Dysregulation of glutamine/glutamate metabolism in COVID-19 patients: A metabolism study in African population and mini meta-analysis.

Coronavirus disease 2019 (COVID-19) remains a serious global threat. The metabolic analysis had been successfully applied in the efforts to uncover the pathological mechanisms and biomarkers of disease severity. Here we performed a quasi-targeted metabolomic analysis on 56 COVID-19 patients from Sierra Leone in western Africa, revealing the metabolomic profiles and the association with disease severity, which was confirmed by the targeted metabolomic analysis of 19 pairs of COVID-19 patients. A meta-analysis was performed on published metabolic data of COVID-19 to verify our findings. Of the 596 identified metabolites, 58 showed significant differences between severe and nonsevere groups. The pathway enrichment of these differential metabolites revealed glutamine and glutamate metabolism as the most significant metabolic pathway (Impact = 0.5; -log10P = 1.959). Further targeted metabolic analysis revealed six metabolites with significant intergroup differences, with glutamine/glutamate ratio significantly associated with severe disease, negatively correlated with 10 clinical parameters and positively correlated with SPO2 (rs = 0.442, p = 0.005). Mini meta-analysis indicated elevated glutamate was related to increased risk of COVID-19 infection (pooled odd ratio [OR] = 2.02; 95% confidence interval [CI]: 1.17-3.50) and severe COVID-19 (pooled OR = 2.28; 95% CI: 1.14-4.56). In contrast, elevated glutamine related to decreased risk of infection and severe COVID-19, the pooled OR were 0.30 (95% CI: 0.20-0.44), and 0.44 (95% CI: 0.19-0.98), respectively. Glutamine and glutamate metabolism are associated with COVID-19 severity in multiple populations, which might confer potential therapeutic target of COVID-19, especially for severe patients.

Exosomal Vaccine Loading T Cell Epitope Peptides of SARS-CoV-2 Induces Robust CD8+ T Cell Response in HLA-A Transgenic Mice.

Purpose: Current vaccines for the SARS-CoV-2 virus mainly induce neutralizing antibodies but overlook the T cell responses. This study aims to generate an exosomal vaccine carrying T cell epitope peptides of SARS-CoV-2 for the induction of CD8+ T cell response. Methods: Thirty-one peptides presented by HLA-A0201 molecule were conjugated to the DMPE-PEG-NHS molecules, and mixed with DSPE-PEG to form the peptide-PEG-lipid micelles, then fused with exosomes to generate the exosomal vaccine, followed by purification using size-exclusion chromatography and validation by Western blotting, liquid nuclear magnetic resonance (NMR) test and transmission electron microscopy. Furthermore, the exosomal vaccine was mixed with Poly (I:C) adjuvant and subcutaneously administered for three times into the hybrid mice of HLA-A0201/DR1 transgenic mice with wild-type mice. Then, the epitope-specific T cell responses were detected by ex vivo ELISPOT assay and intracellular cytokine staining. Results: The exosomal vaccine was purified from the Peak 2 fraction of FPLC and injected into the hybrid mice for three times. The IFN-γ spot forming units and the frequencies of IFN-γ+/CD8+ T cells were 10-82-fold and 13-65-fold, respectively, higher in the exosomal vaccine group compared to the Poly (I:C) control group, without visible organ toxicity. In comparison with the peptides cocktail vaccine generated in our recent work, the exosomal vaccine induced significantly stronger T cell response. Conclusion: Exosomal vaccine loading T cell epitope peptides of SARS-CoV-2 virus was initially generated without pre-modification for both peptides and exosomes, and elicited robust CD8+ T cell response in HLA-A transgenic mice.

Prognostic significance of pretreatment 18F-fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) in patients with primary T cell lymphomas.

OBJECTIVE: T cell lymphomas are associated with an aggressive worse prognosis. This study is designed to assess T cell lymphomas using 18F-FDG PET/CT. METHODS: Sixty-four patients with newly diagnosed T cell lymphomas underwent PET/computed tomography (PET/CT) scans, 47 cases who were fully followed up were retrospectively reviewed and analyzed. Overall survival (OS) and progression-free survival (PFS) were recorded for prognosis. We measured the maximum standardized uptake value (SUVmax) in all cases, analyzed the correlation between SUVmax and survival and other clinicopathologic parameters. Kaplan-Meier log-rank tests were then used to compare the survival of high and low PET/CT parameter groups, and multivariate Cox proportional hazards regression analysis was carried out to identify predictors of OS and PFS. RESULTS: With a median follow-up of 26.5 (range 0.7-117.5) months, the 1-, 2- and 3-year OS were 75.6, 61.7 and 49.2%, and PFS were 49.3, 39.9 and 29.9%, respectively in 47 patients. Among them, 33 cases progressed with a median time of 9.5 (0.7-115.0) months, and 26 patients died with a median survival time of 26.5 (0.7-117.5) months. Multivariate analysis showed the following independent prognostic factors for OS: age >60 years (P = 0.002), SUVmax >9.7 (P = 0.009) and extranodal involvement of more than one site (P = 0.018). In addition, lactate dehydrogenase level (P = 0.003) and B symptoms (P = 0.018) were independent risk factors for PFS. CONCLUSION: Pretherapy SUVmax may serve as an independent predictor of outcome in patients with newly diagnosed T cell lymphomas.

Generation and Characterization of a Nanobody Against SARS-CoV.

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) has caused global panic in 2003, and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available; thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain (RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensin-converting enzyme 2 (ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

Effect of clinical pharmacist intervention on the treatment of acute pancreatitis.

Background The participation of clinical pharmacists in the treatment of acute pancreatitis has rarely been reported. Objective The aim of this study was to retrospectively evaluate the impact of intervention of clinical pharmacists on the treatment of acute pancreatitis. Setting An academic teaching hospital in Taizhou, Jiangsu, China. Method Two hundred and twenty-eight patients with acute pancreatitis were retrospectively enrolled from July 2017 to July 2018 and divided into an intervention group (n = 119) and a control group (n = 109) according to whether a clinical pharmacist was involved. No significant differences in the baseline clinical characteristics were found between the groups. Clinical pharmacists participated in drug formulation and adjustment, pharmaceutical care, and follow-up. Main outcome measure Clinical outcomes, average hospital stays, costs, incidence of adverse drug reactions, 1-month subsequent visit rate, and patient satisfaction between the two groups were measured. Results The clinical symptoms of patients in both groups were relieved after treatment. There were no significant differences between the groups in computed tomography grades after treatment, incidence of adverse drug reactions, or average hospital stays. However, the intervention group had lower total costs of hospitalization, drugs and antibiotics but higher rates of 1-month subsequent visits and satisfaction compared with the control group. Conclusion The intervention of clinical pharmacists in the treatment of acute pancreatitis can effectively reduce costs of hospitalization, drug and antibiotics and improve follow-up compliance and patient satisfaction.

Features of Ebola Virus Disease at the Late Outbreak Stage in Sierra Leone: Clinical, Virological, Immunological, and Evolutionary Analyses.

We performed Ebola virus disease diagnosis and viral load estimation for Ebola cases in Sierra Leone during the late stage of the 2014-2015 outbreak (January-March 2015) and analyzed antibody and cytokine levels and the viral genome sequences. Ebola virus disease was confirmed in 86 of 1001 (9.7%) patients, with an overall case fatality rate of 46.8%. Fatal cases exhibited significantly higher levels of viral loads, cytokines, and chemokines at late stages of infection versus early stage compared with survivors. The viruses converged in a new clade within sublineage 3.2.4, which had a significantly lower case fatality rate.

Noninvasive imaging of c(RGD)2 -9R as a potential delivery carrier for transfection of siRNA in malignant tumors.

The purpose of our study was to develop and evaluate a novel integrin αv ß3 -specific delivery carrier for transfection of siRNA in malignant tumors. We adopted arginine-glycine-aspartate (RGD) motif as a tissue target for specific recognition of integrin αν ß3 . A chimaeric peptide was synthesized by adding nonamer arginine residues (9-arginine [9R]) at the carboxy terminus of cyclic-RGD dimer, designated as c(RGD)2 -9R, to enable small interfering RNA (siRNA) binding. To test the applicability of the delivery carrier in vivo, c(RGD)2 -9R was labeled with radionuclide of technetium-99m. Biodistribution and γ-camera imaging studies were performed in HepG2 xenograft-bearing nude mice. As results, an optimal 10:1 molar ratio of 99m Tc-c(RGD)2 -9R to siRNA was indicated by the electrophoresis on agarose gels. 99m Tc-c(RGD)2 -9R/siRNA remained stable under a set of conditions in vitro. For in vivo study, tumor radioactivity uptake of 99m Tc-c(RGD)2 -9R/siRNA in nude mice bearing HepG2 xenografts was significantly higher than that of control probe (P < .05). The xenografts were clearly visualized at 4 hours till 6 hours noninvasively after intravenous injection of 99m Tc-c(RGD)2 -9R/siRNA, while the xenografts were not visualized at any time after injection of control probe. It was concluded that c(RGD)2 -9R could be an effective siRNA delivery carrier. Technetium-99m radiolabeled-delivery carrier represents a potential imaging strategy for RNAi-based therapy.

PSO-SVM-Based Online Locomotion Mode Identification for Rehabilitation Robotic Exoskeletons.

Locomotion mode identification is essential for the control of a robotic rehabilitation exoskeletons. This paper proposes an online support vector machine (SVM) optimized by particle swarm optimization (PSO) to identify different locomotion modes to realize a smooth and automatic locomotion transition. A PSO algorithm is used to obtain the optimal parameters of SVM for a better overall performance. Signals measured by the foot pressure sensors integrated in the insoles of wearable shoes and the MEMS-based attitude and heading reference systems (AHRS) attached on the shoes and shanks of leg segments are fused together as the input information of SVM. Based on the chosen window whose size is 200 ms (with sampling frequency of 40 Hz), a three-layer wavelet packet analysis (WPA) is used for feature extraction, after which, the kernel principal component analysis (kPCA) is utilized to reduce the dimension of the feature set to reduce computation cost of the SVM. Since the signals are from two types of different sensors, the normalization is conducted to scale the input into the interval of [0, 1]. Five-fold cross validation is adapted to train the classifier, which prevents the classifier over-fitting. Based on the SVM model obtained offline in MATLAB, an online SVM algorithm is constructed for locomotion mode identification. Experiments are performed for different locomotion modes and experimental results show the effectiveness of the proposed algorithm with an accuracy of 96.00% ± 2.45%. To improve its accuracy, majority vote algorithm (MVA) is used for post-processing, with which the identification accuracy is better than 98.35% ± 1.65%. The proposed algorithm can be extended and employed in the field of robotic rehabilitation and assistance.

Upregulation of microRNA-146a by hepatitis B virus X protein contributes to hepatitis development by downregulating complement factor H.

UNLABELLED: Hepatic injuries in hepatitis B virus (HBV) patients are caused by immune responses of the host. In our previous study, microRNA-146a (miR-146a), an innate immunity-related miRNA, and complement factor H (CFH), an important negative regulator of the alternative pathway of complement activation, were differentially expressed in HBV-expressing and HBV-free hepatocytes. Here, the roles of these factors in HBV-related liver inflammation were analyzed in detail. The expression levels of miR-146a and CFH in HBV-expressing hepatocytes were assessed via analyses of hepatocyte cell lines, transgenic mice, adenovirus-infected mice, and HBV-positive human liver samples. The expression level of miR-146a was upregulated in HBV-expressing Huh-7 hepatocytes, HBV-expressing mice, and patients with HBV infection. Further results demonstrated that the HBV X protein (HBx) was responsible for its effects on miR-146a expression through NF-κB-mediated enhancement of miR-146a promoter activity. HBV/HBx also downregulated the expression of CFH mRNA in hepatocyte cell lines and the livers of humans and transgenic mice. Furthermore, overexpression and inhibition of miR-146a in Huh-7 cells downregulated and upregulated CFH mRNA levels, respectively. Luciferase reporter assays demonstrated that miR-146a downregulated CFH mRNA expression in hepatocytes via 3'-untranslated-region (UTR) pairing. The overall effect of this process in vivo is to promote liver inflammation. These results demonstrate that the HBx-miR-146a-CFH-complement activation regulation pathway might play an important role in the immunopathogenesis of chronic HBV infection. These findings have important implications for understanding the immunopathogenesis of chronic hepatitis B and developing effective therapeutic interventions. IMPORTANCE: Hepatitis B virus (HBV) remains an important pathogen and can cause severe liver diseases, including hepatitis, liver cirrhosis, and hepatocellular carcinoma. Although HBV was found in 1966, the molecular mechanisms of pathogenesis are still poorly understood. In the present study, we found that the HBV X protein (HBx) promoted the expression of miR-146a, an innate immunity-related miRNA, through the NF-κB signal pathway and that increasingly expressed miR-146a downregulated its target complement factor H (CFH), an important negative regulator of the complement alternative pathway, leading to the promotion of liver inflammation. We demonstrated that the HBx-miR-146a-CFH-complement activation regulation pathway is potentially an important mechanism of immunopathogenesis caused by chronic HBV infection. Our data provide a novel molecular mechanism of HBV pathogenesis and thus help to understand the correlations between the complement system, an important part of innate immunity, and HBV-associated disease. These findings will also be important to identify potential therapeutic targets for HBV infection.

Live/Real time three-dimensional transesophageal echocardiography in percutaneous closure of atrial septal defects.

OBJECTIVES: This study assessed the ability of live/real time three-dimensional transesophageal echocardiography (3DTEE) in measuring (1) atrial septal defect (ASD) maximum dimension, area, and adjacent rim size, (2) ASD occluder left and right atrial disk size, (3) length of contact between the left atrial (LA) disk and the aorta, and in (4) assessing device related complications such as residual shunt, device embolization, and device encroachment upon adjacent cardiac structures. MATERIALS AND METHODS: 3DTEE images acquired during percutaneous ASD closure by the Amplatzer Septal Occluder in 15 adult patients were retrospectively analyzed. Offline analysis was done using both the Philips 5500 ultrasound system and Philips QLAB software. 3D color flow Doppler images were used to assess residual ASD shunting. RESULTS: The Philips 5500 and Philips QLAB measurements correlated well for ASD maximum dimension and area measurements. The Philips QLAB 3DTEE disk size measurements also correlated well with the manufacturer obtained sizes. The aortic rim was deficient in 7 of the 15 patients, and the mean ASD occluder device size was 4 mm greater than the mean ASD maximum dimension. The LA occluder disk was in contact with the aorta throughout the cardiac cycle in 12 of the 15 patients, and the LA occluder disk size correlated significantly with the contact length with the aorta. CONCLUSION: Most of the patients demonstrated contact between the LA occluder disk and the aorta throughout the cardiac cycle. 3DTEE may be useful in identifying patients at greater risk for aortic erosion.

A novel mutation impairing the tertiary structure and stability of γC-crystallin (CRYGC) leads to cataract formation in humans and zebrafish lens.

Congenital cataract is one of the leading causes of human blindness. In this study, we identified a novel, heterozygous c.385G

[Polysomnographic findings and clinical presentation of adult men with obstructive sleep apnea].

AIM: To determine the age-related differences in the polysomnography (PSG) and clinical presentation of Chinese male adult subjects with obstructive sleep apnea (OSA), and attempt to identify the age-specific effects on the severity of sleep apnea. METHODS: This retrospective study included a cohort of 836 Chinese male a-dult subjects, who were diagnosed with OSA by the initial overnight PSG and recruited from the clinic population. The eligible subjects were classified into three different age groups: 312 young (mean 37. 07 years), 359 middle-aged(mean 52. 14 years) and 165 older (mean 69.43 years),and their polysomnographic findings and clinical presentation were assessed for the age-specific differences. RESULTS: The AHI-TST between the middle-aged and older subjects was similar (P > 0. 05), but less severe than the young(P <0.01). This trend was also observed in obstructive AI,AHI-NREM, and AHI-REM. The minimum SaO2 was higher in middle-aged and older subjects than in the young ones(P < 0.01). Central Al became greater following age in-crease (P <0.05). In sleep architecture, the elderly had lower total sleep time, sleep duration NREM or REM, and sleep efficiency than the younger (P < 0. 01), whereas sleep latency and WASO became longer ( P < 0. 01).Across all study population, age significantly correlated with AHI (P<0.01), obstructive Al (P<0.01), central AI (P<0.01) and minimum SaO2 ( P < 0. 01). Multiple regression analyses identified that age as an independent variable associated with AHI, obstructive Al and central Al respective- ly, after adjusting for confounding factors. CONCLUSION: In Chinese clinic subjects with OSA, age as an independent predictor associates with sleep apnea severity, presenting as decreased OSA and increased CSA with age.

[Immunosuppressant dexamethasone can significantly extend the expression of hepatitis B virus antigens in the HBV mouse model by hydrodynamic transfection method].

To develop a HBV infection mouse model by hydrodynamic-based transfection and further to optimize the method of development of HBV infection mouse model. We first developed a construct which contained inverted terminal repeat elements (ITR) of adeno-associated virus (AAV) and 1. 3 copies of HBV genome (ayw subtype). The pAAV-HBV1. 3 DNA was then injected hydrodynamically into the tail veins of C57BL/6 mice in 5 seconds. The virus load in serum and liver was assayed by ELISA and Real-time PCR. The expression of virus antigen and the pathologic changes of liver were analyzed by HE and immunohistochemical staining. Meanwhile, to develop HBV transfected immunosuppressied mouse, mice were injected intraperitoneally triple with 0.2 ml dexamethason (50 mg/kg) every two days before HBV transfection. The levels of HBsAg and HBeAg were assayed by ELISA. Our data showed: (1) HBsAg and HBeAg were positive (100%) in serum and liver of experimental normal mouse at day 10 after HBV transfection, and became negative at day 30 and day 60. Meanwhile the viral load in serum and liver in experimental group was significantly higher than that in control group at day 10, 30 and 60 after HBV transfection (P < 0.01, P < 0.05, respectively). (2) HBsAg and HBeAg in serum in immunosuppressed mouse model were positive until 60 days. In conclusion, a HBV infection mouse model was developed successfully by hydrodynamic-based transfection. By suppressing the immune status of mice injected with dexamethasone, the expression of HBV antigens was extended longer than that in normal adult mice. These models pave a way for HBV research and evaluation of HBV vaccine and drug development.

Small interfering RNA targeting m2 gene induces effective and long term inhibition of influenza A virus replication.

RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection.

Expression of the LIM-homeodomain gene Lmx1a in the postnatal mouse central nervous system.

The LIM-homeodomain transcription factor Lmx1a plays critical roles in roof plate formation as well as in the cell fate determination of midbrain dopaminergic neurons during embryonic development, but its function in the adult brain remains unknown. In the present study, as the first step in exploring its function in adult brain, we examined the expression of Lmx1a in the mouse central nervous system (CNS) from birth to adulthood by in situ hybridization. Lmx1a was expressed at high levels in the posterior hypothalamic area, supremammillary nucleus, ventral premammillary nucleus, subthalamic nucleus, ventral tegmental area, compact part of the substantia nigra and parabrachial nucleus from birth to adulthood, and co-localized with its paralogue Lmx1b in these regions. On the other hand, Lmx1a expression in the cochlear nuclei, medial cerebellar nucleus and superior vestibular nucleus was only observed until postnatal day (P) 30 and showed no colocalization with Lmx1b. Lmx1a-expressing neurons in the ventral midbrain were dopaminergic as evidenced by co-expression with tyrosine hydroxylase in these regions. Furthermore, Lmx1a expression was also found in the choroid plexuses and ependymal cells, although its expression was only detected during the first two postnatal weeks. These results suggest that Lmx1a may be involved in postnatal development as well as in maintenance of some aspects of normal brain function.

Delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza A/H5N1 virus.

The mortality of human infection by influenza A/H5N1 virus can exceed 80%. The high mortality and its poor response to the neuraminidase inhibitor oseltamivir have been attributed to uncontrolled virus-induced cytokine storm. We challenged BALB/c mice with 1,000 LD50 of influenza A/Vietnam/1194/04. Survival, body weight, histopathology, inflammatory markers, viral loads, T lymphocyte counts, and neutralizing antibody response were documented in infected mice treated individually or in combination with zanamvir, celecoxib, gemfibrozil, and mesalazine. To imitate the real-life scenario, treatment was initiated at 48 h after viral challenge. There were significant improvements in survival rate (P = 0.02), survival time (P < 0.02), and inflammatory markers (P < 0.01) in the group treated with a triple combination of zanamivir, celecoxib, and mesalazine when compared with zanamivir alone. Zanamivir with or without immunomodulators reduced viral load to a similar extent. Insignificant prolongation of survival was observed when individual agents were used alone. Significantly higher levels of CD4+ and CD8+ T lymphocytes and less pulmonary inflammation were also found in the group receiving triple therapy. Zanamivir alone reduced viral load but not inflammation and mortality. The survival benefits of adding celecoxib and mesalazine to zanamivir could be caused by their synergistic effects in reducing cytokine dysfunction and preventing apoptosis. Combinations of a neuraminidase inhibitor with these immunomodulators should be considered in randomized controlled treatment trials of patients suffering from H5N1 infection.

Expression of the transcription factor GATA3 in the postnatal mouse central nervous system.

GATA binding protein 3 (GATA3) is an important regulator of central nervous system (CNS) development, but its expression pattern in the postnatal CNS has not been studied. In the present study, we examined the distribution of GATA3 mRNA in the mouse CNS at different postnatal stages by in situ hybridization. During the first 2 weeks of postnatal development, numerous GATA3-expressing cells were found in the intergeniculate leaf, ventral lateral geniculate nucleus, pretectal nucleus, nucleus of the posterior commissure, superior colliculus, inferior colliculus, periaqueductal grey, substantia nigra and raphe nuclei. Few notable changes in the profile of GATA3 expression occurred over this time period. As postnatal development progressed, however, GATA3 expression weakened, and was maintained in only a few regions of the adult CNS. Throughout the brain, we found that GATA3-expressing cells were NeuN-positive, and no colocalization with glial fibrillary acidic protein (GFAP) was observed. In the substantia nigra, GATA3 was exclusively expressed in cells of the reticulate part and some of which were found to be GABAergic. This study presents a comprehensive overview of GATA3 expression in the CNS throughout postnatal life, and the dynamics that we observed provide insights for further investigations of the roles of GATA3 in postnatal development and the maintenance of the mature CNS.

Inhibitors of the proteasome suppress homologous DNA recombination in mammalian cells.

Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA double-strand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, gammaH2AX, or Mre11. These data show that the proteasome-mediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways.

A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination.

The ubiquitin (Ub)-conjugating enzyme Ubc13 is implicated in Rad6/Rad18-dependent postreplication repair (PRR) in budding yeast, but its function in vertebrates is not known. We show here that disruption or siRNA depletion of UBC13 in chicken DT40 or human cells confers severe growth defects due to chromosome instability, and hypersensitivity to both UV and ionizing radiation, consistent with a conserved role for Ubc13 in PRR. Remarkably, Ubc13-deficient cells are also compromised for DNA double-strand break (DSB) repair by homologous recombination (HR). Recruitment and activation of the E3 Ub ligase function of BRCA1 and the subsequent formation of the Rad51 nucleoprotein filament at DSBs are abolished in Ubc13-deficient cells. Furthermore, generation of ssDNA/RPA complexes at DSBs is severely attenuated in the absence of Ubc13. These data reveal a critical and unexpected role for vertebrate Ubc13 in the initiation of HR at the level of DSB processing.

Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair.

One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (gammaH2AX) at DNA double-strand breaks (DSBs). However, the role of gammaH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX(-/S139A)) and examined their DNA repair capacity. H2AX(-/S139A) cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and gammaH2AX, we disrupted XRCC3 in H2AX(-/S139A) cells. XRCC3(-/-)/H2AX(-/S139A) mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3(-/-)/H2AX(-/S139A) cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.