Effects of additive dosage and coagulation bath pH on amphoteric fluorocarbon special surfactant (FS-50) blend PVDF membranes.

Amphiphilic copolymers containing hydrophilic and hydrophobic blocks represented by surfactants have proven to be more effective for modifying membranes than hydrophilic copolymers. However, studies on the effects of additive and coagulation bath pH on the morphology and properties of surfactant-modified membranes have rarely been reported. Hence, this study aims to investigate the effects of the additive dosage and the coagulation bath pH on the mechanisms of phase inversion and performance improvement of amphoteric fluorocarbon special surfactant (FS-50) blended PVDF membranes. It was observed that the pure water flux increased from 114.68 LMH/bar of the original membrane M0 to 205.02 LMH/bar of the blend membrane M1, and then to 615.88 LMH/bar of the coagulation-bath-regulated membrane MPH9 with a high BSA rejection rate of 90.86%, showing a two-stage jump. The addition of FS-50 promoted the instantaneous phase inversion of the membrane, allowing the blend membrane to exhibit a higher proportion of pore characteristics and stronger permeability. After that, the mechanisms of the membrane phase inversion process affected by the coagulation bath pH were interpreted according to the pH-response characteristics of FS-50 in terms of charge repulsion effect and compressed double-electron layer effect. Furthermore, the cross-sectional morphology and the surface structure of the membrane prepared in acidic and alkaline coagulation baths were significantly affected by the pH of the coagulation bath, exhibiting different features. For one, the porosity of the membranes gradually decreased as the acidity and alkalinity of the coagulation bath increased, and the membrane MPH9 exhibited both maximum surface and overall porosity. For another, the coagulation bath pH did not negatively affect the contact angle, surface roughness and tensile strength of the membranes. Overall, adjusting the dosage of FS-50 and the pH of the coagulation bath is a promising approach to greatly enhance membrane performance.

Extracellular Production, Characterization, and Engineering of a Polyextremotolerant Subtilisin-Like Protease From Feather-Degrading Thermoactinomyces vulgaris Strain CDF.

Here, the gene encoding a subtilisin-like protease (protease Als) was cloned from Thermoactinomyces vulgaris strain CDF and expressed in Escherichia coli. The recombinant enzyme was released into the culture medium of E. coli as a mature form (mAls). Purified mAls displayed optimal activity at 60-70°C and pH 10.0 using azo-casein as the substrate, and showed a half-life of 13.8 h at 70°C. Moreover, the activity of thermostable mAls was comparable to or higher than those of mesophilic subtilisin Carlsberg and proteinase K at low temperatures (10-30°C). Protease Als was also stable in several organic solvents and showed high compatibility with commercial laundry detergents. Notably, mAls exhibited approximately 100% of its activity at 3 M NaCl, and showed enhanced thermostability with the increase of NaCl concentration up to 3 M. Protease Als possesses an excess of solvent-accessible acidic amino acid residues, which may account for the high halotolerance of the enzyme. Compared with homologous protease C2 from the same strain, protease Als exhibits substantially lower activity toward insoluble keratin substrates but efficiently hydrolyzes soluble keratin released from chicken feathers. Additionally, direct substitution of the substrate-binding site of protease Als with that of protease C2 improves its activity against insoluble keratin substrates. By virtue of its polyextremotolerant attribute and kerationolytic capacity, protease Als may find broad applications in various industries such as laundry detergents, food processing, non-aqueous biocatalysis, and feather processing.

Complete Genome Sequence of Thermoactinomyces vulgaris Strain CDF, a Thermophilic Bacterium Capable of Degrading Chicken Feathers.

Thermoactinomyces vulgaris strain CDF was isolated from soil and shown to have the ability to degrade chicken feathers at high temperatures. Here, we report the complete genome sequence of this bacterium, which is 2,595,509 bp long with 2,642 predicted genes and an average G+C content of 48.14%.

Parallel Analyses of Somatic Mutations in Plasma Circulating Tumor DNA (ctDNA) and Matched Tumor Tissues in Early-Stage Breast Cancer.

PURPOSE: Early detection and intervention can decrease the mortality of breast cancer significantly. Assessments of genetic/genomic variants in circulating tumor DNA (ctDNA) have generated great enthusiasm for their potential application as clinically actionable biomarkers in the management of early-stage breast cancer.Experimental Design: In this study, 861 serial plasma and matched tissue specimens from 102 patients with early-stage breast cancer who need chemotherapy and 50 individuals with benign breast tumors were deeply sequenced via next-generation sequencing (NGS) techniques using large gene panels. RESULTS: Cancer tissues in this cohort of patients showed profound intratumor heterogeneities (ITHGs) that were properly reflected by ctDNA testing. Integrating the ctDNA detection rate of 74.2% in this cohort with the corresponding predictive results based on Breast Imaging Reporting and Data System classification (BI-RADS) could increase the positive predictive value up to 92% and potentially dramatically reduce surgical overtreatment. Patients with positive ctDNA after surgery showed a higher percentage of lymph node metastasis, indicating potential recurrence and remote metastasis. The ctDNA-positive rates were significantly decreased after chemotherapy in basal-like and Her2+ tumor subtypes, but were persistent despite chemotherapy in luminal type. The tumor mutation burden in blood (bTMB) assessed on the basis of ctDNA testing was positively correlated with the TMB in tumor tissues (tTMB), providing a candidate biomarker warranting further study of its potentials used for precise immunotherapy in cancer. CONCLUSIONS: These data showed that ctDNA evaluation is a feasible, sensitive, and specific biomarker for diagnosis and differential diagnosis of patients with early-stage breast cancer who need chemotherapy.

[Enhanced Coagulation as a Pretreatment for Low Temperature Wastewater].

The effects of low temperature on enhanced coagulation were studied. A new composite coagulant called SynthA was synthesized. The effects of enhanced coagulation on the removals of dissolved organic matter, dissolved organic nitrogen, and so on under room temperature or low temperature (2-5℃) were determined, and their influences on biological treatments were investigated by using membrane fractionation distribution, three-dimensional fluorescence spectrum (3DEEM), and differential ultraviolent absorbance. The results showed that, under room temperature, the removals of particulate COD, particulate nitrogen, colloidal COD, and colloidal nitrogen were highly correlated with turbidity reduction by coagulation using aluminum chloride (AlCl3), poly aluminum chloride (PACl), and SynthA as coagulants separately, while the relationship was not clear between the dissolved parameters and turbidity reduction. The reduction of fluorescence value of dissolved organic matter after coagulation was much higher than that of dissolved COD. Dissolved organic nitrogen (DON) is removed to the greatest extent by preset coagulation along with particulate nitrogen (PN) and colloidal nitrogen (CN). Low temperature affected enhanced coagulation in many aspects. It inhibited turbidity reduction and COD removal by the three coagulants with the order being AlCl3 > PACl > SynthA. It exhibited differential influences on the removals of particulate, colloidal and dissolved COD, and nitrogen, and it showed greater adverse effects on particulate and colloidal COD and nitrogen. The fluorescence value of dissolved organic matter in low temperature water showed a significant increase, and its reduction by coagulation was high, compared with that in room temperature water. Low temperature coagulation exerted greater impacts on ultraviolet differential absorbance than did room temperature. Under low temperatures, slight increases of total nitrogen (TN) removal, DN, and DON removals were achieved by using SynthA as coagulant, and removals of PN and CN were maintained, compared with room temperature. As an example, when SynthA dosage was above 30 mg ·L-1, DON removal reached 28.5%-41.7% at low temperature, while the removal was only 17%-31.4% at room temperature. A large portion of the COD and some TN were removed by coagulation as a pretreatment, indicating that a large amount of the time in an aeration pond could be reduced, and the removal efficiency of TN would be stabilized. Therefore, in winter, the decrease of biological treatment efficiency could be alleviated to some extent by using enhanced coagulation with an adaptable coagulant, such as SynthA as a pretreatment, which would relieve the stress of denitrogen and stabilize treatment efficiency.

Autocatalytic activation of a thermostable glutamyl endopeptidase capable of hydrolyzing proteins at high temperatures.

Glutamyl endopeptidases (GSEs) specifically hydrolyze peptide bonds formed by α-carboxyl groups of Glu and Asp residues. We cloned the gene for a thermophilic GSE (designated TS-GSE) from Thermoactinomyces sp. CDF. A proform of TS-GSE that contained a 61-amino acid N-terminal propeptide and a 218-amino acid mature domain was produced in Escherichia coli. We found that the proform possessed two processing sites and was capable of autocatalytic activation via multiple pathways. The N-terminal propeptide could be autoprocessed at the Glu-1-Ser1 bond to directly generate the mature enzyme. It could also be autoprocessed at the Glu-12-Lys-11 bond to yield an intermediate, which was then converted into the mature form after removal of the remaining part of the propeptide. The segment surrounding the two processing sites was flexible, which allowed the proform and the intermediate form to be trans-processed into the mature form by either active TS-GSE or heterogeneous proteases. Deletion analysis revealed that the N-terminal propeptide is important for the correct folding and maturation of TS-GSE. The propeptide, even its last 11-amino acid peptide segment, could inhibit the activity of its cognate mature domain. The mature TS-GSE displayed a temperature optimum of 85 °C and retained approximately 90 % of its original activity after incubation at 70 °C for 6 h, representing the most thermostable GSE reported to date. Mutational analysis suggested that the disulfide bonds Cys32-Cys48 and Cys180-Cys183 cumulatively contributed to the thermostability of TS-GSE.

Degradation of intact chicken feathers by Thermoactinomyces sp. CDF and characterization of its keratinolytic protease.

Thermoactinomyces is known for its resistance to extreme environmental conditions and its ability to digest a wide range of hard-to-degrade compounds. Here, Thermoactinomyces sp. strain CDF isolated from soil was found to completely degrade intact chicken feathers at 55 °C, with the resulting degradation products sufficient to support growth as the primary source of both carbon and nitrogen. Although feathers were not essential for the expression of keratinase, the use of this substrate led to a further 50-300 % increase in enzyme production level under different nutrition conditions, with extracellular keratinolytic activity reaching its highest level (∼400 U/mL) during the late-log phase. Full degradation of feathers required the presence of living cells, which are thought to supply reducing agents necessary for the cleavage of keratin disulfide bonds. Direct contact between the hyphae and substrate may enhance the reducing power and protease concentrations present in the local microenvironment, thereby facilitating keratin degradation. The gene encoding the major keratinolytic protease (protease C2) of strain CDF was cloned, revealing an amino acid sequence identical to that of subtilisin-like E79 protease from Thermoactinomyces sp. E79, albeit with significant differences in the upstream flanking region. Exogenous expression of protease C2 in Escherichia coli resulted in the production of inclusion bodies with proteolytic activity, which could be solubilized to an alkaline solution to produce mature protease C2. Purified protease C2 was able to efficiently hydrolyze α- and ß-keratins at 60-80 °C and pH 11.0, representing a promising candidate for enzymatic processing of hard-to-degrade proteins such as keratinous wastes.

Expression plasmids for use in Candida glabrata.

We describe a series of CEN/ARS episomal plasmids containing different Candida glabrata promoters, allowing for a range of constitutive or regulated expression of proteins in C. glabrata. The set of promoters includes three constitutive promoters (EGD2pr, HHT2pr, PDC1pr), two macrophage/phagocytosis-induced promoters (ACO2pr, LYS21pr), and one nutritionally regulated promoter (MET3pr). Each promoter was cloned into two plasmid backbones that differ in their selectable marker, URA3, or the dominant-selectable NAT1 gene, which confers resistance to the drug nourseothricin. Expression from the 12 resulting plasmids was assessed using GFP as a reporter and flow cytometry or quantitative reverse-transcription polymerase chain reaction to assess expression levels. Together this set of plasmids expands the toolkit of expression vectors available for use with C. glabrata.