Oxalobacter formigenes and Its Potential Role in Human Health

Oxalate degradation by the anaerobic bacterium Oxalobacter formigenes is important for human health, helping to prevent hyperoxaluria and disorders such as the development of kidney stones. Oxalate-degrading activity cannot be detected in the gut flora of some individuals, possibly because Oxalobacter is susceptible to commonly used antimicrobials. Here, clarithromycin, doxycycline, and some other antibiotics inhibited oxalate degradation by two human strains of O. formigenes. These strains varied in their response to gut environmental factors, including exposure to gastric acidity and bile salts. O. formigenes strains established oxalate breakdown in fermentors which were preinoculated with fecal bacteria from individuals lacking oxalate-degrading activity. Reducing the concentration of oxalate in the medium reduced the numbers of O. formigenes bacteria. Oxalate degradation was established and maintained at dilution rates comparable to colonic transit times in healthy individuals. A single oral ingestion of O. formigenes by adult volunteers was, for the first time, shown to result in (i) reduced urinary oxalate excretion following administration of an oxalate load, (ii) the recovery of oxalate-degrading activity in feces, and (iii) prolonged retention of colonization.     

Improving the quality of industrially important enzymes by directed evolution

Directed evolution is a new process for developing industrially viable biocatalysts. This technique does not require a comprehensive knowledge of the relationships between sequence structure and function of proteins as required by protein engineering. It mimics the process of Darwinian evolution in a test tube combining random mutagenesis and recombination with screening or selection for enzyme variants that have the desired properties. Directed evolution helps in enhancing the enzyme performance both in natural and synthetic environments. This article reviews the process of directed evolution and its application to improve substrate specificity, activity, enantioselectivity and thermal stability.     

Global diversity and balancing selection of 23 leading Plasmodium falciparum candidate vaccine antigens

Investigation of the diversity of malaria parasite antigens can help prioritize and validate them as vaccine candidates and identify the most common variants for inclusion in vaccine formulations. Studies of vaccine candidates of the most virulent human malaria parasite, Plasmodium falciparum, have focused on a handful of well-known antigens, while several others have never been studied. Here we examine the global diversity and population structure of leading vaccine candidate antigens of P. falciparum using the MalariaGEN Pf3K (version 5.1) resource, comprising more than 2600 genomes from 15 malaria endemic countries. A stringent variant calling pipeline was used to extract high quality antigen gene ‘haplotypes’ from the global dataset and a new R-package named VaxPack was used to streamline population genetic analyses. In addition, a newly developed algorithm that enables spatial averaging of selection pressure on 3D protein structures was applied to the dataset. We analysed the genes encoding 23 leading and novel candidate malaria vaccine antigens including csp, trap, eba175, ama1, rh5, and CelTOS. Our analysis shows that current malaria vaccine formulations are based on rare haplotypes and thus may have limited efficacy against natural parasite populations. High levels of diversity with evidence of balancing selection was detected for most of the erythrocytic and pre-erythrocytic antigens. Measures of natural selection were then mapped to 3D protein structures to predict targets of functional antibodies. For some antigens, geographical variation in the intensity and distribution of these signals on the 3D structure suggests adaptation to different human host or mosquito vector populations. This study provides an essential framework for the diversity of P. falciparum antigens to be considered in the design of the next generation of malaria vaccines.     

Studies on the removal of dyes from a synthetic textile effluent using barley husk in static-batch mode and in a continuous flow,packed-bed,reactor

Yarrowia lipolytica LGAM S(7)1 presented remarkable growth on industrial glycerol used as sole carbon substrate. Nitrogen-limited flask cultures were accompanied by restricted synthesis of reserve lipid, whilst amounts of citric acid were produced extracellularly. On the contrary, high amounts of reserve lipid (up to 3.5 g/l, 43% w/w of lipids in dry biomass) were produced in highly aerated continuous cultures. Lipid production was favoured at low specific dilution rates whilst fat-free material yield increased over the whole range of D (h(-1)). The maximum volumetric productivity obtained was 0.12 g lipid/1 h. Storage lipid composition did not present remarkable changes in the specific dilution rates tested. Oleate and linoleate were the dominant cellular fatty acids.     

LncRNA OIP5-AS1-directed miR-7 degradation promotes MYMX production during human myogenesis

Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) modulate gene expression programs in physiology and disease. Here, we report a noncoding RNA regulatory network that modulates myoblast fusion into multinucleated myotubes, a process that occurs during muscle development and muscle regeneration after injury. In early stages of human myogenesis, the levels of lncRNA OIP5-AS1 increased, while the levels of miR-7 decreased. Moreover, OIP5-AS1 bound and induced miR-7 decay via target RNA-directed miRNA decay; accordingly, loss of OIP5-AS1 attenuated, while antagonizing miR-7 accelerated, myotube formation. We found that the OIP5-AS1-mediated miR-7 degradation promoted myoblast fusion, as it derepressed the miR-7 target MYMX mRNA, which encodes the fusogenic protein myomixer (MYMX). Remarkably, an oligonucleotide site blocker interfered with the OIP5-AS1-directed miR-7 degradation, allowing miR-7 to accumulate, lowering MYMX production and suppressing myotube formation. These results highlight a mechanism whereby lncRNA OIP5-AS1-mediated miR-7 decay promotes myotube formation by stimulating a myogenic fusion program.     

Dextrose modified bilosomes for peroral delivery: improved therapeutic potential and stability of silymarin in diethylnitrosamine-induced hepatic carcinoma in rats

Abstract

Hepatic carcinoma (HC) is one of the most prevalent cancers, ranked as the second most common cause of cancer-related deaths worldwide. Silymarin (SYL) has been reported for its anticarcinogenic activity against various types of cancer such as prostate, breast, ovary, colon, lung, bladder and liver. Due to poor solubility and low bioavailability SYL lacks satisfactory therapeutic value thus designing a suitable and effective delivery system of SYL can led to improved therapeutic potential. The present study was aimed to develop SYL-loaded dextrose (DEX) modified bilosomes for targeted delivery to HC cells. The DEX-modified bilosomes were prepared through thin-film hydration method and optimized employing Box Behnken design. The bilosomes were evaluated for percent entrapment, drug loading, in vitro release and cytotoxicity on Hep-G2 cells. The optimized DEX-SYL-BL exhibited a particle size of 219.3?±?2.99?nm, percent entrapment of 62.32?±?4.23%, drug loading of 34.56?±?1.23% and 84.96?±?2.76% drug release respectively over a period of 24?hr. The stability of bilosomes was ascertained in simulated gastric and intestinal fluids. Cytotoxicity studies revealed greater performance of DEX-SYL-BL in terms of reduced viability in Hep-G2 cell lines when compared with pure SYL and SYL-BL. Further DEX-modified bilosomes were evaluated in vivo for their therapeutic efficacy in DEN-induced (Diethylnitrosamine) hepatic carcinoma in animal model. The DEX-SYL-BL displayed higher therapeutic potential as revealed from enhanced survival and reduced tumour burden in animals. DEX-SYL-BL also displayed significant restoration of altered oxidative markers and SGOT, SGPT levels towards normal value when compared with pure SYL.     

Computational prediction of blend time in a large-scale viral inactivation process for monoclonal antibodies biomanufacturing

Viral inactivation (VI) is a process widely used across the pharmaceutical industry to eliminate the cytotoxicity resulting from trace levels of viruses introduced by adventitious agents. This process requires adding Triton X-100, a non-ionic detergent solution, to the protein solution and allowing sufficient time for this agent to inactivate the viruses. Differences in process parameters associated with vessel designs, aeration rate, and many other physical attributes can introduce variability in the process, thus making predicting the required blending time to achieve the desired homogeneity of Triton X-100 more critical and complex. In this study we utilized a CFD model based on the lattice Boltzmann method (LBM) to predict the blend time to homogenize a Triton X-100 solution added during a typical full-scale commercial VI process in a vessel equipped with an HE-3-impeller for different modalities of the Triton X-100 addition (batch vs. continuous). Although direct experimental progress of the blending process was not possible because of GMP restrictions, the degree of homogeneity measured at the end of the process confirmed that Triton X-100 was appropriately dispersed, as required, and as computationally predicted here. The results obtained in this study were used to support actual production at the biomanufacturing site.     

The Replication Protein A Binding Site in Simian Virus 40 (SV40) T Antigen and Its Role in the Initial Steps of SV40 DNA Replication

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase α-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.