Lamellipodia and Membrane Blebs Drive Efficient Electrotactic Migration of Rat Walker Carcinosarcoma Cells WC 256

The endogenous electric field (EF) may provide an important signal for directional cell migration during wound healing, embryonic development and cancer metastasis but the mechanism of cell electrotaxis is poorly understood. Additionally, there is no research addressing the question on the difference in electrotactic motility of cells representing various strategies of cell movement—specifically blebbing vs. lamellipodial migration. In the current study we constructed a unique experimental model which allowed for the investigation of electrotactic movement of cells of the same origin but representing different modes of cell migration: weakly adherent, spontaneously blebbing (BC) and lamellipodia forming (LC) WC256 cells. We report that both BC and LC sublines show robust cathodal migration in a physiological EF (1–3 V/cm). The directionality of cell movement was completely reversible upon reversing the field polarity. However, the full reversal of cell direction after the change of EF polarity was much faster in the case of BC (10 minutes) than LC cells (30 minutes). We also investigated the distinct requirements for Rac, Cdc42 and Rho pathways and intracellular Ca²⁺ in electrotaxis of WC256 sublines forming different types of cell protrusions. It was found that Rac1 is required for directional movement of LC to a much greater extent than for BC, but Cdc42 and RhoA are more crucial for BC than for LC cells. The inhibition of ROCK did not affect electrotaxis of LC in contrast to BC cells. The results also showed that intracellular Ca²⁺ is essential only for the electrotactic reaction of BC cells. Moreover, inhibition of MLCK and myosin II did not affect the electrotaxis of LC in contrast to BC cells. In conclusion, our results revealed that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of WC 256 carcinosarcoma cells, however directional migration is mediated by different signalling pathways.     

A Unique Set of the Burkholderia Collagen-Like Proteins Provides Insight into Pathogenesis,Genome Evolution and Niche Adaptation,and Infection Detection

Burkholderia pseudomallei and Burkholderia mallei, classified as category B priority pathogens, are significant human and animal pathogens that are highly infectious and broad-spectrum antibiotic resistant. Currently, the pathogenicity mechanisms utilized by Burkholderia are not fully understood, and correct diagnosis of B. pseudomallei and B. mallei infection remains a challenge due to limited detection methods. Here, we provide a comprehensive analysis of a set of 13 novel Burkholderia collagen-like proteins (Bucl) that were identified among B. pseudomallei and B. mallei select agents. We infer that several Bucl proteins participate in pathogenesis based on their noncollagenous domains that are associated with the components of a type III secretion apparatus and membrane transport systems. Homology modeling of the outer membrane efflux domain of Bucl8 points to a role in multi-drug resistance. We determined that bucl genes are widespread in B. pseudomallei and B. mallei; Fischer’s exact test and Cramer’s V² values indicate that the majority of bucl genes are highly associated with these pathogenic species versus nonpathogenic B. thailandensis. We designed a bucl-based quantitative PCR assay which was able to detect B. pseudomallei infection in a mouse with a detection limit of 50 CFU. Finally, chromosomal mapping and phylogenetic analysis of bucl loci revealed considerable genomic plasticity and adaptation of Burkholderia spp. to host and environmental niches. In this study, we identified a large set of phylogenetically unrelated bucl genes commonly found in Burkholderia select agents, encoding predicted pathogenicity factors, detection targets, and vaccine candidates.     

Endogenous Prostaglandins and Afferent Sensory Nerves in Gastroprotective Effect of Hydrogen Sulfide against Stress-Induced Gastric Lesions

Hydrogen sulfide (H₂S) plays an important role in human physiology, exerting vasodilatory, neuromodulatory and anti-inflammatory effects. H₂S has been implicated in the mechanism of gastrointestinal integrity but whether this gaseous mediator can affect hemorrhagic lesions induced by stress has been little elucidated. We studied the effect of the H₂S precursor L-cysteine, H₂S-donor NaHS, the H₂S synthesizing enzyme (CSE) activity inhibitor- D,L-propargylglycine (PAG) and the gastric H₂S production by CSE/CBS/3-MST activity in water immersion and restraint stress (WRS) ulcerogenesis and the accompanying changes in gastric blood flow (GBF). The role of endogenous prostaglandins (PGs) and sensory afferent nerves releasing calcitonin gene-related peptide (CGRP) in the mechanism of gastroprotection induced by H₂S was examined in capsaicin-denervated rats and those pretreated with capsazepine to inhibit activity of vanilloid receptors (VR-1). Rats were pretreated with vehicle, NaHS, the donor of H₂S and or L-cysteine, the H₂S precursor, with or without the concurrent treatment with 1) nonselective (indomethacin) and selective cyclooxygenase (COX)-1 (SC-560) or COX-2 (rofecoxib) inhibitors. The expression of mRNA and protein for COX-1 and COX-2 were analyzed in gastric mucosa pretreated with NaHS with or without PAG. Both NaHS and L-cysteine dose-dependently attenuated severity of WRS-induced gastric lesions and significantly increased GBF. These effects were significantly reduced by pretreatment with PAG and capsaicin denervation. NaHS increased gastric H₂S production via CSE/CBS but not 3-MST activity. Inhibition of COX-1 and COX-2 activity significantly diminished NaHS- and L-cysteine-induced protection and hyperemia. NaHS increased expression of COX-1, COX-2 mRNAs and proteins and raised CGRP mRNA expression. These effects of NaHS on COX-1 and COX-2 protein contents were reversed by PAG and capsaicin denervation. We conclude that H₂S exerts gastroprotection against WRS-induced gastric lesions by the mechanism involving enhancement in gastric microcirculation mediated by endogenous PGs, sensory afferent nerves releasing CGRP and the activation of VR-1 receptors.     

Maternal Plasma and Amniotic Fluid Sphingolipids Profiling in Fetal Down Syndrome

IntroductionSphingolipids can be potentially involved in the formation of the central and peripheral nervous systems, which are particularly connected with the pathogenesis of Down syndrome. The aim of the study was to determine the concentration of selected sphingolipids in the plasma and amniotic fluid of pregnant patients with fetal Down syndrome.ResultsWe showed a significant increase in the concentration of 2 ceramides, C22-Cer and C24:1-Cer, in the plasma of women with fetal Down syndrome. Furthermore we showed a decrease in the concentration of 7 ceramides—C16-Cer, C18-Cer, C18:1-Cer, C20-Cer, C22-Cer, C24:1-Cer, and C24-Cer—in the amniotic fluid of women with fetal Down syndrome. We created ROC curves for all significant sphingolipids in maternal plasma, which set the threshold values and allowed for predicting the likelihood of Down syndrome in the fetus with specific sensitivity and specificity. We demonstrated a significantly higher risk of Down syndrome when the plasma concentration of C22-Cer > 12.66 ng/100ul (sens. 0.9, sp. 0.79, P value = 0.0007) and C24:1-Cer > 33,19 ng/100ul (sens. 0.6, sp. 0.86, P value = 0.0194).ConclusionOn the basis of our findings, it seems that the sphingolipids may play a role in the pathogenesis of Down syndrome. Defining their potential as biochemical markers of Down syndrome requires further investigation on a larger group of patients.     

Proteomic characterization of biogenesis and functions of matrix vesicles released from mineralizing human osteoblast-like cells

Matrix vesicles (MVs), released by budding from apical microvilli of osteoblasts during bone formation and development, are involved in the initiation of mineralization by promoting the formation of hydroxyapatite in their lumen. To gain additional insights into MV biogenesis and functions, MVs and apical microvilli were co-isolated from mineralizing osteoblast-like Saos-2 cells and their proteomes were characterized using LC-ESI-MS/MS and compared. In total, 282 MV and 451 microvillar proteins were identified. Of those, 262 were common in both preparations, confirming that MVs originate from apical microvilli. The occurrence of vesicular trafficking molecules (e.g. Rab proteins) and of the on-site protein synthetic machinery suggests that cell polarization and apical targeting are required for the incorporation of specific lipids and proteins at the site of MV formation. MV release from microvilli may be driven by actions of actin-severing proteins (gelsolin, cofilin 1) and contractile motor proteins (myosins). In addition to the already known proteins involved in MV-mediated mineralization, new MV residents were detected, such as inorganic pyrophosphatase 1, SLC4A7 sodium bicarbonate cotransporter or sphingomyelin phosphodiesterase 3, providing additional insights into MV functions.     

Aspergillus Collagen-Like Genes (acl): Identification,Sequence Polymorphism,and Assessment for PCR-Based Pathogen Detection

The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects.