The Possible Role of Staphylococcus epidermidis LPxTG Surface Protein SesC in Biofilm Formation

Staphylococcus epidermidis is the most common cause of device-associated infections. It has been shown that active and passive immunization in an animal model against protein SesC significantly reduces S. epidermidis biofilm-associated infections. In order to elucidate its role, knock-out of sesC or isolation of S. epidermidis sesC-negative mutants were attempted, however, without success. As an alternative strategy, sesC was introduced into Staphylococcus aureus 8325–4 and its isogenic icaADBC and srtA mutants, into the clinical methicillin-sensitive S. aureus isolate MSSA4 and the MRSA S. aureus isolate BH1CC, which all lack sesC. Transformation of these strains with sesC i) changed the biofilm phenotype of strains 8325–4 and MSSA4 from PIA-dependent to proteinaceous even though PIA synthesis was not affected, ii) converted the non-biofilm-forming strain 8325–4 ica::tet to a proteinaceous biofilm-forming strain, iii) impaired PIA-dependent biofilm formation by 8325–4 srtA::tet, iv) had no impact on protein-mediated biofilm formation of BH1CC and v) increased in vivo catheter and organ colonization by strain 8325–4. Furthermore, treatment with anti-SesC antibodies significantly reduced in vitro biofilm formation and in vivo colonization by these transformants expressing sesC. These findings strongly suggest that SesC is involved in S. epidermidis attachment to and subsequent biofilm formation on a substrate.     

Vascular tissue engineering: Fabrication and characterization of acetylsalicylic acid-loaded electrospun scaffolds coated with amniotic membrane lysate

As the incidence of small-diameter vascular graft (SDVG) occlusion is considerably high, a great amount of research is focused on constructing a more biocompatible graft. The absence of a biocompatible surface in the lumen of the engineered grafts that can support confluent lining with endothelial cells (ECs) can cause thrombosis and graft failure. Blood clot formation is mainly because of the lack of an integrated endothelium. The most effective approach to combat this problem would be using natural extracellular matrix constituents as a mimic of endothelial basement membrane along with applying anticoagulant agents to provide local antithrombotic effects. In this study, we fabricated aligned and random electrospun poly-L-lactic acid (PLLA) scaffolds containing acetylsalicylic acid (ASA) as the anticoagulation agent and surface coated them with amniotic membrane (AM) lysate. Vascular scaffolds were structurally and mechanically characterized and assessed for cyto- and hemocompatibility and their ability to support endothelial differentiation was examined. All the scaffolds showed appropriate tensile strength as expected for vascular grafts. Lack of cytotoxicity, cellular attachment, growth, and infiltration were proved using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scanning electron microscopy. The blood compatibilities of different scaffolds examined by in vitro hemolysis and blood coagulation assays elucidated the excellent hemocompatibility of our novel AM-coated ASA-loaded nanofibers. Drug-loaded scaffolds showed a sustained release profile of ASA in 7 days. AM-coated electrospun PLLA fibers showed enhanced cytocompatibility for human umbilical vein ECs, making a confluent endothelial-like lining. In addition, AM lysate-coated ASA-PLLA-aligned scaffold proved to support endothelial differentiation of Wharton's jelly-derived mesenchymal stem cells. Our results together indicated that AM lysate-coated ASA releasing scaffolds have promising potentials for development of a biocompatible SDVG.     

The role of nitric oxide signaling in renoprotective effects of hydrogen sulfide against chronic kidney disease in rats: Involvement of oxidative stress,autophagy and apoptosis

The interplay between H₂S and nitric oxide (NO) is thought to contribute to renal functions. The current study was designed to assess the role of NO in mediating the renoprotective effects of hydrogen sulfide in the 5/6 nephrectomy (5/6 Nx) animal model. Forty rats were randomly assigned to 5 experimental groups: (a) Sham; (b) 5/6 Nx; (c) 5/6Nx+sodium hydrosulfide-a donor of H ₂S, (5/6Nx+sodium hydrosulfide [NaHS]); (d) 5/6Nx+NaHS+ L -NAME (a nonspecific nitric oxide synthase [NOS] inhibitor); (e) 5/6Nx+NaHS+aminoguanidine (a selective inhibitor of inducible NOS [iNOS]). Twelve weeks after 5/6 Nx, we assessed the expressions of iNOS and endothelial NOS (eNOS), oxidative/antioxidant status, renal fibrosis, urine N-acetyl-b-glucosaminidase (NAG) activity as the markers of kidney injury and various markers of apoptosis, inflammation, remodeling, and autophagy. NaHS treatment protected the animals against chronic kidney injury as depicted by improved oxidative/antioxidant status, reduced apoptosis, and autophagy and attenuated messenger RNA (mRNA) expression of genes associated with inflammation, remodeling, and NAG activity. Eight weeks Nω-nitro-l-arginine methyl ester ( L -NAME) administration reduced the protective effects of hydrogen sulfide. In contrast, aminoguanidine augmented the beneficial effects of hydrogen sulfide. Our finding revealed some fascinating interactions between NO and H ₂S in the kidney. Moreover, the study suggests that NO, in an isoform-dependent manner, can exert renoprotective effects in 5/6 Nx model of CKD.     

Regulation of AR mRNA translation in response to acute AR pathway inhibition

We report a new mechanism of androgen receptor (AR) mRNA regulation and cytoprotection in response to AR pathway inhibition (ARPI) stress in prostate cancer (PCA). AR mRNA translation is coordinately regulated by RNA binding proteins, YTHDF3 and G3BP1. Under ambient conditions m6A-modified AR mRNA is bound by YTHDF3 and translationally stimulated, while m6A-unmodified AR mRNA is bound by G3BP1 and translationally repressed. When AR-regulated PCA cell lines are subjected to ARPI stress, m6A-modified AR mRNA is recruited from actively translating polysomes (PSs) to RNA-protein stress granules (SGs), leading to reduced AR mRNA translation. After ARPI stress, m6A-modified AR mRNA liquid–liquid phase separated with YTHDF3, while m6A-unmodified AR mRNA phase separated with G3BP1. Accordingly, these AR mRNA messages form two distinct YTHDF3-enriched or G3BP1-enriched clusters in SGs. ARPI-induced SG formation is cell-protective, which when blocked by YTHDF3 or G3BP1 silencing increases PCA cell death in response to ARPI stress. Interestingly, AR mRNA silencing also delays ARPI stress-induced SG formation, highlighting its supportive role in triggering this stress response. Our results define a new mechanism for stress adaptive cell survival after ARPI stress involving SG-regulated translation of AR mRNA, mediated by m6A RNA modification and their respective regulatory proteins.     

Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo

New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.     

Perception of Body Ownership Is Driven by Bayesian Sensory Inference

Recent studies have shown that human perception of body ownership is highly malleable. A well-known example is the rubber hand illusion (RHI) wherein ownership over a dummy hand is experienced, and is generally believed to require synchronized stroking of real and dummy hands. Our goal was to elucidate the computational principles governing this phenomenon. We adopted the Bayesian causal inference model of multisensory perception and applied it to visual, proprioceptive, and tactile stimuli. The model reproduced the RHI, predicted that it can occur without tactile stimulation, and that synchronous stroking would enhance it. Various measures of ownership across two experiments confirmed the predictions: a large percentage of individuals experienced the illusion in the absence of any tactile stimulation, and synchronous stroking strengthened the illusion. Altogether, these findings suggest that perception of body ownership is governed by Bayesian causal inference—i.e., the same rule that appears to govern the perception of outside world.     

Biases in Visual,Auditory, and Audiovisual Perception of Space

Localization of objects and events in the environment is critical for survival, as many perceptual and motor tasks rely on estimation of spatial location. Therefore, it seems reasonable to assume that spatial localizations should generally be accurate. Curiously, some previous studies have reported biases in visual and auditory localizations, but these studies have used small sample sizes and the results have been mixed. Therefore, it is not clear (1) if the reported biases in localization responses are real (or due to outliers, sampling bias, or other factors), and (2) whether these putative biases reflect a bias in sensory representations of space or a priori expectations (which may be due to the experimental setup, instructions, or distribution of stimuli). Here, to address these questions, a dataset of unprecedented size (obtained from 384 observers) was analyzed to examine presence, direction, and magnitude of sensory biases, and quantitative computational modeling was used to probe the underlying mechanism(s) driving these effects. Data revealed that, on average, observers were biased towards the center when localizing visual stimuli, and biased towards the periphery when localizing auditory stimuli. Moreover, quantitative analysis using a Bayesian Causal Inference framework suggests that while pre-existing spatial biases for central locations exert some influence, biases in the sensory representations of both visual and auditory space are necessary to fully explain the behavioral data. How are these opposing visual and auditory biases reconciled in conditions in which both auditory and visual stimuli are produced by a single event? Potentially, the bias in one modality could dominate, or the biases could interact/cancel out. The data revealed that when integration occurred in these conditions, the visual bias dominated, but the magnitude of this bias was reduced compared to unisensory conditions. Therefore, multisensory integration not only improves the precision of perceptual estimates, but also the accuracy.     

Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm²) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm² format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.     

Recombinant functional multidomain hemoglobin from the gastropod Biomphalaria glabrata

The extracellular hemoglobin multimer of the planorbid snail Biomphalaria glabrata, intermediate host of the human parasite Schistosoma mansoni, is presumed to be a 1.44 MDa complex of six 240 kDa polypeptide subunits, arranged as three disulfide-bridged dimers. The complete amino acid sequence of two subunit types (BgHb1 and BgHb2), and the partial sequence of a third type (BgHb3) are known. Each subunit encompasses 13 paralogus heme domains, and N-terminally a smaller plug domain responsible for subunit dimerization. We report here the recombinant expression of different functional fragments of BgHb2 in Escherichia coli, and of the complete functional subunits BgHb1 and BgHb2 in insect cells; BgHb1 was also expressed as disulfide-bridged dimer (480 kDa). Oxygen-binding measurements of the recombinant products show a P(50) of about 7 mmHg and the absence of a significant cooperativity or Bohr effect. The covalently linked dimer of BgHb1, but not the monomer, is capable to form aggregates closely resembling native BgHb molecules in the electron microscope.     

Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells

We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34(+) stem cells (up to 1.95 x 10(6)-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.