Cellular Process Pathways Research Articles

High-throughput sequencing reveals differences in microbial community structure and diversity in the conjunctival tissue of healthy and type 2 diabetic mice

BackgroundTo investigate the differences in bacterial and fungal community structure and diversity in conjunctival tissue of healthy and diabetic mice.MethodsRNA-seq assays and high-throughput sequencing of bacterial 16 S rDNA and fungal internal transcribed spacer (ITS) gene sequences were used to identify differentially expressed host genes and fungal composition profiles in conjunctival tissues of diabetic BKS-db/db mice and BKS (control) mice. Functional enrichment analysis of differentially expressed genes and the correlation between the relative abundance of bacterial and fungal taxa in the intestinal mucosa were also performed.ResultsTotally, 449 differential up-regulated genes and 1,006 down-regulated genes were identified in the conjunctival tissues of diabetic mice. The differentially expressed genes were mainly enriched in metabolism-related functions and pathways. A decrease in conjunctival bacterial species diversity and abundance in diabetic mice compared to control mice. In contrast, fungal species richness and diversity were not affected by diabetes. The microbial colonies were mainly associated with cellular process pathways regulating carbohydrate and lipid metabolism, as well as cell growth and death. Additionally, some interactions between bacteria and fungi at different taxonomic levels were also observed.ConclusionThe present study revealed significant differences in the abundance and composition of bacterial and fungal communities in the conjunctival tissue of diabetic mice compared to control mice. The study also highlighted interactions between bacteria and fungi at different taxonomic levels. These findings may have implications for the diagnosis and treatment of diabetes.

Transcriptome Analysis Reveals Mycelial and Fruiting Responses to Lithium Chloride in Coprinopsis cinerea.

Lithium chloride (LiCl) has been used in signalling and molecular studies of animals, plants, and yeast. However, information on its roles in basidiomycetous fungi is still limited. In this study, we used RNA-Seq to study the effects of LiCl on Coprinopsis cinerea. LiCl enhanced mycelial growth and inhibited fruiting body formation in C. cinerea. RNA-Seq of the LiCl-treated C. cinerea resulted in a total of 14,128 genes. There were 1199 differentially expressed genes (DEGs) between the LiCl-treated samples and control samples in the mycelium stage (the first time point), and 1391 DEGs were detected when the control samples were forming hyphal knots while the treated samples were still in the mycelium (the second time point). Pathway enrichment analysis of the DEGs revealed a significant association between enhanced mycelium growth in the LiCl-treated C. cinerea and metabolic pathways. In addition, the DEGs involved in cellular process pathways, including "cell cycle-yeast" and "meiosis-yeast", were identified in suppressed C. cinerea fruiting body formation by LiCl under favourable environmental conditions. As LiCl can predominantly inhibit the activity of glycogen synthase kinase3 (GSK3), our findings suggest that LiCl affects the expression of genes involved in fruiting body initiation and cellular processes by inhibiting GSK3 activity which is essential for fruiting body formation.

Probiotics mitigate kidney damage after exposure to Sri Lanka's local groundwater from chronic kidney disease with uncertain etiology (CKDu) prevalent area in zebrafish

Groundwater in Sri Lanka, contaminated with environmental toxins, is suspected to potentially induce chronic kidney disease of uncertain etiology (CKDu) in humans. This study aims to elucidate the potential mitigating effects of probiotics on kidney damage induced by exposure to this local groundwater (LW) in zebrafish. We used zebrafish as a model organism and exposed them to local groundwater to evaluate the risk of CKDu. Probiotics were then added at a concentration of 108 colony-forming units per milliliter (CFU/mL). Our findings revealed that exposure to local groundwater resulted in abnormalities, such as tail deletion and spinal curvature in zebrafish larvae. However, the addition of probiotics mitigated these effects, improving the hatching rate, heart rate, length, weight, deformity rate, survival rate, and abnormal behavior of zebrafish. It also positively influenced the differential expression levels of kidney development and immunity-related genes (dync2h1, foxj1, pkd2, gata3, slc20a1, il1β, and lyso). Furthermore, exposure to LW decreased both the diversity and abundance of microbiota in zebrafish larvae. However, treatment with probiotics, such as L. plantarum and L. rhamnosus partially restored the disrupted gut microbiota and significantly impacted the cellular process pathways of the microbial community, as determined by KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. In conclusion, this study highlights the risks associated with Sri Lanka's local groundwater from a CKDu prevalent area and confirms the beneficial effects of different probiotics. These findings may provide new insights into bacterial function in host kidney health.

Enrofloxacin induces intestinal disorders of metabolome and microbiome in American shad ( Alosa sapidissima )

Enrofloxacin (ENR) has been often detected in the environment and organisms due to its widespread use in aquaculture, medical cosmetology and animal husbandry. Though the effects of ENR exposure on the gut microbiome of aquaculture fish have been reported, there is little information on the long-term impact on adult American shad. In this study, ENR (100 mg/kg) exposed to American shad for 30 days. Then, we monitored the alterations in the gut microbiome, the responses on metabolomic and the change in the body weight. ENR exposure affected the composition of the gut microbiota community and altered community diversity. The LC-MS analysis showed the changes in several metabolic pathways, including lipid metabolism, the cellular process pathways, immune system and amino acid metabolism pathway. These metabolic pathways were related to increasing body weight gain through fat accumulation. In conclusion, this study provides evidence that ENR exposure can increase the body weight in American shad and yield new information on the effects of ENR exposure on the aquatic system.

Assessment of cellular cobalamin metabolism in Gaucher disease

BackgroundGaucher disease (GD) is a lysosomal disorder caused by biallelic pathogenic mutations in the GBA1 gene that encodes beta-glucosidase (GCase), and more rarely, by a deficiency in the GCase activator, saposin C. Clinically, GD manifests with heterogeneous multiorgan involvement mainly affecting hematological, hepatic and neurological axes. This disorder is divided into three types, based on the absence (type I) or presence and severity (types II and III) of involvement of the central nervous system. At the cellular level, deficiency of GBA1 disturbs lysosomal storage with buildup of glucocerebroside. The consequences of disturbed lysosomal metabolism on biochemical pathways that require lysosomal processing are unknown. Abnormal systemic markers of cobalamin (Cbl, B12) metabolism have been reported in patients with GD, suggesting impairments in lysosomal handling of Cbl or in its downstream utilization events.MethodsCultured skin fibroblasts from control humans (n = 3), from patients with GD types I (n = 1), II (n = 1) and III (n = 1) and an asymptomatic carrier of GD were examined for their GCase enzymatic activity and lysosomal compartment intactness. Control human and GD fibroblasts were cultured in growth medium with and without 500 nM hydroxocobalamin supplementation. Cellular cobalamin status was examined via determination of metabolomic markers in cell lysate (intracellular) and conditioned culture medium (extracellular). The presence of transcobalamin (TC) in whole cell lysates was examined by Western blot.ResultsCultured skin fibroblasts from GD patients exhibited reduced GCase activity compared to healthy individuals and an asymptomatic carrier of GD, demonstrating a preserved disease phenotype in this cell type. The concentrations of total homocysteine (tHcy), methylmalonic acid (MMA), cysteine (Cys) and methionine (Met) in GD cells were comparable to control levels, except in one patient with GD III. The response of these metabolomic markers to supplementation with hydroxocobalamin (HOCbl) yielded variable results. The content of transcobalamin in whole cell lysates was comparable in control human and GD patients.ConclusionsOur results indicate that cobalamin transport and cellular processing pathways are overall protected from lysosomal storage damage in GD fibroblasts. Extending these studies to hepatocytes, macrophages and plasma will shed light on cell- and compartment-specific vitamin B12 metabolism in Gaucher disease.

Exposure to heat-stress environment affects the physiology, circulation levels of cytokines, and microbiome in dairy cows

The microbiome has emerged as a new player on behavior, physiology and stress because of its significant effects on the brain-gut axis. The aim of this study was to increase our understanding of brain-gut function in dairy cows. We investigated the effects of a heat-stress (HS) environment and individual differences of heat sensitivity (IH) on bovine physiological characteristics and microbial composition. Results indicate that both HS and IH increased rectal temperature (RT) (P < 0.05). An HS environment increased plasma, as well as milk cortisol and cytokines in plasma; however, it decreased plasma, and milk oxytocin, triiodothyronine, and thyroxine (P < 0.05) levels. Exposure to an HS environment reduced the diversity of the fecal microbial population, and resulted in a higher expression of diseases, the environmental adaptation pathway, and the immune related pathway, whereas it lowered the expression of metabolic pathways (P < 0.05). High heat sensitive cows have upregulated metabolisms, environmental adaptation and cellular process pathways, and a downregulated neurodegenerative disease pathway (P < 0.05). Thus, we conclude that exposure to an HS environment modulates physiological characteristics, which may interplay with microbial activity, and in turn, alter the circulation levels of cytokines, implicating the role of the brain-gut axis in dairy cows. The HS environment affected physiological characteristics, cytokine levels, and microbial composition, but IH influenced RT and fecal microbial functions.

POMC: The Physiological Power of Hormone Processing.

Pro-opiomelanocortin (POMC) is the archetypal polypeptide precursor of hormones and neuropeptides. In this review, we examine the variability in the individual peptides produced in different tissues and the impact of the simultaneous presence of their precursors or fragments. We also discuss the problems inherent in accurately measuring which of the precursors and their derived peptides are present in biological samples. We address how not being able to measure all the combinations of precursors and fragments quantitatively has affected our understanding of the pathophysiology associated with POMC processing. To understand how different ratios of peptides arise, we describe the role of the pro-hormone convertases (PCs) and their tissue specificities and consider the cellular processing pathways which enable regulated secretion of different peptides that play crucial roles in integrating a range of vital physiological functions. In the pituitary, correct processing of POMC peptides is essential to maintain the hypothalamic-pituitary-adrenal axis, and this processing can be disrupted in POMC-expressing tumors. In hypothalamic neurons expressing POMC, abnormalities in processing critically impact on the regulation of appetite, energy homeostasis, and body composition. More work is needed to understand whether expression of the POMC gene in a tissue equates to release of bioactive peptides. We suggest that this comprehensive view of POMC processing, with a focus on gaining a better understanding of the combination of peptides produced and their relative bioactivity, is a necessity for all involved in studying this fascinating physiological regulatory phenomenon.

ITRAQ and PRM-based quantitative proteomics in T2DM-susceptible and -tolerant models of Bama mini-pig

Type 2 diabetes mellitus (T2DM) is a complex, multifactorial metabolic disease, and the number of patients with T2DM has continued to increase in recent years. Large-scale proteomic studies on animal models of T2DM are of great importance to understand the pathophysiology of T2DM. Therefore, in our study, Isobaric tags for relative and absolute quantification (iTRAQ) and Parallel reaction monitoring (PRM) were used for proteomic analysis of skeletal muscles from T2DM-susceptible and -tolerant Bama mini-pig models induced by a high-fat, high-sugar diet. In our proteomic analysis, a total of 1646 proteins and 13 differentially expressed proteins (DEPs) were identified by iTRAQ-mass spectrometry, and 6 differentially expressed proteins were validated by PRM. Gene Ontology (GO) analysis revealed that most DEPs were extracellular matrix (ECM) proteins and participated in several biological processes, such as negative regulation of JAK-STAT cascade, negative regulation of STAT cascade, roundabout signaling pathway and peptide cross-linking via chondroitin 4-sulfate glycosaminoglycan, and the molecular functions of roundabout binding, glycosaminoglycan binding, heparin binding, sulfur compound binding, collagen binding, and kinase inhibitor activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis results showed that the differentially expressed proteins were involved in 14 pathways, including human disease pathways, metabolic pathways, signal transduction pathways, signaling molecules and interaction pathways, and the cellular process pathways associated with phagosomes and focal adhesion. In conclusion, the proteomics based on iTRAQ and PRM in T2DM-susceptible and -tolerant Bama mini-pig models showed that changes in amino acid metabolism, inflammation-associated pathways and the impaired function and environment of extracellular matrix are risk factors associated with increased pathogenesis of T2DM in Bama mini-pig.

Characterizing Conformational Ensemble of H‐, K‐, and N‐Ras Using Accelerated Molecular Dynamics and Wide‐Angle X‐ray Solution Scattering (WAXS)

Ras GTPase is involved in cell‐signaling pathways that control cell proliferation, differentiation, and apoptosis. There are three main isoforms of Ras, H‐, K‐, and N‐Ras, which share about 95% sequence identity in their soluble domain. They have very similar three‐dimensional folds, however they have been suggested to display functional specificity. For example, in pancreatic cancer K‐Ras mutations occur at much higher frequency than either H‐ or N‐Ras, whereas in leukemia N‐Ras mutations play the dominant role. These isoforms have also been shown to follow distinct cellular processing pathways and localize to different subcellular compartments. In this study, we combine accelerated Molecular Dynamics (aMD) and wide‐angle X‐ray scattering (WAXS) methods to characterize the structural ensemble of Ras isoforms and measure their dynamic properties. Using aMD, we reduce the energetic barriers to enhance conformational sampling of these proteins. A representative subset of structures from aMD is then experimentally tested using WAXS patterns to quantify their relative population. Each isoform shows at least two major conformations, where one is characterized as a relaxed state and another one as a reactive state, based on their relative potential energy calculated from aMD. These conformations are unique for each of the isoform, where they differ primarily around the active site and regions away from the active site, including helix 3/loop 7. This study will provide significant insights into the conformational features of these small proteins that give rise to their functional specificity.Support or Funding InformationNSF MCB‐1244203

Targeted Protein Degradation of Outer Membrane Decaheme Cytochrome MtrC Metal Reductase inShewanella oneidensisMR-1 Measured Using Biarsenical Probe CrAsH-EDT2

Development of efficient microbial biofuel cells requires an ability to exploit interfacial electron transfer reactions to external electron acceptors, such as metal oxides; such reactions occur in the facultative anaerobic Gram-negative bacterium Shewanella oneidensis MR-1 through the catalytic activity of the outer membrane decaheme c-type cytochrome MtrC. Central to the utility of this pathway to synthetic biology is an understanding of cellular mechanisms that maintain optimal MtrC function, cellular localization, and renewal by degradation and resynthesis. In order to monitor trafficking to the outer membrane, and the environmental sensitivity of MtrC, we have engineered a tetracysteine tag (i.e., CCPGCC) at its C-terminus that permits labeling by the cell impermeable biarsenical fluorophore carboxy-FlAsH (CrAsH) of MtrC at the surface of living Shewanella oneidensis MR-1 cells. In comparison, the cell permeable reagent FlAsH permits labeling of the entire population of MtrC, including proteolytic fragments resulting from incorrect maturation. We demonstrate specific labeling by CrAsH of engineered MtrC (MtrC*) which is dependent on the presence of a functional type 2 secretion system (T2S), as evidenced by T2S system gspD or gspG deletion mutants which are incapable of CrAsH labeling. Under these latter conditions, MtrC* undergoes proteolytic degradation to form a large 35-38 kDa fragment; this degradation product is also resolved during normal turnover of the CrAsH-labeled MtrC protein. No MtrC protein is released into the medium during turnover, suggesting the presence of cellular turnover systems involving MtrC reuptake and degradation. The mature MtrC localized on the outer membrane is a long-lived protein, with a turnover rate of 0.043 h(-1) that is insensitive to O(2) concentration. Maturation of MtrC is relatively inefficient, with substantial rates of turnover of the immature protein prior to export to the outer membrane (i.e., 0.028 h(-1)) that are consistent with the inherent complexity associated with correct heme insertion and acylation of MtrC that occurs in the periplasm prior to its targeting to the outer membrane. These latter results suggest that MtrC protein trafficking to the outer membrane and its subsequent degradation are tightly regulated, which is consistent with cellular processing pathways that target MtrC to extracellular structures and their possible role in promoting electron transfer from Shewanella to extracellular acceptors.

Cellular processing pathways contribute to the activation of etoposide-induced DNA damage responses

Cytotoxic action (tumor cell killing) and carcinogenic side effect (therapy-related secondary leukemia) of etoposide are closely related to its ability in stabilizing topoisomerase II cleavable complex (TOP2cc), a unique form of protein-linked DNA break. How cells process and detect TOP2-concealed DNA damage for the activation of downstream cellular responses remains unclear. Here, we showed proteasomal degradation of both TOP2 isozymes in a transcription-dependent manner upon etoposide treatment. Downregulation of TOP2 was preferentially associated with proteasomal removal of TOP2 in TOP2cc rather than proteolysis of free TOP2. Interestingly, blockage of TOP2 downregulation in TOP2cc also caused reduction in etoposide-induced activation of DNA damage molecules, an observation suggesting that the processing pathways of TOP2cc are involved in activation of etoposide-induced cellular responses. In this regard, we observed two TOP2cc processing pathways, replication- and transcription-initiated processing (RIP and TIP) with proteasome involved in the latter. Importantly, two processing pathways contributed to differential activation of various DNA damage signaling and downstream cellular responses. Etoposide-induced phosphorylation of p53 relied mainly on RIP, whereas activation of Chk1, Chk2 depended largely on TIP. Both RIP and TIP played roles in activating non-homologous end joining pathway, while only RIP modulated etoposide-induced cell killing in a p53-dependent manner. Collectively, our results are consistent with the notion that protein-linked DNA breakage (e.g., TOP2cc) requires processing pathways for initiating downstream DNA damage detection, repair as well as cell death programs.

Beta-Amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms.

We have analyzed the cellular processing pathways which produce the 4-kDa amyloid beta-peptide (A beta) and a 3-kDa derivative (p3) of the beta-amyloid precursor protein (beta APP) found in conditioned media of tissue culture cells and in cerebrospinal fluid. Pulse-chase experiments reveal that both peptides are secreted in parallel with soluble beta APP (APPs); no precursor-product relation between A beta and p3 was found. The protease inhibitor leupeptin did not influence the production of either peptide. In contrast, the weak base ammonium chloride (NH4Cl) showed a dose-dependent inhibition of A beta production with less decrease in p3. A similar effect was observed using the monovalent ionophore monensin. Brefeldin A completely inhibited the generation of both peptides, indicating that proteases located in the endoplasmic reticulum or early Golgi are not sufficient for the production of the small peptides. Deletion of the beta APP cytoplasmic domain, which removes a consensus sequence that probably mediates reinternalization, caused an increase in secretion of both APPs and p3 and did not abolish A beta production. These observations suggest that completely mature beta APP within the late Golgi and/or at the cell surface is a prerequisite for A beta production but processing within the lysosome might not be directly required. p3 appears to derive from the 10-kDa C-terminal stub of beta APP following secretion of APPs.