Carbon doping enhances the fluoride removal performance of aluminum-based adsorbents.

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 (pHpzc). 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.

The effects of immortalization on the N-glycome and proteome of CDK4-transformed lung cancer cells.

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.

Glycomic, Glycoproteomic, and Proteomic Profiling of Philippine Lung Cancer and Peritumoral Tissues: Case Series Study of Patients Stages I-III.

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.

Comparative proteomics reveals anticancer compounds from Lansium domesticum against NSCLC cells target mitochondrial processes.

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 IC50 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.

Multi-glycomic analysis of spheroid glycocalyx differentiates 2- and 3-dimensional cell models.

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.

N-Glycan and Glycopeptide Serum Biomarkers in Philippine Lung Cancer Patients Identified Using Liquid Chromatography-Tandem Mass Spectrometry.

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.

An Integrated Mass Spectrometry-Based Glycomics-Driven Glycoproteomics Analytical Platform to Functionally Characterize Glycosylation Inhibitors.

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.

Enhanced analytical performance of disposable 3D carbon electrodes prepared with stainless steel wire mesh.

This paper aims to use low-cost stainless steel wire mesh (SSWM) as uniform templates to prepare disposable three-dimensional (3D) carbon electrodes to improve their analytical performance. Native SSWM electrodes were prepared with lamination and then coated with carbon cement for bulk preparation of disposable 3D carbon electrodes with drop-casting. The electrodes were then coupled in paper-based analytical devices. Meanwhile, disposable 2D carbon electrodes were prepared with the stainless steel sheets (SSSs) for comparison under the same condition using stripping analysis of heavy metals as a model. Our results demonstrated that the sensitivity of the 3D carbon electrodes was about three times as high as that of the 2D carbon electrodes on stripping analysis of both heavy metals. The electrochemical responses of 1 µg L-1 Pb2+ at the 3D carbon electrodes were about 6 times as high as those at the 2D carbon electrodes. The improved analytical performance of disposable 3D carbon electrodes could be attributed to their increased electrochemical effective area, which was brought by replacing SSSs with SSWM. The obtained disposable 3D carbon electrodes could be used for differentiation of Pb in teethers and corns. This study not only presented the potential of SSWM in the preparation of disposable 3D carbon electrodes but also suggested a simple and effective strategy for the preparation of disposable 3D electrodes for practical applications.

Antitumor activity of a lectibody targeting cancer-associated high-mannose glycans.

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.

Mesenchymal Stromal Cells Regulate Sialylations of N-Glycans, Affecting Cell Migration and Survival.

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.

UDP-glucose pyrophosphorylase 2, a regulator of glycogen synthesis and glycosylation, is critical for pancreatic cancer growth.

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.

Boron-modulated surface of hollow nickel framework for improved hydrogen evolution.

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.

Midkine Is a Potential Urinary Biomarker for Non-Invasive Detection of Bladder Cancer with Microscopic Hematuria.

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.

Approaches to Understanding the Mediator Function of Brh2 in Ustilago maydis.

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.

Dss1 Regulates Association of Brh2 with Rad51.

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.

Electrochemical Fabrication of Monolithic Electrodes with Core/Shell Sandwiched Transition Metal Oxide/Oxyhydroxide for High-Performance Energy Storage.

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.

Dual DNA-binding domains shape the interaction of Brh2 with DNA.

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.

Dss1 release activates DNA binding potential in Brh2.

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.

Improved neuronal survival of focal ischemia after delipid extracorporeal lipoprotein treatment in hyperlipidemic rabbits.

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.

Mutational analysis of Brh2 reveals requirements for compensating mediator functions.

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.