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Objective: PD-L1 and PD-L2 expression levels determine immune evasion and the therapeutic efficacy of immune checkpoint blockade. The factors that drive inducible PD-L1 expression have been extensively studied, but mechanisms that result in constitutive PD-L1 expression in cancer cells are largely unknown. Methods: DNA elements were deleted in cells by CRISPR/Cas9-mediated knockout. Protein function was inhibited by chemical inhibitors. Protein levels were examined by Western blot, mRNA levels were examined by real-time RT-PCR, and surface protein expression was determined by cellular immunofluorescence and flow cytometry. Immune evasion was examined by in vitro T cell-mediated killing. Results: We determined the core regions (chr9: 5, 496, 378–5, 499, 663) of a previously identified PD-L1L2-super-enhancer (SE). Through systematic analysis, we found that the E26 transformation-specific (ETS) variant transcription factor (ETV4) bound to this core DNA region but not to DNA surrounding PD-L1L2SE. Genetic knockout of ETV4 dramatically reduced the expressions of both PD-L1 and PD-L2. ETV4 transcription was dependent on ERK activation, and BRAF/TAK1-induced ERK activation was dependent on extracellular signaling from αvβ3 integrin, which profoundly affected ETV4 transcription and PD-L1/L2 expression. Genetic silencing or pharmacological inhibition of components of the PD-L1L2-SE-associated pathway rendered cancer cells susceptible to T cell-mediated killing. Conclusions: We identified a pathway originating from the extracellular matrix that signaled via integrin/BRAF/TAK1/ERK/ETV4 to PD-L1L2-SE to induce PD-L1-mediated immune evasion. These results provided new insights into PD-L1L2-SE activation and pathways associated with immune checkpoint regulation in cancer.

Lead (Pb) and copper (Cu) are ubiquitous metal contaminants and can pose a threat to ecosystem and human health. Bile acids have recently received considerable attention for their role in the maintenance of health. However, there were few studies on whether Pb and Cu affect bile acid metabolism in amphibians. In this study, a combination approach of histological analysis, targeted metabolomics, 16S rDNA sequencing and qPCR was used to explore the impacts of Pb, Cu and their mixture (Mix) on bile acid in Bufo gargarizans tadpoles. The results showed that Pb, Cu, and Mix resulted in intestinal damage and altered the bile acid profiles. Specifically, Pb and Mix exposure decreased total bile acid concentrations while increased toxic bile acid levels; in contrast, Cu exposure increased total bile acid levels. And hydrophilic bile acids were reduced in all treated tadpoles. Moreover, Pb and/or Cu changed the composition of intestinal microbiota, especially Clostridia, Bacteroides and Eubacterium involved in bile acid biotransformation. qPCR revealed that the decreased total bile acid concentrations in Pb- and Mix-treated tadpoles were most likely attributed to the activation of intestinal farnesoid X receptor (Fxr), which suppressed bile acid synthesis and reabsorption. While activated fxr in the Cu treatment group may be a regulatory mechanism in response to increased bile excretion, which is a detoxification route of tadpoles under Cu stress. Collectively, Pb, Cu and Mix changed bile acid profiles by affecting intestinal microbial composition and activating Fxr signaling. This study provided insight into the impacts of Pb and Cu on bile acid metabolism and contributed to the assessment of the potential ecotoxicity of heavy metals on amphibians.

There is a close relationship between land use/cover change and regional development in new urban areas. In this paper, based on the land-use data before (2010, 2013) and after (2015, 2018) the establishment of Xixian New Area and the economic development data of the corresponding period, with the help of indexes such as the land use transfer matrix, relative change rate/net change rate and the composite index of land use degree, the temporal and spatial differences of land-use change and land use degree in the ten years before and after the establishment of Xixian New Area were analyzed, and the driving factors of land-use change and their relationship with the level of social and economic development were discussed. The results indicate that cultivated land and construction land are the main land-use types in Xixian New Area in the whole studying period. From 2010 to 2018, the area of cultivated land and forest land in the region decreased over time, but the construction land area continuously increased dramatically (increased by 36.5% from 2010 to 2018) and the land-use change corresponds basically to the construction and development process of the New Area. In the spatial transfer of land use, the transformation of cultivated land to construction land was the most evident, and the urbanization construction in the New Area occupied the most cultivated land. The composite index of land use degree in the New Area was generally high and increased with time. Geographical location and policy guidance are the main reasons for the spatial difference of land use degree. The land use benefits in the New Area increased significantly, and the land use degree was positively correlated with the level of regional, social and economic development. The main driving factor of land-use change was human activities guided by policy.

With the development of human-computer interaction technology, how to use intelligent, natural and efficient interaction to promote the development of medicine has gradually become a hot topic of research. Mental and nervous system diseases have a great impact on the quality of people’s daily life. The use of human-computer interaction technology to rehabilitate mental and nervous system diseases can improve the treatment effect and reduce the work intensity of doctors, so it has far-reaching clinical significance. This paper first describes the development process of human-computer interaction technology, and then focuses on the application of human-computer interaction technology such as interactive pen, voice interaction, gait/gesture interaction and physiological computing in the rehabilitation treatment of mental and nervous system diseases, which has important practical significance for improving the use of computer technology to improve traditional medical treatment methods.

Bile acids, as metabolic regulators and signaling molecules, play key roles in the regulation of host metabolism and immune responses. Heavy metals such as lead (Pb) and copper (Cu) are widespread environmental pollutants that threaten public health. However, the effects of heavy metals on bile acid metabolism and the underlying molecular mechanisms remain unclear, particularly for ecologically important amphibian species. In the present research, the effects of exposure to environmentally-relevant concentrations of Pb (250 μg/L), Cu (50 μg/L), and a mixture of both (Mix) on bile acid metabolism and the underlying molecular mechanisms in the intestines of Bufo gargarizans larvae were comprehensively investigated using histopathology, metabolomics and transcriptomics analysis. Our results suggested that Pb and/or Cu caused histopathological damage to the intestine and liver, such as decreased intestinal epithelial cell height and dilated hepatic sinusoid. The total bile acid level was decreased in the Pb and Mix exposure groups but elevated in the Cu treatment. A significant decrease in the ratio of conjugated to unconjugated bile acids was present in all treatment groups. Also, the level of GCA was increased while TCA and TCDCA were decreased in all exposure groups. In addition, exposure to Pb and Cu altered the expression levels of genes related to intestinal absorption. For example, mrp2, mrp3 and aqp4 had higher expression in the Pb and Mix treatment groups, and aqp1 and mrp4 were increased in the Cu treatment group. Overall, we speculated that the dysregulation of bile acid homeostasis induced by Pb and Cu exposure may be due to impaired intestinal absorption. These findings raise further concerns about the hazards of Pb and/or Cu in influencing bile acid metabolism that might lead to the development of metabolic diseases and inflammatory disorders.

The effects of different oxygen uptake rates (OUR) on the physiological metabolism of Rhodobacter sphaeroides were investigated systematically in 50 L fermenters due to the significant influence on industrial coenzyme Q10 production under oxygen supply limitation. Meanwhile, the seriously decreased oxygen transfer rate caused by the increased broth viscosity was successfully prevented with OUR-directed continuous ammonium sulfate feeding in the late fermentation phase. The statistical analysis results showed that controlling OUR constantly at 45 ± 2.2 mmol/L/h by the oxygen supply level adjustment and the continuous ammonium sulfate feeding could greatly enhance Q10 production. This OUR-Stat controlling strategy successfully achieved the maximal coenzyme Q10 production (2584 ± 82 mg/L), which was 15.4% higher than that of the control. The highest specific CoQ10 content was 25.9 mg/(g DCW)), and the yield of CoQ10 to glucose consumption was up to 19.37 mg/g. These results demonstrated that the optimal OUR-Stat controlling strategy would be effective and economical for improving the industrial CoQ10 production.

Metabolomics aims to address what and how regulatory mechanisms are coordinated to achieve flux optimality, different metabolic objectives as well as appropriate adaptations to dynamic nutrient availability. Recent decades have witnessed that the integration of metabolomics and fluxomics within the goal of synthetic biology has arrived at generating the desired bioproducts with improved bioconversion efficiency. Absolute metabolite quantification by isotope dilution mass spectrometry represents a functional readout of cellular biochemistry and contributes to the establishment of metabolic (structured) models required in systems metabolic engineering. In industrial practices, population heterogeneity arising from fluctuating nutrient availability frequently leads to performance losses, that is reduced commercial metrics (titer, rate, and yield). Hence, the development of more stable producers and more predictable bioprocesses can benefit from a quantitative understanding of spatial and temporal cell-to-cell heterogeneity within industrial bioprocesses. Quantitative metabolomics analysis and metabolic modeling applied in computational fluid dynamics (CFD)-assisted scale-down simulators that mimic industrial heterogeneity such as fluctuations in nutrients, dissolved gases, and other stresses can procure informative clues for coping with issues during bioprocessing scale-up. In previous studies, only limited insights into the hydrodynamic conditions inside the industrial-scale bioreactor have been obtained, which makes case-by-case scale-up far from straightforward. Tracking the flow paths of cells circulating in large-scale bioreactors is a highly valuable tool for evaluating cellular performance in production tanks. The "lifelines" or "trajectories" of cells in industrial-scale bioreactors can be captured using Euler-Lagrange CFD simulation. This novel methodology can be further coupled with metabolic (structured) models to provide not only a statistical analysis of cell lifelines triggered by the environmental fluctuations but also a global assessment of the metabolic response to heterogeneity inside an industrial bioreactor. For the future, the industrial design should be dependent on the computational framework, and this integration work will allow bioprocess scale-up to the industrial scale with an end in mind.

Pharmaceutically important polyketides such as avermectin are mainly produced as secondary metabolites during the stationary phase of growth of Streptomyces species in fermenters. The source of intracellular metabolites that are funneled into polyketide biosynthesis has proven elusive. We applied multi-omics to reveal that intracellular triacylglycerols (TAGs), which accumulates in primary metabolism, are degraded during stationary phase. This process could channel carbon flux from both intracellular TAGs and extracellular substrates into polyketide biosynthesis. We devised a strategy named ‘dynamic degradation of TAG’ (ddTAG) to mobilize the TAG pool and increase polyketide biosynthesis. Using ddTAG we increased the titers of actinorhodin, jadomycin B, oxytetracycline and avermectin B1a in Streptomyces coelicolor, Streptomyces venezuelae, Streptomyces rimosus and Streptomyces avermitilis. Application of ddTAG increased the titer of avermectin B1a by 50% to 9.31 g l−1 in a 180-m3 industrial-scale fermentation, which is the highest titer ever reported. Our strategy could improve polyketide titers for pharmaceutical production. Polyketide yields in Streptomyces are boosted by routing stored intracellular triacylglycerol into pathways that make industrially relevant products.

Secretory expression is most often desired but usually hampered by limitations of signal peptide processing and protein folding in the methylotrophic yeast Pichia pastoris. To alleviate such limitations, novel endogenous signal peptides (Dan4, Gas1, Msb2, and Fre2) and folding factors (Mpd1p, Pdi2p, and Sil1p) were predicted based on the reported P. pastoris secretome and genome. Their effects were investigated using three reporter proteins: yeast-enhanced green fluorescent protein (yEGFP), β-galactosidase (Gal) and cephalosporin C acylase (SECA), in comparison with the commonly used Saccharomyces cerevisiae alpha-mating factor pre-pro leader sequence (α-MF) or folding factors (Pdi1p, BiP, and Hac1p). The newly identified signal sequences were superior over α-MF for production of heterologous proteins. The signal peptide Msb2 increased the specific extracellular production of all reporter proteins, ranging from 1.5- to 8.0-fold, and Dan4 enhanced all total protein production up to 172-fold. Co-expression of folding factors exhibited a protein-specific effect on cell growth, transcription and expression of different reporter genes. All of the novel folding factors enhanced total production of SECA, and Sil1p performed best in the extracellular SECA production, showing a 3.3-fold increase. These novel signal peptides and folding factors can be used for promoting secretion of heterologous proteins in P. pastoris.

Coenzyme Q10 (CoQ10) is an important component of the respiratory chain in humans and some bacteria. As a high-value-added nutraceutical antioxidant, CoQ10 has excellent capacity to prevent cardiovascular disease. The content of CoQ10 in the industrial Rhodobacter sphaeroides HY01 is hundreds of folds higher than normal physiological levels. In this study, we found that overexpression or optimization of the synthetic pathway failed CoQ10 overproduction in the HY01 strain. Moreover, under phosphate- limited conditions (decreased phosphate or in the absence of inorganic phosphate addition), CoQ10 production increased significantly by 12% to220 mg/L, biomass decreased by 12%, and the CoQ10 productivity of unit cells increased by 27%. In subsequent fed-batch fermentation, CoQ10 production reached 272 mg/L in the shake-flask fermentation and 1.95 g/L in a 100-L bioreactor under phosphate limitation. Furthermore, to understand the mechanism associated with CoQ10 overproduction under phosphate- limited conditions, the comparatve transcriptome analysis was performed. These results indicated that phosphate limitation combined with glucose fed-batch fermentation represented an effective strategy for CoQ10 production in the HY01. Phosphate limitation induced a pleiotropic effect on cell metabolism, and that improved CoQ10 biosynthesis efficiency was possibly related to the disturbance of energy metabolism and redox potential. Keywords: R sphaeroides, CoQ10, Phosphate limitation, Overproduction, Scale-up, Transcriptome

PD-L1 and PD-L2 expression levels determine immune evasion and the therapeutic efficacy of immune checkpoint blockade. The factors that drive inducible PD-L1 expression have been extensively studied, but mechanisms that result in constitutive PD-L1 expression in cancer cells are largely unknown.DNA elements were deleted in cells by CRISPR/Cas9-mediated knockout. Protein function was inhibited by chemical inhibitors. Protein levels were examined by Western blot, mRNA levels were examined by real-time RT-PCR, and surface protein expression was determined by cellular immunofluorescence and flow cytometry. Immune evasion was examined byWe determined the core regions (chr9: 5, 496, 378-5, 499, 663) of a previously identified PD-L1L2-super-enhancer (SE). Through systematic analysis, we found that the E26 transformation-specific (ETS) variant transcription factor (ETV4) bound to this core DNA region but not to DNA surrounding PD-L1L2SE. Genetic knockout of ETV4 dramatically reduced the expressions of both PD-L1 and PD-L2. ETV4 transcription was dependent on ERK activation, and BRAF/TAK1-induced ERK activation was dependent on extracellular signaling from αvβ3 integrin, which profoundly affected ETV4 transcription and PD-L1/L2 expression. Genetic silencing or pharmacological inhibition of components of the PD-L1L2-SE-associated pathway rendered cancer cells susceptible to T cell-mediated killing.We identified a pathway originating from the extracellular matrix that signaled

Purpose Hyperuricemia is a common disease that may lead to gout, renal damage, and cardiovascular events. Oral medication is the main treatment for hyperuricemia patients when lifestyle intervention fails. An evaluation of the safety of various urate-lowering therapies (ULTs) is integral to clinical decision-making. We constructed a network meta-analysis (NMA) to evaluate the safety of oral ULTs. Methods MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials were searched up to April 1, 2021, for randomized controlled trials that examined the safety of ULTs. The language restriction was English. The three outcomes used to assess the safety of uric acid lowering medications were treatment-related adverse events, liver damage, and major adverse cardiovascular events (MACE). Results Thirty-two trials enrolling 23,868 individuals were included in the study. In terms of treatment-related adverse events, there were no statistically significant differences between five uric acid lowering medications and placebo: allopurinol (risk ratio (RR): 1.08; 95% credible interval (CrI): 0.91, 1.29), febuxostat (RR: 1.05; 95% CrI: 0.89, 1.25), lesinurad (RR: 1.19; 95% CrI: 0.85, 1.67), lesinurad combined with xanthine oxidase inhibitor (XOI, RR: 1.05; 95% CrI: 0.83, 1.32), and topiroxostat (RR: 1.01; 95% CrI: 0.83, 1.23). Topiroxostat likely increases risk of liver damage (RR: 2.65; 95%CI: 1.24, 5.70; NNH: 33.40) as compared with placebo. With regard to MACE, there were no statistically significant differences between three uric acid lowering medications and placebo: allopurinol (RR: 0.63; 95% CrI: 0.36, 1.34), febuxostat (RR: 0.69; 95% CrI: 0.38, 1.66), and lesinurad combined with XOI (RR: 0.56; 95% CrI: 0.23, 1.85). The rankings of different interventions were depicted by cumulative ranking curve (SUCRA). Conclusions Through NMA, we provide some evidence for the safety of ULTs. We found no statistically significant differences in their effects on treatment-related adverse events and MACE. However, topiroxostat likely increases the risk of liver damage.

–Latent factor models are widely used to measure unobserved latent traits in social and behavioral sciences, including psychology, education, and marketing. When used in a confirmatory manner, design information is incorporated as zero constraints on corresponding parameters, yielding structured (confirmatory) latent factor models. In this article, we study how such design information affects the identifiability and the estimation of a structured latent factor model. Insights are gained through both asymptotic and nonasymptotic analyses. Our asymptotic results are established under a regime where both the number of manifest variables and the sample size diverge, motivated by applications to large-scale data. Under this regime, we define the structural identifiability of the latent factors and establish necessary and sufficient conditions that ensure structural identifiability. In addition, we propose an estimator which is shown to be consistent and rate optimal when structural identifiability holds. Finally, a nonasymptotic error bound is derived for this estimator, through which the effect of design information is further quantified. Our results shed lights on the design of large-scale measurement in education and psychology and have important implications on measurement validity and reliability.

Shear stress is one of the key factors affecting the large-scale culture of mammalian cells. In this study, numerical simulation based on computational fluid dynamics was used to conduct a flow-field analysis of 7, 50, 200, and 1200 L inverted frusto-conical shaking bioreactors. The results show that the shear rate, specific mass transfer area (a), and volumetric oxygen mass transfer coefficient (k(L)a) gradually decreased as the scale of the bioreactor increased. Through application of BHK21 and CHO cells in 7, 200, and 1200 L bioreactors, it was found that the cell density and antibody expression level increased as the volume of the bioreactor increased. Moreover, the antibody expression level in a 1200 L bioreactor was nearly 30% and 35% higher than that of 7 and 200 L bioreactors, respectively. The results demonstrate that the environment with a larger volume is more suitable for the growth and antibody expression of CHO cells, indicating shear stress might be the most critical factor affecting the scale-up of mammalian cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10616-019-00308-3) contains supplementary material, which is available to authorized users.

In the past three decades, various computational frameworks for the modelling of the intracellular isotopic distribution have been proposed by different research groups. Among them the cascaded linear systems are the most popular approaches since they can transfer the nonlinear isotopomer labeling system into cascaded linear sub-systems. In this work, a novel two-step decomposition algorithm to model isotopomer labeling system is proposed. It can be utilized to decompose large metabolic networks. For convenience purpose, a new concept of isotopomer decomposition units (IDUs) is defined to distinguish the approach from others. Comparing to other cascaded linear systems, the IDU method follows a different decomposition procedure and results in another set of cascaded linear systems with different coefficients. Two variants of IDU algorithms are proposed, while one variant of them (IDU-B) has fewer number of balance equations than that of EMU balance equations. The efficiency of the IDU approach is demonstrated through two simulated examples.

Half-bridge LLC dc–dc converter is popular in electric vehicle on-board charger due to its soft switching, high-power density, and low cost. But, it also has serious electromagnetic interference problems in that the half-bridge structure brings asymmetry common mode (CM) noise source by switching. So, this letter proposes an asymmetric center tapped rectifier (ACTR) to reduce the CM noise in half-bridge LLC without additional components. With ACTR, the CM noise source on primary winding is cancelled by CM noise sources on secondary windings. The CM noise reduction of the proposed ACTR is verified by a 1.1-kW half-bridge LLC prototype with 800 V input and 300–450 V output.

The selection of informative variables in partial least squares (PLS) is important in process analytical technology (PAT) applications in the pharmaceutical industry, for example, the calibration of spectrometers. In the past, numerous approaches have been proposed to select the variables in partial least squares. In this work, a new variable selection method for PLS with the weighted variable contribution (PLS-WVC) to the first singular value of the covariance matrix for each PLS component is proposed. Several variants of PLS-WVC with different weighting factors are proposed. One variant of PLS-WVC is equivalent to the PLS with variable importance in projection (PLS-VIP). However, the variants with the correlation between X γ w γ and Y γ q γ as the weighting factor are preferred based on the results of the simulation cases studies. The proposed PLS-WVCs are integrated with interval PLS (iPLS) further to select the informative wavelength intervals for spectroscopic modelling. The utility of the proposed WVC based variable selection methods in PLS is demonstrated with the real spectral data sets.

Currently, biomanufacturing technology and industry are receiving worldwide attention. However, there are still great challenges on bioprocess optimization and scale-up, including: lacing the process detection methods, which makes it difficult to meet the requirement of monitoring of key indicators and parameters; poor understanding of cell metabolism, which arouses problems to rationally achieve process optimization and regulation; the reactor environment is very different across the scales, resulting in low efficiency of stepwise scale-up. Considering the above key issues that need to be resolved, here we summarize the key technological innovations of the whole chain of fermentation process, i.e., real-time detection-dynamic regulation-rational scale-up, through case analysis. In the future, bioprocess design will be guided by a full lifecycle in-silico model integrating cellular physiology (spatiotemporal multiscale metabolic models) and fluid dynamics (CFD models). This will promote computer-aided design and development, accelerate the realization of large-scale intelligent production and serve to open a new era of green biomanufacturing.当前，生物制造技术和产业是世界关注的热点。然而，生物过程优化与放大过程中普遍面临以下几个难题，包括：过程检测手段缺乏，难以满足关键指标参数的监控；细胞代谢认知匮乏，无法理性实现过程最优化调控；反应器环境差异大，导致逐级放大效率低下。文中针对以上亟待解决的关键问题，通过案例分析介绍发酵过程实时检测-动态调控-理性放大全链条关键技术创新。在未来，生物过程设计将以集成细胞生理学 (时空多尺度细胞代谢模型) 和流体动力学 (CFD模型) 的全生命周期模型为指导，推进计算机辅助设计与开发，加速生物过程实现大规模智能化生产，开启绿色生物制造新时代。.

Item factor analysis (IFA) refers to the factor models and statistical inference procedures for analyzing multivariate categorical data. IFA techniques are commonly used in social and behavioral sciences for analyzing item-level response data. Such models summarize and interpret the dependence structure among a set of categorical variables by a small number of latent factors. In this chapter, we review the IFA modeling technique and commonly used IFA models. Then we discuss estimation methods for IFA models and their computation, with a focus on the situation where the sample size, the number of items, and the number of factors are all large. Existing statistical softwares for IFA are surveyed. This chapter is concluded with suggestions for practical applications of IFA methods and discussions of future directions.

Glucocorticoids are widely used in the treatment of nephritis, however, its dose-dependent side effects, such as the increased risk of infection and metabolic disturbances, hamper its clinical use. This study reports a visualized podocyte-targeting and focused ultrasound responsive glucocorticoid nano-delivery system (named as Dex/PFP@LIPs-BMS-α), which specific delivers dexamethasone (Dex) to podocyte targets and reduces systemic side effects. Methods: The glucocorticoid nano-delivery system was synthesized by a lipid thin film and a simple facile acoustic-emulsification method. This glucocorticoid nano-delivery system used BMS-470539 (BMS-α), a synthetic compound, as a "navigator" to specifically identify and target the melanocortin-1 receptor (MC-1R) on podocytes. The loaded perfluoropentane (PFP) realizes the directed "explosion effect" through ultrasound-targeted microbubble destruction (UTMD) technology under the coordination of low intensity focused ultrasound (LIFU) to completely release Dex. Results: Both in vitro and in vivo experiments have demonstrated that Dex/PFP@LIPs-BMs-α accurately gathered to podocyte targets and improved podocyte morphology. Moreover, in vivo, proteinuria and serum creatinine levels were significantly reduced in the group treated with Dex/PFP@LIPs-BMS-α, and no severe side effects were detected. Furthermore, Dex/PFP@LIPs-BMS-α, with capabilities of ultrasound, photoacoustic and fluorescence imaging, provided individualized visual guidance and the monitoring of treatment. Conclusion: This study provides a promising strategy of Dex/PFP@LIPs-BMS-α as effective and safe against immune-associated nephropathy.