Yeast Pichia pastoris is a widely used system for heterologous protein expression. However, post-translational modifications, especially glycosylation, usually impede pharmaceutical application of recombinant proteins because of unexpected alterations in protein structure and function. The aim of this study was to identify glycosylation sites on recombinant human platelet-derived growth factor-BB (rhPDGF-BB) secreted by P. pastoris, and investigate possible effects of O-linked glycans on PDGF-BB functional activity. PDGF-BB secreted by P. pastoris is very heterogeneous and contains multiple isoforms. We demonstrated that PDGF-BB was O-glycosylated during the secretion process and detected putative O-glycosylation sites using glycosylation staining and immunoblotting. By site-directed mutagenesis and high-resolution LC/MS analysis, we, for the first time, identified two threonine residues at the C-terminus as the major O-glycosylation sites on rhPDGF-BB produced in P. pastoris. Although O-glycosylation resulted in heterogeneous protein expression, the removal of glycosylation sites did not affect rhPDGF-BB mitogenic activity. In addition, the unglycosylated PDGF-BBΔGly mutant exhibited the immunogenicity comparable to that of the wild-type form. Furthermore, antiserum against PDGF-BBΔGly also recognized glycosylated PDGF-BB, indicating that protein immunogenicity was unaltered by glycosylation. These findings elucidate the effect of glycosylation on PDGF-BB structure and biological activity, and can potentially contribute to the design and production of homogeneously expressed unglycosylated or human-type glycosylated PDGF-BB in P. pastoris for pharmaceutical applications.

Intraflagellar transport (IFT) particles are composed of polyprotein complexes IFT-A and IFT-B as well as cargo adaptors such as the BBSome. Two IFT-B subunits, IFT25 and IFT27 were found to form a heterodimer, which is essential in exporting the BBSome out of the cilium but not involved in flagellar assembly and cytokinesis in vertebrates. Controversial results were, however, recorded to show that defects in IFT, flagellar assembly and even cytokinesis were caused by IFT27 knockdown in Chlamydomonas reinhardtii Using C. reinhardtii as a model organism, we report that depletion of IFT25 has no effect on flagellar assembly and does not affect the entry of the BBSome into the flagellum, but IFT25 depletion did impair BBSome movement out of the flagellum, clarifying the evolutionally conserved role of IFT25 in regulating the exit of the BBSome from the flagellum cross species. Interestingly, depletion of IFT25 causes dramatic reduction of IFT27 as expected, which does not cause defects in flagellar assembly and cytokinesis in C. reinhardtii Our data thus support that Chlamydomonas IFT27, like its vertebrate homologues, is not involved in flagellar assembly and cytokinesis.

Spondyloepiphyseal dysplasia congenita (SEDC) is an extremely rare autosomal dominant chondrodysplasia that is usually caused by substitution of glycine with another amino acid in the triple helical region of COL2A1. Herein, we describe a case of SEDC in a Chinese family with a novel de novo mutation in the COL2A1 gene, c.1150G>A (p.Gly384Ser), which may impair protein stability and lead to dysfunction of type II collagen.

Corn bran is a major agro-industrial byproduct from corn starch processing. It contains abundant arabinoxylan that can be converted into value-added chemicals via biotechnology. Corn bran arabinoxylan (CBAX) is one of the most recalcitrant xylans for enzymatic degradation due to its particular heterogeneous nature. The present study aimed to investigate the capability of the filamentous fungus Penicillium parvum 4-14 to enzymatically saccharify CBAX and reveal the fungal carbohydrate-active enzyme (CAZyme) repertoire by genome sequencing and secretome analysis.

β-Glucosidase is claimed as a key enzyme in cellulose hydrolysis. The cellulosic fibers are usually entrapped with hemicelluloses containing xylose. So there is ongoing interest in searching for glucose- and xylose-stimulated β-glucosidases to increase the efficiency of hydrolysis of cellulosic biomass.

Zinc is essential for almost all living organisms, since it serves as a crucial cofactor for transcription factors and enzymes. However, it is toxic to cell growth when present in excess. The present work aims to investigate the toxicity mechanisms induced by zinc stress in yeast cells. To this end, 108 yeast single-gene deletion mutants were identified sensitive to 6 mM ZnCl2 through a genome-wide screen. These genes were predominantly related to the biological processes of vacuolar acidification and transport, polyphosphate metabolic process, cytosolic transport, the process utilizing autophagic mechanism. A result from the measurement of intracellular zinc content showed that 64 mutants accumulated higher intracellular zinc under zinc stress than the wild-type cells. We further measured the intracellular ROS (reactive oxygen species) levels of 108 zinc-sensitive mutants treated with 3 mM ZnCl2 We showed that the intracellular ROS levels in 51 mutants were increased by high zinc stress, suggesting their possible involvement in regulating ROS homeostasis in response to high zinc. The results also revealed that excess zinc could generate oxidative damage and then activate the expression of several antioxidant defenses genes. Taken together, the data obtained indicated that excess zinc toxicity might be mainly due to the high intracellular zinc levels and ROS levels induced by zinc stress in yeast cells. Our current findings would provide a basis to understand the molecular mechanisms of zinc toxicity in yeast cells.

The responses of broiler chickens to dietary protein reduction were investigated in the presence of glycine and cysteine inclusion at the marginal deficiency of sulfur-containing amino acids. A total of 432 broiler chickens were allotted to six dietary treatments; SP1 is standard protein diet with 100% total sulfur amino acids (TSAA), SP2 is standard protein diet with 85% TSAA, RP is reduced protein diet without glycine and cysteine supplementation, RPC is reduced protein diet with cysteine supplementation at 0.1%, and RPG is reduced protein diet with 1% glycine supplementation, while RPGC is reduced protein diet with 0.1% cysteine and 1% glycine supplementation. In this study, 4.5% protein is reduced in diets-thus, 17.5% CP (crude protein) for starter phase and 15.5% CP for the grower phase. Reduced protein diets contained 85% TSAA. Broiler chickens fed standard protein diet SP2 had superior bodyweight (BW) (p ≤ 0.05) in the starter and grower phase, average daily gain (ADG) (p ≤ 0.05) in the starter and entire feeding period, average daily feed intake (ADFI) (p ≤ 0.05) in the starter phase, and better feed conversion ratio (FCR) (p ≤ 0.05) in the starter, grower and entire feeding period; however, RPGC showed higher ADG (p ≤ 0.05) in the grower phase, and ADFI (p ≤ 0.05) in the grower and entire feeding period. RPC and RPG diet improved BW (p ≤ 0.05), ADG (p ≤ 0.05), ADFI (p ≤ 0.05), and better FCR (p ≤ 0.05) in starter, grower, entire feeding period compared to RP. The RPGC group had higher BW (p ≤ 0.05), ADG (p ≤ 0.05), ADFI (p ≤ 0.05) and better FCR (p ≤ 0.05) compared to the RPC group. Blood biochemical parameters showed that Broiler chickens fed on the SP2 diet had higher levels of total protein (TP) (p ≤ 0.05), albumin (ALB) (p ≤ 0.05), creatinine (CRE) (p ≤ 0.05), and aspartate aminotransferase (AST) (p ≤ 0.05) and, lower level of uric acid (UA) (p ≤ 0.05), blood urea nitrogen (BUN) (p ≤ 0.05), glucose (GLU) (p ≤ 0.05), and alanine aminotransferase (ALT) (p ≤ 0.05) in the starter phase; however, higher level of TP (p ≤ 0.05), GLU (p ≤ 0.05), CRE (p ≤ 0.05), and AST (p ≤ 0.05), and lower level of ALB (p ≤ 0.05), UA (p ≤ 0.05), and ALT (p ≤ 0.05) in the grower phase; RPGC had higher level of TP (p ≤ 0.05), UA (p ≤ 0.05), GLU (p ≤ 0.05), ALT (p ≤ 0.05) and AST (p ≤ 0.05), and lower level of ALB (p ≤ 0.05), BUN (p ≤ 0.05), and CRE (p ≤ 0.05) in the starter phase; however, in grower phase, RPGC had higher level of TP (p ≤ 0.05), and ALB (p ≤ 0.05), and lower level of UA (p ≤ 0.05), CRE (p ≤ 0.05), ALT (p ≤ 0.05), and AST (p ≤ 0.05). Free amino acids profile showed that broiler fed on standard protein diet SP2 had reduced the methionine (p ≤ 0.05) concentration; RPC increased the concentrations of taurine (p ≤ 0.05), phosphoethanolamine (p ≤ 0.05), threonine (p ≤ 0.05), valine (p ≤ 0.05), isoleucine (p ≤ 0.05), phenylalanine (p ≤ 0.05), ornithine (p ≤ 0.05), and lysine (p ≤ 0.05) and reduced the citrulline (p ≤ 0.05) concentration; RPG increased the concentration of glutamate (p ≤ 0.05), glycine (p ≤ 0.05), cysteine (p ≤ 0.05), and arginine (p ≤ 0.05), and decreased the concentration of tyrosine (p ≤ 0.05); and RPGC increased the concentration of serine (p ≤ 0.05) and reduced the concentration of hydroxyproline (p ≤ 0.05). Serum metabolites analysis showed that reduced protein downregulated the 54 metabolites; however, glycine fortification up-regulated the Benzamide, Pro-Ser, N-Carbamylglutamate, D-gluconate, and Gamma-Glutamylcysteine. Carcass quality showed that SP2 decreased the abdominal fat percentage (p ≤ 0.05). Nitrogen digestibility was higher by the diet RP (p ≤ 0.05). This study demonstrated that protein content could be reduced up to 4.5% with 1% glycine and 0.1% cysteine fortification in diet, which has the potential to inhibit the adverse effect of reduced protein and attain the standard growth performance.

Biosurfactants are attracting much interest due to their potential application as therapeutic agents in the medical and cosmetic field. Previous studies have demonstrated that biosurfactant such as sophorolipid (SL) exhibits immunomodulatory effects. In this article, we found the potential of sophorolipid for inhibiting histamine-induced itch and preliminarily explored its molecular basis. First, behavioral tests indicated that SL can remit the histamine-induced scratching behaviors of mice. Second, SL can suppress the the calcium influx induced by histamine, HTMT and VUF8430 in HaCaT cells. RT-PCR analysis showed that the histamine-induced upregulation of mRNA levels of phospholipase Cγ1, 1,4,5-trisphosphate receptor (IP3R), and protein kinase Cα can be inhibted by SL, suggesting that SL may impede the PLC/IP3R signaling pathway activated by histamine. In further tests, the capsaicin-induced calcium influx can also be inhibited by SL. The immunofluorescence and molecular docking analysis indicated that SL acts as an inhibitor of transient receptor potential vanilloid-1 (TRPV1) activation to decrease calcium influx against stimuli. In summary, these results revealed that SL may inhibit histamine-induced itch by decreasing PLC/IP3R signaling pathway activation and modulating TRPV1 activity. This paper indicates that SL can be a useful treatment for histamine-dependent itch.

Sepsis, a common critical disease, has high morbidity and mortality. Acute lung injury (ALI) is one of the important complications of sepsis, its effective treatment measures remain scarce. The purpose of the present study was to search for the biomarker and effective treatment measures. Lipopolysaccharide (LPS) was used to establish sepsis induced ALI model in vivo and in vitro. Proteomics, immunoprecipitation, molecular docking techniques, and Sirt3 knockout (KO) mice and silence MLE-12 cells were used to search for biomarker and treatment measures for sepsis ALI. 38 differentially expressed proteins were found in the lung tissues of sepsis ALI mice, among which Sirt3 changed most. Further study found that Sirt3 could inhibit NLRP3 activation. Sirt3 KO or silence significantly aggravated sepsis induced ALI and MLE-12 cell injury. Plantainoside D (PD), an effective component of Plantago asiatica L., significantly improved sepsis induced ALI by regulation of Sirt3/NLRP3 pathway. In conclusion, Sirt3 may be the important molecular targets for sepsis ALI. PD could protect sepsis ALI via Sirt3/NLRP3 signal pathway. The findings provide a new treatment target for sepsis ALI and a potential treatment measure.

The coronavirus disease 2019 (COVID-19) has penetrated every populated patch of the globe and sows destruction in our daily life. Reliable and sensitive virus sensing systems are therefore of vital importance for timely infection detection and transmission prevention. Here we present a thermoplasmonic-assisted dual-mode transducing (TP-DMT) concept, where an amplification-free-based direct viral RNA detection and an amplification-based cyclic fluorescence probe cleavage (CFPC) detection collaborated to provide a sensitive and self-validating plasmonic nanoplatform for quantifying trace amounts of SARS-CoV-2 within 30 min. In the CFPC detection, endonuclease IV recognized the synthetic abasic site and cleaved the fluorescent probes in the hybridized duplex. The nanoscale thermoplasmonic heating dehybridized the shortened fluorescent probes and facilitated the cyclical binding-cleavage-dissociation (BCD) process, which could deliver a highly sensitive amplification-based response. This TP-DMT approach was successfully validated by testing clinical COVID-19 patient samples, which indicated its potential applications in fast clinical infection screening and real-time environmental monitoring.

China is facing a rapidly expanding aging population. Insights into the health status of older adults are of great significance for health resource allocation and health care provision to this population.

Neurotrophin-3 growth factor can improve cochlear neuron survival, and localized delivery of this protein to the round window membrane in the middle ear may be able to reverse sensorineural hearing loss. Thus, the goal of this work was to develop an injectable hydrogel delivery system that can allow localized release of neurotrophin-3 in a controlled and sustained manner. We identified a PEG hydrogel formulation that uses thiol-vinyl sulfone Michael addition for crosslinking. This injectable formulation provides elastic hydrogels with higher mechanical rigidity, better bio-adhesion and longer residence time than Poloxamer hydrogels currently being investigated clinically for hearing loss. In vivo, PEG hydrogels induce local immune responses comparable to biocompatible Poloxamer hydrogels, yet they released payloads at a ~5-fold slower rate in the subcutaneous area. Based on this injectable hydrogel formulation, we designed an affinity-based protein release system by modifying PEG hydrogels with affinity peptides specific to neurotrophin-3 proteins. We verified the sustained release of neurotrophin-3 from peptide-conjugated PEG hydrogels resulting from the reversible interaction between peptides and proteins. The rate of affinity-controlled release depends on the polymer concentrations, the affinity of peptides and the peptide-to-protein ratios. Collectively, we developed an injectable hydrogel formulation for localized delivery of neurotrophin-3, which provides affinity-controlled release and longer delivery time compared to Poloxamer hydrogels.

This study explored the effect of 70-mT magnetic field on wastewater treatment capacity for activated sludge in long-term laboratory-scale experiments. Metagenomic sequencing were conducted based on Illumina HiSeq 2000 platform after DNA extraction of the activated sludge. Then the effect of the magnetic field on the microbial unigene and metabolic pathways in activated sludge was investigated. As a result, higher pollutant removal was observed at 70 mT, with which the elimination of total nitrogen (TN) was the most effective. Functional genes annotated based on eggNOG database showed that unigenes related to information storage and processing were enhanced by the magnetic field. For CAZy classification, category such as glycosyl transferases was more abundant in the reactor with magnetic field, which has been shown to promote the entire energy supply pathway. Additionally, in the KEGG categories, unigenes related to signaling molecules and interaction were significantly inhibited. Through the enrichment analysis of the nitrogen metabolism pathway, the magnetic field inhibited anabolic nitrate reduction by significantly inhibiting enzymes such as [EC:1.7.7.2], [EC:1.7.7.1], [EC:3.5.5.1], [EC:1.4.1.2] and [EC:4.2.1.1], which are related to the improvement of the denitrification ability. This study can provide insight for future research on the response mechanism of activated sludge to magnetic fields.

The lipase-producing strain Bacillus subtilis SPZ1 is isolated from the medium by tributyrin as the sole carbon source. Here, we present a 4.13-Mb assembly of its genome sequence, which may provide various kinds of useful information related to Bacillus spp., such as mechanisms and control of the substrate uptake and protein secretion pathways.

Human enterovirus 71 (EV71) is the major pathogen that causes hand, foot and mouth disease that particularly affects young children. Growing hand, foot and mouth disease outbreaks were observed worldwide in recent years and caused devastating losses both economically and politically. However, vaccines or effective drugs are unavailable to date. The genome of EV71 consists of a positive sense, single-stranded RNA of ∼7400 bp, encoding a large precursor polyprotein that requires proteolytic processing to generate mature viral proteins. The proteolytic processing mainly depends on EV71 3C protease (3C(pro)) that possesses both proteolysis and RNA binding activities, which enable the protease to perform multiple tasks in viral replication and pathogen-host interactions. The central roles played by EV71 3C(pro) make it an appealing target for antiviral drug development. We determined the first crystal structure of EV71 3C(pro) and analyzed its enzymatic activity. The crystal structure shows that EV71 3C(pro) has a typical chymotrypsin-like fold that is common in picornaviral 3C(pro). Strikingly, we found an important surface loop, also denoted as β-ribbon, which adopts a novel open conformation in EV71 3C(pro). We identified two important residues located at the base of the β-ribbon, Gly123 and His133, which form hinges that govern the intrinsic flexibility of the ribbon. Structure-guided mutagenesis studies revealed that the hinge residues are important to EV71 3C(pro) proteolytic activities. In summary, our work provides the first structural insight into EV71 3C(pro), including a mobile β-ribbon, which is relevant to the proteolytic mechanism. Our data also provides a framework for structure-guided inhibitor design against EV71 3C(pro).

As a red tide algal toxin with intense neurotoxicity distributed worldwide, domoic acid (DA) has attracted increasing concerns. In this work, the integrative analysis of metagenome and metabolome are applied to investigate the impact of DA on nitrogen cycling in coastal sediments. Here we show that DA can act as a stressor to induce the variation of nitrogen (N) cycling by altering the abundance of functional genes and electron supply. Moreover, microecology theory revealed that DA can increase the role of deterministic assembly in microbial dynamic succession, resulting in the shift of niches and, ultimately, the alteration in N cycling. Notably, denitrification and Anammox, the important process for sediment N removal, are markedly limited by DA. Also, variation of N cycling implies the modification in cycles of other associated elements. Overall, DA is capable of ecosystem-level effects, which require further evaluation of its potential cascading effects.

Over the past few years, the antitumor activity exhibited by 5-caffeylquinic acid (5-CQA), especially its inhibitory effect on hepatocellular carcinoma (HCC) proliferation and metastasis, has been recognized as a new research hotspot. However, impacted by the weak antitumor toxicity of 5-CQA, its clinical application has been limited. In this study, the antitumor effect arising from 5-CQA on HCC cells was evaluated through cell viability assay. In addition, proteomics, flow cytometry, qRT-PCR and western blotting were adopted to investigate the drug resistance mechanism of HCC cells to 5-CQA. As indicated by the results, 5-CQA significantly inhibited the proliferation of HCC cell lines MHCC97H and HCCLM3 with IC5048 h of 546.8 μM and 452 μM, respectively. According to the in-depth studies, the abnormal activation of HIF-1α/glucose transporters/glycolysis pathway of 5-CQA could be a key molecular mechanism leading to drug resistance of HCC cells. Thus, this study found that glucose starvation, glucose analogue 2-DG, hexokinase inhibitor bromopyruvic acid and PKM2 inhibitor compound 3k inhibited HCC cell proliferation in synergy with 5-CQA. Furthermore, though the 5-CQA derivatives methyl chlorogenate (MCGA) and 3,5-dicaffeoylquinic acid (3,5-diCQA) exhibited more potent antiproliferation activity in HCC cells than 5-CQA, they also up-regulated the expression of GLUT1/3, whereas they had no effect on hepatocytes. To be specific, under low-glucose culture conditions, the order of sensitivity of HCC cells to CQAs was 3,5-diCQA > MCGA > 5-CQA. In brief, the above results revealed that intervention in glucose metabolism can facilitate the effects of 5-CQA and its derivatives for treating HCC.

Phycocyanin (PC), derived from cyanobacteria and Spirulina cells, is a type of natural antineoplastic marine protein. It has been reported that phycocyanin exerts an antitumor function in non-small cell lung cancer (NSCLC) cells, but the underlying mechanism has not been elucidated. In this research, a transcriptome study was performed to investigate the regulatory mechanisms of phycocyanin on human NSCLC A549 cells. The survival rate and proliferation ability of A549 cells were markedly reduced by phycocyanin, along with abnormal morphologic changes. The transcriptome analysis showed that 2970 genes were differentially expressed after phycocyanin treatment in A549 cells, including 1431 down-regulated and 1539 up-regulated genes. Gene ontology and KEGG analysis suggested that some classical pathways, such as Wnt, NF-κB, and PI3K-AKT signaling, were significantly enriched. Strikingly, protein⁻protein interaction (PPI) analysis showed that ubiquitin-C (UBC) occupied the highest degree (the highest number of interactions) in differential genes, indicating that it might play a key role in the phycocyanin-mediated regulatory process in A549 cells. Moreover, qRT-PCR results showed consistent expression trends of differential genes with transcriptome analysis. Consequently, this study has provided a theoretical basis for regulation of phycocyanin in A549 cells, which lays a foundation for the treatment of NSCLC.

Disinfection byproducts (DBPs) are produced during the disinfection of drinking water and pose a hazard to human health. As a typical type of DBPs, iodoacetic acid (IAA) exhibits prominent cytotoxicity in mammalian cell systems which links with oxidative stress. However, little is known about the relationship of catalase (CAT) with the cytotoxicity of IAA and the adverse effects of IAA to CAT. This study investigated the effects of IAA on the cell viability and CAT activity in the mouse primary hepatocytes. It was shown that IAA exposure induced the loss of cell viability and the increase of intracellular CAT activity. Intracellular CAT activity significantly increased due to the stimulation of CAT production under IAA exposure. The molecular CAT activity was inhibited due to the direct interaction of IAA with HIS 74 and TYR 357 around the active sites of CAT. IAA binds to CAT with (4.05 ± 1.98) sites via van der Waals and hydrogen bonding interactions, resulting in the loosening of protein skeletons and the change of protein size.

Cadmium is a carcinogen that can induce ER stress, DNA damage, oxidative stress and cell death. The yeast mitogen-activated protein kinase (MAPK) signalling pathways paly crucial roles in response to various stresses. Here, we demonstrate that the unfolded protein response (UPR) pathway, the high osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway are all essential for yeast cells to defend against the cadmium-induced toxicity, including the elevated ROS and cell death levels induced by cadmium. We show that the UPR pathway is required for the cadmium-induced phosphorylation of HOG_MAPK Hog1 but not for CWI_MAPK Slt2, while Slt2 but not Hog1 is required for the activation of the UPR pathway through the transcription factors of Swi6 and Rlm1. Moreover, deletion of HAC1 and IRE1 could promote the nuclear accumulation of Hog1, and increase the cytosolic and bud neck localisation of Slt2, indicating crucial roles of Hog1 and Slt2 in regulating the cellular process in the absence of UPR pathway. Altogether, our findings highlight the significance of these two MAPK pathways of HOG and CWI and their interrelationship with the UPR pathway in responding to cadmium-induced toxicity in budding yeast.