Identification and distribution of anaerobic bacteria isolated from clinical specimens in a University Hospital: 4 years’ experience

Anaerobes, which are components of microbiota, can cause life-threatening infections. Because of their fastidious nature, they are difficult to isolate and are often overlooked. The goal of this study was to identify the anaerobic bacteria isolated from clinical specimens at the Central Laboratory of Hacettepe University Hospital in 2015-2018 and to evaluate the distribution of the isolated bacterial species among the different specimen types. The anaerobic bacteria isolated from the specimens were identified by the conventional methods and MALDI-TOF MS.Overall, 15,300 anaerobic cultures were studied. Of these, 14,434 (94.3%) were blood samples and 866 (5.7%) were other clinical specimens. A total of 138 anaerobic bacteria were isolated: 62 (44.9%) were isolated from blood samples and 76 (55.1%) from other specimens. The most isolated anaerobes from blood cultures were Bacteroides spp. (41.9%), followed by Cutibacterium acnes (25.8%) and Clostridium spp. (9.7%). The most isolated anaerobes from the other specimens were Gram-negative bacilli, including Bacteroides spp. (15.8%), Fusobacterium spp. (14.5%), Prevotella spp. (14.5%), and Porphyromonas spp. (2.6%). Anaerobic Finegoldia magna represented the major species among the isolated Gram-positive bacteria (10.5%). Anaerobic growth was observed in 0.4% of all the blood cultures and in 5.8% of the positive blood cultures. The results of our study showed that the incidence of anaerobic bacteremia was stable during the 2015-2018 period.Anaerobes, which are components of microbiota, can cause life-threatening infections. Because of their fastidious nature, they are difficult to isolate and are often overlooked. The goal of this study was to identify the anaerobic bacteria isolated from clinical specimens at the Central Laboratory of Hacettepe University Hospital in 2015-2018 and to evaluate the distribution of the isolated bacterial species among the different specimen types. The anaerobic bacteria isolated from the specimens were identified by the conventional methods and MALDI-TOF MS.Overall, 15,300 anaerobic cultures were studied. Of these, 14,434 (94.3%) were blood samples and 866 (5.7%) were other clinical specimens. A total of 138 anaerobic bacteria were isolated: 62 (44.9%) were isolated from blood samples and 76 (55.1%) from other specimens. The most isolated anaerobes from blood cultures were Bacteroides spp. (41.9%), followed by Cutibacterium acnes (25.8%) and Clostridium spp. (9.7%). The most isolated anaerobes from the other specimens were Gram-negative bacilli, including Bacteroides spp. (15.8%), Fusobacterium spp. (14.5%), Prevotella spp. (14.5%), and Porphyromonas spp. (2.6%). Anaerobic Finegoldia magna represented the major species among the isolated Gram-positive bacteria (10.5%). Anaerobic growth was observed in 0.4% of all the blood cultures and in 5.8% of the positive blood cultures. The results of our study showed that the incidence of anaerobic bacteremia was stable during the 2015-2018 period

Multi-aspect analysis of ureteral access sheath usage in retrograde intrarenal surgery: A RIRSearch group study

Objective: To evaluate the effect of ureteral access sheath (UAS) use and calibration change on stone-free rate and complications of retrograde intrarenal surgery (RIRS). Methods: Data from 568 patients undergoing RIRS for kidney or upper ureteral stones were retrospectively included. Firstly, patients were compared after 1:1 propensity score matching, according to UAS usage during RIRS (UAS used [+] 87 and UAS non-used [?] 87 patients). Then all UAS+ patients (n=481) were subdivided according to UAS calibration: 9.5–11.5 Fr, 10–12 Fr, 11–13 Fr, and 13–15 Fr. Primary outcomes of the study were the success and complications of RIRS. Results: Stone-free rate of UAS+ patients (86.2%) was significantly higher than UAS? patients (70.1%) after propensity score matching (p=0.01). Stone-free rate increased with higher caliber UAS (9.5–11.5 Fr: 66.7%; 10–12 Fr: 87.3%; 11–13 Fr: 91.3%; 13–15 Fr: 100%; p<0.0001). Postoperative complications of UAS+ patients (11.5%) were significantly lower than UAS? patients (27.6%) (p=0.01). Complications (8.7%) with 9.5–11.5 Fr UAS was lower than thicker UAS (17.3%) but was not statistically significant (p=0.08). UAS usage was an independent factor predicting stone-free status or peri- and post-operative complications (odds ratio [OR] 3.654, 95% confidence interval [CI] 1.314–10.162; OR 4.443, 95% CI 1.350–14.552; OR 4.107, 95% CI 1.366–12.344, respectively). Conclusion: Use of UAS in RIRS may increase stone-free rates, which also increase with higher caliber UAS. UAS usage may reduce complications; however, complications seemingly increase with higher UAS calibration. © 2022 Editorial Office of Asian Journal of Urolog

Analyses of the mechanical, electrical and electromagnetic shielding properties of thermoplastic composites doped with conductive nanofillers

The purpose of this study is to observe effect of incorporating vapor-grown carbon nanofibers with various amounts in polyvinylidene fluoride matrix in terms of mechanical strength and electromagnetic shielding effectiveness. Thermoplastic conductive nanocomposites were prepared by heat-pressed compression molding. Vapor-grown carbon nanofibers were utilized at various weight ratios (1 wt.%, 3 wt.%, 5 wt.%, and 8 wt.%) as conductive and reinforcing materials. Polyvinylidene fluoride was used as a thermoplastic polymer matrix. Scanning electron microscopic analysis was conducted in order to characterize the morphology and structural properties of the nanocomposites and results revealed well dispersion of carbon nanofibers within the matrix for all concentrations. Mechanical characteristics were investigated according to standards. Findings proved that overall increments of 16%, 37.5%, and 56% were achieved in terms of tensile strength, elasticity modulus, and impact energy, respectively, where a total reduction of 44.8% was observed in terms of elongation for 8 wt.% vapor-grown nanofiber matrix compared to that of 0 wt.%. Electromagnetic shielding effectiveness's of the nanocomposites were determined by standard protocol using coaxial transmission line measurement technique in the frequency range of 15–3000 MHz. It was observed that resistance, sheet resistance, and resistivity of nanocomposites depicted substantial reduction with the increment in nanofiber content. Nevertheless, it was observed that nanofiber content, dispersion, and network formation within the composites were highly influent on the electromagnetic shielding effectiveness performance of the structures

Transcriptional regulation of the IGF signaling pathway by amino acids and insulin-like growth factors during myogenesis in Atlantic salmon

The insulin-like growth factor signalling pathway is an important regulator of skeletal muscle growth. We examined the mRNA expression of components of the insulin-like growth factor (IGF) signalling pathway as well as Fibroblast Growth Factor 2 (FGF2) during maturation of myotubes in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon (Salmo salar). The transcriptional regulation of IGFs and IGFBP expression by amino acids and insulin-like growth factors was also investigated. Proliferation of cells was 15% d(-1) at days 2 and 3 of the culture, increasing to 66% d(-1) at day 6. Three clusters of elevated gene expression were observed during the maturation of the culture associated with mono-nucleic cells (IGFBP5.1 and 5.2, IGFBP-6, IGFBP-rP1, IGFBP-2.2 and IGF-II), the initial proliferation phase (IGF-I, IGFBP-4, FGF2 and IGF-IRb) and terminal differentiation and myotube production (IGF2R, IGF-IRa). In cells starved of amino acids and serum for 72 h, IGF-I mRNA decreased 10-fold which was reversed by amino acid replacement. Addition of IGF-I and amino acids to starved cells resulted in an 18-fold increase in IGF-I mRNA indicating synergistic effects and the activation of additional pathway(s) leading to IGF-I production via a positive feedback mechanism. IGF-II, IGFBP-5.1 and IGFBP-5.2 expression was unchanged in starved cells, but increased with amino acid replacement. Synergistic increases in expression of IGFBP5.2 and IGFBP-4, but not IGFBP5.1 were observed with addition of IGF-I, IGF-II or insulin and amino acids to the medium. IGF-I and IGF-II directly stimulated IGFBP-6 expression, but not when amino acids were present. These findings indicate that amino acids alone are sufficient to stimulate myogenesis in myoblasts and that IGF-I production is controlled by both endocrine and paracrine pathways. A model depicting the transcriptional regulation of the IGF pathway in Atlantic salmon muscle following feeding is proposed.Publisher PDFPeer reviewe

Functional characterisation of the TSC1–TSC2 complex to assess multiple TSC2 variants identified in single families affected by tuberous sclerosis complex

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by seizures, mental retardation and the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, interact to form a protein complex that inhibits signal transduction to the downstream effectors of the mammalian target of rapamycin (mTOR). METHODS: We have used a combination of different assays to characterise the effects of a number of pathogenic TSC2 amino acid substitutions on TSC1-TSC2 complex formation and mTOR signalling. RESULTS: We used these assays to compare the effects of 9 different TSC2 variants (S132C, F143L, A196T, C244R, Y598H, I820del, T993M, L1511H and R1772C) identified in individuals with symptoms of TSC from 4 different families. In each case we were able to identify the pathogenic mutation. CONCLUSION: Functional characterisation of TSC2 variants can help identify pathogenic changes in individuals with TSC, and assist in the diagnosis and genetic counselling of the index cases and/or other family members

The Mitochondrial Ca(2+) Uniporter: Structure, Function, and Pharmacology.

Mitochondrial Ca(2+) uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca(2+) uptake and our current understanding of mitochondrial Ca(2+) homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca(2+) uniporter complex

Proton-Assisted Amino Acid Transporter PAT1 Complexes with Rag GTPases and Activates TORC1 on Late Endosomal and Lysosomal Membranes

Mammalian Target of Rapamycin Complex 1 (mTORC1) is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K)/Akt/Rheb signalling and extracellular amino acids (AAs) to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs), subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. We have recently shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1)/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. Here we demonstrate in Human Embryonic Kidney (HEK-293) cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. We also use the powerful in vivo genetic methodologies available in Drosophila to investigate the regulation of the PAT1/Rag/Ragulator complex. We show that GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H+-ATPase as another Rag-interacting component, we propose a model in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments

Regulation of mTORC1 Signaling by pH

BACKGROUND: Acidification of the cytoplasm and the extracellular environment is associated with many physiological and pathological conditions, such as intense exercise, hypoxia and tumourigenesis. Acidification affects important cellular functions including protein synthesis, growth, and proliferation. Many of these vital functions are controlled by mTORC1, a master regulator protein kinase that is activated by various growth-stimulating signals and inactivated by starvation conditions. Whether mTORC1 can also respond to changes in extracellular or cytoplasmic pH and play a role in limiting anabolic processes in acidic conditions is not known. METHODOLOGY/FINDINGS: We examined the effects of acidifying the extracellular medium from pH 7.4 to 6.4 on human breast carcinoma MCF-7 cells and immortalized mouse embryo fibroblasts. Decreasing the extracellular pH caused intracellular acidification and rapid, graded and reversible inhibition of mTORC1, assessed by measuring the phosphorylation of the mTORC1 substrate S6K. Fibroblasts deleted of the tuberous sclerosis complex TSC2 gene, a major negative regulator of mTORC1, were unable to inhibit mTORC1 in acidic extracellular conditions, showing that the TSC1-TSC2 complex is required for this response. Examination of the major upstream pathways converging on the TSC1-TSC2 complex showed that Akt signaling was unaffected by pH but that the Raf/MEK/ERK pathway was inhibited. Inhibition of MEK with drugs caused only modest mTORC1 inhibition, implying that other unidentified pathways also play major roles. CONCLUSIONS: This study reveals a novel role for the TSC1/TSC2 complex and mTORC1 in sensing variations in ambient pH. As a common feature of low tissue perfusion, low glucose availability and high energy expenditure, acidic pH may serve as a signal for mTORC1 to downregulate energy-consuming anabolic processes such as protein synthesis as an adaptive response to metabolically stressful conditions

Diet and Energy-Sensing Inputs Affect TorC1-Mediated Axon Misrouting but Not TorC2-Directed Synapse Growth in a Drosophila Model of Tuberous Sclerosis

The Target of Rapamycin (TOR) growth regulatory system is influenced by a number of different inputs, including growth factor signaling, nutrient availability, and cellular energy levels. While the effects of TOR on cell and organismal growth have been well characterized, this pathway also has profound effects on neural development and behavior. Hyperactivation of the TOR pathway by mutations in the upstream TOR inhibitors TSC1 (tuberous sclerosis complex 1) or TSC2 promotes benign tumors and neurological and behavioral deficits, a syndrome known as tuberous sclerosis (TS). In Drosophila, neuron-specific overexpression of Rheb, the direct downstream target inhibited by Tsc1/Tsc2, produced significant synapse overgrowth, axon misrouting, and phototaxis deficits. To understand how misregulation of Tor signaling affects neural and behavioral development, we examined the influence of growth factor, nutrient, and energy sensing inputs on these neurodevelopmental phenotypes. Neural expression of Pi3K, a principal mediator of growth factor inputs to Tor, caused synapse overgrowth similar to Rheb, but did not disrupt axon guidance or phototaxis. Dietary restriction rescued Rheb-mediated behavioral and axon guidance deficits, as did overexpression of AMPK, a component of the cellular energy sensing pathway, but neither was able to rescue synapse overgrowth. While axon guidance and behavioral phenotypes were affected by altering the function of a Tor complex 1 (TorC1) component, Raptor, or a TORC1 downstream element (S6k), synapse overgrowth was only suppressed by reducing the function of Tor complex 2 (TorC2) components (Rictor, Sin1). These findings demonstrate that different inputs to Tor signaling have distinct activities in nervous system development, and that Tor provides an important connection between nutrient-energy sensing systems and patterning of the nervous system

A Genome-Wide Immunodetection Screen in S. cerevisiae Uncovers Novel Genes Involved in Lysosomal Vacuole Function and Morphology

Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface – ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY). Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes – MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events