Antibacterial activity of hydroxyapatite/silver nanocomposite

Hydroxyapatite containing silver nanoparticles (HAp/Ag) was synthesized by homogeneous sonochemical metod. Morphology of obtained particles was formed of micrometer-sized rod-like HAp with nanosized silver sphere-like particles attached to their surface. So obtained material was tested for interaction with bacteria. For that purpose composites with three different contents of silver were prepared and their interactions with two different types of bacteria were studied. Escherichia coli was used as a representative of Gram negative while Staphylococcus aureus was applied as a representative of Gram positive bacteria. In the case of E. coli wider inhibition zone without presence of bacteria and with a layer of inactive bacteria near the surface of material was obtained. In this case obtained antibacterial effect was not concentration dependant. In the case of S. aureus, inhibition zone was narrower with the presence of modified bacteria in inhibition zone. Obtained antibacterial response pronounced dependence on concentration of silver within composite. According to results achieved with this investigation we concluded that HAp/Ag composite is effective against both, Gram positive and Gram negative bacteria and shows stronger activity against E. coli

Bacteroides muris sp. nov. isolated from the cecum of wild-derived house mice

Two bacterial strains, KH365_2T and KH569_7, were isolated from the cecum contents of wild-derived house mice. The strains were characterized as Gram-negative, rod-shaped, strictly anaerobic, and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains were most closely related to Bacteroides uniformis ATCC 8492T. Whole genome sequences of KH365_2T and KH569_7 strains have a DNA G + C content of 46.02% and 46.03% mol, respectively. Most morphological and biochemical characteristics did not differ between the newly isolated strains and classified Bacteroides strains. However, the average nucleotide identity (ANI) and dDNA–DNA hybridization (dDDH) values clearly distinguished the two strains from described members of the genus Bacteroides. Here, we present the phylogeny, morphology, and physiology of a novel species of the genus Bacteroides and propose the name Bacteroides muris sp. nov., with KH365_2T (DSM 114231T = CCUG 76277T) as type strain

Dual-RNAseq Analysis Unravels Virus-Host Interactions of MetSV and Methanosarcina mazei

Methanosarcina spherical virus (MetSV), infecting Methanosarcina species, encodes 22 genes, but their role in the infection process in combination with host genes has remained unknown. To study the infection process in detail, infected and uninfected M. mazei cultures were compared using dual-RNAseq, qRT-PCRs, and transmission electron microscopy (TEM). The transcriptome analysis strongly indicates a combined role of virus and host genes in replication, virus assembly, and lysis. Thereby, 285 host and virus genes were significantly regulated. Within these 285 regulated genes, a network of the viral polymerase, MetSVORF6, MetSVORF5, MetSVORF2, and the host genes encoding NrdD, NrdG, a CDC48 family protein, and a SSB protein with a role in viral replication was postulated. Ultrastructural analysis at 180 min p.i. revealed many infected cells with virus particles randomly scattered throughout the cytoplasm or attached at the cell surface, and membrane fragments indicating cell lysis. Dual-RNAseq and qRT-PCR analyses suggested a multifactorial lysis reaction in potential connection to the regulation of a cysteine proteinase, a pirin-like protein and a HicB-solo protein. Our study's results led to the first preliminary infection model of MetSV infecting M. mazei, summarizing the key infection steps as follows: replication, assembly, and host cell lysis

Antibacterial activity and cytotoxicity of hydroxyapatite/silver nanocomposites synthesized by a sonochemical approach

Hydroxyapatite, as a major constituent of the inorganic part of natural human bones, is a natural choice and one of the most frequently applied materials for the reparation and regeneration of bone tissue. The formation of composites based on HAp, the incorporation of foreign ions within its structure and/or the functionalization of their surfaces allow improvements to the morphological, structural and surface properties. Such improvements can be utilized to increase the material quality for applications in dentistry and orthopedics as well to extend the potential applicability in other fields of biomedicine. Hydroxyapatite/silver (HAp/Ag) nanocomposite particles were formed using the sonochemical method. Particles were formed from HAp rods with silver (present in the form of ions) incorporated within the apatite structure and/or metallic particles, embedded into apatite rods and metallic particles attached to the surfaces of these rods. The so obtained materials were tested for antibacterial activity. The p presence of the bacterial growth-inhibition zone around the discs made of composite material and the fluorescence dye detection on the surface of the HAp/Ag composite (corresponding to the labeled live and dead bacteria) proved that the composite was active against both Gram-positive and Gram-negative bacteria. In addition, it was determined that the minimal inhibition concentration (MIC), i.e., the minimal concentration of HAp/Ag composite, which is able to inhibit the growth of bacteria, was higher for Grampositive (MIC = 0.7-0.8 mg/ml) when compared to Gram-negative (MIC = 0.6-0.7 mg/ml) bacteria. The same effect was determined during the investigation of the minimal bactericidal concentration (MBC) of the HAp/Ag composite against the same representatives of Grampositive and Gram-negative bacteria. The obtained concentrations of the HAp/Ag composite which were able to manifest the bactericidal effect were used for an investigation of the toxicity against the representatives of healthy and cancer-modified cell lines. The toxicity of a composite was investigated in vitro and for that purpose the IMR-90 human lung embryonic fibroblast and U-2 OS human osteosarcoma cell lines were applied. In the presentation the antibacterial activity of the HAp/Ag composite and its toxicity against human cells will be discussed

A Rab20-dependent membrane trafficking pathway controls M. tuberculosis replication by regulating phagosome spaciousness and integrity

The intracellular pathogen Mycobacterium tuberculosis (Mtb) lives within phagosomes and also disrupts these organelles to access the cytosol. The host pathways and mechanisms that contribute to maintaining Mtb phagosome integrity have not been investigated. Here, we examined the spatiotemporal dynamics of Mtb-containing phagosomes and identified an interferon-gamma-stimulated and Rab20-dependent membrane trafficking pathway in macrophages that maintains Mtb in spacious proteolytic phagolysosomes. This pathway functions to promote endosomal membrane influx in infected macrophages, and is required to preserve Mtb phagosome integrity and control Mtb replication. Rab20 is specifically and significantly upregulated in the sputum of human patients with active tuberculosis. Altogether, we uncover an immune-regulated cellular pathway of defense that promotes maintenance of Mtb within intact membrane-bound compartments for efficient elimination

Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads

Lumen morphogenesis results from the interplay between molecular pathways and mechanical forces. In several organs, epithelial cells share their apical surfaces to form a tubular lumen. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here, by studying lumenogenesis in differentiating mouse hepatoblasts in vitro, we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen and ensuring its anisotropic expansion. These previously unrecognized structures form a pattern, reminiscent of the bulkheads of boats, also present in the developing and adult liver. Silencing of Rab35 resulted in loss of apical bulkheads and lumen anisotropy, leading to cyst formation. Strikingly, we could reengineer hepatocyte polarity in embryonic liver tissue, converting BC into epithelial tubes. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis

Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. CatD inhibitors could therefore enhance acetate-mediated cancer cell death, presenting a novel strategy for prevention or therapy of CRC.FEDER through POFC – COMPETE and by Fundação para a Ciência e Tecnologia through projects PEst-OE/BIA/UI4050/2014 and FCT ANR/BEX-BCM/0175/201

Subcellular fractionation method to study endosomal trafficking of Kaposi’s sarcoma-associated herpesvirus

Background Virus entry involves multiple steps and is a highly orchestrated process on which successful infection collectively depends. Entry processes are commonly analyzed by monitoring internalized virus particles via Western blotting, polymerase chain reaction, and imaging techniques that allow scientist to track the intracellular location of the pathogen. Such studies have provided abundant direct evidence on how viruses interact with receptor molecules on the cell surface, induce cell signaling at the point of initial contact with the cell to facilitate internalization, and exploit existing endocytic mechanisms of the cell for their ultimate infectious agenda. However, there is dearth of knowledge in regards to trafficking of a virus via endosomes. Herein, we describe an optimized laboratory procedure to isolate individual organelles during different stages of endocytosis by performing subcellular fractionation. This methodology is established using Kaposi’s sarcoma-associated herpesvirus (KSHV) infection of human foreskin fibroblast (HFF) cells as a model. With KSHV and other herpesviruses alike, envelope glycoproteins have been widely reported to physically engage target cell surface receptors, such as integrins, in interactions leading to entry and subsequent infection. Results Subcellular fractionation was used to isolate early and late endosomes (EEs and LEs) by performing a series of centrifugations steps. Specifically, a centrifugation step post-homogenization was utilized to obtain the post-nuclear supernatant containing intact intracellular organelles in suspension. Successive fractionation via sucrose density gradient centrifugation was performed to isolate specific organelles including EEs and LEs. Intracellular KSHV trafficking was directly traced in the isolated endosomal fractions. Additionally, the subcellular fractionation approach demonstrates a key role for integrins in the endosomal trafficking of KSHV. The results obtained from fractionation studies corroborated those obtained by traditional imaging studies. Conclusions This study is the first of its kind to employ a sucrose flotation gradient assay to map intracellular KSHV trafficking in HFF cells. We are confident that such an approach will serve as a powerful tool to directly study intracellular trafficking of a virus, signaling events occurring on endosomal membranes, and dynamics of molecular events within endosomes that are crucial for uncoating and virus escape into the cytosol