A cytotoxic leachable compound from single‐use bioprocess equipment that causes poor cell growth performance

A current trend in the production of biopharmaceuticals is the replacement of fixed stainless steel fluid‐handling units with disposable plastic bags. Such single‐use systems (SUS) offer numerous advantages, but also introduce a new set of materials into the production process and consequently expose biomanufacturers to a new set of risks related to those materials, not to mention reliance on an entirely new supply chain. In the course of developing and conducting a cell‐growth‐based test for suitability of disposable plastic components destined for use in cell culture operations, we discovered that the cytotoxic compound bis(2,4‐di‐tert‐butylphenyl)phosphate (bDtBPP) leaches out of certain bags and into cell culture media in concentrations that are deleterious to cell growth. Specifically, media held in certain bags for several days at 37°C was found to contain bDtBPP, and use of those held‐media samples in cell growth experiments provides data that overlap neatly with cell growth experiments using media spiked directly with bDtBPP, proving that bDtBPP leaching is responsible for the reduced growth attributable to those SUS bags. Overall, this issue represents a risk to the production of biopharmaceuticals in SUS, a risk that must be managed by diligent collaboration among companies along the entire supply chain for SUS components. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:332–337, 2014     

Human Placental Mesenchymal Stem/Stromal cells (pMSCs) inhibit agonist-induced platelet functions reducing atherosclerosis and thrombosis phenotypes

Mesenchymal stem/stromal cells isolated from human term placenta (pMSCs) have potential to treat clinically manifested inflammatory diseases. Atherosclerosis is a chronic inflammatory disease, and platelets play a contributory role towards its pathogenesis. During transplantation, MSCs interact with platelets and exert influence on their functional outcome. In this study, we investigated the consequences of interaction between pMSCs and platelets, and its impact on platelet-mediated atherosclerosis in vitro. Human platelets were treated with various types of pMSCs either directly or with their secretome, and their effect on agonist-mediated platelet activation and functional characteristics were evaluated. Human umbilical vein endothelial cells (HUVECs) were used as control. The impact of pMSCs treatment on platelets was evaluated by the expression of activation markers and by platelet functional analysis. A subset of pMSCs reduced agonist-induced activation of platelets, both via direct contact and with secretome treatments. Decrease in platelet activation translated into diminished spreading, limited adhesion and minimized aggregation. In addition, pMSCs decreased oxidized LDL (ox-LDL)-inducedCD36-mediated platelet activation, establishing their protective role in atherosclerosis. Gene expression and protein analysis show that pMSCs express pro- and anti-thrombotic proteins, which might be responsible for the modulation of agonist-induced platelet functions. These data suggest the therapeutic benefits of pMSCs in atherosclerosis.     

Assessment of the potential of temporin peptides from the frog Rana temporaria (Ranidae) as anti‐diabetic agents

Temporin A (FLPLIGRVLSGIL‐NH₂), temporin F (FLPLIGKVLSGIL‐NH₂), and temporin G (FFPVIGRILNGIL‐NH₂), first identified in skin secretions of the frog Rana temporaria, produced concentration‐dependent stimulation of insulin release from BRIN‐BD11 rat clonal β‐cells at concentrations ≥1 nM, without cytotoxicity at concentrations up to 3 μM. Temporin A was the most effective. The mechanism of insulinotropic action did not involve an increase in intracellular Ca²⁺ concentrations. Temporins B, C, E, H, and K were either inactive or only weakly active. Temporins A, F, and G also produced a concentration‐dependent stimulation of insulin release from 1.1B4 human‐derived pancreatic β‐cells, with temporin G being the most potent and effective, and from isolated mouse islets. The data indicate that cationicity, hydrophobicity, and the angle subtended by the charged residues in the temporin molecule are important determinants for in vitro insulinotropic activity. Temporin A and F (1 μM), but not temporin G, protected BRIN‐BD11 cells against cytokine‐induced apoptosis (P < 0.001) and augmented (P < 0.001) proliferation of the cells to a similar extent as glucagon‐like peptide‐1. Intraperitoneal injection of temporin G (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion whereas temporin A and F administration was without significant effect on plasma glucose levels. The study suggests that combination therapy involving agents developed from the temporin A and G sequences may find application in Type 2 diabetes treatment.     

Phylogenetic,toxigenic and virulence profiles of <Emphasis Type="Italic">Alternaria</Emphasis> species causing leaf blight of tomato in Egypt

Species of Alternaria are serious plant pathogens, causing major losses on a wide range of crops. Leaf blight symptoms were observed on tomato leaves, and samples were collected from various regions. Isolation was done from symptomatic tomato leaves, and 15 representatives were selected from a collection of 65 isolates of Alternaria species. The virulence of Alternaria isolates was investigated on detached leaves (DL) and whole plants of tomato cv. Super strain B. A phylogenetic analysis was performed based on three partial gene regions, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the RNA polymerase second largest subunit (RPB2) and the Alternaria major allergen gene (Alt a 1). The potentiality of Alternaria isolates to produce toxins was also investigated on the basis of thin-layer chromatography (TLC). Our investigations revealed that Alternaria isolates showed different levels of virulence either on tomato plants or DL. Based on the phylogeny of three genes, Alternaria isolates encompassed two species of small-spored morphospecies: A. alternata (14 isolates) and A. arborescens (single isolate). The produced toxins varied among Alternaria isolates with tenuazonic acid (TeA) being the most abundant mycotoxin produced by most isolates. This study highlighted on other Alternaria species in Egypt that might represent a serious concern for tomato producers as causal agents of leaf blight over other species, i.e. A. solani.     

Bioremoval of heavy metals and nutrients from sewage plant by Anabaena oryzae and Cyanosarcina fontana

The present study demonstrated the growth of two species of cyanobacteria on wastewater isolated from sewage plant in Aswan, Egypt. We evaluated their efficiency for eliminating nitrogen, phosphorus, chemical oxygen demand (COD) and heavy metals (Fe²⁺, Pb²⁺, Cu²⁺, and Mn²⁺). The growth of Cyanosarcina fontana has supported wastewater as a growth medium than Anabaena oryzae compared to standard medium. The nutrients concentration such as COD, NO₃–N and PO₄–P were decreased by the growth of A. oryzae and C. fontana in the wastewater after primary settling and centrate. However, the reduction of COD was less efficient than the other nutrients. The reduction percentage of COD, NO₃–N and PO₄–P reached 39.3, 84.1 and 90.7% as well as 54.6, 83.1, and 89.8%, in cultures of A. oryzae and C. fontana grown in the wastewater after primary settling, respectively. The reduction amounted to 10.1, 76.8, and 63.0% by A. oryzae and 43.2, 62.1, and 74.8% by C. fontana, grown in the centrate, respectively. Cyanobacteria species have the ability to accumulate the heavy metals from the wastewater to level far than the exceeding metal level in the water. Whereas, the heavy metals biosorption performance of C. fontana was higher in accumulating Fe²⁺ (93.95%), Pb²⁺ (81.21%), Cu²⁺ (63.9%), and Mn²⁺ (48.49%) compared to A. oryzae. The biosorption ability is dependent on the nature of the adsorbent studied and the type of wastewater treated. Therefore, removal of heavy metals and nutrients by the tested algae is strongly recommended as a powerful technique for the removal of pollutants from wastewater.     

Polarized Cell Division of Chlamydia trachomatis

Bacterial cell division predominantly occurs by a highly conserved process, termed binary fission, that requires the bacterial homologue of tubulin, FtsZ. Other mechanisms of bacterial cell division that are independent of FtsZ are rare. Although the obligate intracellular human pathogen Chlamydia trachomatis, the leading bacterial cause of sexually transmitted infections and trachoma, lacks FtsZ, it has been assumed to divide by binary fission. We show here that Chlamydia divides by a polarized cell division process similar to the budding process of a subset of the Planctomycetes that also lack FtsZ. Prior to cell division, the major outer-membrane protein of Chlamydia is restricted to one pole of the cell, and the nascent daughter cell emerges from this pole by an asymmetric expansion of the membrane. Components of the chlamydial cell division machinery accumulate at the site of polar growth prior to the initiation of asymmetric membrane expansion and inhibitors that disrupt the polarity of C. trachomatis prevent cell division. The polarized cell division of C. trachomatis is the result of the unipolar growth and FtsZ-independent fission of this coccoid organism. This mechanism of cell division has not been documented in other human bacterial pathogens suggesting the potential for developing Chlamydia-specific therapeutic treatments.     

Lipid lowering activity of novel N-(benzoylphenyl)pyridine-3-carboxamide derivatives in Triton WR-1339-induced hyperlipidemic rats

Abstract

Context: Dyslipidemia is a major risk factor for the development of cardiovascular diseases. Many dyslipidemic patients do not achieve their target lipid levels with the currently available medications, and most of them may experience many side effects.

Objective: The present work aimed toward identifying a new class of novel nicotinic acid-carboxamide derivatives as promising antihyperlipidemic compounds.

Materials and methods: Six novel N-(benzoylphenyl)pyridine-3-carboxamide derivatives were synthesized using acid chloride pathways. All structures were confirmed using ¹H-NMR, ¹³C-NMR, IR, and HRMS. The evaluation of biological activity was conducted using Triton WR-1339-induced hyperlipidemic rats model.

Results: This study revealed that some of the newly synthesized novel N-(benzoylphenyl)pyridine-3-carboxamide derivatives mainly C4 and C6 possessed significant antihyperlipidemic activities on lipid components TG and TC (p value?<0.05).

Discussion and conclusion: This research opens the door for new potential antihyperlipidemic compounds derived from nicotinic acid that need further optimization of their biological activities.