ResearchPad - cellular-structures-and-organelles https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Genetic diversity of <i>Echinococcus multilocularis</i> and <i>Echinococcus granulosus sensu lato</i> in Kyrgyzstan: The A2 haplotype of <i>E</i>. <i>multilocularis</i> is the predominant variant infecting humans]]> https://www.researchpad.co/article/elastic_article_13871 Analysis of the genetic variability in Echinococcus species from different endemic countries have contributed to the knowledge in the taxonomy and phylogeography of these parasites. The most important species of this genus, Echinococcus granulosus sensu lato and Echinococcus multilocularis, co-exist in Kyrgyzstan causing serious public health issues. E. granulosus s.l. causes cystic echinococcosis and E. multilocularis is the causative agent of alveolar echinococcosis. The most relevant finding of our study is the identification of the cob/nad2/cox1 A2 haplotype of E. multilocularis as the most commonly found in humans and dogs. However, it remains unknown if this variant of E. multilocularis, based on genetic differences in mitochondrial genes, presents differences in virulence which could have contributed to the emergence of alveolar echinococcosis in Kyrgyzstan. The results also show a number of non-previously described genetic variants of E. multilocularis and E. granulosus s.s.

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<![CDATA[Betanin purification from red beetroots and evaluation of its anti-oxidant and anti-inflammatory activity on LPS-activated microglial cells]]> https://www.researchpad.co/article/elastic_article_13861 Microglial activation can release free radicals and various pro-inflammatory cytokines, which implicates the progress of a neurodegenerative disease. Therefore suppression of microglial activation can be an appropriate strategy for combating neurodegenerative diseases. Betanin is a red food dye that acts as free radical scavenger and can be a promising candidate for this purpose. In this study, purification of betanin from red beetroots was carried out by normal phase colum chromatography, yielding 500 mg of betanin from 100 g of red beetroot. The purified betanin was evaluated by TLC, UV-visible, HPLC, ESI-MASS, FT-IR spectroscopy. Investigation on the inhibitory effect of betanin on activated microglia was performed using primary microglial culture. The results showed that betanin significantly inhibited lipopolysaccharide induced microglial function including the production of nitric oxide free radicals, reactive oxygen species, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β). Moreover, betanin modulated mitochondrial membrane potential, lysosomal membrane permeabilization and adenosine triphosphate. We further investigated the interaction of betanin with TNF-α, IL-6 and Nitric oxide synthase (iNOS or NOS2) using in silico molecular docking analysis. The docking results demonstrated that betanin have significant negative binding energy against active sites of TNF-α, IL-6 and iNOS.

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<![CDATA[Host interactors of effector proteins of the lettuce downy mildew <i>Bremia lactucae</i> obtained by yeast two-hybrid screening]]> https://www.researchpad.co/article/elastic_article_13834 Plant pathogenic bacteria, fungi and oomycetes secrete effector proteins to manipulate host cell processes to establish a successful infection. Over the last decade the genomes and transcriptomes of many agriculturally important plant pathogens have been sequenced and vast candidate effector repertoires were identified using bioinformatic analyses. Elucidating the contribution of individual effectors to pathogenicity is the next major hurdle. To advance our understanding of the molecular mechanisms underlying lettuce susceptibility to the downy mildew Bremia lactucae, we mapped physical interactions between B. lactucae effectors and lettuce candidate target proteins. Using a lettuce cDNA library-based yeast-two-hybrid system, 61 protein-protein interactions were identified, involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten interactors based on the number of independent colonies identified in the Y2H and two interactors that belong to gene families involved in plant immunity, were further characterized. We determined the subcellular localization of the fluorescently tagged lettuce proteins and their interacting effectors. Importantly, relocalization of effectors or their interactors to the nucleus was observed for four protein-pairs upon their co-expression, supporting their interaction in planta.

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<![CDATA[Extending thermotolerance to tomato seedlings by inoculation with SA1 isolate of <i>Bacillus cereus</i> and comparison with exogenous humic acid application]]> https://www.researchpad.co/article/elastic_article_11229 Heat stress is one of the major abiotic stresses that impair plant growth and crop productivity. Plant growth-promoting endophytic bacteria (PGPEB) and humic acid (HA) are used as bio-stimulants and ecofriendly approaches to improve agriculture crop production and counteract the negative effects of heat stress. Current study aimed to analyze the effect of thermotolerant SA1 an isolate of Bacillus cereus and HA on tomato seedlings. The results showed that combine application of SA1+HA significantly improved the biomass and chlorophyll fluorescence of tomato plants under normal and heat stress conditions. Heat stress increased abscisic acid (ABA) and reduced salicylic acid (SA) content; however, combined application of SA1+HA markedly reduced ABA and increased SA. Antioxidant enzymes activities revealed that SA1 and HA treated plants exhibited increased levels of ascorbate peroxidase (APX), superoxide dismutase (SOD), and reduced glutathione (GSH). In addition, heat stress markedly reduced the amino acid contents; however, the amino acids were increased with co-application of SA1+HA. Similarly, inductively-coupled plasma mass-spectrometry results showed that plants treated with SA1+HA exhibited significantly higher iron (Fe+), phosphorus (P), and potassium (K+) uptake during heat stress. Heat stress increased the relative expression of SlWRKY33b and autophagy-related (SlATG5) genes, whereas co-application of SA1+HA augmented the heat stress response and reduced SlWRKY33b and SlATG5 expression. The heat stress-responsive transcription factor (SlHsfA1a) and high-affinity potassium transporter (SlHKT1) were upregulated in SA1+HA-treated plants. In conclusion, current findings suggest that co-application with SA1+HA can be used for the mitigation of heat stress damage in tomato plants and can be commercialized as a biofertilizer.

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<![CDATA[Substantial improvement of tetraene macrolide production in <i>Streptomyces diastatochromogenes</i> by cumulative drug resistance mutations]]> https://www.researchpad.co/article/elastic_article_7861 Tetraene macrolides remain one of the most reliable fungicidal agents as resistance of fungal pathogens to these antibiotics is relatively rare. The modes of action and biosynthesis of polyene macrolides had been the focus of research over the past few years. However, few studies have been carried out on the overproduction of polyene macrolides. In the present study, cumulative drug-resistance mutation was used to obtain a quintuple mutant G5-59 with huge tetraene macrolide overproduction from the starting strain Streptomyces diastatochromogenes 1628. Through DNA sequence analysis, the mutation points in the genes of rsmG, rpsL and rpoB were identified. Additionally, the growth characteristic and expression level of tetrRI gene (belonging to the large ATP binding regulator of LuxR family) involved in the biosynthesis of tetraene macrolides were analyzed. As examined with 5L fermentor, the quintuple mutant G5-59 grew very well and the maximum productivity of tetramycin A, tetramycin P and tetrin B was as high as 1735, 2811 and 1500 mg/L, which was 8.7-, 16- and 25-fold higher than that of the wild-type strain 1628, respectively. The quintuple mutant G5-59 could be useful for further improvement of tetraene macrolides production at industrial level.

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<![CDATA[Functional and structural consequences of epithelial cell invasion by <i>Bordetella pertussis</i> adenylate cyclase toxin]]> https://www.researchpad.co/article/elastic_article_7693 Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at low concentrations (0.5 nM), CyaA could significantly impair the migration and wound healing capacities of the intoxicated alveolar epithelial cells. As such concentrations might be reached locally during B. pertussis infection, our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, a hallmark of pertussis.

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<![CDATA[An electrodiffusive, ion conserving Pinsky-Rinzel model with homeostatic mechanisms]]> https://www.researchpad.co/article/elastic_article_7780 Neurons generate their electrical signals by letting ions pass through their membranes. Despite this fact, most models of neurons apply the simplifying assumption that ion concentrations remain effectively constant during neural activity. This assumption is often quite good, as neurons contain a set of homeostatic mechanisms that make sure that ion concentrations vary quite little under normal circumstances. However, under some conditions, these mechanisms can fail, and ion concentrations can vary quite dramatically. Standard models are thus not able to simulate such conditions. Here, we present what to our knowledge is the first multicompartmental neuron model that accounts for ion concentration variations in a way that ensures complete and consistent ion concentration and charge conservation. In this work, we use the model to explore under which activity conditions the ion concentration variations become important for predicting the neurodynamics. We expect the model to be of great value for the field of neuroscience, as it can be used to simulate a range of pathological conditions, such as spreading depression or epilepsy, which are associated with large changes in extracellular ion concentrations.

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<![CDATA[A mathematical model coupling polarity signaling to cell adhesion explains diverse cell migration patterns]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be0154

Protrusion and retraction of lamellipodia are common features of eukaryotic cell motility. As a cell migrates through its extracellular matrix (ECM), lamellipod growth increases cell-ECM contact area and enhances engagement of integrin receptors, locally amplifying ECM input to internal signaling cascades. In contrast, contraction of lamellipodia results in reduced integrin engagement that dampens the level of ECM-induced signaling. These changes in cell shape are both influenced by, and feed back onto ECM signaling. Motivated by experimental observations on melanoma cells lines (1205Lu and SBcl2) migrating on fibronectin (FN) coated topographic substrates (anisotropic post-density arrays), we probe this interplay between intracellular and ECM signaling. Experimentally, cells exhibited one of three lamellipodial dynamics: persistently polarized, random, or oscillatory, with competing lamellipodia oscillating out of phase (Park et al., 2017). Pharmacological treatments, changes in FN density, and substrate topography all affected the fraction of cells exhibiting these behaviours. We use these observations as constraints to test a sequence of hypotheses for how intracellular (GTPase) and ECM signaling jointly regulate lamellipodial dynamics. The models encoding these hypotheses are predicated on mutually antagonistic Rac-Rho signaling, Rac-mediated protrusion (via activation of Arp2/3 actin nucleation) and Rho-mediated contraction (via ROCK phosphorylation of myosin light chain), which are coupled to ECM signaling that is modulated by protrusion/contraction. By testing each model against experimental observations, we identify how the signaling layers interact to generate the diverse range of cell behaviors, and how various molecular perturbations and changes in ECM signaling modulate the fraction of cells exhibiting each. We identify several factors that play distinct but critical roles in generating the observed dynamic: (1) competition between lamellipodia for shared pools of Rac and Rho, (2) activation of RhoA by ECM signaling, and (3) feedback from lamellipodial growth or contraction to cell-ECM contact area and therefore to the ECM signaling level.

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<![CDATA[Aspirin-triggered resolvin D1 attenuates PDGF-induced vascular smooth muscle cell migration via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc0e7

Background and objectives

Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration.

Methods

VSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity.

Results

Treatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor.

Conclusions

Our results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

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<![CDATA[The tetraspanin CD9 facilitates MERS-coronavirus entry by scaffolding host cell receptors and proteases]]> https://www.researchpad.co/article/598bdfb5fa495b7488185485

Infection by enveloped coronaviruses (CoVs) initiates with viral spike (S) proteins binding to cellular receptors, and is followed by proteolytic cleavage of receptor-bound S proteins, which prompts S protein-mediated virus-cell membrane fusion. Infection therefore requires close proximity of receptors and proteases. We considered whether tetraspanins, scaffolding proteins known to facilitate CoV infections, hold receptors and proteases together on cell membranes. Using knockout cell lines, we found that the tetraspanin CD9, but not the tetraspanin CD81, formed cell-surface complexes of dipeptidyl peptidase 4 (DPP4), the MERS-CoV receptor, and the type II transmembrane serine protease (TTSP) member TMPRSS2, a CoV-activating protease. This CD9-facilitated condensation of receptors and proteases allowed MERS-CoV pseudoviruses to enter cells rapidly and efficiently. Without CD9, MERS-CoV viruses were not activated by TTSPs, and they trafficked into endosomes to be cleaved much later and less efficiently by cathepsins. Thus, we identified DPP4:CD9:TTSP as the protein complexes necessary for early, efficient MERS-CoV entry. To evaluate the importance of these complexes in an in vivo CoV infection model, we used recombinant Adenovirus 5 (rAd5) vectors to express human DPP4 in mouse lungs, thereby sensitizing the animals to MERS-CoV infection. When the rAd5-hDPP4 vectors co-expressed small RNAs silencing Cd9 or Tmprss2, the animals were significantly less susceptible, indicating that CD9 and TMPRSS2 facilitated robust in vivo MERS-CoV infection of mouse lungs. Furthermore, the S proteins of virulent mouse-adapted MERS-CoVs acquired a CD9-dependent cell entry character, suggesting that CD9 is a selective agent in the evolution of CoV virulence.

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<![CDATA[Recapitulation of the accessible interface of biopsy-derived canine intestinal organoids to study epithelial-luminal interactions]]> https://www.researchpad.co/article/N24a1d01a-2f11-47b7-a628-8330af6f7455

Recent advances in canine intestinal organoids have expanded the option for building a better in vitro model to investigate translational science of intestinal physiology and pathology between humans and animals. However, the three-dimensional geometry and the enclosed lumen of canine intestinal organoids considerably hinder the access to the apical side of epithelium for investigating the nutrient and drug absorption, host-microbiome crosstalk, and pharmaceutical toxicity testing. Thus, the creation of a polarized epithelial interface accessible from apical or basolateral side is critical. Here, we demonstrated the generation of an intestinal epithelial monolayer using canine biopsy-derived colonic organoids (colonoids). We optimized the culture condition to form an intact monolayer of the canine colonic epithelium on a nanoporous membrane insert using the canine colonoids over 14 days. Transmission and scanning electron microscopy revealed a physiological brush border interface covered by the microvilli with glycocalyx, as well as the presence of mucin granules, tight junctions, and desmosomes. The population of stem cells as well as differentiated lineage-dependent epithelial cells were verified by immunofluorescence staining and RNA in situ hybridization. The polarized expression of P-glycoprotein efflux pump was confirmed at the apical membrane. Also, the epithelial monolayer formed tight- and adherence-junctional barrier within 4 days, where the transepithelial electrical resistance and apparent permeability were inversely correlated. Hence, we verified the stable creation, maintenance, differentiation, and physiological function of a canine intestinal epithelial barrier, which can be useful for pharmaceutical and biomedical researches.

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<![CDATA[Influence of the tubular network on the characteristics of calcium transients in cardiac myocytes]]> https://www.researchpad.co/article/N7f446290-780e-4486-a1de-95187c6060a1

Transverse and axial tubules (TATS) are an essential ingredient of the excitation-contraction machinery that allow the effective coupling of L-type Calcium Channels (LCC) and ryanodine receptors (RyR2). They form a regular network in ventricular cells, while their presence in atrial myocytes is variable regionally and among animal species We have studied the effect of variations in the TAT network using a bidomain computational model of an atrial myocyte with variable density of tubules. At each z-line the t-tubule length is obtained from an exponential distribution, with a given mean penetration length. This gives rise to a distribution of t-tubules in the cell that is characterized by the fractional area (F.A.) occupied by the t-tubules. To obtain consistent results, we average over different realizations of the same mean penetration length. To this, in some simulations we add the effect of a network of axial tubules. Then we study global properties of calcium signaling, as well as regional heterogeneities and local properties of sparks and RyR2 openings. In agreement with recent experiments in detubulated ventricular and atrial cells, we find that detubulation reduces the calcium transient and synchronization in release. However, it does not affect sarcoplasmic reticulum (SR) load, so the decrease in SR calcium release is due to regional differences in Ca2+ release, that is restricted to the cell periphery in detubulated cells. Despite the decrease in release, the release gain is larger in detubulated cells, due to recruitment of orphaned RyR2s, i.e, those that are not confronting a cluster of LCCs. This probably provides a safeguard mechanism, allowing physiological values to be maintained upon small changes in the t-tubule density. Finally, we do not find any relevant change in spark properties between tubulated and detubulated cells, suggesting that the differences found in experiments could be due to differential properties of the RyR2s in the membrane and in the t-tubules, not incorporated in the present model. This work will help understand the effect of detubulation, that has been shown to occur in disease conditions such as heart failure (HF) in ventricular cells, or atrial fibrillation (AF) in atrial cells.

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<![CDATA[Proteomic analysis of protein composition of rat hippocampus exposed to morphine for 10 days; comparison with animals after 20 days of morphine withdrawal]]> https://www.researchpad.co/article/N2838fdc6-dc33-429a-ba0d-e2e831e6a950

Opioid addiction is recognized as a chronic relapsing brain disease resulting from repeated exposure to opioid drugs. Cellular and molecular mechanisms underlying the ability of organism to return back to the physiological norm after cessation of drug supply are not fully understood. The aim of this work was to extend our previous studies of morphine-induced alteration of rat forebrain cortex protein composition to the hippocampus. Rats were exposed to morphine for 10 days and sacrificed 24 h (groups +M10 and −M10) or 20 days after the last dose of morphine (groups +M10/−M20 and −M10/−M20). The six altered proteins (≥2-fold) were identified in group (+M10) when compared with group (−M10) by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). The number of differentially expressed proteins was increased to thirteen after 20 days of the drug withdrawal. Noticeably, the altered level of α-synuclein, β-synuclein, α-enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also determined in both (±M10) and (±M10/−M20) samples of hippocampus. Immunoblot analysis of 2D gels by specific antibodies oriented against α/β-synucleins and GAPDH confirmed the data obtained by 2D-DIGE analysis. Label-free quantification identified nineteen differentially expressed proteins in group (+M10) when compared with group (−M10). After 20 days of morphine withdrawal (±M10/−M20), the number of altered proteins was increased to twenty. We conclude that the morphine-induced alteration of protein composition in rat hippocampus after cessation of drug supply proceeds in a different manner when compared with the forebrain cortex. In forebrain cortex, the total number of altered proteins was decreased after 20 days without morphine, whilst in hippocampus, it was increased.

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<![CDATA[The draft mitochondrial genome of Magnolia biondii and mitochondrial phylogenomics of angiosperms]]> https://www.researchpad.co/article/N1f661d3e-d0c0-407e-92c0-bb72cd78029d

The mitochondrial genomes of flowering plants are well known for their large size, variable coding-gene set and fluid genome structure. The available mitochondrial genomes of the early angiosperms show extreme genetic diversity in genome size, structure, and sequences, such as rampant HGTs in Amborella mt genome, numerous repeated sequences in Nymphaea mt genome, and conserved gene evolution in Liriodendron mt genome. However, currently available early angiosperm mt genomes are still limited, hampering us from obtaining an overall picture of the mitogenomic evolution in angiosperms. Here we sequenced and assembled the draft mitochondrial genome of Magnolia biondii Pamp. from Magnoliaceae (magnoliids) using Oxford Nanopore sequencing technology. We recovered a single linear mitochondrial contig of 967,100 bp with an average read coverage of 122 × and a GC content of 46.6%. This draft mitochondrial genome contains a rich 64-gene set, similar to those of Liriodendron and Nymphaea, including 41 protein-coding genes, 20 tRNAs, and 3 rRNAs. Twenty cis-spliced and five trans-spliced introns break ten protein-coding genes in the Magnolia mt genome. Repeated sequences account for 27% of the draft genome, with 17 out of the 1,145 repeats showing recombination evidence. Although partially assembled, the approximately 1-Mb mt genome of Magnolia is still among the largest in angiosperms, which is possibly due to the expansion of repeated sequences, retention of ancestral mtDNAs, and the incorporation of nuclear genome sequences. Mitochondrial phylogenomic analysis of the concatenated datasets of 38 conserved protein-coding genes from 91 representatives of angiosperm species supports the sister relationship of magnoliids with monocots and eudicots, which is congruent with plastid evidence.

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<![CDATA[Variants encoding a restricted carboxy-terminal domain of SLC12A2 cause hereditary hearing loss in humans]]> https://www.researchpad.co/article/Nd1837fa5-7737-42fc-aa07-ce2092d99c03

Hereditary hearing loss is challenging to diagnose because of the heterogeneity of the causative genes. Further, some genes involved in hereditary hearing loss have yet to be identified. Using whole-exome analysis of three families with congenital, severe-to-profound hearing loss, we identified a missense variant of SLC12A2 in five affected members of one family showing a dominant inheritance mode, along with de novo splice-site and missense variants of SLC12A2 in two sporadic cases, as promising candidates associated with hearing loss. Furthermore, we detected another de novo missense variant of SLC12A2 in a sporadic case. SLC12A2 encodes Na+, K+, 2Cl cotransporter (NKCC) 1 and plays critical roles in the homeostasis of K+-enriched endolymph. Slc12a2-deficient mice have congenital, profound deafness; however, no human variant of SLC12A2 has been reported as associated with hearing loss. All identified SLC12A2 variants mapped to exon 21 or its 3’-splice site. In vitro analysis indicated that the splice-site variant generates an exon 21-skipped SLC12A2 mRNA transcript expressed at much lower levels than the exon 21-included transcript in the cochlea, suggesting a tissue-specific role for the exon 21-encoded region in the carboy-terminal domain. In vitro functional analysis demonstrated that Cl influx was significantly decreased in all SLC12A2 variants studied. Immunohistochemistry revealed that SLC12A2 is located on the plasma membrane of several types of cells in the cochlea, including the strial marginal cells, which are critical for endolymph homeostasis. Overall, this study suggests that variants affecting exon 21 of the SLC12A2 transcript are responsible for hereditary hearing loss in humans.

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<![CDATA[Transcriptomic analysis of polyketide synthases in a highly ciguatoxic dinoflagellate, Gambierdiscus polynesiensis and low toxicity Gambierdiscus pacificus, from French Polynesia]]> https://www.researchpad.co/article/Nca210627-69b7-4a50-96ce-ecb4ce1a2ae1

Marine dinoflagellates produce a diversity of polyketide toxins that are accumulated in marine food webs and are responsible for a variety of seafood poisonings. Reef-associated dinoflagellates of the genus Gambierdiscus produce toxins responsible for ciguatera poisoning (CP), which causes over 50,000 cases of illness annually worldwide. The biosynthetic machinery for dinoflagellate polyketides remains poorly understood. Recent transcriptomic and genomic sequencing projects have revealed the presence of Type I modular polyketide synthases in dinoflagellates, as well as a plethora of single domain transcripts with Type I sequence homology. The current transcriptome analysis compares polyketide synthase (PKS) gene transcripts expressed in two species of Gambierdiscus from French Polynesia: a highly toxic ciguatoxin producer, G. polynesiensis, versus a non-ciguatoxic species G. pacificus, each assembled from approximately 180 million Illumina 125 nt reads using Trinity, and compares their PKS content with previously published data from other Gambierdiscus species and more distantly related dinoflagellates. Both modular and single-domain PKS transcripts were present. Single domain β-ketoacyl synthase (KS) transcripts were highly amplified in both species (98 in G. polynesiensis, 99 in G. pacificus), with smaller numbers of standalone acyl transferase (AT), ketoacyl reductase (KR), dehydratase (DH), enoyl reductase (ER), and thioesterase (TE) domains. G. polynesiensis expressed both a larger number of multidomain PKSs, and larger numbers of modules per transcript, than the non-ciguatoxic G. pacificus. The largest PKS transcript in G. polynesiensis encoded a 10,516 aa, 7 module protein, predicted to synthesize part of the polyether backbone. Transcripts and gene models representing portions of this PKS are present in other species, suggesting that its function may be performed in those species by multiple interacting proteins. This study contributes to the building consensus that dinoflagellates utilize a combination of Type I modular and single domain PKS proteins, in an as yet undefined manner, to synthesize polyketides.

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<![CDATA[Cobalt ion interaction with TMEM16A calcium-activated chloride channel: Inhibition and potentiation]]> https://www.researchpad.co/article/Nba3bff3f-41a4-460d-bc9b-3a7adada8996

TMEM16A, a Ca2+-sensitive Cl- channel, plays key roles in many physiological functions related to Cl- transport across lipid membranes. Activation of this channel is mediated via binding intracellular Ca2+ to the channel with a relatively high apparent affinity, roughly in the sub-μM to low μM concentration range. Recently available high-resolution structures of TMEM16 molecules reveal that the high-affinity Ca2+ activation sites are formed by several acidic amino acids, using their negatively charged sidechain carboxylates to coordinate the bound Ca2+. In this study, we examine the interaction of TMEM16A with a divalent cation, Co2+, which by itself cannot activate current in TMEM16A. This divalent cation, however, has two effects when applied intracellularly. It inhibits the Ca2+-induced TMEM16A current by competing with Ca2+ for the aforementioned high-affinity activation sites. In addition, Co2+ also potentiates the Ca2+-induced current with a low affinity. This potentiation effect requires high concentration (mM) of Co2+, similar to our previous findings that high concentrations (mM) of intracellular Ca2+ ([Ca2+]i) can induce more TMEM16A current after the Ca2+-activation sites are saturated by tens of μM [Ca2+]i. The degrees of potentiation by Co2+ and Ca2+ also roughly correlate with each other. Interestingly, mutating a pore residue of TMEM16A, Y589, alters the degree of potentiation in that the smaller the sidechain of the replaced residue, the larger the potentiation induced by divalent cations. We suggest that the Co2+ potentiation and the Ca2+ potentiation share a similar mechanism by increasing Cl- flux through the channel pore, perhaps due to an increase of positive pore potential after the binding of divalent cations to phospholipids in the pore. A smaller sidechain of a pore residue may allow the pore to accommodate more phospholipids, thus enhancing the current potentiation caused by high concentrations of divalent cations.

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<![CDATA[Podocyte RNA sequencing reveals Wnt- and ECM-associated genes as central in FSGS]]> https://www.researchpad.co/article/Nff231b2e-f2d8-47eb-acf2-c510faf35a1a

Loss of podocyte differentiation can cause nephrotic-range proteinuria and Focal and Segmental Glomerulosclerosis (FSGS). As specific therapy is still lacking, FSGS frequently progresses to end-stage renal disease. The exact molecular mechanisms of FSGS and gene expression changes in podocytes are complex and widely unknown as marker changes have mostly been assessed on the glomerular level. To gain a better insight, we isolated podocytes of miR-193a overexpressing mice, which suffer from FSGS due to suppression of the podocyte master regulator Wt1. We characterised the podocytic gene expression changes by RNAseq and identified many novel candidate genes not linked to FSGS so far. This included strong upregulation of the receptor tyrosine kinase EphA6 and a massive dysregulation of circadian genes including the loss of the transcriptional activator Arntl. By comparison with podocyte-specific changes in other FSGS models we found a shared dysregulation of genes associated with the Wnt signaling cascade, while classical podocyte-specific genes appeared widely unaltered. An overlap with gene expression screens from human FSGS patients revealed a strong enrichment in genes associated with extra-cellular matrix (ECM) and metabolism. Our data suggest that FSGS progression might frequently depend on pathways that are often overlooked when considering podocyte homeostasis.

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<![CDATA[Potential combinations of endocannabinoid/endocannabinoid-like compounds and antibiotics against methicillin-resistant Staphylococcus aureus]]> https://www.researchpad.co/article/Ne8a72c2e-13c7-43d3-9f49-0ed6410d9d0b

Infections caused by antibiotic-resistant strains of Staphylococcus aureus have reached epidemic proportions globally. Our previous study showed antimicrobial effects of anandamide (AEA) and arachidonoyl serine (AraS) against methicillin (MET)-resistant S. aureus (MRSA) strains, proposing the therapeutic potential of these endocannabinoid/endocannabinoid-like (EC/EC-like) agents for the treatment of MRSA. Here, we investigated the potential synergism of combinations of AEA and AraS with different types of antibiotics against MRSA grown under planktonic growth or biofilm formation. The most effective combinations under planktonic conditions were mixtures of AEA and ampicillin (AMP), and of AraS and gentamicin (GEN). The combination with the highest synergy in the biofilm formation against all tested bacterial strains was AEA and MET. Moreover, the combination of AraS and MET synergistically caused default of biofilm formation. Slime production of MRSA was also dramatically impaired by AEA or AraS combined with MET. Our data suggest the novel potential activity of combinations of EC/EC-like agents and antibiotics in the prevention of MRSA biofilm formation.

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<![CDATA[Reduction of osteoarthritis severity in the temporomandibular joint of rabbits treated with chondroitin sulfate and glucosamine]]> https://www.researchpad.co/article/N10336f10-2066-4958-9182-9e228dac929f

Osteoarthritis is a degenerative disease that causes substantial changes in joint tissues, such as cartilage degeneration and subchondral bone sclerosis. Chondroitin sulfate and glucosamine are commonly used products for the symptomatic treatment of osteoarthritis. The aim of the present study was to investigate the effects of these products when used as structure-modifying drugs on the progression of osteoarthritis in the rabbit temporomandibular joint. Thirty-six New Zealand rabbits were divided into 3 groups (n = 12/group): control (no disease); osteoarthritis (disease induction); and treatment (disease induction and administration of chondroitin sulfate and glucosamine). Osteoarthritis was induced by intra-articular injection of monosodium iodoacetate. Animals were killed at 30 and 90 days after initiation of therapy. The treatment was effective in reducing disease severity, with late effects and changes in the concentration of glycosaminoglycans in the articular disc. The results indicate that chondroitin sulfate and glucosamine may have a structure-modifying effect on the tissues of rabbit temporomandibular joints altered by osteoarthritis.

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