Gene Expression Studies Research Articles

Identification and characterization of zinc importers in Corynebacterium diphtheriae.

Corynebacterium diphtheriae is the causative agent of diphtheria, a severe respiratory disease in humans. C. diphtheriae colonizes the human upper respiratory tract, where it acquires zinc, an essential metal required for survival in the host. While the mechanisms for zinc transport by C. diphtheriae are not well characterized, four putative zinc ABC-type transporter loci were recently identified in strain 1737: iutABCD/E (iut), znuACB (znu), nikABCD1 (nik1), and nikABCD2 (nik2). A mutant deleted for all four loci (Δ4) exhibited similar growth to that of the wild-type strain in a zinc-limited medium, suggesting there are additional zinc transporters. Two additional gene loci predicted to be associated with metal import, mntABCD (mnt) and sidAB (sid), were deleted in the Δ4 mutant to construct a new mutant designated Δ6. The C. diphtheriae Δ6 mutant exhibited significantly reduced growth under zinc limitation relative to the wild type, suggesting a deficiency in zinc acquisition. Strains retaining the iut, znu, mnt, or sid loci grew to near-wild-type levels in the absence of the other five loci, indicating that each of these transporters may be involved in zinc uptake. Plasmid complementation with cloned iut, znu, mnt, or nik1 loci also enhanced the growth of the Δ6 mutant. Quantification of intracellular zinc content by inductively coupled plasma mass spectrometry was consistent with reduced zinc uptake by Δ6 relative to the wild type and further supports a zinc uptake function for the transporters encoded by iut, znu, and mnt. This study demonstrates that C. diphtheriae zinc transport is complex and involves multiple zinc uptake systems.IMPORTANCEZinc is a critical nutrient for all forms of life, including human bacterial pathogens. Thus, the tools that bacteria use to acquire zinc from host sources are crucial for pathogenesis. While potential candidates for zinc importers have been identified in Corynebacterium diphtheriae from gene expression studies, to date, no study has clearly demonstrated this function for any of the putative transporters. We show that C. diphtheriae encodes at least six loci associated with zinc import, underscoring the extent of redundancy for zinc acquisition. Furthermore, we provide evidence that a previously studied manganese-regulated importer can also function in zinc import. This study builds upon our knowledge of bacterial zinc transport mechanisms and identifies potential targets for future diphtheria vaccine candidates.

Differential Mitochondrial Genome Expression of Four Hylid Frog Species under Low-Temperature Stress and Its Relationship with Amphibian Temperature Adaptation.

Extreme weather poses huge challenges for animals that must adapt to wide variations in environmental temperature and, in many cases, it can lead to the local extirpation of populations or even the extinction of an entire species. Previous studies have found that one element of amphibian adaptation to environmental stress involves changes in mitochondrial gene expression at low temperatures. However, to date, comparative studies of gene expression in organisms living at extreme temperatures have focused mainly on nuclear genes. This study sequenced the complete mitochondrial genomes of five Asian hylid frog species: Dryophytes japonicus, D. immaculata, Hyla annectans, H. chinensis and H. zhaopingensis. It compared the phylogenetic relationships within the Hylidae family and explored the association between mitochondrial gene expression and evolutionary adaptations to cold stress. The present results showed that in D. immaculata, transcript levels of 12 out of 13 mitochondria genes were significantly reduced under cold exposure (p < 0.05); hence, we put forward the conjecture that D. immaculata adapts by entering a hibernation state at low temperature. In H. annectans, the transcripts of 10 genes (ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COX1, COX2 and ATP8) were significantly reduced in response to cold exposure, and five mitochondrial genes in H. chinensis (ND1, ND2, ND3, ND4L and ATP6) also showed significantly reduced expression and transcript levels under cold conditions. By contrast, transcript levels of ND2 and ATP6 in H. zhaopingensis were significantly increased at low temperatures, possibly related to the narrow distribution of this species primarily at low latitudes. Indeed, H. zhaopingensis has little ability to adapt to low temperature (4 °C), or maybe to enter into hibernation, and it shows metabolic disorder in the cold. The present study demonstrates that the regulatory trend of mitochondrial gene expression in amphibians is correlated with their ability to adapt to variable climates in extreme environments. These results can predict which species are more likely to undergo extirpation or extinction with climate change and, thereby, provide new ideas for the study of species extinction in highly variable winter climates.

MousiPLIER: A Mouse Pathway-Level Information Extractor Model.

High-throughput gene expression profiling measures individual gene expression across conditions. However, genes are regulated in complex networks, not as individual entities, limiting the interpretability of gene expression data. Machine learning models that incorporate prior biological knowledge are a powerful tool to extract meaningful biology from gene expression data. Pathway-level information extractor (PLIER) is an unsupervised machine learning method that defines biological pathways by leveraging the vast amount of published transcriptomic data. PLIER converts gene expression data into known pathway gene sets, termed latent variables (LVs), to substantially reduce data dimensionality and improve interpretability. In the current study, we trained the first mouse PLIER model on 190,111 mouse brain RNA-sequencing samples, the greatest amount of training data ever used by PLIER. We then validated the mousiPLIER approach in a study of microglia and astrocyte gene expression across mouse brain aging. mousiPLIER identified biological pathways that are significantly associated with aging, including one latent variable (LV41) corresponding to striatal signal. To gain further insight into the genes contained in LV41, we performed k-means clustering on the training data to identify studies that respond strongly to LV41. We found that the variable was relevant to striatum and aging across the scientific literature. Finally, we built a Web server (http://mousiplier.greenelab.com/) for users to easily explore the learned latent variables. Taken together, this study defines mousiPLIER as a method to uncover meaningful biological processes in mouse brain transcriptomic studies.

Cyclic di-peptide Cyclo (L-Phe-L-Pro) mitigates the quorum-sensing mediated virulence in Salmonella typhi and biofilm formation in poultry and plastic system

Salmonella typhi is a predominant foodborne pathogen, posing a significant threat to food quality and safety through typhoid outbreaks. Quorum sensing (QS) and its pathways are crucial for typhoid antigen regulation and S. typhi-mediated spoilage and pathogenesis. Therefore, this study intends to identify secondary metabolites from extracts of 15 bacterial isolates sourced from wild Mangifera indica (mango) brine pickles as novel anti-QS agents against S. typhi. Cell-free supernatants (CFS) of 15 bacterial isolates from fermented mango brine pickles were assessed and identified for anti-QS positive metabolites using GC-MS. Anti-QS metabolite was tested on early and mature biofilms, as well as violacein inhibition, EPS, cellular adherence, motility, antibiotics, and immune resistance of S. typhi. Gene expression and in-silico studies were also conducted to validate the compound's efficacy. The results depicted that among the 15 isolates, Bacillus subtilis P89 exhibited potent anti-QS properties. Gas chromatography-mass spectrometry (GC-MS) analysis identified twelve potential quorum-sensing inhibiting compounds, with Cyclo (L-Phenylalanine-L-Proline) (CPP) demonstrating the maximum anti-QS activity against S. typhi. CPP disrupted the pathogen's early and mature biofilms, reducing exo-polymeric substance production, cell hydrophobicity, and motility at sub-lethal concentrations. It also enhanced the immune response against S. typhi and improved traditional efficacy. Real-time qPCR analyses revealed CPP's modulation of essential quorum-sensing regulatory genes. Molecular docking studies indicated competitive binding of CPP with the SdiA receptor. CPP inhibited S. typhi biofilm formation on chicken skin, plastics, and eggshells. To our knowledge, this study is the first to highlight CPP's potential in food and clinical environments to combat S. typhi via quorum sensing-based mechanisms.

#2347 Multi-omics analysis identifies traditional Chinese medicine targets for chronic kidney disease

Abstract Background and Aims As a major public health problem, chronic kidney disease (CKD) has substantial comorbidities and disease burden. It is necessary to discover new therapeutic targets considering its irreversibly. Traditional Chinese medicine (TCM) has become indispensable for the integrated treatment of CKD. The current study aims to explore potential new TCM targets in high-throughput experiment- and reference-guided database (HERB) that are related to renal phenotypes utilizing multi-omics. Method We conducted comprehensive Mendelian Randomization and Bayesian colocalization on more than 2,000 HERB target integrating genomes and transcriptomes, using summary statistics of genome-wide association study and cis gene expression quantitative trait loci. The results were validated through differential gene expression in public database, previous literature reports and clinical kidney biopsy specimens. Results In all, we discovered 34 targets associated with renal phenotypes among the 2,421 HERB TCM targets in the data mining part. Among them, most of the targets can be validated by differential gene expression analysis in GEO and Nephroseq, and have been reported by basic research, thus confirming the reliability of this method. In addition, PRKCI, KNG1, PKN3, GGT7, GBA, APEH and GSTA2 are expected to be potential new therapeutic targets for CKD. Immunohistochemical staining using clinical kidney biopsy specimens of PKN3 and KNG1, two targets with the highest evidence level, further confirm the results. Conclusion Integration of multi-omics can extensively and efficiently screen potential drug targets related to CKD. The new targets discovered in the current study, especially PKN3 and KNG1, need to be further validated in basic research.

#1331 PCSK9 inhibitor addition during hypothermic perfusion protects kidney from ischemic damage in a rat model of donation after circulatory death

Abstract Background and Aims PCSK9 inhibitors (PCSK9i) are a new class of cholesterol-lowering drugs that is gaining market share in treating hypercholesterolemia because of their effectiveness in lowering LDL cholesterol and cardiovascular risk, even in patients with kidney disease. In addition to the well-known effect of inhibiting the LDL receptor catabolism, new pharmacological properties are being discovered. Experimental cellular and animal models have showed that PCSK9i reduce apoptosis, pyroptosis and in acute myocardial infarction decrease the infarct area, suggesting their protective role in tissue ischemia. However, so far PCSK9i effect on renal ischemic damage has not yet been studied. The aim of this study was to evaluate the effects of PEP 2-8 (PCSK9i) addition during hypothermic perfusion (HP) on ischemic kidney damage (ID) in a rat model of donation after circulatory death (DCD). Method n. 15 Sprague-Dawley rats were used as DCD donors. Briefly after a midline laparotomy, the left and right retroperitoneal renal areas were exposed, and lumbar arteries were isolated and sectioned; subsequently, the renal arteries and veins were isolated. After 30 min of warm ischemia, induced by aorta clamping, left and right nephrectomies were completed with preservation of the renal hilums. Then the kidneys were perfused with Perf-Gen Solution (100 ml) (Institut Georges Lopez, Lissieu, France) or Perf-Gen solution (100 ml) supplemented with 3ug (10ug/kg) of PEP 2-8. Continuous HP was performed for 6 h at 4 °C. DCD right and left kidneys of each rat were randomized to the following experimental groups: CTRL group: n = 15 DCD rat kidneys perfused with Perf-Gen PEP group: n = 15 DCD rat kidneys perfused with Pef-Gen supplemented with PEP 2-8. Effluent fluid was collected at the beginning (T0) and at the end (T6h) of HP. Glucose and potassium levels were measured in the effluent fluid at T0 and T6h. At T6h the kidneys were splitted into three aliquots; one was fixed in 10% formalin for morphological studies, and the other two were stored in trizol for gene expression studies and at −80 °C for biochemical assays respectively. Tubular ischemic damage (TID) was evaluated at T6 by scoring all tubules observed in at least 10 non-consecutive high-powered fields, as described by Paller (Paller Score). NOX- 4 gene expression was studied by RT-PCR assay. Tissue LDH and ATP levels were measured at T6h. Results PEP 2-8 treated kidneys showed a lower TID score compared to CTRL (PEP 107.0 IQR 90.75-116.0 vs CTRL 134.5 IQR 131.3-155.3 p &amp;lt; 0.005) (Fig. 1). The percentage of tubules with score zero (normal tubules) was higher in PEP than CTRL (PEP 29.0% range % 24.2-39.5 vs CTRL 9.5% range % 5.5-12.0, p &amp;lt; 0.001). The percentage of tubules with necrosis was lower in PEP than CTRL (PEP 5.5% range % 3.2-10.5 vs CTRL 12.5% range % 9.0-15.7, p &amp;lt; 0.005). Expression of NOX-4 gene, known inducer of apoptosis, was lower in PEP compared to CTRL (p &amp;lt; 0.05) Tissue ATP content was higher in PEP than CTRL (PEP 0.031 ± 0.01 vs CTRL 0.025 ± 0.006 p &amp;lt; 0.05). In tissue samples at T6 LDH was lower in PEP than CTRL (PEP 554.8 ± 164.7 vs CTRL 742.4 ± 256.7 p &amp;lt; 0.05) conversely the potassium release in effluent was significant only in CTRL (T6 vs T0 p &amp;lt; 0.01), according to TID score results. During HP, PEP 2-8 treated kidneys released more glucose in the effluent than CTRL kidneys (PEP 35.4 IQR 12.3-82.9 vs CTRL 12.3 IQR 7.1-70.5 p &amp;lt; 0.01). Conclusion Our preliminary results show that PEP 2-8 added to Perf-Gen solution during HP protects kidneys from ID, preserving energy metabolism in rat DCD model. To the best of our knowledge, this is the first evidence of protective role of PCSK9 inhibition in pre-transplant renal conditioning and potentially it could be used in conditioning other organs (liver, lung). However, further studies are necessary to understand the mechanisms underlying PCSK9i effect on energy metabolism and neoglucogenesis.

#1446 PCSK9 inhibitor addition during hypothermic perfusion protects kidney from ischemic damage in a rat model of donation after circulatory death

Abstract Background and Aims PCSK9 inhibitors (PCSK9i) are a new class of cholesterol-lowering drugs that is gaining market share in treating hypercholesterolemia because of their effectiveness in lowering LDL cholesterol and cardiovascular risk, even in patients with kidney disease. In addition to the well-known effect of inhibiting the LDL receptor catabolism, new pharmacological properties are being discovered. Experimental cellular and animal models have showed that PCSK9i reduce apoptosis, pyroptosis and in acute myocardial infarction decrease the infarct area, suggesting their protective role in tissue ischemia. However, so far PCSK9i effect on renal ischemic damage has not yet been studied. The aim of this study was to evaluate the effects of PEP 2-8(PCSK9i) addition during hypothermic perfusion (HP) on ischemic kidney damage (ID) in a rat model of donation after circulatory death (DCD). Method n. 15 Sprague-Dawley rats were used as DCD donors. Briefly after a midline laparotomy, the left and right retroperitoneal renal areas were exposed, and lumbar arteries were isolated and sectioned; subsequently, the renal arteries and veins were isolated. After 30 min of warm ischemia, induced by aorta clamping, left and right nephrectomies were completed with preservation of the renal hilums. Then the kidneys were perfused with Perf-Gen Solution (100 ml) (Institut Georges Lopez, Lissieu, France) or Perf-Gen solution (100 ml) supplemented with 3ug (10ug/kg) of PEP 2-8. Continuous HP was performed for 6 h at 4 °C. DCD right and left kidneys of each rat were randomized to the following experimental groups: CTRL group: n = 15 DCD rat kidneys perfused with Perf-Gen PEP group: n = 15 DCD rat kidneys perfused with Pef-Gen supplemented with PEP 2-8. Effluent fluid was collected at the beginning (T0) and at the end (T6h) of HP. Glucose and potassium levels were measured in the effluent fluid at T0 and T6h. At T6h the kidneys were splitted into three aliquots; one was fixed in 10% formalin for morphological studies, and the other two were stored in trizol for gene expression studies and at −80 °C for biochemical assays respectively. Tubular ischemic damage (TID) was evaluated at T6 by scoring all tubules observed in at least 10 non-consecutive high-powered fields, as described by Paller (Paller Score). NOX- 4 gene expression was studied by RT-PCR assay. Tissue LDH and ATP levels were measured at T6h. Results PEP 2-8 treated kidneys showed a lower TID score compared to CTRL (PEP 107.0 IQR 90.75-116.0 vs CTRL 134.5 IQR 131.3-155.3 p &amp;lt; 0.005) (Fig. 1). The percentage of tubules with score zero (normal tubules) was higher in PEP than CTRL (PEP 29.0% range % 24.2-39.5 vs CTRL 9.5% range % 5.5-12.0, p &amp;lt; 0.001). The percentage of tubules with necrosis was lower in PEP than CTRL (PEP 5.5% range % 3.2-10.5 vs CTRL 12.5% range % 9.0-15.7, p &amp;lt; 0.005). Expression of NOX-4 gene, known inducer of apoptosis, was lower in PEP compared to CTRL (p &amp;lt; 0.05) Tissue ATP content was higher in PEP than CTRL (PEP 0.031 ± 0.01 vs CTRL 0.025 ± 0.006 p &amp;lt; 0.05). In tissue samples at T6 LDH was lower in PEP than CTRL (PEP 554.8 ± 164.7 vs CTRL 742.4 ± 256.7 p &amp;lt; 0.05) conversely the potassium release in effluent was significant only in CTRL (T6 vs T0 p &amp;lt; 0.01), according to TID score results. During HP, PEP 2-8 treated kidneys released more glucose in the effluent than CTRL kidneys (PEP 35.4 IQR 12.3-82.9 vs CTRL 12.3 IQR 7.1-70.5 p &amp;lt; 0.01). Conclusion Our preliminary results show that PEP 2-8 added to Perf-Gen solution during HP protects kidneys from ID, preserving energy metabolism in rat DCD model. To the best of our knowledge, this is the first evidence of protective role of PCSK9 inhibition in pre-transplant renal conditioning and potentially it could be used in conditioning other organs (liver, lung). However, further studies are necessary to understand the mechanisms underlying PCSK9i effect on energy metabolism and neoglucogenesis.

#2048 Exploring somatic mutation mechanisms in pre-cystic renal cells of autosomal dominant polycystic kidney disease patients

Abstract Background and Aims Genetic changes accumulate in our cells during a lifetime, at a rate that varies according to cell type and mutagen exposure [1]. Somatic mutations are the driving force of cancer and contribute to “second hit” disorders, such as Autosomal Dominant Polycystic Kidney Disease (ADPKD). ADPKD is caused by inherited mutations that disrupt one allele of PKD1 or PKD2 genes. Somatic loss of the second allele triggers tubule cell clonal expansion and the formation of renal cysts. Given the high number of cysts that form during a lifetime, somatic mutation rates may be abnormally high in the kidney of ADPKD patients. The identification of factors enhancing somatic mutation rates can lead to preventive strategies to limit cyst formation. Metabolic alterations, i.e. increased glutamine utilization and reduced urea cycle, have been shown to induce somatic mutations in cancer [2]. Loss of urea cycle increases pyrimidine synthesis, which unbalances the nucleotide pools and results in a distinct signature of single base substitutions (SBSs) [1]. Similar metabolic changes have been observed in ADPKD [3, 4]. Thus, we sought to test metabolism-driven somatic mutagenesis in pre-cystic kidneys and its contribution to second hit mutations in ADPKD. Method We clonally expanded normal kidney cells from human urine samples and performed a gene expression study by qPCR. We studied 31 clones from 4 ADPKD patients (age range: 25-45) with truncating mutations in PKD1 and normal kidney function, despite a clear cystic phenotype. ADPKD cells were compared to 55 clones from 5 healthy volunteers (age range: 24-53), and 32 clones from 6 patients with another cystic kidney disease, Von Hippel Lindau disease (VHL; age range: 29-56). A subset of clones (n = 19 controls, n = 12 ADPKD) was subjected to whole genome sequencing and somatic mutation analysis. Results Urines of ADPKD patients contained higher numbers of cells that expanded in vitro, compared to both control (p = 0.0008) and VHL individuals (p = 0.01). Gene expression analyses showed that irrespective of genetic background, all cultured clones originated from the kidney tubule epithelium (high PAX2 and PAX8), and some originated from damaged tubules (VCAM1, KIM1). We tested the hypothesized metabolic rewiring by analyzing expression levels of enzymes involved in glutamine utilization, urea cycle, and pyrimidine biosynthesis. Except for ASNS (Asparagine Synthetase), which was higher in ADPKD vs control clones (p = 0.0098), no gene-expression differences were observed. However, clones from ADPKD patients showed signs of the metabolic rewiring responsible for increased pyrimidine production, i.e. a positive correlation (r = 0.691; p &amp;lt; 0.0001) between urea cycle enzymes downregulation and upregulation of the pyrimidine synthesis enzyme CAD. Since excessive pyrimidines lead to mutation [2], we analyzed the number of somatic SBSs per genome, after filtering for germline variants. We did not find increased mutation rates in ADPKD compared to controls, but an analogous, linear increase of mutations with age and similar levels of the pyrimidine-rich mutational signature. Conclusion Urine-derived kidney tubule epithelial cells with heterozygous truncating mutations in PKD1 exhibit certain characteristics of metabolic reprogramming typical of kidneys from ADPKD patients with advanced pathology. Nevertheless, in the limited number of pre-cystic cells that we have analyzed, this metabolic reprogramming did not translate into an excess of somatic mutations.

Environmental enrichment and the combined interventions of EE and metformin enhance hippocampal neuron survival and hippocampal-dependent memory in type 2 diabetic rats under stress through the BDNF-TrkB signaling pathways

BackgroundType 2 diabetes (T2D) with depression causes severe cognitive impairments. The devastating conditions will further compromise the overall quality of life. The overconsumption of high-fat and high-sucrose (HFS) diet is one of the modifiable risk factors for T2D, depression, and cognitive impairments. Thus, it is essential to identify effective therapeutic strategies to overcome the cognitive impairments in T2D with depression. We proposed environmental enrichment (EE) which encompasses social, cognitive, and physical components as the alternative treatment for such impairments. We also investigated the potential neuroprotective properties of the antidiabetic drug metformin. This study aimed to investigate the effects of EE and metformin interventions on hippocampal neuronal death, and hippocampal-dependent memory impairment in T2D rats under stress. MethodsThirty-two male rats (200–250 g) were divided into four groups: C group (standard diet + conventional cage), DS group [HFS-induced T2D + restraint stress (RS)], DSE group [HFS-induced T2D + RS + EE] and DSEM group [HFS + RS + EE + metformin]. Serum corticosterone (CORT) was measured to evaluate stress levels. The serum Free Oxygen Radicals Testing (FORT) and Free Oxygen Radicals Defence Test (FORD) were measured to evaluate the systemic oxidative status (OS). Serum brain-derived neurotrophic factor (BDNF) and T-maze tasks were performed to evaluate cognitive functions. Rats were humanely sacrificed to collect brains for histological, morphometric, and hippocampal gene expression studies. ResultsThe CORT and the serum FORT levels in the DSE and DSEM groups were lower than in the DS group. Meanwhile, the serum BDNF, T-maze scores, histological, and morphometric analysis were improved in the DSE and DSEM groups than in the DS group. These findings supported that EE and the combined interventions of EE and metformin had neuroprotective properties. The hippocampal gene expression analysis revealed that the DSE and DSEM groups showed improved regulation of BDNF-TrkB signalling pathways, including the BDNF/TrkB binding, PI3K – Akt pathway, Ras–MAPK pathway, PLCγ–Ca2+ pathway, and CREB transcription. ConclusionEE and the combined interventions of EE and metformin improved hippocampal neuron survival and hippocampal-dependent memory in T2D rats under stress by enhancing gene expression regulation of neurogenesis and synaptic plasticity.

Analysis of gene expression in the postmortem brain of neurotypical Black Americans reveals contributions of genetic ancestry

Ancestral differences in genomic variation affect the regulation of gene expression; however, most gene expression studies have been limited to European ancestry samples or adjusted to identify ancestry-independent associations. Here, we instead examined the impact of genetic ancestry on gene expression and DNA methylation in the postmortem brain tissue of admixed Black American neurotypical individuals to identify ancestry-dependent and ancestry-independent contributions. Ancestry-associated differentially expressed genes (DEGs), transcripts and gene networks, while notably not implicating neurons, are enriched for genes related to the immune response and vascular tissue and explain up to 26% of heritability for ischemic stroke, 27% of heritability for Parkinson disease and 30% of heritability for Alzheimer’s disease. Ancestry-associated DEGs also show general enrichment for the heritability of diverse immune-related traits but depletion for psychiatric-related traits. We also compared Black and non-Hispanic white Americans, confirming most ancestry-associated DEGs. Our results delineate the extent to which genetic ancestry affects differences in gene expression in the human brain and the implications for brain illness risk.

Aegeline attenuates TNBS-induced colitis by suppressing the NFƙB-mediated NLRP3 inflammasome pathway in mice.

A chronic inflammatory condition of the intestine, ulcerative colitis (UC), is challenging to successfully manage once diagnosed. Currently, available medical therapies for UC exhibit minimal efficacy with unacceptable side effects, while inventive biological agents are expensive and yetnotwell accepted by patients. Discovering more effective and safer treatments to treat UC is therefore essential. One of the primary alkaloids found in Aegle marmelos, aegeline, has anti-inflammatory and antioxidant properties as well as being able to suppress severalpro-inflammatory cytokines responsible for inflammation. The study aimed to investigate the effectiveness of aegeline in alleviating 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis through the NFƙB-mediated NLRP3 inflammasome pathway. Mice were randomly allocated into six groups, Normal control (NC), Model control (MC-TNBS, 2,4,6-trinitrobenzene sulfonic acid), STD (TNBS + sulfasalazine 100mg/kg), AG1, AG2, and AG3 (TNBS + aegeline 5, 10, 20mg/kg) respectively. Physical parameters such as a change in body weight, stool consistency, rectal bleeding, colon length, myeloperoxidase (MPO) levels and nitric oxide (NO) levels, and disease activity index (DAI) were assessed and supporting gene expression studies of various pro-inflammatory cytokines and enzymes were evaluated and histopathological changes observed. Administration of aegeline (10, 20mg/kg) was found to be effective in colon protection by lowering the disease activity score and myeloperoxidase level and improving other physical parameters. Aegeline in high dose significantly downregulated the gene expression of NFƙB, iNOS, COX-2, NLRP3, IL-1β, and IL-18, conferring great anti-inflammatory potential. Suggestive of the findings, aegeline reduced the damage to the colon by downregulating transcriptional genes and enzymes leading to inflammation and mitigated TNBS-induced colitis probably through the NFƙB-mediated NLRP3 inflammasome pathway.

Quantitative RUBY reporter assay for gene regulation analysis.

Various reporter genes have been developed to study gene expression pattern and gene regulation. The RUBY reporter gene was recently developed and widely used, because of its visible and noninvasive advantages. However, quantitative analysis of RUBY gene expression levels was lacking. In this study, we introduce a novel betalain quantification method in combination with the tobacco transient expression system. The betalain produced in tobacco leaves was extracted and purified, and its concentration was quantitatively measured. We successfully applied this approach in studying the transcriptional regulation of ARC5 gene by transcription factors CPD25 and CPD45. Furthermore, with this method, we showed that the gene expression of RCA and Rbcs1A gene were regulated by light, transcription factors HY5 and PIFs through G-box and I-box elements. The development of this betalain quantification approach with the tobacco transient expression system offers a cost-effective and intuitive strategy for studying the regulatory mechanism of gene expression.

Diuretic effects of Hecogenin and Hecogenin acetate via aldosterone synthase inhibition

Hecogenin (HEC) is a steroidal saponin found in many plant species and serves as a precursor for steroidal drugs. The diuretic effects of HEC and its derivative, hecogenin acetate (HA), remain largely unexplored. The present study aimed to explore the potential diuretic effects of HEC and HA compared to furosemide (FUR) and spironolactone (SPIR). Additionally, the study aimed to explore the underlying mechanism particularly focusing on aldosterone synthase gene expression. Fifty-four Sprague-Dawley rats were allocated into nine groups (Group 1–9). Group 1 (control) received the vehicle, Groups 2 received FUR 10 mg/kg, Group 3, 4, and 5 were given HEC, while Groups 6, 7 and 8 received HA i.p at doses of 5, 10, and 25 mg/kg, respectively. Group 9 received SPIR i.p at the dose of 25 mg/kg. Urine volume, diuretic index and diuretic activity were monitored at 1, 2, 3, 4, 5, 6, and 24 h post-administration. Treatment was given daily for seven days. After that, rats were sacrificed and blood was collected for serum electrolytes determination. Adrenal glands were dissected out for gene expression studies. The results revealed that HEC and HA at the administered doses significantly and dose-dependently increased urine and electrolyte excretion. These results were primarily observed at 25 mg/kg of each compound. Gene expression studies demonstrated a dose-dependent reduction in aldosterone synthase gene expression, suggesting aldosterone synthesis inhibition as a potential mechanism for their diuretic activity. Notably, HA exhibited more pronounced diuretic effects surpassing those of HEC. This enhanced diuretic activity of HA can be attributed to its stronger impact on aldosterone synthase inhibition. These findings offer valuable insights into the diuretic effects of both HEC and HA along with their underlying molecular mechanisms.

Sequencing complex plants on a budget: The development of Kalanchoë blossfeldiana as a C3, CAM comparative tool

Societal Impact StatementResearch efforts in plant biology have often been focused on sequenced and well‐studied ‘model’ organisms. Despite the advent of relatively inexpensive genome sequencing, most plant taxonomic groups are underrepresented, with few species that ‘represent’ the diversity of whole genera. This study describes an economical guide to sequencing a non‐model organism, which may be useful in reducing the cost of sequencing more species within genera and across plant life. This method was used to develop Kalanchoë blossfeldiana as a resource for comparing C3 and the water‐conserving mode of photosynthesis known as Crassulacean acid metabolism (CAM) within the same plant.Summary Despite the increasing number of well‐studied plant species with well‐annotated genomes across plant life, there are few densely sampled genera with more than a couple of genome sequences representing the diversity of whole genera. Here, we develop an economic approach to full‐genome sequencing that could be used to sequence many species within a genus. We made use of the Nanopore rapid sequencing kit to assist in plant genome assembly, dramatically reducing the cost. Here we applied this method to cost‐effectively develop genomic resources for Kalanchoë blossfeldiana, a commercially important ornamental, in which Crassulacean Acid Metabolism (CAM), a water‐conserving mode of photosynthesis can be induced. We present a physiological and biochemical characterisation of Kalanchoe blossfeldiana with its nuclear and chloroplastic genome and a comparative C3, CAM dusk transcriptome. We apply this approach to a complex tetraploid genome, making use of a relative species for chromosomal scaffolding to reduce assembly ploidy, we provide a resource for future gene expression studies. We highlight its limitations, e.g. the need for deeper sequencing to accurately resolve genome structure and haplotypes without using a relative species for scaffolding. The study demonstrates the merits of K. blossfeldiana as a comparative system for studying C3 and CAM within a plant and has identified substantial changes in the dusk transcriptome between young C3 and mature CAM K. blossfeldiana leaves in response to age‐induced CAM, and shows that in the absence of abiotic stress, CAM induction still involves the engagement of drought and abscisic acid (ABA) response pathways.

Comparing chemical transfection, electroporation, and lentiviral vector transduction to achieve optimal transfection conditions in the Vero cell line

BackgroundTransfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells.MethodsIn the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells.ResultsAmong the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 104 Vero cells.ConclusionTurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.

Significant improvement of cardiac outflow tract septation defects in a DiGeorge syndrome model after minoxidil treatment

The T-BOX transcription factor TBX1 is essential for the development of the pharyngeal apparatus and it is haploinsufficient in DiGeorge syndrome (DGS), a developmental anomaly associated with congenital heart disease and other abnormalities. The murine model recapitulates the heart phenotype and showed collagen accumulation.We first used a cellular model to study gene expression during cardiogenic differentiation of WT and Tbx1−/− mouse embryonic stem cells. Then we used a mouse model of DGS to test whether interfering with collagen accumulation using an inhibitor of lysyl hydroxylase would modify the cardiac phenotype of the mutant.We found that loss of Tbx1 in a precardiac differentiation model was associated with up regulation of a subset of ECM-related genes, including several collagen genes. In the in vivo model, early prenatal treatment with Minoxidil, a lysyl hydroxylase inhibitor, ameliorated the cardiac outflow tract septation phenotype in Tbx1 mutant fetuses, but it had no effect on septation in WT fetuses. We conclude that TBX1 suppresses a defined subset of ECM-related genes. This function is critical for OFT septation because the inhibition of collagen cross-linking in the mutant reduces significantly the penetrance of septation defects.

Identification of CD5/SOX11 double-negative pleomorphic mantle cell lymphoma.

Cyclin D1 protein-positive diffuse large B cell lymphoma (DLBCL) has an immunophenotype of CD5(-) cyclin D1(+) SOX11(-), and most cases lack a CCND1 rearrangement and have a gene expression profile of DLBCL. Rarely, cyclin D1 protein-positive DLBCL harbors a CCND1 rearrangement, and some genetic copy number features typical of mantle cell lymphoma (MCL) have been detected. Since gene expression studies have not been performed, whether such CCND1-rearranged cases represent cyclin D1 protein-positive DLBCL or CD5/SOX11 double-negative pleomorphic MCL remains unclear. To date, no cases of CD5/SOX11 double-negative MCL have been reported. In this study, we collected eight cases initially diagnosed as cyclin D1 protein-positive DLBCL, including four with a CCND1 rearrangement and four without. Immunohistochemically, all four CCND1-rearranged cases had >50% of tumor cells positive for cyclin D1 protein, whereas only one (25%) non-rearranged case had >50% positive tumor cells. Analysis of genome-wide copy number, mutational, and gene expression profiles revealed that CCND1-rearranged cases were similar to MCL, whereas CCND1-non-rearranged cases resembled DLBCL. Despite the SOX11 negativity by immunohistochemistry, CCND1-rearranged cases had a notable trend (P = 0.064) of higher SOX11 mRNA levels compared to non-rearranged cases. Here, we show for the first time that CCND1 rearrangement could be useful for identifying CD5/SOX11 double-negative pleomorphic MCL in cases diagnosed as cyclin D1 protein-positive DLBCL. Cases with >50% cyclin D1 protein-positive tumor cells immunohistochemically and higher SOX11 mRNA levels are more likely to have a CCND1 rearrangement, and fluorescence in situ hybridization can be used to detect the rearrangement.

Effect of the Protein Kinase B (PKB) Gene on the Carcinogenesis of Oral Squamous Cell Carcinoma in the South Indian Population.

The most common head and neck cancer is oral squamous cell carcinoma (OSCC). It is also one of the most prevalent forms of cancer globally. The current pharmacological treatment strategy for oral cancer lacks specificity and is capable of causing various side effects. This fact highlights the increasing need for targeted therapy. Interestingly, protein kinase B (PKB), commonly referred to as the AKT serine/threonine kinase, is an oncogenic protein that controls cell development, proliferation, apoptosis, and glycogen metabolism. Thus, the present study analyzed the AKT gene expression in OSCC patient samples. A total of 25 OSCC tissue samples and normal tissue samples were collected from the patients who reported to the Department of Oral and Maxillofacial Surgery, Saveetha Dental College and Hospitals in Chennai, India. The tissues were processed for H&E staining for histopathological confirmation, and expression studies of the AKT gene were done on both healthy and proven OSCC tissue samples. The data were shown as mean ± standard deviation, and p<0.05* was considered to be statistically significant. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the AKT gene had been significantly upregulated in the OSCC tissue samples when compared to normal tissues (p<0.05). Moreover, upregulated AKT is postulated to be involved in increased cell proliferation and reduced apoptosis in OSCC. The gene expression analysis was done in the samples of histologically confirmed OSCC, and it revealed that the AKT gene was significantly upregulated in OSCC tissues. Thus, AKT could be postulated as a potential therapeutic target for OSCC.

ECM-mimetic, NSAIDs loaded thermo-responsive, immunomodulatory hydrogel for rheumatoid arthritis treatment

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, and it leads to irreversible inflammation in intra-articular joints. Current treatment approaches for RA include non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), corticosteroids, and biological agents. To overcome the drug-associated toxicity of conventional therapy and transdermal tissue barrier, an injectable NSAID-loaded hydrogel system was developed and explored its efficacy.ResultsThe surface morphology and porosity of the hydrogels indicate that they mimic the natural ECM, which is greatly beneficial for tissue healing. Further, NSAIDs, i.e., diclofenac sodium, were loaded into the hydrogel, and the in vitro drug release pattern was found to be burst release for 24 h and subsequently sustainable release of 50% drug up to 10 days. The DPPH assay revealed that the hydrogels have good radical scavenging activity. The biocompatibility study carried out by MTT assay proved good biocompatibility and anti-inflammatory activity of the hydrogels was carried out by gene expression study in RAW 264.7 cells, which indicate the downregulation of several key inflammatory genes such as COX-2, TNF-α & 18s.ConclusionIn summary, the proposed ECM-mimetic, thermo-sensitive in situ hydrogels may be utilized for intra-articular inflammation modulation and can be beneficial by reducing the frequency of medication and providing optimum lubrication at intra-articular joints.Graphical

Effect of dietary vitamin E supplement on the reproductive performance and gene expression profiling in Ompok bimaculatus during breeding period

AbstractThis study aimed to evaluate the effect of vitamin E on the reproductive responses and the expression of reproductive genes in butter catfish (Ompok bimaculatus). Diets with different levels of vitamin E supplementation (0, 100, and 150 mg/kg) were provided to groups of fish for 60 days. After rearing for 60 days fish were induced with synthetic hormone, that is, Ovatide to study the breeding performance. Ovatide was administrated intramuscularly at the rate of 0.5 and 0.25 mL/kg of body weight for females and males, respectively, in all the treatments. Breeding performance result shows that supplements of vitamin E positively influenced the GSI%, fertilization rate, fecundity, and hatching rate. To understand the relationship between vitamin E and the breeding performance of the fish at the molecular level, a gene expression study was conducted. This study employed real‐time PCR for the selected genes critical for reproductive function. In the brain, testis, and ovary dietary vitamin E supplementation of 100 mg/kg significantly enhanced the mRNA transcription of FSHR, Brdt, ESR1, 17beta2, and LHR. Hence, it can be said that 100 mg/kg of vitamin E supplementation in the diet during the reproductive period of O. bimaculatus could improve breeding performance and the mRNA expression of reproductive hormone receptor genes in both males and females of O. bimaculatus.