47,767 research outputs found

    Identification and characterization of proteins involved in the cytoskeletal rearrangements caused by bacterial pathogens

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    Bacterial pathogens have evolved to alter the cytoskeleton of their hosts during their respective infection processes. The extracellular bacterium, enteropathogenic Escherichia coli (EPEC), generates an actin-rich pedestal to ‚Äúsurf‚ÄĚ along the host cell surface. In contrast, L. monocytogenes (L. monocytogenes) invades its host and polymerizes actin filaments to generate a comet tail for movement within and among host cells of epithelia. Salmonella enterica serovar Typhimurium (S. Typhimurium) induces actin-rich membrane-ruffles to invade its host cell. These bacteria have evolved to generate their respective actin-rich structures to colonize the intestinal epithelia. To further characterize the actin-rich structures generated by these bacteria, I selected four proteins from a mass spectrometry analysis of EPEC pedestals previously conducted in our laboratory. I found that the known actin-bundling proteins calponin 1 and calponin 2 decorated all the actin-rich structures formed by these three bacteria. Another actin-stabilizing protein transgelin (SM22) also decorated EPEC pedestals and L. monocytogenes comet tails. Moreover, the formation of pedestals and comet tails were dependent on SM22 protein levels. Aside from these three members of the calponin family, I found that a ubiquitin conjugating enzyme Ube2N was enriched at the invasion events and at the plasma membrane-bound comet tails formed by L. monocytogenes. This novel association of Ube2N with actin structures at the plasma membrane led to my discovering that Ube2N binds directly to actin, and that Ube2N function influences actin-based whole cell motility. Another bacterial pathogen, Klebsiella pneumoniae (K. pneumoniae), has been shown by others to alter the host actin cytoskeleton. I have found that the disassembly of the host microtubule networks precedes these actin cytoskeletal alterations in lung epithelial cells, and show that the Klebsiella pneumoniae gene ytfL (Kp ytfL) initiates this microtubule disassembly and that the katanin catalytic subunit A like 1 protein (KATNAL1) as well as the katanin regulatory subunit B1 protein (KATNB1) are activated to cause microtubule severing. Through this, I identified the bacterial initiator and the host cell effector proteins responsible for K. pneumoniae-induced microtubule disassembly. From these, I identified proteins that are novel to the actin structures of EPEC, L. monocytogenes and S. Typhimurium as well as effector proteins that are crucial for the novel host microtubule alterations of K. pneumoniae

    Papel da acetilação N-terminal na fertilidade e no envelhecimento

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    A acetila√ß√£o do N-terminal das prote√≠nas tem um papel crucial em diferentes eventos celulares e moleculares, sendo umas das modifica√ß√Ķes proteicas mais abundantes em c√©lulas eucariotas. As acetitltranferases N-terminal (NATs) s√£o constitu√≠das por uma subunidade catal√≠tica e em alguns casos por uma ou mais unidades reguladoras possuindo, portanto, caracter√≠sticas individuais e atuando num conjunto de substratos espec√≠ficos. A acetila√ß√£o do N-terminal pode influenciar a fun√ß√£o e a localiza√ß√£o proteicas de diferentes formas, podendo afetar o normal desenvolvimento de organismos e a progress√£o tumoral. Este trabalho tem por objetivo caracterizar a fun√ß√£o do complexo NatC (Naa30) em Drosophila melanogaster. Naa30 √© necess√°rio para a longevidade e a perda de motilidade com a idade de moscas adultas. Naa30 √© tamb√©m necess√°rio para a fertilidade dos machos uma vez que √© essencial √† copula√ß√£o. A perda da fun√ß√£o de Naa30 est√° associado a um aumento de agregados de prote√≠nas poli-ubiquitinadas nos m√ļsculos, o que sugere que os fen√≥tipos observados est√£o associados a defeitos de proteostase. No entanto, como em moscas velhas, o grau de perda de motilidade n√£o est√° associado a n√≠veis mais elevados de agregados, esta acumula√ß√£o n√£o √© provavelmente causal para os fen√≥tipos observados. Apesar das mitoc√īndrias serem aparentemente morfologicamente normais foi igualmente observado uma redu√ß√£o dos n√≠veis de ATP em mutantes de Naa30. Conclu√≠mos ent√£o que a redu√ß√£o de longevidade, fertilidade e motilidade em mutantes de Naa30 est√£o potencialmente relacionados com defeitos de proteostase e uma redu√ß√£o dos n√≠veis celulares de ATP. Em linhas celulares humanas, NatF (Naa60) √© importante para a modifica√ß√£o de prote√≠nas transmembranares e para a integridade do Golgi. Uma vez que a fun√ß√£o desta NAT em organismos multicelulares √© pouco conhecida, um outro objetivo deste trabalho foi fazer uma caracteriza√ß√£o fenot√≠pica de um mutante do gene Naa60. Observou-se que em Drosophila, Naa60 n√£o √© essencial para a viabilidade de moscas adultas, nem para a fertilidade dos machos. Apesar da dele√ß√£o desta NAT estar associada a uma ligeira redu√ß√£o da fertilidade das f√™meas, n√£o foram detetados fen√≥tipos √≥bvios durante a oog√©nese e os est√°dios iniciais de desenvolvimento do embri√£o.N-terminal acetylation of proteins plays a crucial role in different cellular and molecular events, being one of the most abundant protein modifications in eukaryotic cells. N-terminal acetityltransferases (NATs) are made up of a catalytic subunit and in some cases one or more regulatory units having therefore individual characteristics and acting on a set of specific substrates. N-terminal acetylation may influence protein function and localization in different ways and may affect normal organism development and tumour progression. One of the aims of this work was to characterize the function of NatC (Naa30) in Drosophila melanogaster. Naa30 is required for the longevity and age-dependent motility of adult flies. Naa30 is also required for male fertility as it is essential for copulation. The loss of Naa30 function is associated with an increase in polyubiquitinated protein like-aggregates in the muscles, suggesting that the observed phenotypes result due to proteostasis defects. However, as the degree of motility loss is not associated with higher levels of polyubiquitinated protein like-aggregates, their accumulation is probably not causal for the observed phenotypes. Although the mitochondria were morphological normal, a reduction in ATP levels was observed in Naa30 mutants. We conclude that the reduction in longevity, fertility and motility in Naa30 mutants are potentially related to proteostasis defects and a reduction in cellular ATP levels. NatF (Naa60) has been previously described in human cell lines as being important for transmembrane protein modification and Golgi integrity. Since the role of this NAT during the development of multicellular organisms is poorly described, another aim of this work was to make a phenotypic characterization of a Naa60 deletion mutant. It was observed that in Drosophila, Naa60 is not essential for the viability of adult flies, nor for male fertility. Although deletion of this NAT is associated with a slight reduction in female fertility, no obvious phenotypes were detected during oogenesis and early stages of embryo development

    The Effect of Flagella Stiffness on the Locomotion of a Multi-Flagellated Robot at Low Reynolds Environment

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    Microorganisms such as algae and bacteria move in a viscous environment with extremely low Reynolds (ReRe), where the viscous drag dominates the inertial forces. They have adapted to this environment by developing specialized features such as whole-body deformations and flexible structures such as flagella (with various shapes, sizes, and numbers) that break the symmetry during the motion. In this study, we hypothesize that the changes in the flexibility of the flagella during a cycle of movement impact locomotion dynamics of flagellated locomotion. To test our hypothesis, we developed an autonomous, self-propelled robot with four flexible, multi-segmented flagella actuated together by a single DC motor. The stiffness of the flagella during the locomotion is controlled via a cable-driven mechanism attached to the center of the robot. Experimental assessments of the robot's swimming demonstrate that increasing the flexibility of the flagella during recovery stroke and reducing the flexibility during power stroke improves the swimming performance of the robot. Our results give insight into how these microorganisms manipulate their biological features to propel themselves in low viscous media and are of great interest to biomedical and research applications

    Antimicrobial resistance and molecular characterization of Escherichia coli isolated from bovine mastitis samples in Nghe An province, Vietnam

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    Background and Aim: Vietnam’s dairy sector is in its early phase of large-scale farming development. Therefore, mastitis in cows is always a concern to farm owners. This study aimed to determine the antimicrobial susceptibility, resistance, and virulence-related genes of Escherichia coli isolated from bovine mastitis in Nghe An province of Vietnam. Materials and Methods: Fifty E. coli strains were isolated from the clinical cases and subjected to this study. All isolates were tested for antimicrobial susceptibility by the disk-diffusion method, as described by the Clinical and Laboratory Standards Institute. Antimicrobial and virulence genes were confirmed by polymerase chain reaction with specific primers. Results: All isolates were resistant to lincomycin and sulfamethoxazole and sensitive to gentamicin, while other antimicrobials showed resistance from 2% to 90%. Multidrug resistance was confirmed in 46% of isolates, and none of them were identified as extended-spectrum beta-lactamase producers. From fifty strains tested for antimicrobial and virulence genes, six isolates harbored tetA, 6 tetB, 13 sul1, 15 sul2, 2 Intimin (eae), 1 iutA, and 3 stx2. Conclusion: Antimicrobial and multidrug resistances are the main virulence factors of E. coli isolated from bovine mastitis in Vietnam. The virulence genes encoding adhesion, siderophore, Shiga-toxin-producing, and antimicrobials resistant were first reported in Vietnam with low prevalence and contributed to the pathogenesis

    <i>Cn</i>Cel1 affects melanization and capsule architecture.

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    (A) Melanization in the absence and presence of exogenous copper. Indicated strains were incubated on L-DOPA plates at 30¬įC or 37¬įC, with and without supplementation with exogenous CuSO4. Melanin formation was assessed at 1‚Äď2 days as indicated. (B-E) Surface capsule formation and architecture. The WT and cel1őĒ mutant were incubated for 3 days in CIM tissue culture medium to induce capsule. Surface capsule characteristics were assessed by (B) India ink counter-staining and (C) SEM. Representative images are shown from 3 independent experiments. Quantification of capsule size (D) and cell body size (E) was performed using ImageJ/Fiji from static images of india ink-stained cells. Mean values from >100 cells per sample (+/- SEM) from 3 biological replicates are shown as bar graph. Data were plotted, and an unpaired t-test was performed using GraphPad Prism. (F) Electromobility of exopolysaccharide. The WT and cel1őĒ mutant were incubated in CIM for 3 days. Exopolysaccharide in the culture supernatant was assessed for relative size and gel motility by agarose gel electrophoresis, transferred to a Nylon membrane, and probed with anti-GXM antibody 18B7. Representative image of multiple replicates is shown.</p

    Holistic analysis of lysine acetylation in aquaculture pathogenic bacteria Vibrio alginolyticus under bile salt stress

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    Lysine acetylation modification is a dynamic and reversible post-translational modification, which plays an important role in the metabolism and pathogenicity of pathogenic bacteria. Vibrio alginolyticus is a common pathogenic bacterium in aquaculture, and bile salt can trigger the expression of bacterial virulence. However, little is known about the function of lysine acetylation in V. alginolyticus under bile salt stress. In this study, 1,315 acetylated peptides on 689 proteins were identified in V. alginolyticus under bile salt stress by acetyl-lysine antibody enrichment and high-resolution mass spectrometry. Bioinformatics analysis found that the peptides motif ****A*Kac**** and *******Kac****A* were highly conserved, and protein lysine acetylation was involved in regulating various cellular biological processes and maintaining the normal life activities of bacteria, such as ribosome, aminoacyl-tRNA biosynthesis, fatty acid metabolism, two-component system, and bacterial secretion system. Further, 22 acetylated proteins were also found to be related to the virulence of V. alginolyticus under bile salt stress through secretion system, chemotaxis and motility, and adherence. Finally, comparing un-treated and treated with bile salt stress lysine acetylated proteins, it was found that there were 240 overlapping proteins, and found amino sugar and nucleotide sugar metabolism, beta-Lactam resistance, fatty acid degradation, carbon metabolism, and microbial metabolism in diverse environments pathways were significantly enriched in bile salt stress alone. In conclusion, this study is a holistic analysis of lysine acetylation in V. alginolyticus under bile salt stress, especially many virulence factors have also acetylated

    Transcriptome sequencing reveals the effects of circRNA on testicular development and spermatogenesis in Qianbei Ma goats

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    Circular RNAs (circRNAs) play an important role in regulating the mammalian reproductive system, especially testicular development and spermatogenesis. However, their functions in testicular development and spermatogenesis in the Qianbei Ma goat, the Guizhou endemic breed are still unclear. In this study, tissue sectioning and circRNAs transcriptome analysis were conducted to compare the changes of morphology and circular RNAs gene expression profile at four different developmental stages (0Y, 0-month-old; 6Y, 6-month-old; 12Y, 12-month-old; 18Y, 18-month-old). The results showed that the circumferences and area of the seminiferous tubule gradually increased with age, and the lumen of the seminiferous tubule in the testis differentiated significantly. 12,784 circRNAs were detected from testicular tissues at four different developmental stages by RNA sequencing, and 8,140 DEcircRNAs (differentially expressed circRNAs) were found in 0Y vs. 6Y, 6Y vs. 12Y, 12Y vs. 18Y and 0Y vs. 18Y, 0Y vs. 12Y, 6Y vs. 18Y Functional enrichment analysis of the source genes showed that they were mainly enriched in testicular development and spermatogenesis. In addition, the miRNAs and mRNAs associated with DECircRNAs in 6 control groups were predicted by bioinformatics, and 81 highly expressed DECircRNAs and their associated miRNAs and mRNAs were selected to construct the ceRNA network. Through functional enrichment analysis of the target genes of circRNAs in the network, some candidate circRNAs related to testicular development and spermatogenesis were obtained. Such as circRNA_07172, circRNA_04859, circRNA_07832, circRNA_00032 and circRNA_07510. These results will help to reveal the mechanism of circRNAs in testicular development and spermatogenesis, and also provide some guidance for goat reproduction

    Unravelling the Effects of Syndecan-4 Knockdown on Skeletal Muscle Functions

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    The remodelling of the extracellular matrix plays an important role in skeletal muscle development and regeneration. Syndecan-4 is a cell surface proteoglycan crucial for muscle differentiation. Syndecan-4‚ąí/‚ąí mice have been reported to be unable to regenerate following muscle damage. To investigate the consequences of the decreased expression of Syndecan-4, we have studied the in vivo and in vitro muscle performance and the excitation‚Äďcontraction coupling machinery in young and aged Syndecan-4+/‚ąí (SDC4) mice. In vivo grip force was decreased significantly as well as the average and maximal speed of voluntary running in SDC4 mice, regardless of their age. The maximal in vitro twitch force was reduced in both EDL and soleus muscles from young and aged SDC4 mice. Ca2+ release from the sarcoplasmic reticulum decreased significantly in the FDB fibres of young SDC4 mice, while its voltage dependence was unchanged regardless of age. These findings were present in muscles from young and aged mice as well. On C2C12 murine skeletal muscle cells, we have also found altered calcium homeostasis upon Syndecan-4 silencing. The decreased expression of Syndecan-4 leads to reduced skeletal muscle performance in mice and altered motility in C2C12 myoblasts via altered calcium homeostasis. The altered muscle force performance develops at an early age and is maintained throughout the life course of the animal until old age

    Development of In Vitro Assays with the Canine Hookworm <i>Uncinaria stenocephala</i> and Assessment of Natural Plant Products for Anti-Parasitic Activity

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    Enteric helminth infection is an increasing concern in companion animals due to reports of resistance to commonly used anthelmintic drugs. Thus, the assessment of new therapeutic options such as bioactive dietary additives is of high importance. Here, we adapted egg hatch, larval migration, and larval motility assays to screen extracts of several natural ingredients against the canine hookworm Uncinaria stenocephala, a prevalent parasite of dogs in northern Europe. Egg hatch and larval migration assays were established showing that the anthelmintic drugs levamisole and albendazole had strong anti-parasitic activity against U. stenocephala, validating the use of these assays for the assessment of novel anti-parasitic substances. Subsequently, we identified that extracts from the seaweed Saccharina latissima, but not extracts from grape seed or chicory, significantly inhibited both hatching and larval migration. Finally, we showed that őĪ-linolenic acid, a putative anti-parasitic compound from S. latissima, also exhibited anti-parasitic activity. Collectively, our results established a platform for the screening for anthelmintic resistance or novel drug candidates against U. stenocephala and highlighted the potential use of seaweed extracts as a functional food component to help control hookworm infection in dogs
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