Biosynthesis of animal-free recombinant chondroitin sulfate E using a functional chondroitin sulfotransferase in E. coli.

Chondroitin sulfate E (CS-E) is a vital sulfated glycosaminoglycan with diverse biological functions and therapeutic potential. This study marks a significant milestone by achieving the first successful microbial production of chondroitin 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) in Escherichia coli, enabling recombinant CS-E biosynthesis. Initially, we identified sulfotransferases capable of converting chondroitin sulfate A (CS-A) to CS-E, but these enzymes were non-functional when expressed in E.

Synthesis of bioengineered heparin chemically and biologically similar to porcine-derived products and convertible to low MW heparin.

Heparins have been invaluable therapeutic anticoagulant polysaccharides for over a century, whether used as unfractionated heparin or as low molecular weight heparin (LMWH) derivatives. However, heparin production by extraction from animal tissues presents multiple challenges, including the risk of adulteration, contamination, prion and viral impurities, limited supply, insecure supply chain, and significant batch-to-batch variability. The use of animal-derived heparin also raises ethical and religious concerns, as well as carries the risk of transmitting zoonotic diseases.

Improved soluble expression and use of recombinant human renalase.

Electrochemical bioreactor systems have enjoyed significant attention in the past few decades, particularly because of their applications to biobatteries, artificial photosynthetic systems, and microbial electrosynthesis. A key opportunity with electrochemical bioreactors is the ability to employ cofactor regeneration strategies critical in oxidative and reductive enzymatic and cell-based biotransformations. Electrochemical cofactor regeneration presents several advantages over other current cofactor regeneration systems, such as chemoenzymatic multi-enzyme reactions, because there is no need for a sacrificial substrate and a recycling enzyme.

Elucidating the unusual reaction kinetics of D-glucuronyl C5-epimerase.

The chemoenzymatic synthesis of heparin, through a multienzyme process, represents a critical challenge in providing a safe and effective substitute for this animal-sourced anticoagulant drug. D-glucuronyl C5-epimerase (C5-epi) is an enzyme acting on a heparin precursor, N-sulfoheparosan, catalyzing the reversible epimerization of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). The absence of reliable assays for C5-epi has limited elucidation of the enzymatic reaction and kinetic mechanisms.

Newly identified bacteriolytic enzymes that target a wide range of clinical isolates of Clostridium difficile.

Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora.

Wall Teichoic Acids Are Involved in the Medium-Induced Loss of Function of the Autolysin CD11 against Clostridium difficile.

Bacterial lysins are potent antibacterial enzymes with potential applications in the treatment of bacterial infections. Some lysins lose activity in the growth media of target bacteria, and the underlying mechanism remains unclear. Here we use CD11, an autolysin of Clostridium difficile, as a model lysin to demonstrate that the inability of this enzyme to kill C.

Binding domains of Bacillus anthracis phage endolysins recognize cell culture age-related features on the bacterial surface.

Bacteriolytic enzymes often possess a C-terminal binding domain that recognizes specific motifs on the bacterial surface and a catalytic domain that cleaves covalent linkages within the cell wall peptidoglycan. PlyPH, one such lytic enzyme of bacteriophage origin, has been reported to be highly effective against Bacillus anthracis, and can kill up to 99.99% of the viable bacteria.

Characterization of the activity of the spore cortex lytic enzyme CwlJ1.

The germination enzyme CwlJ1 plays an important role in degrading the cortex during the germination of Bacillus anthracis spores. However, the specific function and catalytic activity of CwlJ1 remain elusive. Here we report for the first time a detailed in vitro mechanistic study of CwlJ1 expressed in Escherichia coli and its activity against the spore cortical fragments of B.

Growth inhibition of Mycobacterium smegmatis by mycobacteriophage-derived enzymes.

We report the ability of mycobacteriophage-derived endolysins to inhibit the growth of Mycobacterium smegmatis. We expressed and purified LysB from mycobacteriophage Bxz2 and compared its activity with that of a previously reported LysB from mycobacteriophage Ms6. The esterase activity of Bxz2 LysB with pNP esters was 10-fold higher than that of the previously reported LysB but its lipolytic activity was significantly lower.

Evaluation of potential genotoxicity of HIV entry inhibitors derived from natural sources.

AIDS is a global pandemic that has seen the development of novel and effective treatments to improve the quality of life of those infected and reduction of spread of the disease. Palmitic Acid (PA), which we identified and isolated from Sargassum fusiforme, is a naturally occurring fatty acid that specifically inhibits HIV entry by binding to a novel pocket on the CD4 receptor. We also identified a structural analogue, 2-bromopalmitate (2-BP), as a more effective HIV entry inhibitor with a 20-fold increase in efficacy.

Enzyme-driven Bacillus spore coat degradation leading to spore killing.

The bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment.

Characterization of AmiBA2446, a novel bacteriolytic enzyme active against Bacillus species.

There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls.

Enzyme-based listericidal nanocomposites.

Cell lytic enzymes represent an alternative to chemical decontamination or use of antibiotics to kill pathogenic bacteria, such as listeria. A number of phage cell lytic enzymes against listeria have been isolated and possess listericidal activity; however, there has been no attempt to incorporate these enzymes onto surfaces. We report three facile routes for the surface incorporation of the listeria bacteriophage endolysin Ply500: covalent attachment onto FDA approved silica nanoparticles (SNPs), incorporation of SNP-Ply500 conjugates into a thin poly(hydroxyethyl methacrylate) film; and affinity binding to edible crosslinked starch nanoparticles via construction of a maltose binding protein fusion.

Lysostaphin-functionalized cellulose fibers with antistaphylococcal activity for wound healing applications.

With the emergence of "super bacteria" that are resistant to antibiotics, e.g., methicillin-resistant Staphylococcus aureus, novel antimicrobial therapies are needed to prevent associated hospitalizations and deaths.

Inhibition of HIV-1 infection in ex vivo cervical tissue model of human vagina by palmitic acid; implications for a microbicide development.

Background: Approximately 80% of all new HIV-1 infections are acquired through sexual contact. Currently, there is no clinically approved microbicide, indicating a clear and urgent therapeutic need. We recently reported that palmitic acid (PA) is a novel and specific inhibitor of HIV-1 fusion and entry.

Palmitic acid analogs exhibit nanomolar binding affinity for the HIV-1 CD4 receptor and nanomolar inhibition of gp120-to-CD4 fusion.

Background: We recently reported that palmitic acid (PA) is a novel and efficient CD4 fusion inhibitor to HIV-1 entry and infection. In the present report, based on in silico modeling of the novel CD4 pocket that binds PA, we describe discovery of highly potent PA analogs with increased CD4 receptor binding affinities (K(d)) and gp120-to-CD4 inhibition constants (K(i)). The PA analogs were selected to satisfy Lipinski's rule of drug-likeness, increased solubility, and to avoid potential cytotoxicity.

Palmitic Acid Is a Novel CD4 Fusion Inhibitor That Blocks HIV Entry and Infection.

The high rate of HIV-1 mutation and the frequent sexual transmission highlight the need for novel therapeutic modalities with broad activity against both CXCR4 (X4) and CCR5 (R5)-tropic viruses. We investigated a large number of natural products, and from Sargassum fusiforme we isolated and identified palmitic acid (PA) as a natural small bioactive molecule with activity against HIV-1 infection. Treatment with 100 microM PA inhibited both X4 and R5 independent infection in the T cell line up to 70%.

BCL11B enhances TCR/CD28-triggered NF-kappaB activation through up-regulation of Cot kinase gene expression in T-lymphocytes.

BCL11B is a transcriptional regulator with an important role in T-cell development and leukaemogenesis. We demonstrated recently that BCL11B controls expression from the IL (interleukin)-2 promoter through direct binding to the US1 (upstream site 1). In the present study, we provide evidence that BCL11B also participates in the activation of IL-2 gene expression by enhancing NF-kappaB (nuclear factor kappaB) activity in the context of TCR (T-cell receptor)/CD28-triggered T-cell activation.

BCL11B is a general transcriptional repressor of the HIV-1 long terminal repeat in T lymphocytes through recruitment of the NuRD complex.

In this study we provide evidence that the transcription factor BCL11B represses expression from the HIV-1 long terminal repeat (LTR) in T lymphocytes through direct association with the HIV-1 LTR. We also demonstrate that the NuRD corepressor complex mediates BCL11B transcriptional repression of the HIV-1 LTR. In addition, BCL11B and the NuRD complex repressed TAT-mediated transactivation of the HIV-1 LTR in T lymphocytes, pointing to a potential role in initiation of silencing.

Sargassum fusiforme fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase.

Sargassum fusiforme (Harvey) Setchell has been shown to be a highly effective inhibitor of HIV-1 infection. To identify its mechanism of action, we performed bioactivity-guided fractionation on Sargassum fusiforme mixture. Here, we report isolation of a bioactive fraction SP4-2 (S.

Inhibition of highly productive HIV-1 infection in T cells, primary human macrophages, microglia, and astrocytes by Sargassum fusiforme.

Background: The high rate of HIV-1 mutation and increasing resistance to currently available antiretroviral (ART) therapies highlight the need for new antiviral agents. Products derived from natural sources have been shown to inhibit HIV-1 replication during various stages of the virus life cycle, and therefore represent a potential source of novel therapeutic agents. To expand our arsenal of therapeutics against HIV-1 infection, we investigated aqueous extract from Sargassum fusiforme (S.