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This study aimed to reveal the roles of the protein kinase A catalytic subunit 1 (pkac1) and carbon catabolite repressor cre1 genes in cellulase production by Trichoderma reesei wild-type strain QM6a. Our strategy might be useful to construct a high-yielding cellulase strain for its wide application.This paper describes cellulase activity, plate conidiation, and yellow pigment synthesis assays of QM6a with the disruption of pkac1 and cre1.Deletion of pkac1 (Δpkac1) had no effect on cellulase production or transcript levels of major cellulase genes in the presence of cellulose. Disruption of cre1 (Δcre1) resulted in a remarkable increase in cellulase production and expression of the four major cellulase genes. Double disruption of pkac1 and cre1 significantly improved enzyme activity and protein production. The double disruption also resulted in a significant reduction in yellow pigment production and abrogated conidial production.Double deletion of pkac1 and cre1 led to increased hydrolytic enzyme production in T. reesei using cellulose as a carbon source.

VGINYW is a highly active angiotensin I-converting enzyme (ACE) inhibitory peptide discovered from α-lactalbumin by an in vitro–in silico high throughput screening strategy.

Background Trichoderma reesei Rut-C30 is a hypercellulolytic mutant strain that degrades abundant sources of lignocellulosic plant biomass into renewable biofuels. Zn2+ is an activator of enzymes in almost all organisms; however, its effect on cellulase activity has not been reported in T. reesei. Results Although high concentrations of Zn2+ severely hampered the extension of T. reesei mycelia, adding 1–4 mM Zn2+ improved cellulase production in T. reesei high-yielding cellulase-producing strain Rut-C30. The expression levels of the major cellulase genes and two critical transcription activators (xyr1 and ace3) increased significantly on Zn2+ addition. Transcriptome analysis revealed that the mRNA level of plc-e encoding phospholipase C, which is involved in the calcium signaling pathway, was enhanced by Zn2+ addition. The disruption of plc-e abolished the cellulase-positive influence of Zn2+ in the early phase, indicating that plc-e is involved in Zn2+-induced cellulase production; furthermore, supplementation with LaCl3 (a plasma membrane Ca2+ channel blocker) and deletion of crz1 (calcineurin-responsive zinc finger transcription factor 1) demonstrated that calcium signaling is partly involved in this process. Moreover, we identified the zinc-responsive transcription factor zafA; the transcriptional level of zafA decreased significantly with Zn2+ stress, and its deletion indicated that zafA mainly mediates Zn2+-induced cellulase production. Conclusions For the first time, we have demonstrated that Zn2+ was toxic to T. reesei and it remarkably promoted cellulase production. This positive influence of Zn2+ was facilitated by the gene plc-e and the transcription factor zafA. These findings provide insights into the role of Zn2+ in T. reesei and a mechanistic study of signal transduction in cellulase synthesis.

The development of biosimilar products or follow-on biologics has been flourishing in recent years because of their lower price than the originators. In this study, a multivariate data analysis method based on JMP software was proposed to assess the glycosylation pattern similarity of antibody candidates from different conditions in optimization experiments with a reference. A specific distance was generated by this method and indicated the glycoform similarity between the biosimilar and the reference. This method can be applied to analyze the similarity of other physicochemical and functional characteristics between follow-on biologics and originators. Then, the design of experimental methods can be realized to optimize the conditions of cell culture to attain similar antibody candidates. A higher concentration of GlcNAc added to the basal media made the glycan of the antibody more similar to the glycan of the reference in this study.

Glycosylation is critical for monoclonal antibody production because of its impact on pharmacokinetics and pharmacodynamics. Modulation of glycan profile is frequently needed in biosimilar development. However, glycosylation profile is not a single value like that of cell culture titer, hence making it challenging for the Design of Experiment (DoE) methodology to be directly applied. In this study, a Her2-binding antibody was developed as a biosimilar to Herceptin. Cluster analysis was introduced to demonstrate the similarity of glycan profiles between the samples and the reference with specific value—distance. The glycosylation was subsequently optimized with the DoE method. Basal medium and feed medium were found to be the significant factors to the glycosylation pattern. Moreover, a combination of medium and feed strategy was developed to attain the most similar glycoprotein molecule to that of the originator biologic drug. This study may provide an additional option to evaluate multivariable factors and assess biosimilarity and/or comparability in monoclonal antibody production.

Background Ascorbic acid (AA), dopamine (DA), uric acid (UA), and nitrite (NO2−) are essential biomarkers for human metabolism and can be used to indicate some chronic diseases and metabolic disorders, including scurvy, Parkinson’s disease, hyperuricemia, and kidney disease. Objective A multifunctional electrochemical sensor that can integrate the detection of these species was constructed using nanoporous gold (NPG) as a recognition element to modify glassy carbon electrode (GCE). Methods The electrochemical performance of the multifunctional electrochemical sensor was investigated toward AA, DA, UA, and NO2− in citrate buffer solution (CBS, 100 mM, pH 4.0) and human serum using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. Results In the quaternary mixture detection, the resulting NPG/GCE electrode displayed four independent oxidation peaks with wide peak separations. Further, the NPG/GCE electrode showed good linear responses with the sensitivities of 32, 1103, 71, and 147 μA/mM/cm2 and the detection limits of 1.58, 0.17, 0.37, and 0.36 μM for AA, DA UA, and NO2−, respectively. Additionally, the NPG/GCE electrode exhibited great anti-interference and was successfully applied in human serum samples. Conclusions These results indicate that the NPG/GCE electrode can simultaneously and selectively detect AA, DA, UA, and NO2−, which has the potential for application and diagnosis in the screening and diagnosis of chronic diseases and metabolic disorders. Highlights A multianalyte electrochemical sensor was fabricated for human metabolites detection. The sensor displayed good performance in the simultaneous detection of AA, DA, UA, and NO2− and applied to human serum samples.

Although Penicillium brevicompactum is a widely used commercial strain for the manufacture of mycophenolic acid, there are few findings of Ca

Medical imaging plays a significant role in clinical practice of medical diagnosis, where the text reports of the images are essential in understanding them and facilitating later treatments. By generating the reports automatically, it is beneficial to help lighten the burden of radiologists and significantly promote clinical automation, which already attracts much attention in applying artificial intelligence to medical domain. Previous studies mainly follow the encoder-decoder paradigm and focus on the aspect of text generation, with few studies considering the importance of cross-modal mappings and explicitly exploit such mappings to facilitate radiology report generation. In this paper, we propose a cross-modal memory networks (CMN) to enhance the encoder-decoder framework for radiology report generation, where a shared memory is designed to record the alignment between images and texts so as to facilitate the interaction and generation across modalities. Experimental results illustrate the effectiveness of our proposed model, where state-of-the-art performance is achieved on two widely used benchmark datasets, i.e., IU X-Ray and MIMIC-CXR. Further analyses also prove that our model is able to better align information from radiology images and texts so as to help generating more accurate reports in terms of clinical indicators., Comment: Natural Language Processing. 11 pages, 6 figures. ACL-IJCNLP 2021

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Immunotoxins are chimeric proteins that combine antibodies and toxins in targeted cancer therapy. Despite the promising application prospects, immunogenicity and nonspecific cytotoxicity of immunotoxins with toxins from bacterial or plant origin have hindered their clinical application. In this study, an anti-HER2 single-chain antibody fragment 4D5, was fused to a human-origin antitumor drug, TRAIL to produce a humanized immunotoxin in Escherichia coli BL21 (DE3). An appropriate linker between the domains was selected using the software Antheprot 5.0. The 4D5 scFv-TRAIL was obtained using a SUMO tag for solubility-enhancing and purified by Ni–NTA affinity chromatography after tag removing. The purity and yield of 4D5 scFv-TRAIL reached 92% and 11.6 mg/L in shake flasks, respectively. Additionally, MTT assays showed 4D5 scFv-TRAIL exhibited selective in vitro cytotoxicity on the breast cancer cell line MDA-MB-231 without cytotoxic effect on the normal cell FBHE. Therefore, 4D5 scFv-TRAIL is a potential humanized immunotoxin for treatment of HER2-overexpressing tumor cells.

A variety of physiological functions such as exopolysaccharide synthesis, antibiotic production, and primary metabolism are tightly controlled by quorum sensing in microorganisms. In this study, a marine-derived bacterium Sphingomonas sp. WG was found to possess a cyclo(L-Pro-L-Phe)-mediated quorum sensing mechanism. The cyclo(L-Pro-L-Phe) produced by Sphingomonas sp. WG functions as a signalling molecule in this quorum sensing system. It is the first attempt to characterize cyclic dipeptides as quorum sensing signalling molecules in Sphingomonas sp. and the results effectively make the classical quorum sensing theory more perfect. Furthermore, the supplementation with isolated cyclo(L-Pro-L-Phe) resulted in a 15% increase in the production of welan gum secreted by Sphingomonas sp. WG in the submerged fermentation. The data presented in this study will provide evidences for exploring the role of cyclic dipeptides in regulating the production of welan gum.

This study aims to investigate the anti-hyperuricemic and nephroprotective effects and the potential mechanisms of the separated gastrointestinal hydrolysates of α-lactalbumin on hyperuricemic mice.The gastrointestinal hydrolysate of α-lactalbumin, the hydrolysate fraction with molecular weight (MW)3 kDa (LH-3k), and the fragments with smallest MW among LH-3K harvested through dextran gel chromatography (F5) are used. Hyperuricemia mice are induced via daily oral gavage of potassium oxonate and hypoxanthine. F5 displays the highest in vitro xanthine oxidase (XO) inhibition among all the fractions separated from LH-3k. Oral administration of F5 significantly reduces the levels of serum uric acid (UA), creatinine, and urea nitrogen. F5 treatment could ameliorate kidney injury through alleviating oxidative stress and inflammation. F5 alleviates hyperuricemia in mice by inhibiting hepatic XO activity and regulating the expression of renal urate transporters. Gut microbiota analysis illustrates that F5 administration increases the abundance of some SCFAs producers, and inhibits the growth of hyperuricemia and inflammation associated genera. LH-3k exhibits similar effects but does not show significance as those of the F5 fraction.The anti-hyperuricemia and nephroprotective functions of F5 are mediated by inhibiting hepatic XO activity, ameliorating oxidative stress and inflammation, regulating renal urate transporters, and modulating the gut microbiota in hyperuricemic mice.

Welan gum, a kind of microbial exopolysaccharides, produced by the genus Sphingomonas, have great potential for application in many fields, such as the food industry, cement production, and enhanced oil recovery. But there are still challenges to reduce the cost, enhance the production and the quality. Herein, the bioinformatics analysis of WelR gene was preformed, and the characterization and function of WelR, welan gum lyase, from Sphingomonas sp. WG were investigated for the first time. The results indicated that 382nd (Asn), 383rd (Met), 494th (Asn), and 568th (Glu) were the key amino acid residues, and C-terminal amino acids were essential to keeping the stability of WelR. The optimal temperature and pH of the enzymatic activity were found to be 25°C and 7.4, respectively. And WelR was good low temperature resistance and alkali resistant. K+, Mg2+, Ca2+, Mn2+, and EDTA increased WelR activities, in contrast to Zn2+. Coupled with the change in glucose concentration and growth profile, the qRT-PCR results indicated that WelR may degrade welan gum existing in the culture to maintain bacterial metabolism when glucose was depleted. This work will lay a theoretical foundation to establish new strategies for the regulation of welan gum biosynthesis.

Additional file 10: Table S4. Whole transcriptome shotgun sequencing data and RT-qPCR verification of the plc-e gene under Mn2+ addition conditions.

Additional file 9: Table S3. The log2 fold changes of the major cellulose degradation-related genes under Mn2+/DMF addition conditions.

Additional file 8: Table S2. Differential transcription of genes in response to Mn2+ and DMF as measured by RNA-sequencing.

Radiology reports play a critical role in communicating medical findings to physicians. In each report, the impression section summarizes essential radiology findings. In clinical practice, writing impression is highly demanded yet time-consuming and prone to errors for radiologists. Therefore, automatic impression generation has emerged as an attractive research direction to facilitate such clinical practice. Existing studies mainly focused on introducing salient word information to the general text summarization framework to guide the selection of the key content in radiology findings. However, for this task, a model needs not only capture the important words in findings but also accurately describe their relations so as to generate high-quality impressions. In this paper, we propose a novel method for automatic impression generation, where a word graph is constructed from the findings to record the critical words and their relations, then a Word Graph guided Summarization model (WGSum) is designed to generate impressions with the help of the word graph. Experimental results on two datasets, OpenI and MIMIC-CXR, confirm the validity and effectiveness of our proposed approach, where the state-of-the-art results are achieved on both datasets. Further experiments are also conducted to analyze the impact of different graph designs to the performance of our method., Comment: 11 pages, 6 figures, ACL2021 Findings

Additional file 4: Figure S4. Relative expression levels of cam (c), cna1 (d), and crz1 (e) in T. reesei QM6a and Δacy1 strains under different conditions. The QM6a and Δacy1 strains were cultured in MM with 2% glucose as the carbon source and then inoculated in fresh MM supplemented with 10 mM Mn2+, 1% DMF, 0.02 mΜ Forskolin, or 5 mM dbcAMP, with 1% Avicel as the carbon source. Cultures that were not supplemented with Mn2+, DMF, Forskolin, or dbcAMP were used as controls. QM6a cells cultured without the addition of Mn2+, DMF, Forskolin, or dbcAMP were used as the reference sample. Values are expressed as the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences from the control (*p < 0.05, Student’s t test).

Additional file 5: Figure S5. Effect of cAMP/LaCl3-induced cellulase overexpression. a and b. pNPCase activity/mg biomass (a) and CMCase activity/mg biomass (b) of T. reesei QM6a strains supplemented with 5 mM dbcAMP and 5 mM LaCl3. Values are the means ± SD of the results from three independent experiments. Asterisks indicate significant differences from the control (*p < 0.05, Student’s t test).

Additional file 3: Figure S3. Effect of Mn2+/DMF/cAMP-induced cellulase overexpression. a and b. pNPCase activity/mg biomass (a) and CMCase activity/mg biomass (b) of T. reesei QM6a and Δacy1 strains supplemented with 5 mM dbcAMP, 10 mM Mn2+ or (and) 1% DMF. Values are the means ± SD of the results from three independent experiments. Asterisks indicate significant differences from the control (*p < 0.05, Student’s t test).

Additional file 1: Table S1. The top 10 pharmaceutical products sold in 2019. Fig. S1. Viable cell density variation of 30 shaker flask runs by the culture time. Fig. S2 Viability variation of 30 shake flask runs by the culture time. Fig. S3 Residual glucose variation of 30 shake flask runs by the culture time.

Additional file 2: Figure S2. Cellulase activities of T. reesei complementary strains Racy1 and Rplc-e under different addition conditions. pNPCase activity/mg biomass of T. reesei QM6a, Racy1 and Rplc-e strains supplemented with 10 mM Mn2+ or 1% DMF. T. reesei QM6a was used as the control. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences from the control (*p < 0.05, Student’s t test).

The development of biosimi lar products or follow on biologics has been flourishing in recent years because of lower price than originators. In th is study, a multivariate data analysis method based on JMP software was proposed to assess the glycosylation pattern similarity of antibo dy candidates from different conditions in optimization experiment with reference. A specific distance was generated by t specific distance was generated by this method and indicated the glycoform similarity his method and indicated the glycoform similarity between biosimilar and reference. This method can be applied to analyze the similaritbetween biosimilar and reference. This method can be applied to analyze the similarity of y of other physicochemical and function characters between followother physicochemical and function characters between follow--on biologics and originators. on biologics and originators. Then the design of experiThen the design of experiments method can be realized to optimize culture conditions of cellments method can be realized to optimize culture conditions of cell culture to attain similar antibody candidatesculture to attain similar antibody candidates..

An endo-1,4-β-mannanase gene (manB) from a Bacillus pumilus Nsic-2 grown in a stinky tofu emulsion was cloned and expressed in Pichia pastoris GS115. After characterized, the endo-1,4-β-mannanase (manB) show maximum activity at pH 6.0 and 50 °C with LBG as substrate and perform high stability at a range of pH 6–8. After applying for a shake flask fermentation, the specific activity of manB reached 3462 U/mg. To produce mannose, the soybean meal (SBM) was pretreated by biological fermentation for 11 days with Penicillium brevicompactum, and then hydrolyzed by manB. As a result, mannose yield reached 3.58 g per 1 kg SBM which indicated that 0.358% SBM was converted into mannose after hydrolyzation, and mean a total 20% mannan of SBM converting into mannose, while the control group demonstrated only 1.78% conversion. An effective β-mannanase for the bioconversion of mannan-rich biomasses and an efficient method to produce mannose with soybean meal were introduced.

Background: Esterases are widely distributed in nature and have important applications in medical, industrial and physiological. Recently, the increased demand for flavor esters has prompted the search of catalysts like lipases and esterases. Esterases from thermophiles also show thermal stability at elevated temperatures and have become enzymes of special interest in biotechnological applications. Although most of esterases catalyzed reactions are carried out in toxic and inflammable organic solvents, the solvent-free system owning many advantages such as low cost and easy downstream processing. Results: The gene estGSU753 from Geobacillus subterraneus DSM13552 was cloned, sequenced, and overexpressed into Escherichia coli BL21 (DE3). The novel gene has an open reading frame of 753 bp, and encoding 250-amino-acid esterase (EstGSU753). The sequence analysis showed that the protein contains a catalytic triad formed by Ser97, Asp196, and His226, and the Ser of the active site is located in the conserved motif Gly95-X-Ser97-X-Gly99 included in most esterases and lipases. The protein catalyzed the hydrolysis of p-nitrophenyl esters of different acyl chain lengths, and the enzyme specific activity was 70 U/mg with the optimum substrate p-nitrophenyl caprylate. The optimum pH and temperature of the recombinant enzyme were 8.0 and 60°C respectively. The resulting EstGSU753 showed remarkable stability against methanol. After the incubation at 50% methanol for 9 days, EstGSU753 retained 50% of its original activity. Even incubation at 90% methanol for 2100 minutes, EstGSU753 retained 50% of its original activity. Also, the preliminary study of the transesterification shows the potential value in synthesis of short-chain flavor esters in a solvent-free system, and more than 99% conversion was obtained in 6 h (substrate: cinnamyl alcohol, 1.0 M). Conclusions: This is the first report of esterase gene cloning from Geobacillus subterraneus with detailed enzymatic properties. This methanol-stable esterase showed potential value in industrial applications especially in the perfume industry.