Systematic Studies on the Anti-SARS-CoV-2 Mechanisms of Tea Polyphenol-Related Natural Products.

The causative pathogen of COVID-19, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), utilizes the receptor-binding domain (RBD) of the spike protein to bind to human receptor angiotensin-converting enzyme 2 (ACE2). Further cleavage of spike by human proteases furin, TMPRSS2, and/or cathepsin L facilitates viral entry into the host cells for replication, where the maturation of polyproteins by 3C-like protease (3CLpro) and papain-like protease (PLpro) yields functional nonstructural proteins (NSPs) such as RNA-dependent RNA polymerase (RdRp) to synthesize mRNA of structural proteins. By testing the tea polyphenol-related natural products through various assays, we found that the active antivirals prevented SARS-CoV-2 entry by blocking the RBD/ACE2 interaction and inhibiting the relevant human proteases, although some also inhibited the viral enzymes essential for replication. Due to their multitargeting properties, these compounds were often misinterpreted for their antiviral mechanisms. In this study, we provide a systematic protocol to check and clarify their anti-SARS-CoV-2 mechanisms, which should be applicable for all of the antivirals.

Synthesis, evaluation, and mechanism of 1-(4-(arylethylenylcarbonyl)phenyl)-4-carboxy-2-pyrrolidinones as potent reversible SARS-CoV-2 entry inhibitors.

A class of 1-(4-(arylethylenylcarbonyl)phenyl)-4-carboxy-2-pyrrolidinones were designed and synthesized via Michael addition, cyclization, aldol condensation, and deprotonation to inhibit the human transmembrane protease serine 2 (TMPRSS2) and Furin, which are involved in priming the SARS-CoV-2 Spike for virus entry. The most potent inhibitor 2f (81) was found to efficiently inhibit the replication of various SARS-CoV-2 delta and omicron variants in VeroE6 and Calu-3 cells, with EC50 range of 0.001-0.026 µM by pre-incubation with the virus to avoid the virus entry. The more potent antiviral activities than the proteases inhibitory activities led to discovery that the synthesized compounds also inhibited Spike's receptor binding domain (RBD):angiotensin converting enzyme 2 (ACE2) interaction as a main target, and their antiviral activities were enhanced by inhibiting TMPRSS2 and/or Furin. To further confirm the blocking effect of 2f (81) on virus entry, SARS-CoV-2 Spike pseudovirus was used in the entry assay and the results showed that the compound inhibited the pseudovirus entry in a ACE2-dependent pathway, via mainly inhibiting RBD:ACE2 interaction and TMPRSS2 activity in Calu-3 cells. Finally, in the in vivo animal model of SARS-CoV-2 infection, the oral administration of 25 mg/kg 2f (81) in hamsters resulted in reduced bodyweight loss and 5-fold lower viral RNA levels in nasal turbinate three days post-infection. Our findings demonstrated the potential of the lead compound for further preclinical investigation as a potential treatment for SARS-CoV-2.

Harnessing microbial phylum-specific molecular markers for assessment of environmental estrogen degradation.

Steroidal estrogens are ubiquitous contaminants that have garnered attention worldwide due to their endocrine-disrupting and carcinogenic activities at sub-nanomolar concentrations. Microbial degradation is one of the main mechanisms through which estrogens can be removed from the environment. Numerous bacteria have been isolated and identified as estrogen degraders; however, little is known about their contribution to environmental estrogen removal. Here, our global metagenomic analysis indicated that estrogen degradation genes are widely distributed among bacteria, especially among aquatic actinobacterial and proteobacterial species. Thus, by using the Rhodococcus sp. strain B50 as the model organism, we identified three actinobacteria-specific estrogen degradation genes, namely aedGHJ, by performing gene disruption experiments and metabolite profile analysis. Among these genes, the product of aedJ was discovered to mediate the conjugation of coenzyme A with a unique actinobacterial C17 estrogenic metabolite, 5-oxo-4-norestrogenic acid. However, proteobacteria were found to exclusively adopt an α-oxoacid ferredoxin oxidoreductase (i.e., the product of edcC) to degrade a proteobacterial C18 estrogenic metabolite, namely 3-oxo-4,5-seco-estrogenic acid. We employed actinobacterial aedJ and proteobacterial edcC as specific biomarkers for quantitative polymerase chain reaction (qPCR) to elucidate the potential of microbes for estrogen biodegradation in contaminated ecosystems. The results indicated that aedJ was more abundant than edcC in most environmental samples. Our results greatly expand the understanding of environmental estrogen degradation. Moreover, our study suggests that qPCR-based functional assays are a simple, cost-effective, and rapid approach for holistically evaluating estrogen biodegradation in the environment.

Circulating androgen regulation by androgen-catabolizing gut bacteria in male mouse gut.

Abnormally high circulating androgen levels have been considered a causative factor for benign prostatic hypertrophy and prostate cancer in men. Recent animal studies on gut microbiome suggested that gut bacteria are involved in sex steroid metabolism; however, the underlying mechanisms and bacterial taxa remain elusive. Denitrifying betaproteobacteria Thauera spp. are metabolically versatile and often distributed in the animal gut. Thauera sp. strain GDN1 is an unusual betaproteobacterium capable of catabolizing androgen under both aerobic and anaerobic conditions. We administered C57BL/6 mice (aged 7 weeks) with strain GDN1 through oral gavage. The strain GDN1 administration caused a minor increase in the relative abundance of Thauera (≤0.1%); however, it has profound effects on the host physiology and gut bacterial community. The results of our ELISA assay and metabolite profile analysis indicated an approximately 50% reduction in serum androgen levels in the strain GDN1-administered male mice. Moreover, androgenic ring-cleaved metabolites were detected in the fecal extracts of the strain GDN1-administered mice. Furthermore, our RT - qPCR results revealed the expression of the androgen catabolism genes in the gut of the strain GDN1-administered mice. We found that the administered strain GDN1 regulated mouse serum androgen levels, possibly because it blocked androgen recycling through enterohepatic circulation. This study discovered that sex steroids serve as a carbon source of gut bacteria; moreover, host circulating androgen levels may be regulated by androgen-catabolizing gut bacteria. Our data thus indicate the possible applicability of androgen-catabolic gut bacteria as potent probiotics in alternative therapy of hyperandrogenism.

Impact of public transport disruption on access to healthcare facility and well-being during the COVID-19 pandemic: A qualitative case study in Metro Manila, Philippines.

The COVID-19 pandemic has forced many governments to halt public transport operations. A consequence of such disruption is the reduction in access to critical facilities by individuals who rely on public transport for their daily mobility. We investigate the impact disparities caused by the restriction of public transportation on the access of healthcare workers and patients to healthcare facilities during the COVID-19 pandemic. Metro Manila is an appropriate case study site because the duration of suspension of public transport in the mega-city is one of the longest in the world. The prolonged duration of the lockdown could have devastating impacts on the well-being of individuals who are reliant on public transport to access essential services. Guided by the Yin-Eisenhardt approach to qualitative research, we examined the data from 55 individuals using within-case and cross-case analyses iteratively for the purpose of building a model on the impact of change in access due to public transport disruption on well-being. We mobilized constructs and concepts known in the literature, such as well-being, access, disruption, resistance, resilience, and vulnerability, in developing our two-step conceptual model. Given the profound impact of the prolonged and system-wide suspension of public transport on the well-being of individuals, it is necessary to provide sufficient public transport and active transport infrastructure and services that can cover their mobility needs. The two-step conceptual model from this study can provide guidance on specific policy interventions.

A unique self-truncation of bacterial GH5 endoglucanases leads to enhanced activity and thermostability.

BACKGROUND: ß-1,4-endoglucanase (EG) is one of the three types of cellulases used in cellulose saccharification during lignocellulosic biofuel/biomaterial production. GsCelA is an EG secreted by the thermophilic bacterium Geobacillus sp. 70PC53 isolated from rice straw compost in southern Taiwan. This enzyme belongs to glycoside hydrolase family 5 (GH5) with a TIM-barrel structure common among all members of this family. GsCelA exhibits excellent lignocellulolytic activity and thermostability. In the course of investigating the regulation of this enzyme, it was fortuitously discovered that GsCelA undergoes a novel self-truncation/activation process that appears to be common among GH5 enzymes. RESULTS: Three diverse Gram-positive bacterial GH5 EGs, but not a GH12 EG, undergo an unexpected self-truncation process by removing a part of their C-terminal region. This unique process has been studied in detail with GsCelA. The purified recombinant GsCelA was capable of removing a 53-amino-acid peptide from the C-terminus. Natural or engineered GsCelA truncated variants, with up to 60-amino-acid deletion from the C-terminus, exhibited higher specific activity and thermostability than the full-length enzyme. Interestingly, the C-terminal part that is removed in this self-truncation process is capable of binding to cellulosic substrates of EGs. The protein truncation, which is pH and temperature dependent, occurred between amino acids 315 and 316, but removal of these two amino acids did not stop the process. Furthermore, mutations of E142A and E231A, which are essential for EG activity, did not affect the protein self-truncation process. Conversely, two single amino acid substitution mutations affected the self-truncation activity without much impact on EG activities. In Geobacillus sp. 70PC53, the full-length GsCelA was first synthesized in the cell but progressively transformed into the truncated form and eventually secreted. The GsCelA self-truncation was not affected by standard protease inhibitors, but could be suppressed by EDTA and EGTA and enhanced by certain divalent ions, such as Ca2+, Mg2+, and Cu2+. CONCLUSIONS: This study reveals novel insights into the strategy of Gram-positive bacteria for directing their GH5 EGs to the substrate, and then releasing the catalytic part for enhanced activity via a spontaneous self-truncation process.

From simple and specific zymographic detections to the annotation of a fungus Daldinia caldariorum D263 that encodes a wide range of highly bioactive cellulolytic enzymes.

BACKGROUND: Lignocellulolytic enzymes are essential for agricultural waste disposal and production of renewable bioenergy. Many commercialized cellulase mixtures have been developed, mostly from saprophytic or endophytic fungal species. The cost of complete cellulose digestion is considerable because a wide range of cellulolytic enzymes is needed. However, most fungi can only produce limited range of highly bioactive cellulolytic enzymes. We aimed to investigate a simple yet specific method for discovering unique enzymes so that fungal species producing a diverse group of cellulolytic enzymes can be identified. RESULTS: The culture medium of an endophytic fungus, Daldinia caldariorum D263, contained a complete set of cellulolytic enzymes capable of effectively digesting cellulose residues into glucose. By taking advantage of the unique product inhibition property of ß-glucosidases, we have established an improved zymography method that can easily distinguish ß-glucosidase and exoglucanase activity. Our zymography method revealed that D263 can secrete a wide range of highly bioactive cellulases. Analyzing the assembled genome of D263, we found over 100 potential genes for cellulolytic enzymes that are distinct from those of the commercially used fungal species Trichoderma reesei and Aspergillus niger. We further identified several of these cellulolytic enzymes by mass spectrometry. CONCLUSIONS: The genome of Daldinia caldariorum D263 has been sequenced and annotated taking advantage of a simple yet specific zymography method followed by mass spectrometry analysis, and it appears to encode and secrete a wide range of bioactive cellulolytic enzymes. The genome and cellulolytic enzyme secretion of this unique endophytic fungus should be of value for identifying active cellulolytic enzymes that can facilitate conversion of agricultural wastes to fermentable sugars for the industrial production of biofuels.

[Application Value of CT Metal Artifact Correction Technology (MAC TM) in CT Review after Total Hip Replacement].

OBJECTIVE: To evaluate the application value of CT metal artifact correction technology (MAC TM) in CT review after total hip replacement. METHODS: A total of 72 patients who underwent CT re-examination after total hip replacement from December 2018 to March 2020 were enrolled, and the original data were reconstructed by filter backup projection （FBP） and MAC. Select three identical levels in the two sets of reconstructed images and place the same ROI. The selected levels were the initial level, central level, and lower edge of acetabulum. Measure the CT and noise (SD) of metal high and low density artifacts of the three levels area, as well as metal hip joint space, metal para-bone tissue, muscle, bladder and subcutaneous fat, and calculate the average value. Subcutaneous fat value was used as a reference to calculate the SNR and CNR of metal implant para-bone tissue, muscle and bladder. Two radiologists scored the two groups of reconstructed images using blinded method, Kappa's test was used to compare the homogeneity. RESULTS: There were differences between the two groups of reconstructed images in high- and low-density artifact areas, joint gap CT values, and image noise. Compared with the FBP group, the CT value of the high-density area and the joint space of the MAC group decreased, the CT value of the low-density area increased, and the noise value of each area decreased. The SNR and CNR of metal adjacent bone tissue, muscle and bladder were higher in the MAC group than those in the FBP group, and the difference was statistically significant ( P<0.05). The difference in subjective scores between the two groups was statistically significant ( Z=-6.564, P<0.05). 2 radiologists had moderate consistency with Kappa value of 0.72 on FBP group, and good consistency with Kappa value of 0.85 on MAC group. CONCLUSION: MAC TM in CT review after total hip replacement can reduce metal artifacts, make the joint space more clear, and improve the quality of CT images.

Hierarchical microstructure strengthening in a single crystal high entropy superalloy.

A hierarchical microstructure strengthened high entropy superalloy (HESA) with superior cost specific yield strength from room temperature up to 1,023 K is presented. By phase transformation pathway through metastability, HESA possesses a hierarchical microstructure containing a dispersion of nano size disordered FCC particles inside ordered L12 precipitates that are within the FCC matrix. The average tensile yield strength of HESA from room temperature to 1,023 K could be 120 MPa higher than that of advanced single crystal superalloy, while HESA could still exhibit an elongation greater than 20%. Furthermore, the cost specific yield strength of HESA can be 8 times that of some superalloys. A template for lighter, stronger, cheaper, and more ductile high temperature alloy is proposed.

CT FFR for Ischemia-Specific CAD With a New Computational Fluid Dynamics Algorithm: A Chinese Multicenter Study.

OBJECTIVES: The aim of this study was to validate the feasibility of a novel structural and computational fluid dynamics-based fractional flow reserve (FFR) algorithm for coronary computed tomography angiography (CTA), using alternative boundary conditions to detect lesion-specific ischemia. BACKGROUND: A new model of computed tomographic (CT) FFR relying on boundary conditions derived from structural deformation of the coronary lumen and aorta with transluminal attenuation gradient and assumptions regarding microvascular resistance has been developed, but its accuracy has not yet been validated. METHODS: A total of 338 consecutive patients with 422 vessels from 9 Chinese medical centers undergoing CTA and invasive FFR were retrospectively analyzed. CT FFR values were obtained on a novel on-site computational fluid dynamics-based CT FFR (uCT-FFR [version 1.5, United-Imaging Healthcare, Shanghai, China]). Performance characteristics of uCT-FFR and CTA in detecting lesion-specific ischemia in all lesions, intermediate lesions (luminal stenosis 30% to 70%), and "gray zone" lesions (FFR 0.75 to 0.80) were calculated with invasive FFR as the reference standard. The effect of coronary calcification on uCT-FFR measurements was also assessed. RESULTS: Per vessel sensitivities, specificities, and accuracies of 0.89, 0.91, and 0.91 with uCT-FFR, 0.92, 0.34, and 0.55 with CTA, and 0.94, 0.37, and 0.58 with invasive coronary angiography, respectively, were found. There was higher specificity, accuracy, and AUC for uCT-FFR compared with CTA and qualitative invasive coronary angiography in all lesions, including intermediate lesions (p < 0.001 for all). No significant difference in diagnostic accuracy was observed in the "gray zone" range versus the other 2 lesion groups (FFR ≤0.75 and >0.80; p = 0.397) and in patients with "gray zone" versus FFR ≤0.75 (p = 0.633) and versus FFR >0.80 (p = 0.364), respectively. No significant difference in the diagnostic performance of uCT-FFR was found between patients with calcium scores ≥400 and <400 (p = 0.393). CONCLUSIONS: This novel computational fluid dynamics-based CT FFR approach demonstrates good performance in detecting lesion-specific ischemia. Additionally, it outperforms CTA and qualitative invasive coronary angiography, most notably in intermediate lesions, and may potentially have diagnostic power in gray zone and highly calcified lesions.

Covered versus uncovered stent insertion for malignant superior vena cava obstruction.

Purpose: To determine whether covered or uncovered stent insertion achieved better clinical efficacy when used to treat malignant superior vena cava (SVC) obstruction (SVCO).Material and methods: A total of 64 patients with malignant SVCO underwent stent insertion between January 2011 and March 2018 at our center. Of these, 34 were treated via uncovered stent insertion while 30 were treated via covered stent insertion. We compared the clinical effectiveness, patency of the stent, and overall survival between these two groups.Results: Both treatments achieved a 100% technical and clinical success rate, without any incidence of complications relating to the procedure. Stent dysfunction was found in one and six patients in the covered and uncovered groups during the follow-up period (1/30 vs. 6/34, p = .153), respectively. The covered stent patency period was significantly longer in the group treated with covered stents (374 vs. 317 days, p = .049), while median survival following stent insertion was 175 and 159 days, respectively, for the covered and uncovered groups (p = .784).Conclusion: Uncovered and covered stent insertion are both safe means of effectively treating patients with malignant SVCO, but covered stents achieve better patency for long-term periods than uncovered stents.

Chaetomella raphigera ß-glucosidase D2-BGL has intriguing structural features and a high substrate affinity that renders it an efficient cellulase supplement for lignocellulosic biomass hydrolysis.

BACKGROUND: To produce second-generation biofuels, enzymatic catalysis is required to convert cellulose from lignocellulosic biomass into fermentable sugars. ß-Glucosidases finalize the process by hydrolyzing cellobiose into glucose, so the efficiency of cellulose hydrolysis largely depends on the quantity and quality of these enzymes used during saccharification. Accordingly, to reduce biofuel production costs, new microbial strains are needed that can produce highly efficient enzymes on a large scale. RESULTS: We heterologously expressed the fungal ß-glucosidase D2-BGL from a Taiwanese indigenous fungus Chaetomella raphigera in Pichia pastoris for constitutive production by fermentation. Recombinant D2-BGL presented significantly higher substrate affinity than the commercial ß-glucosidase Novozyme 188 (N188; K m = 0.2 vs 2.14 mM for p-nitrophenyl ß-d-glucopyranoside and 0.96 vs 2.38 mM for cellobiose). When combined with RUT-C30 cellulases, it hydrolyzed acid-pretreated lignocellulosic biomasses more efficiently than the commercial cellulase mixture CTec3. The extent of conversion from cellulose to glucose was 83% for sugarcane bagasse and 63% for rice straws. Compared to N188, use of D2-BGL halved the time necessary to produce maximal levels of ethanol by a semi-simultaneous saccharification and fermentation process. We upscaled production of recombinant D2-BGL to 33.6 U/mL within 15 days using a 1-ton bioreactor. Crystal structure analysis revealed that D2-BGL belongs to glycoside hydrolase (GH) family 3. Removing the N-glycosylation N68 or O-glycosylation T431 residues by site-directed mutagenesis negatively affected enzyme production in P. pastoris. The F256 substrate-binding residue in D2-BGL is located in a shorter loop surrounding the active site pocket relative to that of Aspergillus ß-glucosidases, and this short loop is responsible for its high substrate affinity toward cellobiose. CONCLUSIONS: D2-BGL is an efficient supplement for lignocellulosic biomass saccharification, and we upscaled production of this enzyme using a 1-ton bioreactor. Enzyme production could be further improved using optimized fermentation, which could reduce biofuel production costs. Our structure analysis of D2-BGL offers new insights into GH3 ß-glucosidases, which will be useful for strain improvements via a structure-based mutagenesis approach.

Total sesquiterpene lactones isolated from Inula helenium L. attenuates 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in mice.

BACKGROUND: Inula helenium L. is an herb whose anti-inflammatory properties are attributed to its active components, the sesquiterpene lactones (SLs). Our previous study demonstrated that the total sesquiterpene lactones isolated from Inula helenium L. (TSL-IHL), consisting mainly of alantolactone (AL) and isoalantolactone (IAL), may have potential in the prevention and treatment of rheumatoid arthritis (RA). However, the effect of TSL-IHL on atopic dermatitis (AD) has not been studied yet. AIM OF THE STUDY: The present study evaluates the potential of TSL-IHL as a treatment for AD. METHODS/STUDY DESIGNS: The effects of TSL-IHL on the expression of inflammatory genes and the activation of NF-κB signaling pathway in HaCat cells were examined by quantitative real-time PCR and western blotting, respectively, and compared with those of AL and IAL. The protective effect of TSL-IHL against AD was tested in a mouse model induced by 2,4-dinitrochlorobenzene (DNCB), in which AD-like skin lesions were induced in ICR mice by sensitizing once with 100 µl of 7% DNCB painted on their shaved back skin and then challenging with 20 µl of 0.2% DNCB five times on their right ears at 3 day intervals starting on day 5 post-sensitization. RESULTS: TSL-IHL, as well as AL and IAL, could all inhibit TNF-α-induced activation of NF-κB and the expression of TNF-α, IL-1, and IL-4 in HaCat cells in a dose-dependent manner in the range of 0.6-2.4 µg/ml. The topical application of TSL-IHL (1% W/W in emollient cream) attenuated DNCB-induced dermatitis severity and right ear swelling. The serum levels of IgE, TNF-α and IFN-γ in TSL-IHL-treated mice were reduced by 81.39%, 89.69%, and 87.85%, respectively, while the mRNA levels of IL-4, IL-5 and IL-13, in the back-skin lesions of TSL-IHL-treated mice were reduced by 39.21%, 40.62% and 48.12%, respectively, compared with the untreated controls. Histopathological examination showed that TSL-IHL treatment reduced epidermis/dermis thickening and dermal inflammatory infiltration in both ear and back skins. CONCLUSIONS: We suggest that TSL-IHL inhibited the development of AD-like skin symptoms by regulating cytokine expression and may be an effective alternative therapy for AD.

Synthesis of oligomeric mannosides and their structure-binding relationship with concanavalin†A.

Small glycodendrimers with α-mannosyl ligands were synthesized by using copper-catalyzed azide-alkyne coupling chemistry and some of these molecules were used as multivalent ligands to study the induction of concanavalin A (Con A) precipitation. The results showed that the monovalent mannose ligand could induce the precipitation of Con A. This unexpected finding initiated a series of studies to characterize the molecular basis of the ligand-lectin interaction. The atypical precipitation is found to be specific to the mannose, fluorescein moiety (FITC), and Con A. Apparently the mannose ligand binds to Con A through hydrogen-bonding interactions, whereas the binding of FITC is mediated by hydrophobic forces.

Multidetector CT and MR imaging cardiac hydatidosis: case report and review of the literature.

Cardiac hydatid cysts are uncommon but potentially fatal. We present a case of isolated unilocular and multivesicular hydatid cysts in the heart, and provide a literature review of this rare condition. A 35-year-old man presented to our cardiology unit with acute chest pain. Computed tomography showed two cystic lesions in the heart. One unilocular cyst was located close to the left atrium and compressed the pulmonary artery. The other multivesicular cyst adhered to the left ventricle and displaced the left coronary arteries. T2-weighted magnetic resonance images revealed lesions in the pericardial cavity with bright signal intensity. Dot immunogold filtration assay was performed, and positive results for anti-EgCF antibody, anti-EgP antibody and anti-EgB antibody for cystic hydatidosis were found. Cardiac hydatidosis was diagnosed because of typical imaging findings and positive serology. The patient underwent surgical excision of the cysts. Postoperatively, symptoms of the patient resolved.

High-level production of a thermoacidophilic beta-glucosidase from Penicillium citrinum YS40-5 by solid-state fermentation with rice bran.

A high yield of beta-glucosidase (EC 3.2.1.21) of 159.1 U/g-solid activity on 4-nitrophenyl beta-d-glucopyranoside (pNPG) was achieved by rice bran-based solid-state fermentation (SSF) of the recently characterized fungus Penicillium citrinum YS40-5. The enzyme was both thermophilic and acidophilic at the optimized temperature and pH of 70 degrees C and 5.0, respectively. Over 95% of the original beta-glucosidase activity was maintained after a prolonged storage at ambient temperature for 4 weeks. The kinetic parameters V(max), K(m) and K(I) were 85.93 U/mg, 1.2 mM and 17.59 mM with pNPG, and 72.49 U/mg, 32.17 mM and 8.29 mM with cellobiose, respectively. The protein band with beta-glucosidase activity was characterized by native PAGE followed by MUG-zymogram analysis, and its identity confirmed by nanoLC-MS/MS. A 3.43-fold synergistic effect by combining this beta-glucosidase with Trichoderma reesei cellulases was observed, indicating this enzyme could potentially be used for improving the efficiency of cellulosic bioconversion.

Identification of Pseudomonas aeruginosa using functional magnetic nanoparticle-based affinity capture combined with MALDI MS analysis.

PA-IL is a galactophilic lectin that is found on the outer membrane of Pseudomonas aeruginosa. Pigeon ovalbumin (POA), a phosphoprotein, contains high levels of terminal Gal alpha(1-->4)Gal units. Thus, magnetic nanoparticles with immobilized POA can be used as affinity probes for P. aeruginosa, functioning via the recognition of galactophilic PA-IL. We fabricated POA-bound nanoparticles (NPs) by immobilizing POA onto the surface of core/shell magnetic iron oxide/alumina NPs via metal-phosphate chelation. We then used the generated NPs to probe target bacteria from complex samples. The trapped bacterial cells were characterized based on their mass peak profiles obtained from MALDI MS analyses. In addition, we confirmed the determination of P. aeruginosa using a proteomic strategy: combining the resultant MALDI MS/MS spectra of its tryptic digest with protein database searching. The feasibility of using this approach to rapidly characterize P. aeruginosa from clinical samples without the need to perform culturing steps was also demonstrated.

Low-concentration 1,2-trans beta-selective glycosylation strategy and its applications in oligosaccharide synthesis.

This study develops an operationally easy, efficient, and general 1,2-trans beta-selective glycosylation reaction that proceeds in the absence of a C2 acyl function. This process employs chemically stable thioglycosyl donors and low substrate concentrations to achieve excellent beta-selectivities in glycosylation reactions. This method is widely applicable to a range of glycosyl substrates irrespective of their structures and hydroxyl-protecting functions. This low-concentration 1,2-trans beta-selective glycosylation in carbohydrate chemistry removes the restriction of using highly reactive thioglycosides to construct 1,2-trans beta-glycosidic bonds. This is beneficial to the design of new strategies for oligosaccharide synthesis, as illustrated in the preparation of the biologically relevant beta-(1-->6)-glucan trisaccharide, beta-linked Gb(3) and isoGb(3) derivatives.

A novel endo-glucanase from the thermophilic bacterium Geobacillus sp. 70PC53 with high activity and stability over a broad range of temperatures.

A thermophilic Geobacillus bacterium secreting high activity of endo-glucanase (EC 3.2.1.4) was isolated from rice straw compost supplemented with pig manure. A full-length gene of 1,104 bp, celA, encoding this glycosyl hydrolase family 5 endo-glucanase of 368 amino acids was isolated. No related gene from Geobacillus has been reported previously. The recombinant CelA expressed in Escherichia coli had an optimal activity at 65 degrees C and pH 5.0, and it exhibited tenfold greater specific activity than the commercially available Trichoderma reesei endo-glucanase. CelA displayed activity over a broad temperature range from 45 to 75 degrees C and was a thermostable enzyme with 90% activity retained after heating at 65 degrees C for 6 h. Interestingly, CelA activity could be enhanced by 100% in the presence of 2 mM MnSO(4). CelA had high specific activity over beta-D-glucan from barley and Lichenan, making it a potentially useful enzyme in biofuel and food industries.