Exploring the therapeutic potential of endolysin CD27L_EAD against Clostridioides difficile infection.

OBJECTIVES: Clostridioides difficile has emerged as a major cause of life-threatening diarrheal disease. Conventional antibiotics used in current standards of care exacerbate the emergence of antibiotic-resistant strains and pose a risk of recurrent C. difficile infection (CDI). Thus, there is an urgent need for alternative therapeutics that selectively eliminate C. difficile without disturbing the commensal microbiota. This study aimed to explore the potential of endolysins as an alternative therapeutic agent to antibiotics. Endolysin is a bacteriophage-derived peptidoglycan hydrolase that aids in the release of phage progeny during the final stage of infection. METHODS: In order to exploit endolysin as a therapeutic agent against CDI, the bactericidal activity of 23 putative endolysins was compared and ΦCD27 endolysin CD27L was selected and modified to CD27L_EAD by cleaving the cell-wall binding domain of CD27L. RESULTS: CD27L_EAD exhibited greater bacteriolytic activity than CD27L and its activity was stable over a wide range of salt concentrations and pH conditions. CD27L_EAD was added to a co-culture of human gut microbiota with C. difficile and the bacterial community structure was analyzed. CD27L_EAD did not impair the richness and diversity of the bacterial population but remarkably attenuated the abundance of C. difficile. Furthermore, the co-administration of vancomycin exerted synergistic bactericidal activity against C. difficile. ß-diversity analysis revealed that CD27L_EAD did not significantly disturb the composition of the microbial community, whereas the abundance of some species belonging to the family Lachnospiraceae decreased after CD27L_EAD treatment. CONCLUSIONS: This study provides insights into endolysin as a prospective therapeutic agent for the treatment of CDI without damaging the normal gut microbiota.

Comparison of trapping methods for use in surveys for potential Culicoides vectors of orbiviruses.

BACKGROUND: Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are orbiviruses that can cause fatal vector-borne diseases in white-tailed deer (Odocoileus virginianus). Trapping methods for collecting potential Culicoides vectors of orbiviruses were compared to optimize surveillance studies. METHODS: The number of captured midges and the virus infection rates of midge pools were compared for dry ice-baited Centers for Disease Control and Prevention (CDC) traps with or without black light. The number of individual midges of different Culicoides species captured at different crepuscular and nocturnal periods using rotator traps also was determined. The number of species/specimens of Culicoides was measured using five different trap methods including three animal-baited methods, a CDC trap with black light, and a CDC trap with no light. RESULTS: In trial one, there was no significant difference (P = 0.37) in the proportion of BTV-infected flies caught in traps with light compared to traps without light. However, there was a significant difference (P = 0.026) for EHDV-infected flies, and 89% were captured in traps with light. In trial two, more specimens of C. debilipalpis were captured in the morning hours (06:00-08:00) than in the evening hours (18:00-20:00). For trial three, the animal-baited traps did not capture any species of Culicoides that were not captured in the CDC light traps. There was no significant difference (P = 0.22) in total specimens captured among all five trap types. CONCLUSIONS: Specimens of Culicoides infected with BTV were not repelled by light traps in the first trial, while the majority of the specimens positive for EHDV were caught in traps with light. For the second trial, specimens of C. debilipalpis were most abundant during early morning hours, and thus spray applications of insecticides for control of that species may be more effective at sunrise rather than sunset. For objective three, no animal-baited trapping method collected different species of midges when compared to the CDC traps with light, which is unlike certain studies conducted in other geographical regions.

Wet in situ transesterification of spent coffee grounds with supercritical methanol for the production of biodiesel.

This work introduces biodiesel production from wet spent coffee grounds (SCGs) with supercritical methanol without any pre-drying process. Supercritical methanol and subcritical water effectively produced biodiesel via in situ transesterification by inducing more porous SCG and enhancing the efficiency of lipid extraction and conversion. It was also found that space loading was one of the critical factors for biodiesel production. An optimal biodiesel yield of 10.17 wt% of dry SCG mass (86.33 w/w% of esterifiable lipids in SCG) was obtained at reaction conditions of 270 °C, 90 bars, methanol to wet SCG ratio 5:1, space loading 58.4 ml/g and reaction time 20 min. Direct use of wet SCG waste as feedstock for supercritical biodiesel production eliminates the conventional dying process and the need of catalyst and also reduces environmental problems caused by landfill accumulation.

Solvo-thermal in situ transesterification of wet spent coffee grounds for the production of biodiesel.

This work addresses non-catalytic biodiesel production from spent coffee ground (SCG) by integrating solvo-thermal effect of 1,2-dichloroethane (DCE) with in situ transesterification over 160 °C. The SCG water content has a positive effect on the DCE hydrolysis up to 60 wt% due to the bimolecular substitution mechanism. The hydrolysis gives an acidic environment favorable for cellulose decomposition, SCG particle size reduction and lipid conversion. The optimal fatty acid ethyl ester yield was 11.8 wt% based on the mass of dried SCG with 3.36 ml ethanol and 3.16 ml DCE at 196.8 °C through the response surface methodology. Using the solvo-thermal effect, direct utilization of wet SCG as a biodiesel feedstock provides not only economic feasibility without using drying process and additional acid catalyst but also environmental advantage of recycling the municipal waste.

Catalyst-free production of alkyl esters from microalgae via combined wet in situ transesterification and hydrothermal liquefaction (iTHL).

This study introduces a process combining wet in situ transesterification and hydrothermal liquefaction (iTHL) for fatty acid ethyl ester (FAEE) production from intact microalgae, Nannochloropsis gaditana without catalyst at temperatures higher than 160°C. It is found that the chlorinated hydrocarbon solvents, SolvCl (dichloromethane, chloroform, and dichloroethane (DCE)), enhances the FAEE production by providing hydrogen chloride in an ionized form that can act as an acid catalyst. The SolvCl effect on iTHL is compared to acid catalyst assisted wet in situ transesterification. The most effective solvent is DCE with the FAEE selectivity in biocrude equal to 91.85% (maximum transesterifiable lipid basis). The optimum point for maximizing the FAEE yield is 185.08°C with 4.69mL ethanol and 1.98mL DCE/g of dry algal cells based on the response surface methodology. iTHL with both Nannochloropsis and Chlorella species provides a possibility of the process applicable to the other algal species.

Wet in situ transesterification of microalgae using ethyl acetate as a co-solvent and reactant.

This study addresses wet in situ transesterification of microalgae for the production of biodiesel by introducing ethyl acetate as both reactant and co-solvent. Ethyl acetate and acid catalyst are mixed with wet microalgae in one pot and the mixture is heated for simultaneous lipid extraction and transesterification. As a single reactant and co-solvent, ethyl acetate can provide higher FAEE yield and more saccharification of carbohydrates than the case of binary ethanol and chloroform as a reactant and a co-solvent. The optimal yield was 97.8wt% at 114°C and 4.06M catalyst with 6.67mlEtOAC/g dried algae based on experimental results and response surface methodology (RSM). This wet in situ transesterification of microalgae using ethyl acetate doesn't require an additional co-solvent and it also promises more economic benefit as combining extraction and transesterification in a single process.

In-situ transesterification of wet spent coffee grounds for sustainable biodiesel production.

This work addresses in-situ transesterification of wet spent coffee grounds (SCGs) for the production of biodiesel. For in-situ transesterification process, the methanol, organic solvent and acid catalyst were mixed with wet SCG in one pot and the mixture was heated for simultaneous lipid extraction and transesterification. Maximum yield of fatty acid methyl esters (FAME) was 16.75wt.% based on the weight of dry SCG at 95°C. Comprehensive experiments were conducted with varying temperatures and various amounts of moisture, methanol, co-solvent and acid catalyst. Moderate polar and alcohol-miscible organic solvent is suitable for the high FAME yield. Unsaturated FAMEs are subject to oxidative cleavage by nitric acid and shorter chain (C6 and C10) FAMEs were mainly produced while sulfuric acid yielded long chain unsaturated FAMEs (C16 and C18). Utilization of wet SCGs as a biodiesel feedstock gives economic and environmental benefits by recycling the municipal waste.

Redox-active charge carriers of conducting polymers as a tuner of conductivity and its potential window.

Electric conductivity of conducting polymers has been steadily enhanced towards a level worthy of being called its alias, "synthetic metal". PEDOT:PSS (poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate)), as a representative conducting polymer, recently reached around 3,000 S cm(-1), the value to open the possibility to replace transparent conductive oxides. The leading strategy to drive the conductivity increase is solvent annealing in which aqueous solution of PEDOT:PSS is treated with an assistant solvent such as DMSO (dimethyl sulfoxide). In addition to the conductivity enhancement, we found that the potential range in which PEDOT:PSS is conductive is tuned wider into a negative potential direction by the DMSO-annealing. Also, the increase in a redox-active fraction of charge carriers is proposed to be responsible for the enhancement of conductivity in the solvent annealing process.

Edge-exfoliated graphites for facile kinetics of delithiation.

As high rate charge and discharge characteristics of energy storage devices become more important with the market of electric vehicles intensively growing, the kinetics of lithiation or delithiation of electrode materials for lithium ion batteries require enhancement. Graphites, the most widely used anode materials, have a limited power density at high discharge rates, while their alternatives, such as silicon and transition metal oxides, show even inferior rate capability. This work was motivated from an idea of what if the edge opening of graphite was zipped more open to lithium ions in the electrolyte. By edge-selective functionalization, the peripheral d-spacing of graphite (d(0)) was locally controlled. Larger values of d(0) led to higher capacity especially at high discharge rates. Around 2-fold enhancement of capacity or energy density was achieved at 50C discharge rate from 110 to 190 mAh g(-1) by exfoliating graphite locally in its edge region. Also, the d(0) dependency of delithiation kinetics confirmed that the electrochemical step of Li(+) influx into or efflux out of the interlayer space of graphite is possibly the rate-determining step of lithiation or delithiation.

Apoptotic and anti-metastatic effects of the whole skin of Venenum bufonis in A549 human lung cancer cells.

In the present study, the effects of the whole skin of Venenum bufonis on apoptotic and anti-invasive activity in A549 human lung cancer cells were investigated. Treatment with extract of the whole skin of V. bufonis (SVB) resulted in a significant decrease in cell growth of A549 cells, depending on dosage, which was associated with apoptosis induction, as proved by chromatin condensation and accumulation of apoptotic fraction. SVB treatment induced expression of death receptor-related proteins, such as death receptor 4, which further triggered activation of caspase-8 and cleavage of Bid. In addition, the increase in apoptosis by SVB treatment was correlated with dysfunction of mitochondria, activation of caspase-9 and -3, downregulation of IAP family proteins, such as XIAP, cIAP-1 and cIAP-2, and concomitant degradation of activated caspase-3-specific target proteins, such as poly (ADP-ribose) polymerase and ß-catenin proteins. However, z-DEVD-fmk, a caspase-3-specific inhibitor, blocked SVB-induced apoptosis and increased the survival rate of SVB-treated cells, indicating that activation of caspase-3 plays a key role in SVB-induced apoptosis. In addition, within concentrations that were not cytotoxic to A549 cells, SVB induced marked inhibition of cell motility and invasiveness. Activities of matrix metalloproteinase (MMP)-2 and MMP-9 in AGS cells were dose-dependently inhibited by treatment with SVB, and this was also correlated with a decrease in expression of their mRNA and proteins, and upregulation of tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 mRNA expression. Further studies are needed; however, the results indicated that SVB induces apoptosis of A549 cells through a signaling cascade of death receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways. Our data also demonstrated that MMPs are critical targets of SVB-induced anti-invasiveness in A549 cells.

Processing of optineurin in neuronal cells.

Optineurin is a gene linked to amyotrophic lateral sclerosis, Paget disease of bone, and glaucoma, a major blinding disease. Mutations such as E50K were identified in glaucoma patients. We investigated herein the involvement of ubiquitin-proteasome pathway (UPP) and autophagy, two major routes for protein clearance, in processing of optineurin in a retinal ganglion cell model line RGC5 and neuronal PC12 cells. It was found that the endogenous optineurin level in neuronal cells was increased by treatment of proteasomal inhibitor but not by autophagic and lysosomal inhibitors. Multiple bands immunoreactive to anti-ubiquitin were seen in the optineurin pulldown, indicating that optineurin was ubiquitinated. In cells overexpressing wild type and E50K optineurin, the level of the proteasome regulatory ß5 subunit (PSMB5, indicative of proteasome activity) was reduced, whereas that for autophagy marker microtubule-associated protein 1 light chain 3 was enhanced compared with controls. Autophagosome formation was detected by electron microscopy. The foci formed after optineurin transfection were increased upon treatment of an autophagic inhibitor but were decreased by treatment of an inducer, rapamycin. Moreover, the level of optineurin-triggered apoptosis was reduced by rapamycin. This study thus provides compelling evidence that in a normal homeostatic situation, the turnover of endogenous optineurin involves mainly UPP. When optineurin is up-regulated or mutated, the UPP function is compromised, and autophagy comes into play. A decreased PSMB5 level and an induced autophagy were also demonstrated in vivo in retinal ganglion cells of E50K transgenic mice, validating and making relevant the in vitro findings.

Impairment of protein trafficking upon overexpression and mutation of optineurin.

BACKGROUND: Glaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu(50) to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis. METHODOLOGY/PRINCIPAL FINDINGS: Human retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential Leu(157) to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization. CONCLUSIONS/SIGNIFICANCE: The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation.

Differential effects of myocilin and optineurin, two glaucoma genes, on neurite outgrowth.

Myocilin and optineurin are two genes linked to glaucoma, a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. To investigate the effects of force-expressed wild-type and mutant myocilin and optineurin on neurite outgrowth in neuronal cells, we transiently transfected cells with pEGFP-N1 (mock control) as well as myocilin and optineurin plasmids including pMYOC(WT)-EGFP, pMYOC(P370L)-EGFP, pMYOC(1-367)-EGFP, pOPTN(WT)-EGFP, and pOPTN(E50K)-EGFP. PC12 cells transfected with pEGFP-N1 produced, as anticipated, long and extensive neuritis on nerve growth factor induction. The neurite length in those cells transfected with myocilin constructs was shortened and the number of neurites was also reduced. A similar inhibitory effect on neurite outgrowth was also elicited by myocilin transfection in RGC5 cells. In contrast, neither transfection of the optineurin constructs pOPTN(WT)-EGFP and pOPTN(E50K)-EGFP nor the myocilin and optineurin small-interfering RNA treatments induced significant alterations in neurite outgrowth. Transfection with the wild-type optineurin construct, but not with that of the wild-type myocilin, increased the apoptotic activity in cells. These results demonstrated that the two glaucoma genes, myocilin and optineurin, exhibited differential effects on neurite outgrowth. They may contribute to the development of neurodegenerative glaucoma via distinct mechanisms.

Effects of Sp1 overexpression on cultured human corneal stromal cells.

Sp1, a transcription factor, is upregulated in keratoconus, a cornea-thinning disease. Keratoconus corneas have also been shown to contain increased levels of degradative enzymes such as cathepsin B and decreased proteinase inhibitors such as alpha1-proteinase inhibitor (alpha1-PI). We transfected cultured human corneal stromal cells to overexpress Sp1. The resulting effects on cathepsin B and alpha1-PI levels as well as the cellular proliferative and apoptotic activities were examined by Western blotting and cytochemical staining. It was found that the Sp1 transfected cells contained a greater amount of cathepsin B than did mock transfected controls. The activity of cathepsin B was also increased. By contrast, the protein level of alpha1-PI was lowered in corneal stromal cells upon Sp1 overexpression. The Sp1-induced alterations thus mimicked closely those observed in keratoconus, supporting the notion that Sp1 upregulation may be a key factor contributing directly to the disease development. Furthermore, the apoptotic activity was unaffected in Sp1 transfectants but the proliferation was inhibited, consistent with the idea that Sp1 may play a role in differentiation of corneal cells.

Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N-glycan by blocking core oligosaccharide assembly.

The initial lipid-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-dolichyl pyrophosphate (Dol-PP) for N-glycan is synthesized and assembled at the membrane of the endoplasmic reticulum (ER) and subsequently transferred to a nascent polypeptide by the oligosaccharide transferase complex. We have identified an ALG3 homolog (HpALG3) coding for a dolichyl-phosphate-mannose dependent alpha-1,3-mannosyltransferase in the methylotrophic yeast Hansenula polymorpha. The detailed analysis of glycan structure by linkage-specific mannosidase digestion showed that HpALG3 is responsible for the conversion of Man5GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP, the first step to attach a mannose to the lipid-linked oligosaccharide in the ER. The N-glycosylation pathway of H. polymorpha has been remodeled by deleting the HpALG3 gene in the Hpoch1 null mutant strain blocked in the yeast-specific outer mannose chain synthesis and by introducing an ER-targeted Aspergillus saitoi alpha-1,2-mannosidase gene. This glycoengineered H. polymorpha strain produced glycoproteins mainly containing trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core backbone of various human-type N-glycans. The results demonstrate the high potential of H. polymorpha to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.

Functional characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 genes as members of the yeast OCH1 mannosyltransferase family involved in protein glycosylation.

The alpha-1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 (ScOCH1) is responsible for the outer chain initiation of N-linked oligosaccharides. To identify the genes involved in the first step of outer chain biosynthesis in the methylotrophic yeast Hansenula polymorpha, we undertook the functional analysis of three H. polymorpha genes, HpHOC1, HpOCH1, and HpOCR1, that belong to the OCH1 family containing seven members with significant sequence identities to ScOCH1. The deletions of these H. polymorpha genes individually resulted in several phenotypes suggestive of cell wall defects. Whereas the deletion of HpHOC1 (Hphoc1Delta) did not generate any detectable changes in N-glycosylation, the null mutant strains of HpOCH1 (Hpoch1Delta) and HpOCR1 (Hpocr1Delta) displayed a remarkable reduction in hypermannosylation. Although the apparent phenotypes of Hpocr1Delta were most similar to those of S. cerevisiae och1 mutants, the detailed structural analysis of N-glycans revealed that the major defect of Hpocr1Delta is not in the initiation step but rather in the subsequent step of outer chain elongation by alpha-1,2-mannose addition. Most interestingly, Hpocr1Delta showed a severe defect in the O-linked glycosylation of extracellular chitinase, representing HpOCR1 as a novel member of the OCH1 family implicated in both N- and O-linked glycosylation. In contrast, addition of the first alpha-1,6-mannose residue onto the core oligosaccharide Man8GlcNAc2 was completely blocked in Hpoch1Delta despite the comparable growth of its wild type under normal growth conditions. The complementation of the S. cerevisiae och1 null mutation by the expression of HpOCH1 and the lack of in vitro alpha-1,6-mannosyltransferase activity in Hpoch1Delta provided supportive evidence that HpOCH1 is the functional orthologue of ScOCH1. The engineered Hpoch1Delta strain with the targeted expression of Aspergillus saitoi alpha-1,2-mannosidase in the endoplasmic reticulum was shown to produce human-compatible high mannose-type Man5GlcNAc2 oligosaccharide as a major N-glycan.