Utility of protein-protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display.

Over the past 3 decades, a diverse collection of small protein domains have been used as scaffolds to generate general purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from reversion-inducing Cysteine-rich Protein with Kazal Motifs and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and next generation sequencing, we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low µM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were inserted into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general purpose protein-binding reagents.

Lactate drives CD38 signaling to promote Epithelial-Mesenchymal Transition through Snail induction in non-small cell lung cancer cells.

CD38 is the main NADase in mammalian cells. It regulates the homeostasis of nicotinamide adenine dinucleotide (NAD+) and extracellular nucleotides. Its function plays an important role in infection and aging. However, its potential functions in tumor cells have not been fully elucidated. In the present study, we demonstrated that lactate, which is derived from tumor metabolism remodeling, upregulates the expression of CD38 through OXPHOS-driven Hippo-TAZ pathway. The highly expressed CD38 converts NAD + to adenosine through the CD203a/CD73 complex and adenosine binds and activates its receptor A2AR, inducing the expression of Snail and promoting the invasion and metastasis of lung cancer cells. This finding elucidates a new perspective on the interplay between NAD + metabolism and glycolysis in tumor development.

Effects of short- and long-term plant functional group loss on alpine meadow community structure and soil nutrients.

The rapid loss of global biodiversity can greatly affect the normal functioning of ecosystems. However, how biodiversity losses affect plant community structure and soil nutrients is unclear. We conducted a field experiment to examine the short- and long-term effects of removing plant functional groups (Gramineae, Cyperaceae, legumes, and forbs) on the interrelationships among the species diversity, productivity, community structure, and soil nutrients in an alpine meadow ecosystem at Menyuan County, Qinghai Province. The variations in the species richness, above- and belowground biomass of the community gradually decreased over time. Species richness and productivity were positively correlated, and this correlation tended to be increasingly significant over time. Removal of the Cyperaceae, legumes, and other forbs resulted in fewer Gramineae species in the community. Soil total nitrogen, phosphorus, organic matter, and moisture contents increased significantly in the legume removal treatment. The removal of other forbs led to the lowest negative cohesion values, suggesting that this community may have difficulty recovering its previous equilibrium state within a short time. The effects of species removal on the ecosystem were likely influenced by the species structure and composition within the community. Changes in the number of Gramineae species indicated that they were more sensitive and less resistant to plant functional group removal. Legume removal may also indirectly cause distinct community responses through starvation and compensation effects. In summary, species loss at the community level led to extensive species niche shifts, which caused community resource redistribution and significant changes in community structure.

Comprehensive environmental performance of bottle-to-bottle recycling of PET bottles based on deposit-refund system in China.

Polyethylene terephthalate (PET) is a widely used packaging material and has high value in recycling. However, under China's dominant informal recycling system, most PET bottles are downcycled into fibers. The deposit-refund system (DRS) is considered a feasible mechanism to facilitate the high-value recycling of PET bottles. To comparatively evaluate the environmental performance [reduction of greenhouse gas (GHG) and pollutant emissions] under different scenarios using life cycle assessments, including the current system based on informal recycling, an improved system with a larger contribution from the source separation of municipal solid waste, and evolving systems with DRS application, five scenarios were set up. The DRS can reduce GHG emissions and the comprehensive environmental impact by 0.538 kg CO2 /kg PET bottles and 1.73 × 10-3 PE/kg PET bottles, respectively, compared to informal recycling. It can be concluded that the DRS-based recycling approach and the bottle-to-bottle recycling provide the substantial emission reduction potential of GHGs and pollutants.

A Systematic Review on Password Guessing Tasks.

Recently, many password guessing algorithms have been proposed, seriously threatening cyber security. In this paper, we systematically review over thirty methods for password guessing published between 2016 and 2023. First, we introduce a taxonomy for classifying the existing methods into trawling guessing and targeted guessing. Second, we present an extensive benchmark dataset that can assist researchers and practitioners in successive works. Third, we conduct a bibliometric analysis to present trends in this field and cross-citation between reviewed papers. Further, we discuss the open challenges of password guessing in terms of diverse application scenarios, guessing efficiency, and the combination of traditional and deep learning methods. Finally, this review presents future research directions to guide successive research and development of password guessing.

Prognostic nomogram models for predicting survival probability in elderly glioblastoma patients.

PURPOSE: To investigate the prognostic factors of survival and develop a predictive nomogram model for elderly GBM patients. METHODS: Elderly patients (> = 65 years) with histologically diagnosed GBM were extracted from the SEER database. Survival analysis of overall survival (OS) was performed by Kaplan-Meier analysis. Univariate and multivariate Cox regression analyses were used to determine independent prognostic factors and these factors were used to further construct the nomogram model. RESULTS: A total of 9068 elderly GBM patients (5122 males and 3946 females) were included, with a median age of 72 years (65-96 years). All patients were divided randomly into the training group (n = 6044) and the validation group (n = 3024) by a ratio of 2:1. Cox regression analyses on OS showed eight independent prognostic factors (race, age, tumor side, tumor size, metastasis, surgery, radiotherapy, and chemotherapy) in the training cohort. Also, seven variables (except for race) were identified on CSS in the training group. By comprising these variables, the nomogram models on OS and CSS for predicting the 6-month, 1-year, and 2-year survival probability were constructed and exhibited moderate consistency, respectively. Then, they could be validated well in the validation cohort and by C-index, time-dependent ROC curve, calibration plot, and DCA curve. CONCLUSIONS: Nomogram models on OS and CSS could provide an applicable tool to predict the survival probability and provide clinical references regarding treatment strategies and prognosis.

Antibody Display Technology (ADbody) to Present Challenging and Unstable Target Proteins on Antibodies.

Antibodies are the major components of adaptive immunity for the recognition of diverse antigens. Six complementarity-determining regions (CDRs) from each heavy chain and light chain present the antigen-binding site, which determines the antigen-binding specificity. Here, we describe the detailed method of a novel display technology termed antibody display technology (ADbody) (Hsieh and Chang, bioRxiv, 2021), based on the novel structure of human antibodies from malaria-endemic regions of Africa (Hsieh and Higgins, eLife 6:e27311, 2017). The principle of ADbody is to insert proteins of interest (POI) into the heavy-chain CDR3 while still retaining the biological function of POI on the antibody. In this chapter, we described how to use the ADbody method to display challenging and unstable POI on the antibody in mammalian cells. Collectively, this method is designed to provide an alternative outside the current display systems and to generate novel synthetic antibodies.

Development and Performance Evaluation of Integrated Hybrid Power Module for Three-Phase Servo Motor Applications.

This study aims to develop a 30 kHz/12 kW silicon carbide (SiC)/Si integrated hybrid power module (iHPM) for variable frequency drive applications, particularly industrial servo motor control, and, additionally, to theoretically and experimentally assess its dynamic characteristics and efficiency during operation. This iHPM integrates a brake circuit, a three-phase Si rectifier, and a three-phase SiC inverter within a single package to achieve a minimal current path. A space-vector pulse width modulation (SVPWM) scheme is used to control the inverter power switches. In order to reduce parasitic inductance and power loss, an inductance cancellation design is implemented in the Si rectifier and SiC inverter. The switching transients and their parasitic effects during a three-phase operation are assessed through an electromagnetic-circuit co-simulation model, by which the power loss and efficiency of the iHPM are estimated. The modeled parasitic inductance of the inverter is validated through inductance measurement, and the effectiveness of the simulated results in terms of switching transients and efficiency is verified using the experimental results of the double pulse test and open-loop inverter operation, respectively. In addition, the power loss and efficiency of the SiC MOSFET inverter are experimentally compared against those of a commercial Si IGBT inverter.

Structure of WNT inhibitor adenomatosis polyposis coli down-regulated 1 (APCDD1), a cell-surface lipid-binding protein.

Diverse extracellular proteins negatively regulate WNT signaling. One such regulator is adenomatosis polyposis coli down-regulated 1 (APCDD1), a conserved single-span transmembrane protein. In response to WNT signaling in a variety of tissues, APCDD1 transcripts are highly up-regulated. We have determined the three-dimensional structure of the extracellular domain of APCDD1, and this structure reveals an unusual architecture consisting of two closely apposed ß-barrel domains (ABD1 and ABD2). ABD2, but not ABD1, has a large hydrophobic pocket that accommodates a bound lipid. The APCDD1 ECD can also bind to WNT7A, presumably via its covalently bound palmitoleate, a modification that is common to all WNTs and is essential for signaling. This work suggests that APCDD1 functions as a negative feedback regulator by titrating WNT ligands at the surface of responding cells.

Integrated Analysis of lncRNA-mRNA Regulatory Networks Related to Lipid Metabolism in High-Oleic-Acid Rapeseed.

A high oleic acid content is considered an essential characteristic in the breeding of high-quality rapeseed in China. Long-chain non-coding RNA (lncRNA) molecules play an important role in the plant's growth and its response to stress. To better understand the role of lncRNAs in regulating plant reproductive development, we analyzed whole-transcriptome and physiological data to characterize the dynamic changes in lncRNA expression during the four representative times of seed development of high- and low-oleic-acid rapeseed in three regions. We identified 21 and 14 lncRNA and mRNA modules, respectively. These modules were divided into three types related to region, development stages, and material. Next, we analyzed the key modules related to the oil content and the oleic acid, linoleic acid, and linolenic acid contents with physiological data and constructed the key functional network analysis on this basis. Genes related to lipid metabolism, such as 3-ketoacyl-CoA synthase 16 (KCS16) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), were present in the co-expression network, suggesting that the effect of these genes on lipid metabolism might be embodied by the expression of these lncRNAs. Our results provide a fresh insight into region-, development-stage-, and material-biased changes in lncRNA expression in the seeds of Brassica napus. Some of these lncRNAs may participate in the regulatory network of lipid accumulation and metabolism, together with regulated genes. These results may help elucidate the regulatory system of lncRNAs in the lipid metabolism of high-oleic-acid rapeseed seeds.

Flavonoid Synthesis-Related Genes Determine the Color of Flower Petals in Brassica napus L.

The color of rapeseed (Brassica napus L.) petal is usually yellow but can be milky-white to orange or pink. Thus, the petal color is a popular target in rapeseed breeding programs. In his study, metabolites and RNA were extracted from the yellow (Y), yellow/purple (YP), light purple (LP), and purple (P) rapeseed petals. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), RNA-Seq, and quantitative real-time (qRT-PCR) analyses were performed to analyze the expression correlation of differential metabolites and differential genes. A total of 223 metabolites were identified in the petals of the three purple and yellow rapeseed varieties by UPLC-MS/MS. A total of 20511 differentially expressed genes (DEGs) between P, LP, YP, versus Y plant petals were detected. This study focused on the co-regulation of 4898 differential genes in the three comparison groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and quantitative RT-PCR analysis showed that the expression of BnaA10g23330D (BnF3'H) affects the synthesis of downstream peonidin and delphinidin and is a key gene regulating the purple color of petals in B. napus. L. The gene may play a key role in regulating rapeseed flower color; however, further studies are needed to verify this. These results deepen our understanding of the molecular mechanisms underlying petal color and provide the theoretical and practical basis for flower breeding targeting petal color.

Structural insights into plasmalemma vesicle-associated protein (PLVAP): Implications for vascular endothelial diaphragms and fenestrae.

In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low-molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration-presumably a coiled coil-held together with interchain disulfide bonds. Overall, ~2/3 of the ~390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP-i.e., the long dimension of the pore-and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.

Effect of ultrasonic vibration on forming force and forming quality in micro-punching with a flexible punch.

In this paper, a special ultrasonic microforming method, Micro Ultrasonic thin Sheetmetal Forming using molten plastic as a flexible punch (short as Micro-USF), was used to conduct micro-punching experiments on stainless steel sheet with thickness of 10 µm and 20 µm. The influence of ultrasonic vibration on forming force and forming quality were investigated. The experimental results showed that the forming force required for punching thin sheet metal decreased gradually as the ultrasonic time or ultrasonic power increased. By applying the ultrasonic vibration effect, the forming force could be decreased dramatically and the maximum value of forming force drop could reach 86%. Moreover, with the application of ultrasonic vibration, the size accuracy and shape accuracy of micro-holes could be increased by 36.92% and 22.65%, but the cross-section quality of micro-holes were not significantly improved.

Comprehensive evaluation of high-oleic rapeseed (Brassica napus) based on quality, resistance, and yield traits: A new method for rapid identification of high-oleic acid rapeseed germplasm.

To scientifically evaluate and utilize high-oleic acid rape germplasm resources and cultivate new varieties suitable for planting in the Hunan Province, 30 local high-oleic acid rape germplasms from Hunan were used as materials. The 12 personality indices of quality, yield, and resistance were comprehensively evaluated by variability, correlation, principal component, and cluster analyses. The results of variability showed that except for oleic acid, the lowest coefficient of variation was oil content, which was 0.06. Correlation analysis showed that oil content was positively correlated with main traits such as yield per plant and oleic acid, which could be used in the early screening of high-oleic rape germplasm. The results of principal component analysis showed that the 12 personality indicators were integrated into four principal components, and the cumulative contribution rate was 62.487%. The value of comprehensive coefficient 'F' was positively correlated with the first, second, and fourth principal components and negatively correlated with the third principal component. Cluster analysis showed that 30 high-oleic rape germplasms could be divided into four categories consisting of 9 (30%), 6 (20%), 7 (23%), and 8 (27%) high-oleic rape germplasms, each with the characteristics of "high disease resistance", "high yield", "high protein", and "more stability". This study not only provides a reference basis for high-oleic rape breeding but also provides a theoretical basis for their early screening.

Fumigant Activity of Bacterial Volatile Organic Compounds against the Nematodes Caenorhabditis elegans and Meloidogyne incognita.

Plant-parasitic nematodes infect a diversity of crops, resulting in severe economic losses in agriculture. Microbial volatile organic compounds (VOCs) are potential agents to control plant-parasitic nematodes and other pests. In this study, VOCs emitted by a dozen bacterial strains were analyzed using solid-phase microextraction followed by gas chromatography-mass spectrometry. Fumigant toxicity of selected VOCs, including dimethyl disulfide (DMDS), 2-butanone, 2-pentanone, 2-nonanone, 2-undecanone, anisole, 2,5-dimethylfuran, glyoxylic acid, and S-methyl thioacetate (MTA) was then tested against Caenorhabditis elegans. DMDS and MTA exhibited much stronger fumigant toxicity than the others. Probit analysis suggested that the values of LC50 were 8.57 and 1.43 µg/cm3 air for DMDS and MTA, respectively. MTA also showed stronger fumigant toxicity than DMDS against the root-knot nematode Meloidogyne incognita, suggesting the application potential of MTA.

Effects of Pedicularis kansuensis Expansion on Plant Community Characteristics and Soil Nutrients in an Alpine Grassland.

Pedicularis kansuensis is an indicator species of grassland degradation. Its population expansion dramatically impacts the production and service function of the grassland ecosystem, but the effects and mechanisms of the expansion are still unclear. In order to understand the ecological effects of P. kansuensis, three P. kansuensis patches of different densities were selected in an alpine grassland, and species diversity indexes, biomasses, soil physicochemical properties, and the mechanism among them were analyzed. The results showed that P. kansuensis expansion increased the richness index, the Shannon−Wiener index significantly, and the aboveground biomass ratio (ABR) of the Weed group (p < 0.05), but reduced the total biomass of the community and the ABR of the Gramineae and Cyperaceae decreased insignificantly (p > 0.05); soil moisture, soil AOC, and NO3−·N decreased significantly (p < 0.05), while soil pH and total soil nutrients did not change significantly, and available phosphorus (AP) decreased at first and then increased (p < 0.05). The structural equation model (SEM) showed that P. kansuensis expansion had a significant positive effect on the community richness index, and a significant negative effect followed on the soil AOC from the increase of the index; the increase of pH had a significant negative effect on the soil AOC, NO3−·N, and AP. It indicated that P. kansuensis expansion resulted in the increase of species richness, the ABR of the Weed group, and the community's water demand, which promoted the over-utilization of soil available nutrients in turn, and finally caused the decline of soil quality. This study elucidated a possible mechanism of poisonous weeds expansion, and provided a scientific and theoretical basis for grassland management.

The Association of Homocysteine and Diabetic Retinopathy in Homocysteine Cycle in Chinese Patients With Type 2 Diabetes.

Objective: This study aimed to explore the relationship between homocysteine (Hcy) and diabetic retinopathy (DR) and the impacts of the Hcy pathway on this relationship against this background. Methods: This study retrieved 1979 patients with type 2 diabetes (T2D) from the First Affiliated Hospital of Liaoning Medical University in Jinzhou, Liaoning Province, China. Multiple logistic regression was used to analyze the effects of Hcy cycle on the relationship between Hcy and DR. Spearman's rank correlation analysis was used to analyze the correlation between risk factors related to DR progression and Hcy. Finally, the results of logistic regression were supplemented by mediation analysis. Results: We found there was a negative correlation between low concentration of Hcy and DR (OR : 0.83, 95%CI: 0.69-1). After stratifying all patients by cysteine (Cys) or Methionine (Met), this relationship remained significant only in low concentration of Cys (OR: 0.75, 95%CI: 0.61-0.94). Through the RCS curve, we found that the effect of Hcy on DR presents a U-shaped curve relationship. Mediating effect in Met and Hcy cycles was also significant [Total effect c (OR: 0.968, 95%CI: 0.938-0.998), Direct effect path c' (OR: 0.969, 95%CI: 0.940-0.999), Path a (OR: 1.047, 95%CI: 1.004-1.091), Path b (OR: 0.964, 95%CI: 0.932-0.998)]. Conclusions: The relationship between Hcy and DR presents a U-shaped curve and the homocysteine cycle pathway has an impact on it. And too low concentration of Hcy indicates a lack of other substances, such as vitamins. It is suggested that the progression of DR is the result of a combination of many risk factors. Further prospective studies are needed to determine the role of Hcy in the pathogenesis of DR.

Biodiversity and Ecosystem Function under Simulated Gradient Warming and Grazing.

Biodiversity and ecosystem functions and their relationship with environmental response constitute a major topic of ecological research. However, the changes in and impact mechanisms of multi-dimensional biodiversity and ecosystem functions in continuously changing environmental gradients and anthropogenic activities remain poorly understood. Here, we analyze the effects of multi-gradient warming and grazing on relationships between the biodiversity of plant and soil microbial with productivity/community stability through a field experiment simulating multi-gradient warming and grazing in alpine grasslands on the Tibetan Plateau. We show the following results: (i) Plant biodiversity, soil microbial diversity and community productivity in alpine grasslands show fluctuating trends with temperature gradients, and a temperature increase below approximately 1 °C is beneficial to alpine grasslands; moderate grazing only increases the fungal diversity of the soil surface layer. (ii) The warming shifted plant biomass underground in alpine grasslands to obtain more water in response to the decrease in soil moisture caused by the temperature rise. Community stability was not affected by warming or grazing. (iii) Community stability was not significantly correlated with productivity, and environmental factors, rather than biodiversity, influenced community stability and productivity.

Hydroxysafflor yellow A improved retinopathy via Nrf2/HO-1 pathway in rats.

The aim of the study was to investigate the inhibitory effect of hydroxysaff yellow A (HSYA) on diabetic retinopathy (DR). For this, a total of 27 rats were randomly divided into normal control, model, and HSYA groups. The body weight, blood glucose, and blood-retinal barrier damage of the rats were observed and compared. The pathological change of retinal tissue were measured using H&E staining. The apoptosis of retinal tissue ganglion cells was detected by TUNEL. The interleukin (IL)-1ß and tumor necrosis fator (TNF)-α levels were detected using enzyme-linked immunosorbent assay. The level of malondialdehyde (MDA) was detected using thiobarbituric acid method. Superoxide dismutase levels were detected using xanthine oxidase method; Nrf2 and total HO-1 protein expressions were detected using western blot assay; Bcl-2 and P53 protein expression was measured using immunohistochemical staining. The body weight and retinal damage of the HYSA group were significantly improved (p < 0.01, respectively). The apoptosis index of the HYSA group was lower than the model group (p < 0.001). The IL-1ß, TNF-α, and MDA levels of the HYSA group were significantly improved in comparison with those of the model group (p < 0.01, respectively). The Nrf-2, HO-1, Bcl-2, and P53 protein expression of HYSA group was significantly improved (p < 0.001, respectively). In conclusion, HYSA can effectively alleviate the apoptosis of retinal ganglion cells in type 2 diabetic rats and improve the progression of DR.