Clinical Features of COVID-19 Patients in Xiaogan City.

On February 6, 2020, Xiaogan City became the second most seriously affected city with coronavirus disease 2019 (COVID-19), outside Wuhan district, Hubei Province, China. The objectives are to study the clinical features of COVID-19 patients and assess the relationship between the severity of COVID-19, age, and C-reactive protein (CRP) levels. The retrospective data of 134 COVID-19 patients hospitalized in 3 hospitals of Xiaogan City, between February 1 and March 1, 2020, was collected. This study documented COVID-19 patients. Clinical data in terms of body temperature, history of travel, and direct contact with COVID-19 patients, and incubation period was collected. Out of the 134 patients, only 5 required intensive care. Moreover, 2 patients succumbed during this period. The median age of patients was 45 (33-56) years. The most common symptoms at the onset of disease were fever (66.4%), cough (33, 6%), and sore throat (14.7%). Amongst the medicines used, antiviral agents (92.3%) followed by the traditional Chinese medicine (89.5%) were most commonly used. In both the crude and adjusted (I to III) models, odds ratio and its 95% confidence interval for both age and CRP levels were > 1. Moreover, the smooth curve fitting graph reflected that the severity of COVID-19 was positively correlated with both age and CRP levels (all P value < 0.05). The signs and symptoms of COVID-19 patients were fairly moderate. The health care professionals treating the COVID-19 patients should be aware of the increased likelihood of progression to severe COVID-19 in elderly patients and those with high CRP levels.

Transformation of Bacillus subtilis.

Bacillus subtilis has tremendous applications in both academic research and industrial production. However, molecular cloning and transformation of B. subtilis are not as easy as those of Escherichia coli. Here we developed a simple protocol based on super-competent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids generated by prolonged overlap extension-PCR. Super-competent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. This new protocol is simple (e.g., restriction enzyme, phosphatase, and ligase free), fast, and highly efficient (i.e., ~10(7) or ~10(4) transformants per µg of multimeric plasmid or ligated plasmid DNA, respectively). Shuttle vectors for E. coli-B. subtilis are not required.

An internal hydrophobic helical domain of Bacillus subtilis enolase is essential but not sufficient as a non-cleavable signal for its secretion.

Many cytoplasmic proteins without a cleavable signal peptide, including enolase, are secreted during the stationary phase in Bacillus subtilis but the molecular mechanism is not yet clear. We previously identified a highly conserved embedded membrane domain in an internal hydrophobic α-helix of enolase that plays an important role in its secretion. In this study, we examined the role of the helix in more detail for the secretion of enolase. Altering this helix by mutations showed that many mutated forms in this domain were not secreted, some of which were not stable as a soluble form in the cytoplasm. On the other hand, mutations on the flanking regions of the helix or the conserved basic residues showed no deleterious effect. Bacillus enolase with the proper hydrophobic helical domain was also exported extracellularly in Escherichia coli, indicating that the requirement of the helix for the secretion of enolase is conserved in these species. GFP fusions with enolase regions showed that the hydrophobic helix domain itself was not sufficient to serve as a functional secretion signal; a minimal length of N-terminus 140 amino acids was required to mediate the secretion of the fused reporter GFP. We conclude that the internal hydrophobic helix of enolase is essential but is not sufficient as a signal for secretion; the intact long N-terminus including the hydrophobic helix domain is required to serve as a non-cleavable signal for the secretion of Bacillus enolase.

Enzymatic transformation of nonfood biomass to starch.

The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world's future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture's environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma.

Characterization of a novel thermostable ß-glucosidase from a metagenomic library of termite gut.

A novel ß-glucosidase-encoding gene, bgl-gs1, which was identified from a positive fosmid clone in a metagenomic library of the gut of Globitermes brachycerastes, [corrected] encodes a 455 amino acid polypeptide that contains a catalytic domain belonging to glycoside hydrolase family 1 (GH1). It was expressed in Escherichia coli BL21 (DE3) and the expression product showed a molecular mass of ∼51.7 kDa by SDS-PAGE. The optimal temperature and pH for the activity of the purified recombinant enzyme Bgl-gs1 with p-nitrophenyl-ß-D-glucoside (pNPG) were 90°C and 6.0, respectively. The specific activities of Bgl-gs1 on pNPG and salicin were 110 and 14U/mg of protein, respectively, and its K(m) values were 0.18 and 2.59 mM, respectively. The residual activity of Bgl-gs1 was maintained above 70% after the recombinant enzyme was incubated at 75°C and pH 6.0 for 2h, and its half-life at 90°C was approximately 1h in the presence of 4mM pNPG. Bgl-gs1 showed synergistic effect with either a crude enzyme mixture of the fungal strain Trichoderma reesei Rut-C30 or a fusion protein (TcE1) created from the cellobiohydrolase cbh1 gene of T. reesei and endoglucanase from Acidothermus cellulolyticus; 87 and 137% increases in hydrolytic efficiency were noted on microcrystalline cellulose, respectively. These results suggest that the thermostable ß-glucosidase Bgl-gs1 is a likely candidate for industrial applications.

Thermophilic Thermotoga maritima ribose-5-phosphate isomerase RpiB: optimized heat treatment purification and basic characterization.

The open reading frame TM1080 from Thermotoga maritima encoding ribose-5-phosphate isomerase type B (RpiB) was cloned and over-expressed in Escherichia coli BL21 (DE3). After optimization of cell culture conditions, more than 30% of intracellular proteins were soluble recombinant RpiB. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 70°C and pH 6.5-8.0. Under its suboptimal conditions (60°C and pH 7.0), k(cat) and K(m) values were 540s(-1) and 7.6mM, respectively; it had a half lifetime of 71h, resulting in its turn-over number of more than 2×10(8)mol of product per mol of enzyme. This study suggests that it is highly feasible to discover thermostable enzymes from exploding genomic DNA database of extremophiles with the desired stability suitable for in vitro synthetic biology projects and produce high-purity thermoenzymes at very low costs.

Enhanced microbial utilization of recalcitrant cellulose by an ex vivo cellulosome-microbe complex.

A cellulosome-microbe complex was assembled ex vivo on the surface of Bacillus subtilis displaying a miniscaffoldin that can bind with three dockerin-containing cellulase components: the endoglucanase Cel5, the processive endoglucanase Cel9, and the cellobiohydrolase Cel48. The hydrolysis performances of the synthetic cellulosome bound to living cells, the synthetic cellulosome, a noncomplexed cellulase mixture with the same catalytic components, and a commercial fungal enzyme mixture were investigated on low-accessibility recalcitrant Avicel and high-accessibility regenerated amorphous cellulose (RAC). The cell-bound cellulosome exhibited 4.5- and 2.3-fold-higher hydrolysis ability than cell-free cellulosome on Avicel and RAC, respectively. The cellulosome-microbe synergy was not completely explained by the removal of hydrolysis products from the bulk fermentation broth by free-living cells and appeared to be due to substrate channeling of long-chain hydrolysis products assimilated by the adjacent cells located in the boundary layer. Our results implied that long-chain hydrolysis products in the boundary layer may inhibit cellulosome activity to a greater extent than the short-chain products in bulk phase. The findings that cell-bound cellulosome expedited the microbial cellulose utilization rate by 2.3- to 4.5-fold would help in the development of better consolidated bioprocessing microorganisms (e.g., B. subtilis) that can hydrolyze recalcitrant cellulose rapidly at low secretory cellulase levels.

Simple cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis.

We developed a general restriction enzyme-free and ligase-free method for subcloning up to three DNA fragments into any location of a plasmid. The DNA multimer generated by prolonged overlap extension PCR was directly transformed in Escherichia coli [e.g., TOP10, DH5α, JM109, and BL21(DE3)] and Bacillus subtilis for obtaining chimeric plasmids.

A minimal set of bacterial cellulases for consolidated bioprocessing of lignocellulose.

Cost-effective release of fermentable sugars from non-food biomass through biomass pretreatment/enzymatic hydrolysis is still the largest obstacle to second-generation biorefineries. Therefore, the hydrolysis performance of 21 bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase (BsCel5), family 9 Clostridium phytofermentans processive endoglucanase (CpCel9), and family 48 C. phytofermentans cellobiohydrolase (CpCel48) was studied on partially ordered low-accessibility microcrystalline cellulose (Avicel) and disordered high-accessibility regenerated amorphous cellulose (RAC). Faster hydrolysis rates and higher digestibilities were obtained on RAC than on Avicel. The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was the most important for high cellulose digestibility regardless of substrate type. This study provides important information for the construction of a minimal set of bacterial cellulases for the consolidated bioprocessing bacteria, such as Bacillus subtilis, for converting lignocellulose to biocommodities in a single step.

Ultra-stable phosphoglucose isomerase through immobilization of cellulose-binding module-tagged thermophilic enzyme on low-cost high-capacity cellulosic adsorbent.

One-step enzyme purification and immobilization were developed based on simple adsorption of a family 3 cellulose-binding module (CBM)-tagged protein on the external surface of high-capacity regenerated amorphous cellulose (RAC). An open reading frame (ORF) Cthe0217 encoding a putative phosphoglucose isomerase (PGI, EC 5.3.1.9) from a thermophilic bacterium Clostridium thermocellum was cloned and the recombinant proteins with or without CBM were over-expressed in Escherichia coli. The rate constant (kcat ) and Michaelis-Menten constant (Km ) of CBM-free PGI at 60°C were 2,765 s(-1) and 2.89 mM, respectively. PGI was stable at a high protein concentration of 0.1 g/L but deactivated rapidly at low concentrations. Immobilized CBM (iCBM)-PGI on RAC was extremely stable at ∼60°C, nearly independent of its mass concentration in bulk solution, because its local concentration on the solid support was constant. iCBM-PGI at a low concentration of 0.001 g/L had a half-life time of 190 h, approximately 80-fold of that of free PGI. Total turn-over number of iCBM-PGI was as high as 1.1×10(9) mole of product per mole of enzyme at 60°C. These results suggest that a combination of low-cost enzyme immobilization and thermoenzyme led to an ultra-stable enzyme building block suitable for cell-free synthetic pathway biotransformation that can implement complicated biochemical reactions in vitro.

One-step production of lactate from cellulose as the sole carbon source without any other organic nutrient by recombinant cellulolytic Bacillus subtilis.

Although intensive efforts have been made to create recombinant cellulolytic microorganisms, real recombinant cellulose-utilizing microorganisms that can produce sufficient secretory active cellulase, hydrolyze cellulose, and utilize released soluble sugars for supporting both cell growth and cellulase synthesis without any other organic nutrient (e.g., yeast extract, peptone, amino acids), are not available. Here we demonstrated that over-expression of Bacillus subtilis endoglucanase BsCel5 enabled B. subtilis to grow on solid cellulosic materials as the sole carbon source for the first time. Furthermore, two-round directed evolution was conducted to increase specific activity of BsCel5 on regenerated amorphous cellulose (RAC) and enhance its expression/secretion level in B. subtilis. To increase lactate yield, the alpha-acetolactate synthase gene (alsS) in the 2,3-butanediol pathway was knocked out. In the chemically defined minimal M9/RAC medium, B. subtilis XZ7(pBscel5-MT2C) strain (ΔalsS), which expressed a BsCel5 mutant MT2C, was able to hydrolyze RAC with cellulose digestibility of 74% and produced about 3.1g/L lactate with a yield of 60% of the theoretical maximum. When 0.1% (w/v) yeast extract was added in the M9/RAC medium, cellulose digestibility and lactate yield were enhanced to 92% and 63% of the theoretical maximum, respectively. The recombinant industrially safe cellulolytic B. subtilis would be a promising consolidated bioprocessing platform for low-cost production of biocommodities from cellulosic materials.

Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis.

Bacillus subtilis can serve as a powerful platform for directed evolution, especially for secretory enzymes. However, cloning and transformation of a DNA mutant library in B. subtilis are not as easy as they are in Escherichia coli. For direct transformation of B. subtilis, here we developed a new protocol based on supercompetent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids. This new protocol is simple (restriction enzyme-, phosphatase- and ligase-free), fast (i.e. 1 day) and of high efficiency (i.e. ~107 or ~104 transformants per mg of multimeric plasmid or ligated plasmid DNA respectively). Supercompetent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. The DNA mutant library was generated through a two-round PCR: (i) the mutagenized DNA fragments were generated by error-prone PCR and linearized plasmids were made using high-fidelity PCR, and (ii) the multimeric plasmids were generated based on these two DNA templates by using overlap PCR. Both protein expression level and specific activity of glycoside hydrolase family 5 endoglucanse on regenerated amorphous cellulose were improved through this new system. To our limited knowledge, this study is the first report for enhancing secretory cellulase performance on insoluble cellulose.

Engineering of Clostridium phytofermentans Endoglucanase Cel5A for improved thermostability.

A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60 degrees C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.

Glycoside hydrolase family 9 processive endoglucanase from Clostridium phytofermentans: heterologous expression, characterization, and synergy with family 48 cellobiohydrolase.

The glycoside hydrolase family 9 cellulase (Cel9) from Clostridium phytofermentans has a multi-modular structure and is essential for cellulose hydrolysis. In order to facilitate production and purification of Cel9, recombinant Cel9 was functionally expressed in Escherichia coli. Cel9 exhibited maximum activity at pH 6.5 and 65 degrees C on carboxymethyl cellulose in a 10-min reaction period. The hydrolysis products on regenerated amorphous cellulose (RAC) were cellotetraose (a major product), cellotriose, cellobiose and glucose, and 71-80% of the reducing sugars produced by Cel9 were in soluble form, suggesting that Cel9 was a processive endoglucanase. The highest synergy between C. phytofermentans Cel9 and C. phytofermentans cellobiohydrolase Cel48 on Avicel was about 1.8 at a ratio of about 1:5. Cel9 alone was sufficient to solublize filter paper while Cel48 was not; however, it enhanced the solublization process along with Cel9 synergistically. This study provided useful information for understanding of the cellulose hydrolysis mechanism of this cellulolytic bacterium with potential industrial importance.

The noncellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg: heterologous expression, characterization, and processivity.

Family 48 glycoside hydrolases (cellobiohydrolases) are among the most important cellulase components for crystalline cellulose hydrolysis mediated by cellulolytic bacteria. Open reading frame (Cphy_3368) of Clostridium phytofermentans ISDg encodes a putative family 48 glycoside hydrolase (CpCel48) with a family 3 cellulose-binding module. CpCel48 was successfully expressed as two soluble intracellular forms with or without a C-terminal His-tag in Escherichia coli and as a secretory active form in Bacillus subtilis. It was found that calcium ion enhanced activity and thermostability of the enzyme. CpCel48 had high activities of 15.1 U micromol(-1) on Avicel and 35.9 U micromol(-1) on regenerated amorphous cellulose (RAC) with cellobiose as a main product and cellotriose and cellotetraose as by-products. By contrast, it had very weak activities on soluble cellulose derivatives (e.g., carboxymethyl cellulose (CMC)) and did not significantly decrease the viscosity of the CMC solution. Cellotetraose was the smallest oligosaccharide substrate for CpCel48. Since processivity is a key characteristic for cellobiohydrolases, the new initial false/right attack model was developed for estimation of processivity by considering the enzyme's substrate specificity, the crystalline structure of homologous Cel48 enzymes, and the configuration of cellulose chains. The processivities of CpCel48 on Avicel and RAC were estimated to be approximately 3.5 and 6.0, respectively. Heterologous expression of secretory active cellobiohydrolase in B. subtilis is an important step for developing recombinant cellulolytic B. subtilis strains for low-cost production of advanced biofuels from cellulosic materials in a single step.

[Piezoelectric property of novel biological piezoelectric ceramic HALNK and its effect on the functional expression of rat osteoblast cells].

OBJECTIVE: To test the Piezoelectric property of novel biological piezoelectric ceramic HALNK and its effect on the proliferation and differentiation of rat osteoblast cells. METHODS: The biological piezoelectric ceramic HALNK1/9 and HALNK5/5 were prepared by mixing Hydroxyapatite (HA) with lithium sodium potassium niobate (LNK) piezoelectric ceramic at a ratio of 1/9 and 5/5 (wt/wt), respectively. After poling treatment, the piezoelectric constants were measured. The osteoblast cells were then seeded on the surfaces of HALNK. The proliferation and differentiation activities of the osteoblast cells were evaluated by MTT assays, ALP activities and scanning electron microscopy examinations. RESULTS: Cells grown on the surfaces of HALNK showed normal morphology, and had better proliferation and differentiation activities than the control. The growth of osteoblastic cells on the surface of HALNK1/9 was significantly better than others. CONCLUSION: The surface of HALNK 1/9 possesses better piezoelectric property and osteogenesis potential than HALNK5/5.

[Biocompatibility of a novel biological piezoelectric ceramic to the rat periosteum derived osteoblast].

OBJECTIVE: A novel biological piezoelectric ceramic was made by beta-tricalcium phosphate (beta-TCP) and lithium sodium potassium niobate (LNK) piezoelectric ceramics. To study its biocompatibility to osteoblast isolated from the cranium of 1-day-old Sprague-Dawley mice. METHODS: The biological piezoelectric ceramic TCPLNK1/10, TCPLNK5/5 respectively mixed by beta-TCP and LNK piezoelectric ceramic at the ratio of 1/10 and 5/5. Then osteoblasts were used and seeded respectively on the negative and positive surfaces of TCPLNK1/10 and TCPLNK5/5. Growth and proliferation of the osteoblasts on TCPLNK1/10 and TCPLNK5/5 surfaces were evaluated in vitro by means of scanning electron microscopy (SEM) examination, fluorescence dyeing of osteoblast skeleton protein and MTT assay. RESULTS: Cell morphology of osteoblast on positive and negative surfaces of TCPLNK1/10 and TCPLNK5/5 was normal, and both adhesion and growth characteristics showed better than control group. The growing osteoblasts on the TCPLNK1/10 negative surface were significantly higher than others. The negative surface of TCPLNK1/10 possessed better osteogenesis potential than others in vitro. CONCLUSION: The surface of TCPLNK may permit the imitation piezoelectric effect of natural bone for bone regeneration.

[Construction of the shuttle expression-secretion vector with the promoters and signal peptide-encoding sequence from Brevibacillus brevis].

The multiple and tandem promoters and signal peptide-encoding sequence of cell wall protein encoding gene was amplified from Brevibacillus brevis B15 total DNA, the PCR fragment was cloned, sequenced and analyzed, then was submitted to GenBank with a Accession No. AY956423. Another pair of primers were designed to amplify the fragment again, BamHI and Pstl sites was introduced flanking the PCR production. BamHI/Pstl digested fragment was cloned into the corresponding site of shuttle vector pP43NMK to generate the expression-secretion vector pP15MK. The inserted fragment was upstream of mpd gene and the signal peptide-encoding sequence was fused in frame with the mpd gene, which its own signal peptide-encoding sequence was deleted. The recombinant vector was transformed into Bacillus subtilis 1A751, under the control of the promoters and signal peptide from Brevibacillus brevis B15, mpd gene was continuously expressed and secreted at a high efficiency throughout the exponential growth phase and into the late stationary phase, the expression production methyl parathion hydrolase (MPH) was attached on the outside of the cell membrane. MPH activity accumulated to a maximum level of 7.79 U/mL after 48 h of cultivation at the late stationary phase, which was 8.1-fold higher than the expression level of the original Plesiomonas strain M6.

mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis.

Here, we present a novel method for the directed genetic manipulation of the Bacillus subtilis chromosome free of any selection marker. Our new approach employed the Escherichia coli toxin gene mazF as a counter-selectable marker. The mazF gene was placed under the control of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible expression system and associated with a spectomycin-resistance gene to form the MazF cassette, which was flanked by two directly-repeated (DR) sequences. A double-crossover event between the linearized delivery vector and the chromosome integrated the MazF cassette into a target locus and yielded an IPTG-sensitive strain with spectomycin-resistance, in which the wild-type chromosome copy had been replaced by the modified copy at the targeted locus. Another single-crossover event between the two DR sequences led to the excision of the MazF cassette and generated a strain with IPTG resistance, thereby realizing the desired alteration to the chromosome without introducing any unwanted selection markers. We used this method repeatedly and successfully to inactivate a specific gene, to introduce a gene of interest and to realize the in-frame deletion of a target gene in the same strain. As there is no prerequisite strain for this method, it will be a powerful and universal tool.

[Recombinant expression and secretion of mpd gene using the promoters and signal peptide-encoding sequences of ytkA and ywoF gene from Bacillus subtilis].

A shuttle promoter-probe vector pNW33N-mpd was constructed with the E. coli-B. subtilis shuttle vector pNW33N and the mature mpd gene without it' s signal peptide-encoding sequence. The promoter fragments of B. subtilis ytkA and ywoF gene were cloned from plasmid pMPDP3 and pMPDP29 then generated the shuttle expression vector pNYTM and pNYWM. Expression vectors pNYTM and pNYWM were transformed into B. subtilis 1A751 to construct the expression strain 1A751 (pNYTM) and 1A751 (pNYTM), in these strains, under the control of the promoters and signal peptides of ytkA and ywoF gene, mpd gene was expressed and secreted with its biological activity; the result showed that the promoter of ytkA gene is much stronger than that of ywoF gene. Then a new shuttle expression-secretion vector pYNMK was constructed using the ytkA gene promoter and the signal peptide-encoding sequence of B. subtilis nprB gene, the expression of mpd gene achieved a higher level using the B. subtilis WB800 as the host, the methyl parathion hydrolase activity accumulated to a maximum level of 10.40 u/mL after 84 h of cultivation at the late stationary phase, which was 10.8-fold higher than the expression level of the original Plesiomonas strain M6, about 91.4% of the recombinant expression production was secreted into the culture medium.