Development of a predictive machine learning model for pathogen profiles in patients with secondary immunodeficiency.

BACKGROUND: Secondary immunodeficiency can arise from various clinical conditions that include HIV infection, chronic diseases, malignancy and long-term use of immunosuppressives, which makes the suffering patients susceptible to all types of pathogenic infections. Other than HIV infection, the possible pathogen profiles in other aetiology-induced secondary immunodeficiency are largely unknown. METHODS: Medical records of the patients with secondary immunodeficiency caused by various aetiologies were collected from the First Affiliated Hospital of Nanchang University, China. Based on these records, models were developed with the machine learning method to predict the potential infectious pathogens that may inflict the patients with secondary immunodeficiency caused by various disease conditions other than HIV infection. RESULTS: Several metrics were used to evaluate the models' performance. A consistent conclusion can be drawn from all the metrics that Gradient Boosting Machine had the best performance with the highest accuracy at 91.01%, exceeding other models by 13.48, 7.14, and 4.49% respectively. CONCLUSIONS: The models developed in our study enable the prediction of potential infectious pathogens that may affect the patients with secondary immunodeficiency caused by various aetiologies except for HIV infection, which will help clinicians make a timely decision on antibiotic use before microorganism culture results return.

Efficacy and safety of camrelizumab combined with albumin-bound paclitaxel as third- or later-line regimen in patients with advanced non-small cell lung cancer.

Background: The clinical efficacy and safety of camrelizumab as a third- or later-line regimen in patients with advanced non-small cell lung cancer (NSCLC) have not been determined in large clinical trials. Objective: This study aimed to evaluate the clinical efficacy and safety of camrelizumab in combination with albumin-bound paclitaxel as a third- or later-line treatment for patients with advanced NSCLC. Methods: A total of 257 patients with advanced NSCLC who were histopathologically confirmed and failed in clinical second-line therapy regimens at Jiangxi Province Cancer hospital from January 2018 to December 2021 were retrospectively selected. Patients with advanced NSCLC were divided into the single treatment group (STG) of camrelizumab, and the combined treatment group (CTG) of camrelizumab in combination with albumin-bound paclitaxel according to the treatment regimen. The primary outcomes of interest were clinical efficacy[objective response rate (ORR) and disease control rate (DCR)], progression-free survival (PFS), and overall survival (OS). Survival data were analyzed using the Kaplan-Meier method, and the log-rank test was performed. Additionally, Cox proportional hazard regression was used to analyze the correlation of prognosis and baseline characteristics between subgroups, to identify the potential independent risk factors for PFS and OS. Furthermore, the occurrence of side effects was assessed according to the Common Terminology Criteria for Adverse Events (CTCAE 4.03). Results: Of the 257 patients with advanced NSCLC included in the research, 135 patients received camrelizumab, and 122 patients received camrelizumab plus albumin-bound paclitaxel. The ORR of CTG and STG was 59.84% and 50.38%, and the DCR was 77.05% and 65.93%, respectively. The median PFS in CTG was higher than that in the STG (5.27 vs. 3.57 months, P = 0.0074), and the median OS was longer (7.09 vs. 6.47 months, P < 0.01). The lines of treatment, metastases, and PD-L1 expression levels were independent risk factors for the mPFS and mOS of patients with advanced NSCLC. The occurrence of adverse events was similar between camrelizumab and camrelizumab plus albumin-bound paclitaxel groups. Conclusion: Camrelizumab combined with albumin-bound paclitaxel as the third- or later-line regimen greatly prolonged PFS and OS of advanced NSCLC patients. A prospective clinical trial is warranted.

Advances in 4-Hydroxyphenylacetate-3-hydroxylase Monooxygenase.

Catechols have important applications in the pharmaceutical, food, cosmetic, and functional material industries. 4-hydroxyphenylacetate-3-hydroxylase (4HPA3H), a two-component enzyme system comprising HpaB (monooxygenase) and HpaC (FAD oxidoreductase), demonstrates significant potential for catechol production because it can be easily expressed, is highly active, and exhibits ortho-hydroxylation activity toward a broad spectrum of phenol substrates. HpaB determines the ortho-hydroxylation efficiency and substrate spectrum of the enzyme; therefore, studying its structure-activity relationship, improving its properties, and developing a robust HpaB-conducting system are of significance and value; indeed, considerable efforts have been made in these areas in recent decades. Here, we review the classification, molecular structure, catalytic mechanism, primary efforts in protein engineering, and industrial applications of HpaB in catechol synthesis. Current trends in the further investigation of HpaB are also discussed.

Hub gene associated with prognosis in bladder cancer is a novel therapeutic target.

Objective: Bladder cancer is a clinical and social conundrum due to its high incidence and recurrence rate. It is urgent to find new targets for the diagnosis and treatment of bladder cancer and improve the prognosis and survival rate of bladder cancer patients. We sought a prognosis-related gene, built related models of evaluated bladder cancer and identified the function of the hub gene in bladder cancer. Methods: We downloaded the data of bladder cancer patients from the TCGA database, and used differentially expressed genes (DEGs), copy number variation (CNV) and survival analysis to scan the hub genes associated with prognosis in bladder cancer. Then, multi-factor cox regression was used to obtain the bladder cancer prognosis correlation model. Then, we analyzed the relationship between the expression of hub gene and immune microenvironment of bladder cancer. The relationship between the expression of hub gene and prognosis in bladder cancer patients was verified by immunohistochemistry. Cell proliferation assay and drug sensitivity test in vivo were used to verify the inhibition of bladder cancer by targeted inhibitors. Results: In bladder cancer, we screened seven hub genes (ACLY, CNP, NKIRAS2, P3H4, PDIA6, VPS25 and XPO1) associated with survival. Moreover, the multifactor regression model constructed with hub gene can well distinguish the prognosis of bladder cancer. Hub gene is mostly associated with immune microenvironment. Immunohistochemical results basically confirmed the importance of XPO1 in bladder cancer. Selinexor (an inhibitor of XPO1) could effectively inhibit the proliferation of bladder cancer in the cell proliferation experiments by CCK-8 assays and it could suppress the growth of bladder cancer in mouse bladder cancer model. Conclusions: In this study, a prognostic model with seven hub genes has provided great help for the prognosis prediction of bladder cancer patients. And XPO1 is an important target affecting the prognosis of bladder cancer, and inhibition of XPO1 can effectively inhibit bladder cancer proliferation and growth.

Modification of the 4-Hydroxyphenylacetate-3-hydroxylase Substrate Pocket to Increase Activity towards Resveratrol.

4-Hydroxyphenylacetate-3-hydroxylase (4HPA3H; EC 1.14.14.9) is a heterodimeric flavin-dependent monooxygenase complex that catalyzes the ortho-hydroxylation of resveratrol to produce piceatannol. Piceatannol has various health benefits and valuable applications in food, medicine, and cosmetics. Enhancing the catalytic activity of 4HPA3H toward resveratrol has the potential to benefit piceatannol production. In this study, the critical amino acid residues in the substrate pocket of 4HPA3H that affect its activity toward resveratrol were identified using semi-rational engineering. Two key amino acid sites (I157 and A211) were discovered and the simultaneous "best" mutant I157L/A211D enabled catalytic efficiency (Kcat/Km-resveratrol) to increase by a factor of 4.7-fold. Molecular dynamics simulations indicated that the increased flexibility of the 4HPA3H substrate pocket has the potential to improve the catalytic activity of the enzyme toward resveratrol. On this basis, we produced 3.78 mM piceatannol by using the mutant I157L/A211D whole cells. In this study, we successfully developed a highly active 4HPA3H variant for the hydroxylation of resveratrol to piceatannol.

Enhancing the organic solvent resistance of ω-amine transaminase for enantioselective synthesis of (R)-(+)-1(1-naphthyl)-ethylamine.

BACKGROUND: Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. An R-selective ω-amine transaminase from Aspergillus terreus (AtATA) exhibited activity toward 1-acetylnaphthalene. However, AtATA displayed unsatisfactory organic solvent resistance, which is required to enhance the solubility of the hydrophobic substrate 1-acetylnaphthalene. So, improving the tolerance of enzymes in organic solvents is essential. MAIN METHODS AND RESULTS: The method of regional random mutation combined with combinatorial mutation was used to improve the resistance of AtATA in organic solvents. Enzyme surface areas are structural elements that undergo reversible conformational transitions, thus affecting the stability of the enzyme in organic solvents. Herein, three surface areas containing three loops were selected as potential mutation regions. And the "best" mutant T23I/T200K/P260S (M3) was acquired. In different concentrations of dimethyl sulfoxide (DMSO), the catalytic efficiency (kcat /Km ) toward 1-acetylnaphthalene and the stability (half-life t1/2 ) were higher than the wild-type (WT) of AtATA. The results of decreased Root Mean Square Fluctuation (RMSF) values via 20-ns molecular dynamics (MD) simulations under 15%, 25%, 35%, and 45% DMSO revealed that mutant M3 had lower flexibility, acquiring a more stable protein structure and contributing to its organic solvents stability than WT. Furthermore, M3 was applied to convert 1-acetylnaphthalene for synthesizing (R)-(+)-1(1-naphthyl)-ethylamine ((R)-NEA), which was an intermediate of Cinacalcet Hydrochloride for the treatment of secondary hyperthyroidism and hypercalcemia. Moreover, in a 20-mL scale-up experiment, 10 mM 1-acetylnaphthalene can be converted to (R)-NEA with 85.2% yield and a strict R-stereoselectivity (enantiomeric excess (e.e.) value >99.5%) within 10 h under 25% DMSO. CONCLUSION: The beneficial mutation sites were identified to tailor AtATA's organic solvents stability via regional random mutation. The "best" mutant T23I/T200K/P260S (M3) holds great potential application for the synthesis of (R)-NEA.

Clinical Significance of Serum Iron Metabolism-Related Markers in Patients with Nasopharyngeal Carcinoma.

INTRODUCTION: It is known that iron metabolism is dysregulated in nasopharyngeal carcinoma (NPC). However, a meaningful assessment of the iron metabolic status in cancer patient is still under debate. This study aims to evaluate the status of iron metabolism, as well as to explore the correlation between those related serum markers and clinicopathological features of patients with NPC. METHODS: Peripheral blood was collected from 191 pretreatment NPC patients and 191 healthy controls. The red blood cell parameters, plasma Epstein-Barr virus (EBV) DNA load, serum iron (SI), total iron-binding capacity (TIBC), transferrin, soluble transferrin receptor (sTFR), ferritin, and hepcidin were quantitatively detected. RESULTS: The mean levels of hemoglobin and red blood cell count in the NPC group were significantly lower than those in the control group, while no statistical differences in mean MCV were found between the two groups. Median levels of SI, TIBC, transferrin, and hepcidin were significantly lower in the NPC group than in the control group. Compared to patients with the T1-T2 classification, patients with the T3-T4 classification exhibited significantly lower expression levels of SI and TIBC. Serum levels of ferritin and sTFR were significantly higher in patients with M1 classification than those with M0 classification. The EBV DNA load was associated with serum levels of sTFR and hepcidin. CONCLUSION: NPC patients had functional iron deficiency. The degree of iron deficiency was related to the tumor burden and metastasis of NPC. EBV might be involved in the regulation of iron metabolism in the host.

Exogenous spermidine improved drought tolerance in Ilex verticillata seedlings.

Winterberry (Ilex verticillata (L.) A. Gray) is a recently introduced ornamental tree species in China that has not been closely investigated for its drought resistance. In this study, we used two-year-old cuttings from I. verticillata (L.) A. Gray and two representative varieties derived from it, I. verticillata 'Oosterwijk' and I. verticillata 'Jim Dandy', as materials to investigate how this plant responds to drought stress and whether exogenous spermidine (SPD) can alleviate the negative effects caused by drought stress. The results showed that as the degree of drought stress increased, the leaves of winterberry seedlings became chlorotic, and their edges became dry. Similarly, the relative water content, specific leaf weight, chlorophyll content, leaf nitrogen content, net photosynthetic rate, stomatal conductance and transpiration rate were significantly reduced, whereas the content of malondialdehyde continuously increased with the degree of drought stress. The activities of superoxide dismutase, peroxidase, and catalase increased under moderate drought stress and then decreased under severe drought stress. The levels of soluble sugar and abscisic acid continued to increase, while those of auxin and gibberellic acid decreased. When compared with individual drought stress, an increase in the amount of external SPD clearly alleviated the effect of drought stress on winterberry seedlings. The combined phenotypes and physiological indices of the winterberry leaves under drought stress conditions revealed that the drought resistance of the native species was significantly higher than its two varieties. This finding serves as an important theoretical foundation for the popularization and application of I. verticillata (L.) A. Gray and the two varieties.

Selinexor demonstrates anti-tumor efficacy in paired patient-derived xenograft models and hydrogel-embedded histoculture drug sensitivity test of penile cancer.

BACKGROUND: Penile cancer is a rare malignancy with a poor prognosis, even with various treatment options. Considering the little progress in the study of the pathogenesis and treatment of penile cancer because of the lack of models that mimic the biological properties of the tumor, we have developed a patient-derived xenograft (PDX) model and paired hydrogel-embedded histoculture drug sensitivity test (HDST) to screen for drugs that can inhibit tumors. The increased expression of XPO1, as a key nuclear export protein involved in the transport of various tumor suppressors and cell cycle regulatory proteins, is associated with the prognosis of a variety of tumors [World J Uroly 27(2):141-150, 2009]. Selinexor is an inhibitor of XPO1, which can treat cancers, such as multiple myeloma, gastric cancer, triple-negative breast cancer, and non-small cell carcinoma [Transl Androl Urol 6(5):785-790, 2017; OncoTargets Therapy 13:6405-6416, 2020]. However, whether XPO1 inhibition has a role in penile cancer remains unknown. Therefore, this article used the PDX and HDST models to investigate whether the inhibition of XPO1 has an effect on penile cancer and its underlying mechanism. METHODS: We used penile cancer tumor tissues to construct a PDX model of penile cancer and paired PDXE model and confirmed the consistency of PDX tumor tissues in source patients. Then, we assessed the ability of Selinexor to inhibit penile cancer tissues in vivo using a PDX model and in vitro by HDST. We also examined the potential mechanism of XPO1 action on penile cancer by IHC and TUNEL. Finally, we assessed the safety of the drug treatment by H&E and biochemical blood analysis. RESULTS: Result showed that the penile cancer PDX model and patient penile cancer tissues were clinically consistent in morphological characteristics and protein expression. In addition, Selinexor could inhibit tumor growth in PDX models and HDST. We found that P53, P21 expression was upregulated; Cyclin D1 expression was downregulated, and apoptosis of tumor cells was increased in the Selinexor-treated PDX model. Moreover, it had no significant effect on liver, kidney, and cardiac function. CONCLUSION: The PDX model of penile cancer was a powerful tool for penile cancer research and new drug development. It showed that Selinexor can effectively inhibit penile cancer in vitro and in vivo. In addition, XPO1 may affect P53, P21, and Cyclin D1 expression to regulate the growth and apoptosis of penile carcinoma.

Turning thermostability of Aspergillus terreus (R)-selective transaminase At-ATA by synthetic shuffling.

As versatile and green biocatalysts for the asymmetric amination of ketones, the insufficient thermostability of transaminases always limits its broad application in the pharmaceutical and fine chemical industries. Here, synthetic shuffling technology was used to enhance stability of (R)-selective transaminase from Aspergillus terreus. The results showed that 30 out of 5000 mutants had improved thermostability by color-based screening method, among which mutants with residual enzyme activity higher than 50% at 45 °C for 10 min were selected for further analysis. Especially, the half-inactivation temperature (T5010), half-life (t1/2), and melting temperature (Tm) of the best mutant M14 (M280C-H210N-M150C-F115L) were 13.7 °C, 165.8 min, and 13.9 °C higher than that of the wild type (WT), respectively. M14 also exhibited a significant biocatalytic efficiency toward acetophenone and 1-acetylnaphthalene, the yield of which were 265.6% and 117.5% higher than WT, respectively. Based on molecular dynamics simulation, improved catalytic efficiency of M14 could be attributed to its increased hydrogen bonds interaction around the mutation sites. Additionally, the introduction of disulfide bond combined with above mutations has a synergistic effect on the improved protein thermostability.

A study on expression level and nutritional status of IGFBP-2 after left neck anastomosis combined with placement of feeding nutritional applicators carrying

Background: To explore the changes and significance of the expression level and nutritional status of human insulin-like growth factor binding protein-2 (IGFBP2) after the treatment of esophageal cancer with left neck anastomosis combined with placement of feeding nutritional applicators carrying ^(125)I particles. Methods: A total of 110 patients with esophageal cancer (observation group: left neck anastomosis combined with placement of feeding nutritional applicators carrying ^(125)I particles) and 100 healthy people (control group) were enrolled at the same period. Then enzyme-linked immunosorbent assay (ELISA) was carried out to detect level of IGFBP-2. Lymphocyte count and serum albumin were measured by immune analyzer and automatic protein analyzer to evaluate nutritional status. Logistic regression analysis was used to analyze the relationship between serum IGFBP-2, nutritional status and prognosis of esophageal cancer after combined treatment. Results: The albumin, lymphocyte absolute value and PNI detection value of the control group were lower than those of the observation group 1 month after treatment, and the difference was statistically significant compared with the control group. The detection value of IGFBP-2 in early patients before and after treatment was lower than that in middle and late patients, and the detection values of albumin, lymphocyte absolute value and PNI were higher than those in middle and late patients, the differences were statistically significant. Serum IGFBP-2 level was negatively correlated with PNI, and albumin and lymphocyte absolute value were positively correlated with PNI. The detection value of IGFBP-2 in patients with good prognosis was significantly lower than that in patients with poor prognosis, and the detection values of albumin, lymphocyte absolute value and PNI were significantly higher than those in patients with poor prognosis. The AUC (0.887,95% CI: 0.799-0.975) of IGFBP-2, albumin, lymphocyte absolute value and PNI in predicting poor prognosis of esophageal cancer was the largest, and the sensitivity and specificity were 94.12% and 92.47%, respectively. Conclusions: Left neck anastomosis combined with ^(125)I particle application nutritional tube is helpful for the decrease of serum IGFBP-2 and the increase of various nutritional status indicators, which is beneficial for the improvement of the patient's condition.

Expression analysis of PIN family genes in Chinese hickory reveals their potential roles during grafting and salt stress.

Grafting is an effective way to improve Chinese hickory while salt stress has caused great damage to the Chinese hickory industry. Grafting and salt stress have been regarded as the main abiotic stress types for Chinese hickory. However, how Chinese hickory responds to grafting and salt stress is less studied. Auxin has been proved to play an essential role in the stress response through its re-distribution regulation mediated by polar auxin transporters, including PIN-formed (PIN) proteins. In this study, the PIN gene family in Chinese hickory (CcPINs) was identified and structurally characterized for the first time. The expression profiles of the genes in response to grafting and salt stress were determined. A total of 11 CcPINs with the open reading frames (ORFs) of 1,026-1,983 bp were identified. Transient transformation in tobacco leaves demonstrated that CcPIN1a, CcPIN3, and CcPIN4 were localized in the plasma membrane. There were varying phylogenetic relationships between CcPINs and homologous genes in different species, but the closest relationships were with those in Carya illinoinensis and Juglans regia. Conserved N- and C-terminal transmembrane regions as well as sites controlling the functions of CcPINs were detected in CcPINs. Five types of cis-acting elements, including hormone- and stress-responsive elements, were detected on the promoters of CcPINs. CcPINs exhibited different expression profiles in different tissues, indicating their varied roles during growth and development. The 11 CcPINs responded differently to grafting and salt stress treatment. CcPIN1a might be involved in the regulation of the grafting process, while CcPIN1a and CcPIN8a were related to the regulation of salt stress in Chinese hickory. Our results will lay the foundation for understanding the potential regulatory functions of CcPIN genes during grafting and under salt stress treatment in Chinese hickory.

Genome-wide identification and expression profiles of ABCB gene family in Chinese hickory (Carya cathayensis Sarg.) during grafting.

Chinese hickory (Carya cathayensis Sarg.) is an important nut tree species native to China. Excessive plant height and long juvenile phase has restricted development of its industry. Recently, grafting has been used increasingly in production practice of this species to solve the problems above. Previous studies have proved the importance of auxin during Chinese hickory grafting. However, the function of ATP-binding cassette subfamily B (ABCB) genes during Chinese hickory grafting is less studied. In this study, 23 ABCB genes were identified and characterized in Chinese hickory (CcABCBs). The expression profiles of auxin-related ABCBs among tissues, under auxin-related phytohormone treatments and during grafting were determined. CcABCB proteins were divided into half-size and full-size transporters. Many phytohormone-related cis-acting regulatory elements were detected on the promoters of CcABCB genes. Four CcABCB genes homologous to auxin-related AtABCB1, 6, 19 and 20 in Arabidopsis were selected for expression analysis. The four genes displayed varying expression patterns in different tissues of Chinese hickory. Expressions of the four CcABCB genes were regulated by auxin-related phytohormones to varying degrees. Expression levels of the four genes were significantly changed at different stages of grafting, especially 7 days after grafting, indicating their involvement of auxin homeostasis regulation during grafting. In addition, the expressions of CcABCB1 were regulated by IAA and NPA treatments during grafting in comparison with CK treatment, while expressions of the other 3 CcABCB genes were slightly affected. This study will lay the foundation for understanding the potential regulatory roles of CcABCB genes during Chinese hickory grafting.

Reconstruction of the glutamate decarboxylase system in Lactococcus lactis for biosynthesis of food-grade γ-aminobutyric acid.

Gamma-aminobutyric acid (GABA), an important bioactive compound, is synthesized through the decarboxylation of L-glutamate (L-Glu) by glutamate decarboxylase (GAD). The use of lactic acid bacteria (LAB) as catalysts opens interesting avenues for the biosynthesis of food-grade GABA. However, a key obstacle involved in the improvement of GABA production is how to resolve the discrepancy of optimal pH between the intracellular GAD activity and cell growth. In this work, a potential GAD candidate (LpGadB) from Lactobacillus plantarum was heterologously expressed in Escherichia coli. Recombinant LpGadB existed as a homodimer under the native conditions with a molecular mass of 109.6 kDa and exhibited maximal activity at 40°C and pH 5.0. The Km value and catalytic efficiency (kcat/Km) of LpGadB for L-Glu was 21.33 mM and 1.19 mM-1s-1, respectively, with the specific activity of 26.67 µM/min/mg protein. Subsequently, four C-terminally truncated LpGadB mutants (GadBΔC10, GadBΔC11, GadBΔC12, GadBΔC13) were constructed based on homology modeling. Among them, the mutant GadBΔC11 with highest catalytic activity at near-neutral pH values was selected. In further, the GadBΔC11 and Glu/GABA antiporter (GadC) of Lactococcus lactis were co-overexpressed in the host L. lactis NZ3900. Finally, after 48 h of batch fermentation, the engineered strain L. lactis NZ3900/pNZ8149-gadBΔC11C yielded GABA concentration up to 33.52 g/L by applying a two-stage pH control strategy. Remarkably, this is the highest yield obtained to date for GABA from fermentation with L. lactis as a microbial cell factory.Key points• The GadB from L. plantarum was heterologously expressed in E. coli and biochemically characterized.• Deletion of the C-plug in GadB shifted its pH-dependent activity toward a higher pH.• Reconstructing the GAD system of L. lactis is an effective approach for improving its GABA production.

The need to belong: A parallel process latent growth curve model of late life negative affect and cognitive function.

OBJECTIVE: Late life negative affect (NA) often co-occurs with poor cognitive function (CF); however, very little is known about the mechanism of the relationship between them. We examined the longitudinal relationship between NA and CF over a 12-year period and the effects of several related risk factors in a general sample. METHODS: Five waves of data on Chinese Longitudinal Healthy Longevity Survey (CLHLS) were collected from a total of 1,314 elderly Chinese, aged 60 and over. A parallel process latent growth curve model with two time-invariant covariates and seven time-varying covariates was used to demonstrate the joint trajectories of NA and CF to assess their related factors in the elderly during a 12-year period. RESULTS: Significant association of negative affect and cognitive decline was found at baseline and over time for our sample. Poorer initial cognitive performance predicted a faster increase in negative affect over time. Being female was associated with worse initial performance and a faster rate of deterioration of NA and CF. Regular exercise, married status, social activities, and Mahjong playing were associated with slower rates of negative affect increase and cognitive decline. CONCLUSION: These findings demonstrated that the late life negative affect co-occurs with cognitive decline and negative affect might be a mutative mental reaction to cognitive dysfunction. Gender difference, exercise benefit, and the "need to belong" effect were observed over time, highlighting the importance of exercise and socialization for older females.

Improving the Thermostability of Glutamate Decarboxylase from Lactobacillus brevis by Consensus Mutagenesis.

γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). In this study, stabilized variants of the GAD from Lactobacillus brevis were constructed by consensus mutagenesis. Using Consensus Finder ( http://cbs-kazlab.oit.umn.edu/ ), eight positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these eight residues were individually mutated to match the consensus sequence using site-directed mutagenesis. Compared to the wild-type, T383K variant displayed the largest shift in thermostability among the single variants, with a 3.0 °C increase in semi-inactivation temperature (T5015), a 1.7-fold improvement of half-life (t1/2) at 55 °C, and a 1.2-fold improvement of t1/2 at 37 °C, respectively, while its catalytic efficiency (kcat/Km) was reduced. To obtain the mutant with improvement in both thermostability and catalytic activity, we performed a site-saturation mutation at T383. Notably, mutants T383V and T383G exhibited an increasement in thermostability and kcat/Km than that of wild-type. This study not only emphasizes the value of consensus mutagenesis for improving the thermostability of GAD but also sheds a powerful guidance to study the thermal stability of other enzymes.

Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase.

Glutamate decarboxylase (GAD; EC 4.1.1.15) is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme that specifically catalyzes the decarboxylation of L-glutamic acid to produce γ-aminobutyric acid (GABA), which exhibits several well-known physiological functions. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, a parallel strategy comprising sequential analysis and free energy calculation was applied to identify critical amino acid sites affecting thermostability of GAD and select proper mutation contributing to improve structure rigidity of the enzyme. Two mutant enzymes, D203E and S325A, with higher thermostability were obtained, and their semi-inactivation temperature (T5015) values were 2.3 °C and 1.4 °C higher than the corresponding value of the wild-type enzyme (WT), respectively. Moreover, the mutant, S325A, exhibited enhanced activity compared to the wild type, with a 1.67-fold increase. The parallel strategy presented in this work proved to be an efficient tool for the reinforcement of protein thermostability.

InSituAnalyze: A Python Framework for Multicomponent Synchronous Analysis of Spectral Imaging.

Spectral imaging is visualization of high precision and high sensitivity and suitable for analyzing the spatial distribution of complex materials. While providing rich and detailed information, it makes higher demands on feature extraction and information mining of high-dimensional data. For the convenience of further utilization, our research team has developed a Python framework for the multicomponent synchronous analysis of spectral imaging based on a characteristic band method and fast-NNLS algorithm, helping to handle spectrum data from complex samples and gaining semiquantitative information on the sample on the scale of pixel based on target components. With the help of the easy-to-use framework, users are leading to choose suitable pretreatment methods for images and spectra, extract spatial information on tissues/structures account of multispace, and conduct analysis on target components in an intuitive and timesaving way. The sophisticated functional architecture also makes the framework expedited to add algorithms and supported data formats.

Identification of antibiotic mycelia residues in cottonseed meal using Fourier transform near-infrared microspectroscopic imaging.

Near-infrared microscopy (NIRM) technology can analyze different components within a sample while also obtaining spatial information about the sample. No rapid detection methods are available for effectively identifying antibiotic mycelia residues (AMRs) in protein feeds materials to date. In this study, the feasibility of using NIRM to identify AMRs (oxytetracycline residue, streptomycin sulfate residue and clay colysin sulfate residue) mixed in cottonseed meals was studied. The samples were scanned by NIRM, then the spectra of images were analyzed by principal component analysis (PCA) to select characteristic bands for further identification with one-class partial least squares analysis (OCPLS). The results showed that: a) AMRs were effectively identified in cottonseed meal; b) screening characteristic bands and increasing the spectral number of the calibration set improved the identification results of the model; and c) the sensitivity, specificity, accuracy and class error of the method were 100%, 95.93%, 99.01% and 2.03%, respectively.

A Single Mutation Increases the Thermostability and Activity of Aspergillus terreus Amine Transaminase.

Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t1/2) at 40 °C and a 5.3 °C increase in T5010 compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (Tm) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.