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SorCS2 Is Important for Astrocytic Function in Neurovascular Signaling
Get PDFIntroduction: The receptor SorCS2 is involved in the trafficking of membrane receptors and transporters. It has been implicated in brain disorders and has previously been reported to be indispensable for ionotropic glutamatergic neurotransmission in the hippocampus.
Aim: We aimed to study the role of SorCS2 in the control of astrocyte-neuron communication, critical for neurovascular coupling.
Methods: Brain slices from P8 and 2-month-old wild-type and SorCS2 knockout (Sorcs2−/−) mice were immunostained for
SorCS2, GFAP, AQP4, IB4, and CD31. Neurovascular coupling was assessed in vivo using laser speckle contrast imaging and ex vivo in live brain slices loaded with calcium-sensitive dye. Bulk and cell surface fraction proteomics was analyzed on freshly isolated and cultured astrocytes, respectively, and validated with Western blot and qPCR.
Results: SorCS2 was strongly expressed in astrocytes, primarily in their endfeet, of P8 mice; however, it was sparsely repre-sented in 2-month-old mice. Sorcs2−/− mice demonstrated reduced neurovascular coupling associated with a reduced astrocytic calcium response to neuronal excitation. No differences in vascularization or endothelium-dependent relaxation ex vivo between the 2-month-old groups were observed. Proteomics suggested changes in glutamatergic signaling and suppressed calcium sign-aling in Sorcs2−/− brains from both P8 and 2-month-old mice. The increased abundance of glutamate metabotropic receptor 3 in
Sorcs2−/− astrocytes was validated by PCR and Western blot. In cultured Sorcs2−/− astrocytes, AQP4 abundance was increased in the bulk lysate but reduced in the cell surface fraction, suggesting impaired trafficking
Impact of diet and nutritional status on gingival crevicular fluid metabolome and microbiome in people with type 1 diabetes: a cross-sectional study
Get PDFPeople with type 1 diabetes (PwT1D) are at higher risk of developing periodontal diseases. We investigated the impact of dietary habits on the gingival crevicular fluid (GCF) microbiome and metabolome in PwT1D. The oral and periodontal examination was performed and GCF sampled. Genomic DNA was extracted, bacterial 16S rRNA sequenced and concentrations of short-chain fatty acids and trimethylamine derivatives determined. Pro-healthy (pHDI) and non-healthy diet indices (nHDI) were calculated using the validated Questionnaire of Eating Behaviours. In total, 110 participants were included (mean age 27.1 ± 6.0 years, 60.0% male). In 65 PwT1D, the mean duration of diabetes was 15.5 ± 8.4 years and mean Hba1c% 6.97 ± 0.95% (53 ± 2.2 mmol/mmol). 22 cases of mild gingivitis (G) were identified, all in the T1D group. There were no significant differences in the frequencies of pHDI categories between study groups (T1D with G, low 19 [86.4%] and moderate 3 [13.6%]; T1D without G, low 28 [66.7%] and moderate 14 [45.2%]; control, low 30 [68.2%], moderate 14 [31.8%]; p = 0.213). GCF microbiome composition did not differ between pHDI categories. In PwT1D and G caproic acid was higher in low vs. moderate pHDI category (3.5 [0.9–4.9] vs. 0.64 [0.49-NA] umol/l, p = 0.04). In people with T1D without G, isocaproic acid and glycerophosphorylcholine were lower in low vs. moderate pHDI category (0.14 [0.13–0.46] vs. 0.45 [0.18–1.24] umol/l, p = 0.032, and 71.23 [32.83–120.40] vs. 129.8 [70.5–228.1] ng/ml, p = 0.013). This was the first study to report on the impact of diet on GCF in PwT1D. The state of periodontal tissues was worse in people with T1D, overweight and with a worse quality diet. Alterations in the concentrations of selected GCF metabolites suggest an indirect association between the quality of diet and contents of pro-healthy and non-healthy products on the state of periodontium
High-Fat Diet with Normal Caloric Intake Elevates TMA and TMAO Production and Reduces Microbial Diversity in Rats
Get PDFBackground/Objectives: Trimethylamine (TMA), produced by gut microbiota, and its derivative trimethylamine N-oxide (TMAO) are both associated with cardiometabolic diseases. While the effects of high-fat diets (HFDs) and high-disaccharide diets (HDDs) on gut microbiota in the context of obesity have been well studied, their impact on TMA/TMAO production, particularly alongside physiological caloric intake, remains obscure. This study investigates how standard HFDs and HDDs alongside physiological caloric intake influence gut microbiota composition and TMA/TMAO production in rats. Methods: Sprague Dawley rats were fed one of three diets a standard diet, an HFD, or an HDD for 12 weeks, with chow availability adjusted by age to maintain physiological caloric intake. Gut bacterial diversity was analyzed using 16S rRNA gene sequencing, and metabolites were quantified via High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) in urine and plasma. Results: The HFD group had significantly higher urinary levels of TMA and TMAO compared to the control and HDD groups. Gut bacterial diversity in the HFD group was markedly reduced, displaying the lowest species richness and phylogenetic diversity among all the groups. Notably, Pasteurellaceae (within the order Pasteurellales) and S24-7 (within the order Bacteroidales) were positively correlated with TMAO levels. The demonstrated HDD group increased microbial diversity compared to both the control and HFD groups. Conclusions: A high-fat diet during controlled and physiological caloric intake increases TMA/TMAO production and reduces gut microbial diversity. This underscores the role of diet composition, beyond caloric excess, in shaping gut microbiota and the related cardiometabolic biomarkers
Genome-Wide Identification of Essential Genes in the Invasive Streptococcus anginosus Strain
Get PDFBackground: Streptococcus anginosus, part of the Streptococcus anginosus group (SAG), is a human commensal increasingly recognized as an opportunistic pathogen responsible for abscesses formation and infections, also invasive ones. Despite its growing clinical importance, the genetic determinants of its pathogenicity remain poorly understood.
Objectives: This study aimed to identify essential genes in S. anginosus 980/01, a bloodstream isolate, under nutrient-rich laboratory conditions using a transposon mutagenesis combined with Transposon-Directed Insertion Site Sequencing (TraDIS).
Methods: A mutant library was generated using the ISS1 transposon delivered via the thermosensitive plasmid pGh9:ISS1. Following transposition, insertions were mapped using Illumina sequencing and subsequently analyzed. Essential genes were identified based on the absence of insertions and statistical filtering.
Results: The library exhibited 98% genome saturation with over 130,000 unique insertion sites. Among 1,825 genes, 348 (19.1%) were essential, 1,446 non-essential, and 30 non-conclusive. Comparative analyses were performed with S. pyogenes MGAS5005 and S. agalactiae A909. Similarly to the latter, essential genes were enriched in functions related to translation, transcription, and cell wall biosynthesis. However, 40 genes uniquely essential to S. anginosus 980/01 were identified, suggesting unique survival strategies in S. anginosus.
Conclusions: This study presents the first genome-wide identification of essential genes for S. anginosus 980/01, highlighting conserved and unique essential genes. These findings provide a basis for understanding its physiology and key genetic determinants of bacterial viability, and may help to uncover the pathogenic potential of S. anginosus in future studies
Plasma Bacterial Metabolites in Crohn’s Disease Pathogenesis and Complications
Get PDFBackground/Objectives: Crohn’s disease is known for being associated with an abnormal composition of the bacterial flora, dysbiosis and intestinal function disorders. Metabolites produced by gut microbiota play a pivotal role in the pathogenesis of CD, and the presence of unspecific extraintestinal manifestations. Methods: The aim of this study was a determination of the level of bacterial metabolites in blood plasma in patients with Crohn’s disease. CD patients (29) and healthy individuals (30) were recruited for this study. Bacterial metabolites (SCFAs and TMAO panel) were measured by a liquid chromatography–mass spectrometry system. Results: A significant correlation (p-value < 0.05) between CD and bacterial metabolites was obtained for three of eight tested SCFAs; acetic acid (reduced in CD; FC 1.7; AUC = 0.714), butyric acid (increased; FC 0.68; AUC = 0.717), 2MeBA (FC 1.168; AUC = 0.702), and indoxyl (FC 0.624). The concentration of CA (FC 0.82) and choline (FC 0.78) in plasma was significantly disturbed according to the biological treatment. Choline level (FC 1.28) was also significantly disturbed in the patients treated with glucocorticoids. In total, 68.97% of Crohn’s patients presented extraintestinal manifestations (EIMs) of Crohn’s disease, mainly osteoarticular complications. The level of BA was statistically significantly elevated in patients with extraintestinal (FC 0.602) manifestations, while in the group of patients with osteoarticular complications, a significant difference in the level of betaine (FC 1.647) was observed. Conclusions: The analyzed bacterial metabolites of plasma may significantly help in the diagnostic process, and in the monitoring of the disease course and treatment, in a lowly invasive way, as biomarkers after additional research on a larger group of patients
Compartmentalization during bacterial spore formation
Get PDFHere, we explore the recent advancements in understanding cellular compartmentalization during bacterial spore formation, primarily focusing on the model organism Bacillus subtilis. The hallmark of sporulation, asymmetric septation, physically separates the mother cell and forespore, enabling distinct developmental fates. We highlight the role of the asymmetric septum as an organizational hub coordinating diverse compartmentalized functions — from gene regulation to metabolism and protein synthesis machinery localization
Phosphoproteomic profiling of Siberian sturgeon (Acipenser baerii, Brandt, 1869) sperm: insights into cryopreservation-induced changes with DMSO and methanol
No full textSemen cryopreservation is a crucial technique in aquaculture, enabling the long-term preservation of genetic material from endangered species such as the Siberian sturgeon (Acipenser baerii, Brandt, 1869). While methanol (MeOH) and dimethyl sulfoxide (DMSO) similarly affect sturgeon semen quality post-thaw, DMSO significantly reduces hatching rates. Given our prior findings on cryoprotectant-specific differences in the sturgeon sperm proteome, we investigated the molecular basis of DMSO-induced dysfunction using label-free quantitative phosphoproteomics. In fresh sperm, 1649 phosphorylation sites were identified on 736 proteins, forming the most extensive database of phosphorylated sperm proteins in fish. Functional enrichment analysis showed these phosphoproteins were primarily involved in cilium organization, energy metabolism, spermatogenesis, transmembrane transport, vesicle-mediated transport and sperm binding to the zona pellucida, predominantly localizing to cytoplasm, cilia and mitochondria. Cryopreservation profoundly altered the phosphoproteome, with 269 phosphosites showing differential phosphorylation between fresh and cryopreserved samples. DMSO induced phosphorylation changes at 231 sites across 114 proteins, whereas MeOH affected 260 sites across 127 proteins. Distinct phosphorylation patterns were identified for each cryoprotectant, with shared alterations involving cilium assembly, glycolysis, phosphatidylinositol metabolism, chromatin structure and ion channel regulation associated with impaired motility, membrane destabilization and reduced acrosomal integrity. DMSO-specific changes disrupted nucleoporin structure and centriole organization while inhibiting Rho signaling, crucial for acrosomal reaction and actin filament dynamics, which likely contribute to low hatching outcomes. In contrast, MeOH uniquely enhanced AMPK signaling, promoting mitochondrial ATP buffering and energy homeostasis. This study provides the first comprehensive phosphoproteomic analysis of sturgeon sperm, linking DMSO-induced disruptions to impaired fertilization outcomes
The complex network of p300/CBP regulation: Interactions, posttranslational modifications, and therapeutic implications
Get PDFTwo closely related acetyltransferases, p300 and its paralog CBP, are important regulators of gene expression and protein modulators in higher eukaryotes, influencing a wide array of cellular processes, including cell division, growth, DNA replication and repair, and apoptosis. The broad cellular impact is underscored by p300/CBP's capacity to interact with hundreds of proteins through various domains and the capability to acetylate numerous substrates and ubiquitinate selected targets. This intricate network of interactions and modifications highlights the essential role of p300/CBP in orchestrating cellular responses to pathological and physiological stimuli, thereby necessitating precise regulatory mechanisms to maintain their activity and substrate specificity. The regulation of p300/CBP is primarily governed by protein interactions and posttranslational modifications, including acetylation and ubiquitination, with autoregulation serving as a vital component in sustaining their enzymatic functions. The significance of tightly controlled p300/CBP activity is further emphasized by its association with various diseases, including Rubinstein-Taybi syndrome, Menke-Hennekam syndrome, and numerous cancers. Furthermore, the potential of p300/CBP as a therapeutic target has sparked interest in developing specific inhibitors. This review aims to elucidate the complex regulatory mechanisms of p300/CBP, focusing on posttranslational modifications, intermolecular interactions, and their implications in disease. A comprehensive understanding of the molecular foundations of p300/CBP regulation is essential for unraveling their roles in cellular processes and advancing targeted therapeutic strategies
NMR methods for studying inclusion complexes focused on chiral hosts
Get PDFHosts, a key component of inclusion complexes, are cyclic oligomeric compounds containing a cavity. Chiral hosts are significant and interesting because they allow the study of specific intermolecular interactions and molecular recognition. The most important classes of chiral
hosts and their physicochemical properties are briefly reviewed. An important part of this Review is the description of selected concepts necessary to understand the properties and behavior of inclusion complexes studied by the most suitable analytical method for studying inclusion complexes—nuclear magnetic resonance
Disorders in the CMG helicase complex increase the proliferative capacity and delay chronological aging of budding yeast
Get PDFThe replication of DNA requires specialized and intricate machinery. This machinery is known as a replisome and is highly evolutionarily conserved, from simple unicellular organisms such as yeast to human cells. The replisome comprises multiple protein complexes responsible for various steps in the replication process. One crucial component of the replisome is the Cdc45-MCM-GINS (CMG) helicase complex, which unwinds double-stranded DNA and coordinates the assembly and function of other replisome components, including DNA polymerases. The genes encoding the CMG helicase components are essential for initiating DNA replication. In this study, we aimed to investigate how the absence of one copy of the CMG complex genes in heterozygous Saccharomyces cerevisiae cells impacts the cells' physiology and aging. Our data revealed that these cells exhibited a significant reduction in transcript levels for the respective CMG helicase complex proteins, as well as disruptions in the cell cycle, extended doubling times, and alterations in their biochemical profile. Notably, this study provided the first demonstration that cells heterozygous for genes encoding subunits of the CMG helicase exhibited a significantly increased reproductive potential and delayed chronological aging. Additionally, we observed a noteworthy correlation between RNA and polysaccharide levels in yeast and their reproductive potential, as well as a correlation between fatty acid levels and cell doubling times. Our findings also shed new light on the potential utility of yeast in investigating potential therapeutic targets for cancer treatment