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A sustainable approach utilizing a low-temperature molten salt strategy is employed in this study to fabricate homogeneous and dense NiFe nitrate hydroxide needle-like arrays on a NiFe foam substrate. The electrode also achieves an ultra-low voltage of 1.77 V at 100 mA cm -2 and maintains stability for more than 120 h at a current density of 100 mA cm -2 , showing excellent overall water splitting (OWS) performance and stability. Notably, the molten salt strategy enables the synthesis of electrodes at a reaction temperature as low as 125 °C. Additionally, the molten salt precursor is repeatable and renewable, thus preventing waste and presenting vast potential for application.

Objective: CA125 is recommended by many countries as the primary screening test for ovarian cancer. But there are patients with ovarian cancer having normal CA125. We hope to identify the types of EOC with normal CA125 levels better by building a refined model based on the ultrasound radiomics, thus providing precise medical treatment for patients., Methods: We included 58 patients with EOC with normal CA125 from 2 centres, who were confirmed by preoperative ultrasound and pathology. We extracted 1130 radiomics features based on the tumour's region of interest from the most typical ultrasound image of each patient. We selected radiomics and clinical features by LASSO and logistic regression to construct Rad-score and clinical models, respectively. Receiver operating characteristic curves judged their test efficacy. On the basis of the combined model, we developed a nomogram., Results: Area under the curves (AUCs) of 0.93 and 0.83 were achieved in both the training and test groups for the combined model. There were similar AUCs between the Rad-score and clinical models of 0.82 and 0.80, respectively. By analysing the calibration curves, it was determined that the nomogram matched actual observations in the training cohort., Conclusion: Ultrasound radiomics can differentiate type I and type II EOC with normal CA125 levels., Advances in Knowledge: This study is the first to focus on EOC cases with normal level of CA125. The subset of patients constituting 20% of the disease population may require more refined radiomics models., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Institute of Radiology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: To assess the effects of inactivated Lactobacillus rhamnosus (ILR) on growth performance, serum biochemical indices, colonic microbiota, and metabolomics in weaned piglets, 120 piglets were randomly divided into five groups. Samples in the control group were fed a basal diet, while the experimental ILR1, ILR2, ILR3, and ILR4 groups were fed basal diets supplemented with 0.1%, 0.2%, 0.3%, and 0.4% ILR, respectively. The prefeeding period lasted for 5 days and was followed by a formal period of 28 days., Results: Compared to the control, the average daily gain increased by 4.38%, 7.98%, 19.32%, and 18.80% for ILR1, ILR2, ILR3, and ILR4, respectively, and the ratio of feed to gain decreased by 0.63%, 3.80%, 12.66%, and 10.76%, respectively. Serum IgA, IgG, IgM, total antioxidant capacity, and glutathione peroxidase levels increased significantly in weaned piglets in the treatment groups. Addition of 0.3% ILR significantly increased the Shannon and Simpson indices of the colonic microbiota in weaned piglets and altered the microbiota composition. Changes in metabolic profiles were observed and were primarily related to the urea cycle, amino acid metabolism, and lipid metabolism., Conclusion: ILR improved growth performance and serum immunological and biochemical indices and optimized the colonic microbiota structure and metabolism of weaned piglets., (© 2024. The Author(s).)

Excessive fluoride presence in water poses significant environmental and public health risks, necessitating the development of effective remediation techniques. Conventional aluminum-based adsorbents face inherent limitations such as limited pH range and low adsorption capacity. To overcome these challenges, we present a facile solvent-thermal method for synthesizing a carbon-doped aluminum-based adsorbent (CDAA). Extensive characterization of CDAA reveals remarkable features including substantial carbon-containing groups, unsaturated aluminum sites, and a high pH at point of zero charge (pH pzc ). CDAA demonstrates superior efficiency and selectivity in removing fluoride contaminants, surpassing other adsorbents. It exhibits exceptional adaptability across a broad pH spectrum from 3 to 12, with a maximum adsorption capacity of 637.4 mg/g, more than 110 times higher than alumina. The applicability of the Langmuir isotherm and pseudo-second-order models effectively supports these findings. Notably, CDAA exhibits rapid kinetics, achieving near-equilibrium within just 5 min. Comprehensive analyses utilizing Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) offer detailed insights into the mechanisms involving electrostatic attraction, ion exchange, and ligand exchange. Carbon-based groups play a role in ligand exchange processes, synergistically interacting with the unsaturated aluminum structure to provide a multitude of adsorption sites. The exceptional attributes of CDAA establish its immense potential as a transformative solution for the pressing challenge of fluoride removal from water sources., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Biological experiments are often conducted in vitro using immortalized cells due to their accessibility and ease of propagation compared to primary cells and live animals. However, immortalized cells may present different proteomic and glycoproteomic characteristics from the primary cell source due to the introduction of genes that enhance proliferation (e.g. CDK4) or enable telomere lengthening. To demonstrate the changes in phenotype upon CDK4-transformation, we performed LC-MS/MS glycomic and proteomic characterizations of a human lung cancer primary cell line (DTW75) and a CDK4-transformed cell line (GL01) derived from DTW75. We observed that the primary and CDK4-transformed cells expressed significantly different levels of sialylated, fucosylated, and sialofucosylated N-glycans. Specifically, the primary cells expressed higher levels of hybrid- and complex-type sialylated N-glycans, while CDK4-transformed cells expressed higher levels of complex-type fucosylated and sialofucosylated N-glycans. Further, we compared the proteomic differences between the cell lines and found that CDK4-transformed cells expressed higher levels of RNA-binding and adhesion proteins. Further, we observed that the CDK4-transformed cells changed N-glycosylation after 31 days in cell culture, with a decrease in high-mannose and increase in fucosylated, sialylated, and sialofucosylated N-glycans. Identifying these changes between primary and CDK4-transformed cells will provide useful insight when adapting cell lines that more closely resemble in vivo physiological conditions., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

(1) Background: the development of new antibiotic substitutes to promote pig growth and health has become an important way to solve the current dilemma and promote the pig industry. (2) Methods: to assess the effects of a fermented Chinese herbal (FCH) formula on the growth and immunity of growing pigs, 100 Duroc × Landrace × Yorshire three-way crossed growing pigs were randomly divided into control and treatment groups that were fed a basal diet, and a basal diet with 1% (group A), 2% (group B), and 3% (group C) FCH formulas, respectively. A sixty-day formal experiment was conducted, and their growth and serum indices, colonic microbiota, and metabolites were analyzed. (3) Results: the daily gain of growing pigs in groups A, B, and C increased by 7.93%, 17.68%, and 19.61%, respectively, and the feed-to-gain ratios decreased by 8.33%, 15.00%, and 14.58%, respectively. Serum immunity and antioxidant activities were significantly increased in all treatment groups. Particularly, adding a 2% FCH formula significantly changed the colon's microbial structure; the Proteobacteria significantly increased and Firmicutes significantly decreased, and the metabolite composition in the colon's contents significantly changed. (4) Conclusions: these results indicate that the FCH formula is a good feed additive for growing pigs, and the recommended addition ratio was 3%.

Recently, production of hydrogen (H 2 ) through the urea oxidation reaction (UOR) and hydrogen evolution reaction (HER) has acquired great attention because it is more environmentally friendly and energy-saving. Herein, an approach of water activation was developed for in situ growth of NiMo LDH nanosheet arrays on NiMo foam without using any binder or pressurizing or heating steps. The obtained NiMo foam electrodes showed exceptional catalytic activity and durability for both the UOR and HER. This work offers a new standpoint on designing electrodes with high activation for efficient and sustainable hydrogen production coupled with urea organic oxidation.

This study explored the potential of utilizing aluminum and yttrium amended (Al/Y amended) sewage sludge biochar (Al/Y-CSBC) for efficient fluoride removal from wastewater. The adsorption kinetics of fluoride on bimetallic modified Al/Y-CSBC followed the pseudo-second-order model, while the adsorption isotherm conformed to the Freundlich equation. Remarkably, the material exhibited excellent fluoride removal performance over a wide pH range, achieving a maximum adsorption capacity of 62.44 mg·g -1 . Moreover, Al/Y-CSBC demonstrated exceptional reusability, maintaining 95% removal efficiency even after six regeneration cycles. The fluoride adsorption mechanism involved ion exchange, surface complexation, and electrostatic adsorption interactions. The activation and modification processes significantly increased the specific surface area of Al/Y-CSBC, leading to a high isoelectric point (pH pzc = 9.14). The incorporation of aluminum and yttrium metals exhibited a novel approach, enhancing the adsorption capacity for fluoride ions due to their strong affinity. Furthermore, the dispersing effect of biochar played a crucial role in improving defluoridation efficiency by enhancing accessibility to active sites. These findings substantiate the significant potential of Al/Y-CSBC for enhanced fluoride removal from wastewater., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In this paper, a facile and ultrafast two-step dipping process was developed to in situ form an electrocatalyst on a NiMo foam substrate without consuming extra energy. The obtained electrode showed a porous coral-like structure decorated with nanosheets and exhibited excellent overall water splitting properties in alkaline solution. This study provides a feasible strategy for developing an environmentally friendly and energy-efficient non-noble metal electrode for hydrogen production from water splitting.

Currently, the fabrication of low cost and high efficiency electrocatalysts is a hotspot in the study of water splitting. Herein, plasma spray (PS) was used to induce a microcosmic explosion ( me ) on Ni mesh to modify the nanoscale Ni for the preparation of me -PS-NM electrodes with excellent hydrogen evolution. We also demonstrated that oxygen evolution could be significantly enhanced after the me -PS-NM electrodes were doped with Fe 3+ . Both electrodes formed a system exhibiting superior activity and stability for overall water splitting without noble metals.

NiFe layered double hydroxide (NiFe LDH) is a promising material with multiple functions. In this communication, a novel method is used to prepare NiFe LDH. This synthesis method is achieved via galvanic-cell corrosion between nickel and iron substrates in aqueous solutions containing a halogen group anion ( e.g. , Cl) at ambient temperature. The as-prepared NiFe LDH electrodes are developed as electrocatalysts for the oxygen evolution reaction (OER) and exhibit excellent catalytic activities and durability. This work provides an energy-efficient, cost-effective, and scaled-up corrosion engineering approach for manufacturing NiFe LDH materials.

A top-down strategy using acid etching followed by water soaking is utilized to in situ synthesize autologous NiFe LDH nanosheets on NiFe foam without other metal ions, oxidizing agents or heating steps. The NiFe foam serves as both the metal source and substrate, and the obtained nanosheets are firmly anchored on the foam. The obtained ultrathin nanosheet arrays could greatly increase the electrocatalytic active sites. This factor together with the synergistic effect between Fe and Ni simultaneously leads to an enhanced catalytic effect for water splitting and urea oxidation. This strategy could be scaled up to pave a viable way for low-cost fabrication of highly efficient electrodes for electrocatalysis.

Lung cancer is the leading cause of cancer death and non-small cell lung carcinoma (NSCLC) accounting for majority of lung cancers. Thus, it is important to find potential biomarkers, such as glycans and glycoproteins, which can be used as diagnostic tools against NSCLC. Here, the N-glycome, proteome, and N-glycosylation distribution maps of tumor and peritumoral tissues of Filipino lung cancer patients (n = 5) were characterized. We present several case studies with varying stages of cancer development (I-III), mutation status ( EGFR, ALK ), and biomarker expression based on a three-gene panel ( CD133 , KRT19 , and MUC1 ). Although the profiles of each patient were unique, specific trends arose that correlated with the role of aberrant glycosylation in cancer progression. Specifically, we observed a general increase in the relative abundance of high-mannose and sialofucosylated N-glycans in tumor samples. Analysis of the glycan distribution per glycosite revealed that these sialofucosylated N-glycans were specifically attached to glycoproteins involved in key cellular processes, including metabolism, cell adhesion, and regulatory pathways. Protein expression profiles showed significant enrichment of dysregulated proteins involved in metabolism, adhesion, cell-ECM interactions, and N-linked glycosylation, supporting the protein glycosylation results. The present case series study provides the first demonstration of a multi-platform mass-spectrometric analysis specifically for Filipino lung cancer patients.

Lansium domesticum is identified as a potential source of anticancer compounds. However, there are minimal studies on its anti-lung cancer properties as well as its mechanism of action. Here, we show the specificity of lanzones hexane (LH) leaf extracts to non-small cell lung cancer cells (A549) compared to normal lung fibroblast cells (CCD19-Lu) and normal epithelial prostate cells (PNT2). Subsequent bioassay-guided fractionation of the hexane leaf extracts identified two bioactive fractions with IC 50 values of 2.694 μg/ml (LH6-6) and 2.883 μg/ml (LH7-6). LH 6-6 treatment (1 μg/ml concentration) also showed a significantly reduced migration potential of A549 relative to the control. Thirty-one phytocompounds were isolated and identified using gas chromatography-mass spectrometric (MS) analysis and were then subjected to network pharmacology analysis to assess its effects on lung cancer target proteins. Using liquid chromatography-tandem mass spectrometry proteomics experiments, we were able to show that these compounds cause cytotoxic effects through targeting mitochondrial processes in A549 lung cancer cells., (© 2023 John Wiley & Sons Ltd.)

A multi-glycomic method for characterizing the glycocalyx was employed to identify the difference between 2-dimensional (2D) and 3-dimensional (3D) culture models with two human colorectal cancer cell lines, HCT116 and HT29. 3D cell cultures are considered more representative of cancer due to their ability to mimic the microenvironment found in tumors. For this reason, they have become an important tool in cancer research. Cell-cell interactions increase in 3D models compared to 2D, indeed significant glycomic changes were observed for each cell line. Analyses included the N-glycome, O-glycome, glycolipidome, glycoproteome, and proteome providing the most extensive characterization of the glycocalyx between 3D and 2D thus far. The different glycoconjugates were affected in different ways. In the N-glycome, the 3D cells increased in high-mannose glycosylation and in core fucosylation. Glycolipids increased in sialylation. Specific glycoproteins were found to increase in the 3D cell, elucidating the pathways that are affected between the two models. The results show large structural and biological changes between the 2 models suggesting that the 2 are indeed very different potentially affecting individual outcomes in the study of diseases., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Designing earth-abundant electrocatalysts that are highly active, low-cost, and stable for the oxygen evolution reaction (OER) is crucial for electrochemical water splitting. However, in conventional electrode fabrication strategies, NiFe layered double hydroxide (NiFe LDH) catalysts are usually coated onto substrates as external components, which suffers from poor conductivity, easily detaches from the substrate, and hinders their long-term utilization. Herein, the surface-reconstruction strategy is used to synthesize in situ autologous NiFe LDH to increase the surficial active sites numbers. The FeNi foam (FNF) serves as both the metal source and substrate, and the obtained NiFe LDH nanosheets (NSs) are firmly anchored in the monolithic FNF. What needs to be emphasized is that the strategy does not involve any high-temperature or high-pressure processes, apart from a cost-effective etching and a specified drying treatment. The nanostructure of NiFe LDH and the synergistic effect between Fe and Ni simultaneously lead to an enhanced catalytic effect for the OER. Remarkably, the sr -FNF46 requires only an ultralow overpotential of 283 mV to achieve a current density of 100 mA cm -2 for the OER in 1 M KOH electrolyte, and exhibits excellent stability. Thus, the obtained electrode holds promise for electrocatalytic applications. Finally, the formation mechanism of NiFe LDH NSs due to surface reconstruction is investigated and discussed in detail.

Aberrant glycosylation has been extensively reported in cancer, with fundamental changes in the glycosylation patterns of cell-surface and secreted proteins largely occurring during cancer progression. As such, serum glycan and glycopeptide biomarkers have been discovered using mass spectrometry and proposed for cancer detection. Here, we report for the first time potential serum N -glycan and glycopeptide biomarkers for Philippine lung cancer patients. The N -glycan and glycoprotein profiles of a cohort ( n = 26 patients, n = 22 age- and gender-matched) of lung cancer patients were analyzed and compared to identify potential N -glycan and glycopeptide serum biomarkers using nano-QToF-MS/MS and ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry dynamic multiple monitoring methods, respectively. Statistical analyses identified differential N -glycan and glycopeptide abundances. The N -glycans were mostly sialylated and sialofucosylated branched structures. The glycopeptides involved proteins in complement and coagulation cascades ( p adj = 6.418 × 10 -4 ), innate immunity ( p adj = 6.094 × 10 -3 ), acute inflammatory response ( p adj = 6.404 × 10 -5 ), defense response ( p adj = 2.082 × 10 -4 ), complement activation pathways ( p adj = 1.895 × 10 -2 ), and immunoglobulin-mediated immune response pathways ( p adj = 4.818 × 10 -2 ). Biomarker models were constructed using serum N -glycans [area under the curve (AUC) = 0.775; 95% CI: 0.617-0.931] and glycopeptides (AUC = 0.959; 95% CI: 0.85-1.0), with glycopeptides having higher accuracies than N -glycans. The results suggest that in the Philippine lung cancer patient sera, specific N -glycans and site-specific glycans are differentially expressed between cases and controls. This report represents the first serum glycan and glycopeptide biomarkers of Philippine lung cancer patients, further demonstrating the utility of mass spectrometry-based glycomic and glycoproteomic methods., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

In this work, we developed a targeted glycoproteomic method to monitor the site-specific glycoprofiles and quantities of the most abundant HDL-associated proteins using Orbitrap LC-MS for (glyco)peptide target discovery and QqQ LC-MS for quantitative analysis. We conducted a pilot study using the workflow to determine whether HDL protein glycoprofiles are altered in healthy human participants in response to dietary glycan supplementation., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Cancer progression is linked to aberrant protein glycosylation due to the overexpression of several glycosylation enzymes. These enzymes are underexploited as potential anticancer drug targets and the development of rapid-screening methods and identification of glycosylation inhibitors are highly sought. An integrated bioinformatics and mass spectrometry-based glycomics-driven glycoproteomics analysis pipeline was performed to identify an N-glycan inhibitor against lung cancer cells. Combined network pharmacology and in silico screening approaches were used to identify a potential inhibitor, pictilisib, against several glycosylation-related proteins, such as Alpha1-6FucT, GlcNAcT-V, and Alpha2,6-ST-I. A glycomics assay of lung cancer cells treated with pictilisib showed a significant reduction in the fucosylation and sialylation of N-glycans, with an increase in high mannose-type glycans. Proteomics analysis and in vitro assays also showed significant upregulation of the proteins involved in apoptosis and cell adhesion, and the downregulation of proteins involved in cell cycle regulation, mRNA processing, and protein translation. Site-specific glycoproteomics analysis further showed that glycoproteins with reduced fucosylation and sialylation were involved in apoptosis, cell adhesion, DNA damage repair, and chemical response processes. To determine how the alterations in N-glycosylation impact glycoprotein dynamics, modeling of changes in glycan interactions of the ITGA5-ITGB1 (Integrin alpha 5-Integrin beta-1) complex revealed specific glycosites at the interface of these proteins that, when highly fucosylated and sialylated, such as in untreated A549 cells, form greater hydrogen bonding interactions compared to the high mannose-types in pictilisib-treated A549 cells. This study highlights the use of mass spectrometry to identify a potential glycosylation inhibitor and assessment of its impact on cell surface glycoprotein abundance and protein-protein interaction.

Introduction: There is an increased need for the development of novel blood-based biomarkers for early detection, prevention, or intervention in Alzheimer's disease (AD). This study sought to determine whether serum glycopeptide analysis holds potential for identifying novel diagnostics and prognostics of AD., Methods: The study involved 195 participants, including 96 patients with an AD diagnosis and 99 controls with no cognitive deficit. Utilizing a validated analytical mass spectrometry method, we monitored the site-specific glycosylation of 52 serum glycoproteins., Results: Partial least-squares discriminant analysis revealed that changes in overall sialylation and fucosylation of serum glycoproteins may be indicators of an AD disease state. Loss of fucosylation of immunoglobulin G1 (IgG1) and IgG2 was indicative of AD diagnosis. Individual glycopeptide analysis found separation between the AD patients and controls on complement proteins and apolipoprotein B., Discussion: The results of this study suggest that serum glycoprofiling may be a promising approach for biomarker discovery., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring published by Wiley Periodicals, LLC on behalf of Alzheimer's Association.)

The molecular complexity of the carbohydrates consumed by humans has been deceptively oversimplified due to a lack of analytical methods that possess the throughput, sensitivity, and resolution required to provide quantitative structural information. However, such information is becoming an integral part of understanding how specific glycan structures impact health through their interaction with the gut microbiome and host physiology. This work presents a detailed catalogue of the glycans present in complementary foods commonly consumed by toddlers during weaning and foods commonly consumed by American adults. The monosaccharide compositions of over 800 foods from diverse food groups including Fruits, Vegetables, Grain Products, Beans, Peas, Other Legumes, Nuts, Seeds; Sugars, Sweets and Beverages; Animal Products, and more were obtained and used to construct the "Davis Food Glycopedia" (DFG), an open-access database that provides quantitative structural information on the carbohydrates in food. While many foods within the same group possessed similar compositions, hierarchical clustering analysis revealed similarities between different groups as well. Such a Glycopedia can be used to formulate diets rich in specific monosaccharide residues to provide a more targeted modulation of the gut microbiome, thereby opening the door for a new class of prophylactic or therapeutic diets.

Aberrant protein glycosylation is a hallmark of cancer, but few drugs targeting cancer glycobiomarkers are currently available. Here, we showed that a lectibody consisting of the high-mannose glycan-binding lectin Avaren and human immunoglobulin G1 (IgG1) Fc (AvFc) selectively recognizes a range of cell lines derived from lung, breast, colon, and blood cancers at nanomolar concentrations. Binding of AvFc to the non-small cell lung cancer (NSCLC) cell lines A549 and H460 was characterized in detail. Co-immunoprecipitation proteomics analysis revealed that epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R) are among the lectibody's common targets in these cells. AvFc blocked the activation of EGFR and IGF1R by their respective ligands in A549 cells and inhibited the migration of A549 and H460 cells upon stimulation with EGF and IGF1. Furthermore, AvFc induced potent Fc-mediated cytotoxic effects and significantly restricted A549 and H460 tumor growth in severe combined immunodeficiency (SCID) mice. Immunohistochemistry analysis of primary lung tissues from NSCLC patients demonstrated that AvFc preferentially binds to tumors over adjacent non-tumor tissues. Our findings provide evidence that increased abundance of high-mannose glycans in the glycocalyx of cancer cells can be a druggable target, and AvFc may provide a new tool to probe and target this tumor-associated glycobiomarker., Competing Interests: Declaration of interest N.M. filed a patent application related to this work (PCT/US2018/017617)., (Copyright © 2022 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Glycomic profiling methods were used to determine the effect of metabolic inhibitors on glycan production. These inhibitors are commonly used to alter the cell surface glycosylation. However, structural analysis of the released glycans has been limited. In this research, the cell membranes were enriched and the glycans were released to obtain the N -glycans of the glycocalyx. Glycomic analysis using liquid chromatography-mass spectrometry (LC-MS) with a PGC chip column was used to profile the structures in the cell membrane. Glycans of untreated cells were compared to glycans of cells treated with inhibitors, including kifunensine, which inhibits the formation of complex- and hybrid-type structures, 2,4,7,8,9-Penta-O-acetyl- N -acetyl-3-fluoro-b-d-neuraminic acid methyl ester for sialylated glycans, 2-deoxy-2-fluorofucose, and 6-alkynyl fucose for fucosylated glycans. Kifunensine was the most effective, converting nearly 95% of glycans to high mannose types. The compound 6-alkynyl fucose inhibited some fucosylation but also incorporated into the glycan structure. Proteomic analysis of the enriched membrane for the four inhibitors showed only small changes in the proteome accompanied by large changes in the N -glycome for Caco-2. Future works may use these inhibitors to study the cellular behavior associated with the alteration of glycosylation in various biological systems, e.g., viral and bacterial infection, drug binding, and cell-cell interactions.

UDP-glucose pyrophosphorylase 2 (UGP2), the enzyme that synthesizes uridine diphosphate (UDP)-glucose, rests at the convergence of multiple metabolic pathways, however, the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an important role for UGP2 in the maintenance of pancreatic ductal adenocarcinoma (PDAC) growth in both in vitro and in vivo tumor models. We found that transcription of UGP2 is directly regulated by the Yes-associated protein 1 (YAP)-TEA domain transcription factor (TEAD) complex, identifying UGP2 as a bona fide YAP target gene. Loss of UGP2 leads to decreased intracellular glycogen levels and defects in N-glycosylation targets that are important for the survival of PDACs, including the epidermal growth factor receptor (EGFR). These critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer insights into therapeutic options for otherwise intractable PDACs., Competing Interests: Competing interest statement: A.L.W. has received research funding from Oncogenuity. M.S. has received research funds from Puma Biotechnology, AstraZeneca, Daiichi-Sankyo, Immunomedics, Targimmune, and Menarini Ricerche. He is on the scientific advisory board of Menarini Ricerche and the Bioscience Institute, a cofounder of medendi.org, and an employee and stockholder of AstraZeneca. F.M. is a consultant for Daiichi-Sankyo, Pfizer, Amgen, BridgeBio, Olema, OPNA-IO, PMV, Quanta, and Remedy Plan and has received research funding from Daiichi-Sankyo., (Copyright © 2021 the Author(s). Published by PNAS.)

N-Glycosylations are an important post-translational modification of proteins that can significantly impact cell function. Terminal sialic acid in hybrid or complex N-glycans has been shown to be relevant in various types of cancer, but its role in non-malignant cells remains poorly understood. We have previously shown that the motility of human bone marrow derived mesenchymal stromal cells (MSCs) can be modified by altering N-glycoforms. The goal of this study was to determine the role of sialylated N-glycans in MSCs. Here, we show that IFN-gamma or exposure to culture media low in fetal bovine serum (FBS) increases sialylated N-glycans, while PDGF-BB reduces them. These stimuli alter mRNA levels of sialyltransferases such as ST3Gal1, ST6Gal1, or ST3Gal4, suggesting that sialylation of N-glycans is regulated by transcriptional control of sialyltransferases. We next show that 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-Neu5Ac) effectively inhibits sialylations in MSCs. Supplementation with 3F-Neu5Ac increases adhesion and migration of MSCs, as assessed by both videomicroscopy and wound/scratch assays. Interestingly, pre-treatment with 3F-Neu5Ac also increases the survival of MSCs in an in vitro ischemia model. We also show that pre-treatment or continuous treatment with 3F-Neu5Ac inhibits both osteogenic and adipogenic differentiation of MSCs. Finally, secretion of key trophic factors by MSCs is variably affected upon exposure to 3F-Neu5Ac. Altogether, our experiments suggest that sialylation of N-glycans is tightly regulated in response to environmental cues and that glycoengineering MSCs to reduce sialylated N-glycans could be beneficial to increase both cell migration and survival, which may positively impact the therapeutic potential of the cells.

A hollow Ni framework (HNF) is designed and prepared to utilize the exterior and interior surface active sites of a Ni-based binder-free and support-free electrode. Furthermore, the more efficient Ni-B active sites are then in situ introduced on the surface of HNF. This micro/nano structure engineering, together with the surface boron modulation, results in a synergistically enhanced catalytic effect for the HER performance.

Self-derivation-behaviour of substrates is utilized to fabricate monolithic electrodes for oxygen evolution, in which the selected substrate functions as both the precursor of the active catalyst and a conductive support. In particular, NiFe layered double hydroxide (LDH) can be directly derived from the surface metal of commercial NiFe foam (NFF). Moreover, the as-prepared monolithic electrode exhibits enhanced activity and durability, originating from the resultant defective nanosheet structure and autologous catalyst-support features.

Electrodeposition induces material syntheses on conductive surfaces, distinguishing it from the widely used solid-state technologies in Li-based batteries. Electrodeposition drives uphill reactions by applying electric energy instead of heating. These features may enable electrodeposition to meet some needs for battery fabrication that conventional technologies can rarely achieve. The latest progress of electrodeposition technologies in Li-based batteries is summarized. Each component of Li-based batteries can be electrodeposited or synthesized with multiple methods. The advantages of electrodeposition are the main focus, and they are discussed in comparison with traditional technologies with the expectation to inspire innovations to build better Li-based batteries. Electrodeposition coats conformal films on surfaces and can control the film thickness, providing an effective approach to enhancing battery performance. Engineering interfaces by electrodeposition can stabilize the solid electrolyte interphase (SEI) and strengthen the adhesion of active materials to substrates, thereby prolonging the battery longevity. Lastly, a perspective of future studies on electrodepositing batteries is provided. The significant merits of electrodeposition should greatly advance the development of Li-based batteries., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: To determine the role of Midkine (MDK) in non-invasive detection of bladder cancer (Bca) and the relationship with Ki67., Methods: Sixty-five Bca patients and 55 non-Bca patients or healthy volunteers were enrolled and voided urine samples were prospectively obtained on the first day of enrollment. Tissue samples were collected by surgery. MDK and Ki67 expressions were analyzed by immunohistochemistry and Western Blot (WB). Specificity and sensitivity of MDK mRNA testing in the detection of Bca were determined by Receiver Operating Characteristic curve (ROC). The relationship between MDK and Ki67 was also assessed., Results: MDK was overexpressed in Bca tissues than that in the non-cancer tissues. The specificity and sensitivity for MDK mRNA testing in urine in the identification of Bca was 80% and 72.3%. MDK detected 85.7% of high-grade tumors, 87.5% of muscle-invasive tumors and 79.4% of tumors larger than 3 cm in patients without gross hematuria. Microscopic hematuria may even increase the detection rate of Bca by MDK testing. Furthermore, the correlation of MDK and Ki67 was found positive., Conclusion: MDK was overexpressed in Bca tissues and positively correlated with Ki67. MDK might be a potential biomarker for the detection of Bca, especially for those without gross hematuria but with microscopic hematuria., Competing Interests: The authors report no conflicts of interest in this work., (© 2019 Lin et al.)

Hydrogen production is the key step for the future hydrogen economy. As a promising H 2 production route, electrolysis of water suffers from high overpotentials and high energy consumption. This study proposes an N-doped CoP as the novel and effective electrocatalyst for hydrogen evolution reaction (HER) and constructs a coupled system for simultaneous hydrogen and sulfur production. Nitrogen doping lowers the d-band of CoP and weakens the H adsorption on the surface of CoP because of the strong electronegativity of nitrogen as compared to phosphorus. The H adsorption that is close to thermos-neutral states enables the effective electrolysis of the HER. Only -42 mV is required to drive a current density of -10 mA cm -2 for the HER. The oxygen evolution reaction in the anode is replaced by the oxidation reaction of Fe 2+ , which is regenerated by a coupled H 2 S absorption reaction. The coupled system can significantly reduce the energy consumption of the HER and recover useful sulfur sources., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Primary components of the homologous recombination pathway in eukaryotes include Rad51 whose function is to search for DNA sequence homology and promote strand exchange, its mediator BRCA2, and Dss1, a key regulator of BRCA2. We seek to understand the role of BRCA2 in governing the activity of Rad51 and to learn how BRCA2 function is regulated by Dss1. We use the microbe Ustilago maydis as a model system for experimentation because it has a well-conserved BRCA2-homolog, Brh2, and is amenable to biochemical and molecular genetic manipulations and analysis. The powerful attributes of this system open the way for gaining insight into BRCA2's molecular mechanism through avenues not immediately approachable in the vertebrate systems. Here we provide protocols for preparing Brh2, Dss1, and Rad51 as reagents for use in biochemical assays to monitor function and present methods for transposon-based mutational analysis of Brh2 for use in genetic dissection of function., (© 2018 Elsevier Inc. All rights reserved.)

A novel spinel Co 2 VO 4 is studied as the Li-ion battery anode material and it is sandwiched with a 3D ultralight porous current collector (PCC) and amorphous carbon. Co 2 VO 4 demonstrates the high capacity and excellent cyclability because of the mixed lithium storage mechanisms. The 3D composite structure requires no binders and replaces the conventional current collector (Cu foil) with a 3D ultralight porous metal scaffold, yielding the high electrode-based capacity. Such a novel composite anode also enables the close adhesion of Co 2 VO 4 to the PCC scaffold. The resulting monolithic electrode has the rapid electron pathway and stable mechanical properties, which lead to the excellent rate capabilities and cycling properties. At a current density of 1 A g -1 , the PCC and carbon sandwiched Co 2 VO 4 anode is able to deliver a stable reversible capacity of about 706.8 mAh g -1 after 1000 cycles. Generally, this study not only develops a new Co 2 VO 4 anode with high capacity and good cyclability, but also demonstrates an alternative approach to improve the electrochemical properties of high capacity anode materials by using ultralight porous metallic current collector instead of heavy copper foil., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Brh2, the BRCA2 ortholog in the fungus Ustilago maydis, mediates delivery of Rad51 to DNA during the course of homology-directed DNA repair. Rad51 interacts with Brh2 through the highly conserved BRC element and through a second region termed CRE located at the extreme carboxy terminus. Dss1, a small intrinsically unstructured protein that interacts with Brh2, is crucial for its activity in DNA repair, but the mechanism of this regulation is uncertain. In previous studies, we found that interaction of Brh2 with DNA was strongly modulated by association with Dss1. Here we report that CRE influences interaction of Dss1 with Brh2 and that Dss1 status markedly alters interaction of Brh2 with Rad51. While it appears that a single Rad51 protomer associates with Brh2 in complex with Dss1, loss of Dss1 is accompanied by a large increase in the number of Rad51 protomers that can associate with Brh2. Concomitant with this buildup of Rad51, Brh2 loses its ability to bind DNA. These observations suggest a feedback circuit in which release of Dss1 from Brh2 as it binds DNA triggers nucleation of a short Rad51 oligomer on Brh2, which in turn promotes dissociation of Brh2 from the DNA.

Lead halide perovskites exhibit outstanding optoelectronic and optical properties. However, some applications of perovskites are hindered by their instability in polar environments; thus, how to balance stability with conductivity is a great challenge. Here, we report a new approach of using X-type ligands to address this issue. Surface treatments containing multi-step ligand exchanges and ion filling were necessary to obtain X-type ligand-protected perovskites. Performances of this material show that: (1) the crystal structure of perovskites is stable in ethanol; (2) surface defects can be fixed by a photoactivation process and photoluminescence intensity can be enhanced to 136%; and (3) electronic devices fabricated from such materials show stabilility even after washing with ethanol. X-type ligand-protected perovskites with high stability and good conductivity are promising new materials for wide applications in electronic and optoelectronics devices.

Transition metal oxides/oxyhydroxides (TMOs) are promising high-capacity materials for electrochemical energy storage. However, the low rate and poor cyclability hinder practical applications. In this work, we developed a general electrochemical route to fabricate monolithic core/shell sandwiched structures, which are able to significantly improve the electrochemical properties of TMO electrodes by electrically wiring the insulating active materials and alleviating the adverse effects caused by volume changes using engineered porous structures. As an example, a lithium ion battery anode of porous MnO sandwiched between CNT and carbon demonstrates a high capacity of 554 mAh g -1 even after 1000 cycles at 2 A g -1 . An all-solid-state symmetric pseudocapacitor consisting of CNT@MnOOH@polypyrrole exhibits a high specific capacitance of 148 F g -1 and excellent capacitance retention (92% after 10000 cycles at 2 A g -1 ). Several other examples and applications have further confirmed the effectiveness of improving the electrochemical properties by core/shell sandwiched structures.

Brh2, the BRCA2 ortholog in the fungus Ustilago maydis, harbors two different DNA-binding domains, one located in the N-terminal region and the other located in the C-terminal region. Here we were interested in comparing the biochemical properties of Brh2 fragments, Brh2(NT) and Brh2(CT), respectively, harboring the two different DNA-binding regions to understand the mechanistic purpose of dual DNA-interaction domains. With oligonucleotide substrates to model different DNA conformations, it was found that the substrate specificity of Brh2(NT) and Brh2(CT) was almost indistinguishable although avidity was different depending on salt concentration. DNA annealing activity inherent in Brh2 was found to be attributable to Brh2(NT). Likewise, activity responsible for a second-end capture reaction modeling a later step in repair of DNA double-strand breaks was found attributable to Brh2(NT). Efficient annealing of DNA strands coated with RPA required full length Brh2 rather than Brh2(NT) suggesting Brh2(CT) contributes to the activity when RPA is present. Brh2(NT) and Brh2(CT) were both found capable of physically interacting with RPA. The results suggest that while the two DNA-binding regions of Brh2 appear functionally redundant in certain aspects of DNA repair, they differ in fundamental properties, and likely contribute in different ways to repair processes involving or arising from stalled DNA replication forks., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The design and development of nanomaterials has become central to the advancement of pseudocapacitive performance. Many one-dimensional nanostructures (1D NSs), two-dimensional nanostructures (2D NSs), and three-dimensional hierarchical structures (3D HSs) composed of these building blocks have been synthesized as pseudocapacitive materials via different methods. However, due to the unclear assembly mechanism of these NSs, reports of HSs simultaneously assembled from two or more types of NSs are rare. In this article, NiCo2O4 multiple hierarchical structures (MHSs) composed of 1D nanowires and 2D nanosheets are simply grown on Ni foam using an ordered two-step hydrothermal synthesis followed by annealing processing. The low-dimensional nanowire is found to hold priority in the growth order, rather than the high-dimensional nanosheet, thus effectively promoting the integration of these different NSs in the assembly of the NiCo2O4 MHSs. With vast electroactive surface area and favorable mesoporous architecture, the NiCo2O4 MHSs exhibit a high specific capacitance of up to 2623.3 F g(-1), scaled to the active mass of the NiCo2O4 sample at a current density of 1 A g(-1). A nearly constant rate performance of 68% is achieved at a current density ranging from 1 to 40 A g(-1), and the sample retains approximately 94% of its maximum capacitance even after 3000 continuous charge-discharge cycles at a consistently high current density of 10 A g(-1).

Dss1 is an intrinsically unstructured polypeptide that partners with the much larger Brh2 protein, the BRCA2 ortholog in Ustilago maydis, to form a tight complex. Mutants lacking Dss1 have essentially the same phenotype as mutants defective in Brh2, implying that through physical interaction Dss1 serves as a positive activator of Brh2. Dss1 associates with Brh2 through an interaction surface in the carboxy-terminal region. Certain derivatives of Brh2 lacking this interaction surface remain highly competent in DNA repair as long as a DNA-binding domain is present. However, the Dss1-independent activity raises the question of what function might be met in the native protein by having Brh2 under Dss1 control. Using a set of Brh2 fusions and truncated derivatives, we show here that Dss1 is capable of exerting control when there is a cognate Dss1-interacting surface present. We find that association of Dss1 attenuates the DNA binding potential of Brh2 and that the amino-terminal domain of Brh2 helps evict Dss1 from its carboxy-terminal interaction surface. The findings presented here add to the notion that Dss1 serves in a regulatory capacity to dictate order in association of Brh2's amino-terminal and carboxy-terminal domains with DNA.

A delipid extracorporeal lipoprotein filter (DELP) system has been used to treat patients with stroke and has shown favorable prognosis. However, the mechanism for the neuronal functional recovery remains unclear. This study aimed to investigate the neuronal histological assessment, and the levels of C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), and oxidized low-density lipoprotein (oxLDL) after ischemic stroke following DELP treatment. Hyperlipidemic rabbits underwent middle cerebral artery occlusion. After 30 min, the animals received an extracorporeal apheresis treatment with a DELP filter. Total cholesterol (TC), triglyceride, and low-density lipoprotein (LDL) of the plasma were measured. The levels of CRP, TNF-α, and oxLDL in brain tissue were also measured by enzyme-linked immunosorbent assay. Hematoxylin and eosin staining, cresyl violet staining, neuron-specific enolase (NSE) and glial fibrillary acidic protein immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed. DELP apheresis reduced TC and LDL by >30%. The number of neurons at day 7 (P < 0.01) and the integrated optical density of NSE at day 1 (P < 0.05) and day 7 (P < 0.01) were significantly increased in the DELP group. TUNEL-positive cells were significantly decreased (P < 0.05). Astrocytes were moderately activated, and this activation persisted up to 7 days. Gliosis was not found in the DELP group. After treatment, the level of CRP declined at day 1 (P < 0.05); TNF-α and oxLDL declined at day 7 (P < 0.05). DELP apheresis decreased neuronal apoptosis, reduced inflammatory markers and oxidative stress in cerebral ischemia, and improved neuronal survival., (© 2011, Copyright the Authors. Artificial Organs © 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Brh2, a member of the BRCA2 family of proteins, governs homologous recombination in the fungus Ustilago maydis through interaction with Rad51. Brh2 serves at an early step in homologous recombination to mediate Rad51 nucleoprotein filament formation and also has the capability to function at a later step in recombination through its inherent DNA annealing activity. Rec2, a Rad51 paralogue, and Rad52 are additional components of the homologous recombination system, but the absence of either is less critical than Brh2 for operational activity. Here we tested a variety of mutant forms of Brh2 for activity in recombinational repair as measured by DNA repair proficiency. We found that a mutant of Brh2 deleted of the non-canonical DNA-binding domain within the N-terminal region is dependent upon the presence of Rad52 for DNA repair activity. We also determined that a motif first identified in human BRCA2 as important in binding DMC1 also contributes to DNA repair proficiency and cooperates with the BRC element in Rad51 binding., (© 2010 Blackwell Publishing Ltd.)

Objective: To provide anatomy evidence of the simple injury of the deep branch of the ulnar nerve for clinical diagnosis and treatments., Methods: Fifteen fresh samples of voluntary intact amputated forearms with no deformity were observed anatomically, which were mutilated from the distal end of forearm. The midpoint of the forth palm fingerweb was defined as dot A, the midpoint of the hook of the hamate bone as dot B, the ulnar margin of the flexor digitorum superficialis of the little finger as OD, and the superficial branch of the ulnar nerve and the forth common finger digital nerve as OE, dot O was the vertex of the triangle, dot C was intersection point of a vertical line passing dot B toward OE; dot F was the intersection point of CB's extension line and OD. OCF formed a triangle. OCF and the deep branch of the ulnar nerve were observed. From May 2000 to June 2007, 3 cases were treated which were all simple injury of the deep branch of the ulnar nerve by glass, diagnosed through anatomical observations. The wounds were all located in the hypothenar muscles, and passed through the distal end of the hamate bone. Muscle power controlled by the ulnar nerve got lower. The double ends was sewed up in 2 cases directly intra operation, and the superficial branch of radial nerve grafted freely in the other 1 case., Results: The distance between dot B and dot O was (19.20 +/- 1.30) mm. The length of BC was (7.80 +/- 1.35) mm. The morphia of OCF was various, and the route of profundus nervi ulnaris was various in OCF. OCF contains opponens canales mainly. The muscle branch of the hypothenar muscles all send out in front of the opponens canales. The wounds of these 3 cases were all located at the distal end of the hook of the hamate bone, intrinsic muscles controlled by the ulnar nerve except hypothenar muscles were restricted without sensory disorder or any other injuries. Three cases were followed up for 2 months to 4 years. Postoperation, the symptoms disappeared, holding power got well, patients' fingers were nimble. According to the trial standard of the function of the upper limb peripheral nerve established by Chinese Medical Surgery of the Hand Association, the synthetical evaluations were excellent., Conclusion: Simple injuries of the deep branch of the ulnar nerve are all located in OCF; it is not easy to be diagnosed at the early time because of the little wounds, the function of the hypothenar muscles in existence and the normal sense.

Brh2, the BRCA2 homologue in Ustilago maydis, plays a crucial role in homologous recombination by controlling Rad51. In turn, Brh2 is governed by Dss1, an intrinsically disordered protein that forms a tight complex with the C-terminal region of Brh2. This region of the protein associating with Dss1 is highly conserved in sequence and by comparison with mammalian BRCA2 corresponds to a part of the DNA binding domain with characteristic OB folds. The N-terminal region of Brh2 harbors a less-defined but powerful DNA binding site, the activity of which is revealed upon deletion of the C-terminal region. Full-length Brh2 complexed with Dss1 binds DNA slowly, while the N-terminal fragment binds quickly. The DNA binding activity of full-length Brh2 appears to correlate with dissociation of Dss1. Addition of Dss1 to the heterotypic Brh2-Dss1 complex attenuates DNA binding activity, but not by direct competition for the N-terminal DNA binding site. Conversely, the Brh2-Dss1 complex dissociates more quickly when DNA is present. These findings suggest a model in which binding of Brh2 to DNA is subject to allosteric regulation by Dss1.

The C-terminal region of Brh2 (Brh2(CT)), the BRCA2 homolog in Ustilago maydis, is highly conserved and aligns with the DSS1/DNA-binding domain (DBD) of mammalian BRCA2, while the N-terminal region (Brh2(NT)) is poorly conserved and has no obvious functional domain except for the single Rad51-interacting BRC element. Paradoxically, Brh2(NT), but not Brh2(CT), complements the DNA repair and recombination deficiency of the brh2 mutant. We show here that Brh2(NT) exhibits an unexpected DNA binding activity with properties similar to that of the full-length protein. Deletion mapping localized the region responsible for the DNA binding activity to a stretch of residues between the BRC element and the canonical DBD. A heterologous DNA-binding domain from the large subunit of replication protein A substituted for the endogenous binding region within Brh2(NT) in supporting DNA repair. Rad51-promoted strand invasion was stimulated by Brh2(NT), but required the presence of the BRC element. The findings suggest a model in which Brh2(NT) serves as the principal site for association with DNA, while the Brh2(CT) provides a means for regulation.

A single Rad52-related protein is evident by blast analysis of the Ustilago maydis genome database. Mutants created by disruption of the structural gene exhibited few discernible defects in resistance to UV, ionizing radiation, chemical alkylating or cross-linking agents. No deficiency was noted in spontaneous mutator activity, allelic recombination or meiosis. GFP-Rad51 foci were formed in rad52 cells following DNA damage, but were initially less intense than normal suggesting a possible role for Rad52 in formation of the Rad51 nucleoprotein filament. A search for interacting genes that confer a synthetic fitness phenotype with rad52 after DNA damage by UV irradiation identified the genes for Mph1, Ercc1 and the Rad51 paralogue Rec2. Testing known mutants in recombinational repair revealed an additional interaction with the BRCA2 orthologue Brh2. Suppression of the rec2 mutant's UV sensitivity by overexpressing Brh2 was found to be dependent on Rad52. The results suggest that Rad52 serves in an overlapping, compensatory role with both Rec2 and Brh2 to promote and maintain formation of the Rad51 nucleoprotein filament.

Brh2, the ortholog of the BRCA2 tumor suppressor in Ustilago maydis, works hand in hand with Rad51 to promote repair of DNA by homologous recombination. Previous studies established that Brh2 can stimulate DNA strand exchange by enabling Rad51 nucleoprotein filament formation on replication protein A-coated ssDNA. But, more recently, it was noted that Brh2 has an inherent DNA annealing activity, raising the notion that it might have roles in recombination in addition to or beyond the mediator function. Here, we found that Brh2 can autonomously promote the formation of D-loops in reactions with plasmid DNA and homologous single-stranded oligonucleotides. The reaction differs from that catalyzed by Rad51 in having no requirement for cofactors or preloading phase on ssDNA. D-loop formation was most effective when Brh2 was mixed with plasmid DNA before addition of single-stranded oligomer. D-loop formation catalyzed by Rad51 was also enhanced when Brh2 was premixed with plasmid DNA. Brh2 rendered defective in Rad51 interaction by mutation in the BRC element was still capable of promoting D-loop formation. However, the mutant protein was unable to enhance the Rad51-catalyzed reaction. The results suggest a model in which Brh2 binding to plasmid DNA attracts and helps capture Rad51-coated ssDNA.

The BRCA2 tumor suppressor functions in repair of DNA by homologous recombination through regulating the action of Rad51. In turn, BRCA2 appears to be regulated by other interacting proteins. Dss1, a small interacting protein that binds to the C-terminal domain, has a profound effect on activity as deduced from studies on the BRCA2-related protein Brh2 in Ustilago maydis. Evidence accumulating in mammalian systems suggests that BCCIP, another small interacting protein that binds to the C-terminal domain of BRCA2, also serves to regulate homologous recombination activity. Here we were interested in testing the role of the putative U. maydis BCCIP ortholog Bcp1 in DNA repair and recombination. In keeping with the mammalian paradigm, Bcp1 bound to the C-terminal region of Brh2. Mutants deleted of the gene were extremely slow growing, showed a delay passing through S phase and exhibited sensitivity to hydroxyurea, but were otherwise normal in DNA repair and homologous recombination. In the absence of Bcp1 cells were unable to maintain the wild type morphology when challenged by a DNA replication stress. These results suggest that Bcp1 could be involved in coordinating morphogenetic events with DNA processing during replication.

Brh2 is the Ustilago maydis ortholog of the BRCA2 tumor suppressor. It functions in repair of DNA by homologous recombination by controlling the action of Rad51. A critical aspect in the control appears to be the recruitment of Rad51 to single-stranded DNA regions exposed as lesions after damage or following a disturbance in DNA synthesis. In previous experimentation, Brh2 was shown to nucleate formation of the Rad51 nucleoprotein filament that becomes the active element in promoting homologous pairing and DNA strand exchange. Nucleation was found to be initiated at junctions of double-stranded and single-stranded DNA. Here we investigated the DNA binding specificity of Brh2 in more detail using oligonucleotide substrates. We observed that Brh2 prefers partially duplex structures with single-stranded branches, flaps, or D-loops. We found also that Brh2 has an inherent ability to promote DNA annealing and strand exchange reactions on free as well as RPA-coated substrates. Unlike Rad51, Brh2 was able to promote DNA strand exchange when preincubated with double-stranded DNA. These findings raise the notion that Brh2 may have roles in homologous recombination beyond the previously established Rad51 mediator activity.

Brh2, the BRCA2 ortholog in Ustilago maydis, enables recombinational repair of DNA by controlling Rad51 and is in turn regulated by Dss1. Interplay with Rad51 is conducted via the BRC element located in the N-terminal region of the protein and through an unrelated domain, CRE, at the C terminus. Mutation in either BRC or CRE severely reduces functional activity, but repair deficiency of the brh2 mutant can be complemented by expressing BRC and CRE on different molecules. This intermolecular complementation is dependent upon the presence of Dss1. Brh2 molecules associate through the region overlapping with the Dss1-interacting domain to form at least dimer-sized complexes, which in turn, can be dissociated by Dss1 to monomer. We propose that cooperation between BRC and CRE domains and the Dss1-provoked dissociation of Brh2 complexes are requisite features of Brh2's molecular mechanism.

Rec2 is the single Rad51 paralog in Ustilago maydis. Here, we find that Rec2 is required for radiation-induced Rad51 nuclear focus formation but that Rec2 foci form independently of Rad51 and Brh2. Brh2 foci also form in the absence of Rad51 and Rec2. By coprecipitation from cleared extracts prepared from Escherichia coli cells expressing the proteins, we found that Rec2 interacts physically not only with Rad51 and itself but also with Brh2. Transgenic expression of Brh2 in rec2 mutants can effectively restore radiation resistance, but the frequencies of spontaneous Rad51 focus formation and allelic recombination are elevated. The Dss1-independent Brh2-RPA70 fusion protein is also active in restoring radiation sensitivity of rec2 but is hyperactive to an extreme degree in allelic recombination and in suppressing the meiotic block of rec2. However, the high frequency of chromosome missegregation in meiotic products is an indicator of a corrupted process. The results demonstrate that the importance of Rec2 function is not only in stimulating recombination activity but also in ensuring that recombination is properly controlled.