Optimization of Fermentation Conditions for Xylanase Production by Aspergillus niger NS-1

In this study, a xylanase-produeing Aspergillus niger strain, NS-1, was screened and isolated from agricultural and forestry wastes. Based on single-fac- tor experiments, the effects of different carbon sources, composite carbon sources, nitrogen sources, calcium carbonate concentrations, initial pH and surfactants on xylanase production by A. niger NS-1 were investigated. The results indicated that the most appropriate carbon source was corncobs ; the best composite carbon source was corncobs ＋ xylan, which was conducive to xylanase secretion; the most suitable nitrogen source was ammonium sulfate. Xylanase activity reached the highest in the medium added with 1.5% calcium carbonate and SDS as a surfactant with an initial pH of 5.0. This study provided the basis for the production of high-activity xylanase.

Optimization of Ultrasonic Extraction Process of Polysaccharides from Polyporus umbellatus

[ Objective] This study aimed to investigate the optimal conditions for ultrasonic extraction of polysaccharides from Polyporus umbeUatus, thus providing a reliable basis for the development and utilization of P. umbellatus. [ Method ] By single-factor experiment, the ultrasonic extraction process of polyperus pelysac- charides was optimized. [Result] The optimal conditions for ultrasonic extraction of polysaccharides from P. umbeUatus were： solid-liquid ratio 1：40 （g： ml）, ex- traction duration 60 rain, extraction temperature 70 ℃, ultrasonic power 100 W. [ Conclusion] Compared with conventional water extraction method, ultrasonic ex- traction could significantly improve the content of polyporus polysaccharides with shorter extraction duration, lower solid-liquid ratio and lower extraction tempera-

Charge-guided masking of a membrane-destabilizing peptide enables efficient endosomal escape for targeted intracellular delivery of proteins

Intracellular delivery of biologicals such as peptides,proteins,and nucleic acids presents a great opportunity for innovative therapeutics.However,the endosome entrapment remains a major bottleneck in the intracellular delivery of biomacromolecules,largely limiting their therapeutic potential.Here,we converted a cell-penetrating peptide(CPP),low molecular weight protamine(LMWP),to endosomal escape peptides(EEPs)by masking LMWP with a pH-responsive counter-ionic peptide.The resulting masked CPPs(mLMWP and mLMWP2)effectively promoted the escape of peptide/protein cargoes from endosomes into the cytoplasm.Consequential lysosome repair and lysophagy were initiated upon the endolysosomal leakage.Minimal reactive oxygen species(ROS)elevation or cell death was observed.Based on mLMWP2,we constructed an intracellular protein delivery system containing an antibody as a targeting module,mLMWP2 as an endosomal escape module,and the desired protein cargo.With the HER2-targeting delivery system,we efficiently translocated cyclization recombination enzyme(Cre)and BH3-interacting domain death agonist(BID)into the cytosol of HER2^(+)cells to exert their biological activity.Thereby,the modular delivery system shows its potential as a promising tool for scientific studies and therapeutic applications.

In situ polymerization on biomacromolecules for nanomedicines

Biopharmaceuticals, including proteins, DNAs, and RNAs, hold vast promise for the treatment of many disorders, such as cancer, diabetes, autoimmune diseases, infectious diseases, and rare diseases. The application of biopharmaceuticals, however, is limited by their poor stability, immunogenicity, suboptimal pharmacokinetic performance, undesired tissue distribution, and low penetration through biological barriers. In situ polymerization provides an appealing and promising platform to improve the pharmacological characteristics of biopharmaceuticals. Instead of the traditional ＂grafting to＂ polymer-biomolecule conjugation, in situ polymerization grows polymers on the surfaces of the biomacromolecules, resulting in easier purification procedures, high conjugation yields, and unique structures. Herein, this review surveys recent advances in the polymerization methodologies. Additionally, we further review improved therapeutic performance of the resultant nanomedicines. Finally, the opportunities, as well as the challenges, of these nanocomposites in the biomedical fields are discussed.

Objective assessment of bradykinesia in Parkinson’s disease using evolutionary algorithms:clinical validation

Background:There is an urgent need for developing objective,effective and convenient measurements to help clinicians accurately identify bradykinesia.The purpose of this study is to evaluate the accuracy of an objective approach assessing bradykinesia in finger tapping(FT)that uses evolutionary algorithms(EAs)and explore whether it can be used to identify early stage Parkinson’s disease(PD).Methods:One hundred and seven PD,41 essential tremor(ET)patients and 49 normal controls(NC)were recruited.Participants performed a standard FT task with two electromagnetic tracking sensors attached to the thumb and index finger.Readings from the sensors were transmitted to a tablet computer and subsequently analyzed by using EAs.The output from the device(referred to as"PD-Monitor")scaled from−1 to+1(where higher scores indicate greater severity of bradykinesia).Meanwhile,the bradykinesia was rated clinically using the Movement Disorder Society-Sponsored Revision of the Unified Parkinson’s Disease Rating Scale(MDS-UPDRS)FT item.Results:With an increasing MDS-UPDRS FT score,the PD-Monitor score from the same hand side increased correspondingly.PD-Monitor score correlated well with MDS-UPDRS FT score(right side:r=0.819,P=0.000;left side:r=0.783,P=0.000).Moreover,PD-Monitor scores in 97 PD patients with MDS-UPDRS FT bradykinesia and each PD subgroup(FT bradykinesia scored from 1 to 3)were all higher than that in NC.Receiver operating characteristic(ROC)curves revealed that PD-Monitor FT scores could detect different severity of bradykinesia with high accuracy(≥89.7%)in the right dominant hand.Furthermore,PD-Monitor scores could discriminate early stage PD from NC,with area under the ROC curve greater than or equal to 0.899.Additionally,ET without bradykinesia could be differentiated from PD by PD-Monitor scores.A positive correlation of PD-Monitor scores with modified Hoehn and Yahr stage was found in the left hand sides.Conclusions:Our study demonstrated that a simple to use device employing classifiers derived from EAs could not only be used to accurately measure different severity of bradykinesia in PD,but also had the potential to differentiate early stage PD from normality.

Synthesis of Protein Nano-Conjugates for Cancer Therapy

A eukaryotic cell contains thousands of proteins that regulate its cellular function; delivering functional proteins into cells to rectify cellular functions holds great promise for treatment of various diseases, especially cancers. In this context, ribonuclease （RNase）, an enzyme that breaks down ribonucleic acid （RNA）, has great potential for cancer therapy. However, its therapeutic application is hampered by poor intracellular delivery efficiency and inhibition by ubiquitous intracellular RNase inhibitors. In this work, by designing and synthesizing RNase nano-conjugates by in situ atom transfer radical polymerization （ATRP）, we demonstrate a simple solution to address both challenges. Compared with native RNase, nano-conjugates exhibit significantly enhanced intracellular delivery efficiency, inhibitor resistance, and a near five-fold increase in cytotoxicity. This work provides a novel platform for delivery of therapeutic proteins for cancer therapy and other applications.

Chiral resolution of N-methyl-d,l-aspartic acid using a predicted N-demethylase from Chloroflexi bacterium 54-19

N-methyl-d-aspartic acid(NMDA),an amino acid existing in human and animal central nervous system,exerts agonist action on one of the glutamate receptor subtypes and is clinically used for treatment of diabetes,Parkinson’s and Alzheimer’s syndrome.In this study,an enzymatic protocol for the chiral resolution of N-methyl-d,l-aspartic acid was built with a predicted N-demethylase(GenBank ID:OJV90073.1)from the genome of Chloroflexi bacterium 54-19.Through sequence alignment,the enzyme shares an identity of 32.19%to 2UZZ(PDB ID)with a conserved catalytic center.Recombinantly expressed in Bacillus subtilis WB600,the N-demethylase was characterized with optimal temperature and pH at 55℃and 7.5,and adaptive temperature and pH were 40-60℃ and 6-8.The effects of organic solvents and metal ions were investigated as well.Compared with other previously reported sarcosine oxidases,the enzyme showed a specific N-demethylation activity against N-methyl-l-aspartic acid according to the analysis by chiral liquid chromatography,LC-MS/MS and a detection by polarimeter.The results demonstrated 76%of 4.5 mM N-methyl-d,l-aspartic acid could be chirally separated by 7 mg·L^(−1) enzyme after reaction of 80 min.This work provided a foundation for mild synthesis of NMDA in industry.