is a biotechnological workhorse used in the production of homologous and heterologous proteins for diverse applications including biofuel production, textile finishing and food additives. This fungus uses a complex adaptation machinery to regulate its cellulases in response to environmental conditions. Detailed understanding of this regulation allows for improvement of enzyme production using the strong enzyme gene promotors. Here, we selected six genes with characteristic transcript levels associated with cellulase production to be tested for their enzyme regulatory function. Machine learning for inference of a gene regulatory network (GRN) was applied to support the association of these genes with cellulase induction. Additionally, we screened available transcriptomic data for genes with strong constitutive transcript levels and selected the promoter of the gene cfe1 , whose transcript levels were above those of tef1 and cDNA1 and near those of cbh1 , for gene overexpression testing. Using this promoter, we explored the relevance to cellulose degradation efficiency of three transporters, two ferric reductases and one gene of unknown function, which were overexpressed in T. reesei grown on cellulose. This promoter enabled up to 400 fold overexpression and yielded transcript levels above those of tef1 or cDNA1. We provide evidence of effects of a ferric reductase, an ammonium permease and a gene of unknown function on the regulatory machinery of cellulase expression in T. reesei . In summary we identified the cfe1 promoter, a novel constitutive promoter with promising efficiency, as well as three genes relevant to cellulase regulation in T. reesei .

1020 – TARGETED PROTEIN DEGRADATION DEFINES DIRECT TARGETS AND MECHANISMS OF CONTROL BY RUNX1/ETO

Loss-of-function germline mutations in the master transcription factor, RUNX1, cause familial platelet disorder with a predisposition to malignancy, while somatic mutations and chromosomal translocations targeting RUNX1 are associated with MDS and AML. Fundamentally, RUNX1 is a sequence-specific transcription factor that regulates the expression of select target genes critical for normal hematopoiesis. Thus, the ability to precisely define downstream targets of RUNX1 activity is critical to our understanding of how RUNX1 regulates hematopoietic cell fate decisions and how disruption of RUNX1 activity predisposes to cancer. However, defining direct transcriptional targets remains a challenge, because while gene expression networks are highly dynamic, our approaches to eliminate transcription factor function through genetic targeting are incredibly slow. These approaches allow the accumulation of secondary/compensatory changes in gene expression that make it difficult to distinguish direct from indirect transcriptional targets, creating a technical hurdle for target identification that must be overcome. Here, we have engineered leukemia cells to integrate an FKBP12-based degron tag into the endogenous RUNX1 or RUNX1-ETO (RE) fusion locus. This allowed the degradation of the RUNX1 or RE fusion proteins within two hours of treatment with the small molecule, dTAG. By combining rapid protein degradation with nascent transcript profiling, we defined direct RUNX1/RE target genes. Further characterization of changes in RUNX1 or RE bound enhancers following dTAG treatment allowed us to identify enhancers regulated by RUNX1 to elicit these rapid changes in gene expression. Integration of a 3xFLAG tag into the endogenous RUNX1 or RE locus allowed the identification of endogenous co-regulatory complexes recruited to RUNX1 and RE-regulated enhancers. Thus, the combination of rapid transcription factor degradation, nascent transcript profiling and mass spectrometry, is allowing us to define detailed mechanisms of transcriptional regulation at RUNX1-regulated enhancer elements. Loss-of-function germline mutations in the master transcription factor, RUNX1, cause familial platelet disorder with a predisposition to malignancy, while somatic mutations and chromosomal translocations targeting RUNX1 are associated with MDS and AML. Fundamentally, RUNX1 is a sequence-specific transcription factor that regulates the expression of select target genes critical for normal hematopoiesis. Thus, the ability to precisely define downstream targets of RUNX1 activity is critical to our understanding of how RUNX1 regulates hematopoietic cell fate decisions and how disruption of RUNX1 activity predisposes to cancer. However, defining direct transcriptional targets remains a challenge, because while gene expression networks are highly dynamic, our approaches to eliminate transcription factor function through genetic targeting are incredibly slow. These approaches allow the accumulation of secondary/compensatory changes in gene expression that make it difficult to distinguish direct from indirect transcriptional targets, creating a technical hurdle for target identification that must be overcome. Here, we have engineered leukemia cells to integrate an FKBP12-based degron tag into the endogenous RUNX1 or RUNX1-ETO (RE) fusion locus. This allowed the degradation of the RUNX1 or RE fusion proteins within two hours of treatment with the small molecule, dTAG. By combining rapid protein degradation with nascent transcript profiling, we defined direct RUNX1/RE target genes. Further characterization of changes in RUNX1 or RE bound enhancers following dTAG treatment allowed us to identify enhancers regulated by RUNX1 to elicit these rapid changes in gene expression. Integration of a 3xFLAG tag into the endogenous RUNX1 or RE locus allowed the identification of endogenous co-regulatory complexes recruited to RUNX1 and RE-regulated enhancers. Thus, the combination of rapid transcription factor degradation, nascent transcript profiling and mass spectrometry, is allowing us to define detailed mechanisms of transcriptional regulation at RUNX1-regulated enhancer elements.

First Evidence of Feeding-Induced RNAi in Banana Weevil via Exogenous Application of dsRNA.

Simple SummaryRNA interference (RNAi), a conserved mechanism in eukaryotic organisms, is initiated by the presence of double-stranded RNA (dsRNA) in the cells, regulating the expression of specific genes by degradation of their mRNA, and it is known as post-transcriptional gene-silencing (PTGS). RNAi can be employed to develop species-specific biopesticides where dsRNA is delivered to a pest insect via the oral route. Banana weevil (Cosmopolites sordidus) is the most devastating pest of banana and plantain worldwide, yet current control measures are neither effective, sustainable, nor environmentally sound. This study reports an artificial diet-based multi-well plate method and the first findings on the potential application of biotechnology in the control of the banana weevil using dietary RNAi, which would arguably provide the most sustainable and practical method for dsRNA delivery. A selection of target genes with effective RNAi leading to high and rapid mortality is presented. These results indicate the potential of RNAi-mediated management to suppress C. sordidus efficiently in the banana crop.Banana weevil (Cosmopolites sordidus) is the most devastating pest of banana and plantain worldwide, yet current control measures are neither effective, sustainable, nor environmentally sound, and no resistant farmer-preferred cultivars are known to date. In this paper, we examined the ability to induce RNA interference (RNAi) in the banana weevil via feeding. We first developed an agar- and banana corm (rhizome) flour-based artificial diet in a multi-well plate setup that allowed the banana weevils to complete their life cycle from egg through the larval instars to the pupal stage in an average period of 53 days. Adults emerged about 20 days later. The artificial diet allowed the tunneling and burrowing habits of the larvae and successful metamorphosis up to adult eclosion. Adding dsRNA for laccase2 to the artificial diet resulted in albino phenotypes, confirming gene-silencing. Finally, C. sordidus was fed with dsRNA against a selection of essential target genes: snf7, rps13, mad1, vha-a, vha-d, and lgl for a period of 45 days. 100% mortality within 9–16 days was realized with dssnf7, dsrps13, and dsmad1 at 200 ng/mL artificial diet, and this corresponded to a strong reduction in gene expression. Feeding the dsRNA targeting the two vha genes resulted in 100% mortality after about 3–4 weeks, while treatment with dslgl resulted in no mortality above the dsgfp-control and the water-control. Our results have implications for the development of RNAi approaches for managing important crop pests, in that banana weevils can be controlled based on the silencing of essential target genes as snf7, rps13, and mad1. They also highlight the need for research into the development of RNAi for banana protection, eventually the engineering of host-induced gene-silencing (HIGS) cultivars, given the high RNAi efficacy and its species-specific mode of action, adding the RNAi approach to the armory of integrated pest management (IPM).

Effects of Qinghuang Powder on Acute Myeloid Leukemia Based on Network Pharmacology, Molecular Docking, and In Vitro Experiments.

Qinghuang powder (QHP) is a traditional Chinese herbal medicine. This is a unique formula that is frequently used to treat malignant hematological diseases such as acute myeloid leukemia (AML) in modern clinical practice. An approach of network pharmacology and experimental validation were applied to investigate the pharmacological mechanisms of QHP in AML treatment. First, public databases for target genes known to be associated with AML are searched and compared to the target genes of the active compounds in QHP. Second, AML-associated genes and QHP target genes are compared to identify overlapping enriched genes, and these were used to predict selected target genes that may be implicated in the effects of QHP on AML. Additionally, we conducted functional enrichment analyses, such as gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The significantly enriched pathway associated with potential target proteins was the PI3K-Akt signaling pathway, suggesting that these potential target proteins and pathways may mediate the beneficial biological effects of QHP on AML. All these following genes were found to occur in the compounds-target-pathway networks: AKT1, MAPK1, MAPK3, PIK3CG, CASP3, CASP9, TNF, TGFB1, MAPK8, and TP53. Then, based on the molecular docking studies, it was suggested that the active compound isovitexin can fit into the binding pockets of the top candidate QHP-AML target proteins (PIK3CG). Subsequently, based on the prediction by network pharmacology analysis, both in vitro AML cells and western blot experiments were performed to validate the curative role of QHP. QHP exerted its antitumor activity on AML in vitro, as it inhibits cells proliferation, reduced the expression of Bcl-2 protein, and downregulated the PI3K-Akt signaling pathway. In conclusion, these results revealed that QHP could treat AML via a “multicomponent, multitarget, multipathway” regulatory network. Furthermore, our study also demonstrated that the combination of network pharmacology with the experimental study is effective in discovering and identifying QHP in the treatment of AML and its underlying pharmacological mechanisms.

Clozapine modulation of zebrafish swimming behavior and gene expression as a case study to investigate effects of atypical drugs on aquatic organisms

Mental illnesses affect more than 150 million people in Europe and lead to an increasing consumption of neuroactive drugs during the last twenty years. The antipsychotic compound, clozapine, is one of the most used psychotropic drugs worldwide, with potentially negative consequences for the aquatic environment. Hence, the objectives of the study presented here were the quantification of clozapine induced changes in swimming behavior of exposed Danio rerio embryos and the elucidation of the molecular effects on the serotonergic and dopaminergic systems. Yolk-sac larvae were exposed to different concentrations (0.2 mg/L, 0.4 mg/L, 0.8 mg/L, 1.6 mg/L, 3.2 mg/L and 6.4 mg/L) of clozapine for 116 h post-fertilization, and changes in the swimming behavior of the larvae were assessed. Further, quantitative real-time PCR was performed to analyze the expression of selected genes. The qualitative evaluation of changes in the swimming behavior of D. rerio larvae revealed a significant decrease of the average swimming distance and velocity in the light-dark transition test, with more than a 36% reduction at the highest exposure concentration of 6.4 mg/L. Furthermore, the total larval body length was reduced at the highest concentration. An in-depth analysis based on expression of selected target genes of the serotonin (slc6a4a) and dopamine (drd2a) system showed an upregulation at a concentration of 1.6 mg/L and above. In addition, a lower increase in expression was detected for biomarkers of general stress (adra1a and cyp1a2). Our data show that exposure to clozapine during development inhibits swimming activity of zebrafish larvae, which could, in part, be due to disruption of the serotonin- and dopamine system.

Shotgun metagenome sequencing of a Sudanese toombak snuff tobacco: genetic attributes of a high tobacco-specific nitrosamine containing smokeless tobacco product.

The most alarming aspect of the Sudanese toombak smokeless tobacco is that it contains high levels of highly toxic tobacco-specific nitrosamines (TSNAs). Understanding the microbiology of toombak is of relevance because TSNAs are an indirect result of microbial-mediated nitrate reductions. We conducted shotgun metagenomic sequencing on a toombak product for which relevant features are presented here. The microbiota was composed of over 99% Bacteria. The most abundant taxa included Actinobacteria, specifically the genera Enteractinococcus and Corynebacterium, while Firmicutes were represented by the family Bacillaceae and the genus Staphylococcus. Selected gene targets were nitrate reduction and transport, antimicrobial resistance, and other genetic transference mechanisms. Canonical nitrate reduction and transport genes (i.e. nar) were found for Enteractinococcus and Corynebacterium while various species of Staphylococcus exhibited a notable number of antimicrobial resistance and genetic transference genes. The nitrate reduction activity of the microbiota in toombak is suspected to be a contributing factor to its high levels of TSNAs. Additionally, the presence of antimicrobial resistance and transference genes could contribute to deleterious effects on oral and gastrointestinal health of the end user. Overall, the high toxicity and increased incidences of cancer and oral disease of toombak users warrants further investigation into the microbiology of toombak.

The palliative effects of folic acid on retinal microvessels in diabetic retinopathy via regulating the metabolism of DNA methylation and hydroxymethylation

ABSTRACT Diabetic retinopathy (DR) is one of the severe microvascular complications of diabetes. The protective effects of FA on retinal vascular endothelial cells against high glucose levels involve in multiple aspects in DR; however, the underlying mechanism is not fully elucidated. In present study, we investigated the transcriptome as well as genome-wide DNA methylation and hydroxymethylation signature in human retinal microvascular endothelial ACBRI 181 cells cultured within high glucose (HG) medium supplemented with or without FA by RNA-seq, MeDIP-seq, and hMeDIP-seq. Total 3308 differential expressed genes (DEGs) were involved in multiple biological processes and molecular functions containing angiogenesis, inflammation, S-adenosyl methionine metabolism, and hypoxia response. Moreover, the global DNA methylation and hydroxymethylation in ACBRI 181 cells with FA treatment were both compromised compared to HG. Combined with transcriptome data, four subclusters of DEGs with hyper- or hypomethylated promoters were further verified. Unexpectedly, promoters of these 487 genes all displayed a pattern of increased DNA hydroxymethylation. Furthermore, hyperglycemia rat model was established and administered with FA. The DNA methylation and hydroxymethylation changes of selected target genes COL1A1, ITGA7, MMP-14, and VEGFB confirmed by MeDIP-qPCR were consistent with the results in human ACBRI 181 cells. Finally, the presence of activated DNMT1 and TET2 induced by FA was determined in ACBRI 181 cells and hyperglycemia rat. Taken together, this research provided a resource of expression and epigenetic profiles in retinal microvascular endothelial cell, emphasizing a pharmacological mechanism of FA on DNA methylation and hydroxymethylation regulation in retinal microvessel cells of DR.

Patulin-producing mold, toxicological, biosynthesis, and molecular detection of patulin

Mycotoxin is one of the food safety problems that raise concern because of its negative health impact and significant economic losses. Patulin is the most common mycotoxin found in fruits and its products like apple, orange, grape, pear, etc. There are 167 species reported to produce patulin; Penicillium expansum is the major patulin producer among those fungi species. Patulin is not classifiable as to its carcinogenicity to humans, but some studies showed the toxicities of patulin. Patulin has a broad spectrum of toxicity such as acute toxicity, sub-acute toxicity, genotoxicity, embryotoxicity and teratogenicity, carcinogenicity, and immunotoxicity. There are several ways to detect mycotoxins, such as HPLC, TLC, ELISA, and PCR. The popular way to detect mycotoxin molecularly is PCR because of its sensitivity and specificity. To detect patulin molecularly require proper selection of target gene. Therefore, the biosynthesis of patulin, its influencing gene, and its underlying factor needs to be understood.

Application of the RNA interference technique to Xenopus embryos: Specific reduction of the β-catenin gene products by short double-stranded RNA produced by recombinant human Dicer.

RNA interference (RNAi) is a technique for suppressing the function of specific genes and is widely used in many organisms, including yeast, nematodes, flies, plants, mice, and cultured mammalian cells. As of date, this technique has not been successfully applied to Xenopus laevis embryos. In this study, we applied RNAi to Xenopus embryos using β-catenin as a model gene. Injection of long double-stranded RNA (dsRNA) corresponding to the 3'-untranslated region of β-catenin mRNA into embryos induced embryonic lethality without any specific phenotype. However, injection of short dsRNA, generated from long dsRNA by treatment with recombinant human Dicer, into embryos resulted in decreased expression of endogenous β-catenin mRNA and protein, as well as decreased Wnt signaling activity in the embryos. The decrease in β-catenin mRNA and protein levels was observed only after mid-blastula transition. Embryos injected with short dsRNA showed a characteristic phenotype of enlarged anterior structures and loss of posterior structures. These phenotypes, as well as the increased expression of the anterior gene and decreased expression of the posterior gene, suggest that RNAi against the β-catenin gene suppresses the "late Wnt signaling" involved in proper anterior-posterior patterning of Xenopus embryos. The effect of RNAi on Xenopus embryos was also found to be sensitive to temperature. These results strongly suggest that the RNAi technique can be applied to Xenopus embryos using short dsRNAs, appropriate temperature control, and proper selection of target genes.

Clinical Value and Potential Mechanism of miRNA-33a-5p in Lung Squamous Cell Carcinoma.

This study is aimed at thoroughly exploring the expression status, clinical significance, and underlying molecular mechanism of miRNA-33a-5p in lung squamous cell carcinoma (LUSC). Here, we detected miRNA-33a-5p in 20 samples from patients with LUSCs and 20 matching non-LUSC specimens by in-house quantitative real-time PCR (RT-qPCR). Relationship between miRNA-33a-5p expression and clinicopathological traits was investigated from materials derived from miRNA sequencing and miRNA microarrays. A pool standard mean difference (SMD) and summary receiver operating characteristic curves (SROC) were calculated to evaluate the integrated expression value of miRNA-33a-5p in LUSC. Twelve online platforms were applied to select potential target genes of miRNA-33a-5p. The differentially expressed genes (DEGs) of LUSC and the candidate target genes of miRNA-33a-5p were overlapped to acquire a set of specific genes for further analyses of the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and protein–protein interaction (PPI) network. miRNA-33a-5p overexpressed in LUSC was supported by 706 LUSC and 261 non-LUSC samples gathering from RT-qPCR, miRNA-seq, and public miRNA microarrays. The pooled SMD was 0.56 (95% CI: -0.01-1.05), and the area under the curve (AUC) of the SROC was 0.78 (95% CI: 0.74-0.82). A total of 240 genes were identified as potential target genes of miRNA-33a-5p for functional enrichment analyses; the results suggested that these target genes may participate in several vital biological processes that promote the proliferation and progression of LUSC. miRNA-33a-5p may play an essential role in the occurrence and development of LUSC by targeting hub genes (ETS1, EDNRB, CYR61, and LRRK2) derived from the PPI network. In summary, our results indicated that miRNA-33a-5p may contribute as a prospective therapeutic target in LUSC.