Repression of Transcription at DNA Breaks Requires Cohesin throughout Interphase and Prevents Genome Instability.

Cohesin subunits are frequently mutated in cancer, but how they function as tumor suppressors is unknown. Cohesin mediates sister chromatid cohesion, but this is not always perturbed in cancer cells. Here, we identify a previously unknown role for cohesin. We find that cohesin is required to repress transcription at DNA double-strand breaks (DSBs). Notably, cohesin represses transcription at DSBs throughout interphase, indicating that this is distinct from its known role in mediating DNA repair through sister chromatid cohesion. We identified a cancer-associated SA2 mutation that supports sister chromatid cohesion but is unable to repress transcription at DSBs. We further show that failure to repress transcription at DSBs leads to large-scale genome rearrangements. Cancer samples lacking SA2 display mutational patterns consistent with loss of this pathway. These findings uncover a new function for cohesin that provides insights into its frequent loss in cancer.

How Tactile Afferents in the Human Fingerpad Encode Tangential Torques Associated with Manipulation: Are Monkeys Better than Us?

Dexterous object manipulation depends critically on information about forces normal and tangential to the fingerpads, and also on torque associated with object orientation at grip surfaces. We investigated how torque information is encoded by human tactile afferents in the fingerpads and compared them to 97 afferents recorded in monkeys (n = 3; 2 females) in our previous study. Human data included slowly-adapting Type-II (SA-II) afferents, which are absent in the glabrous skin of monkeys. Torques of different magnitudes (3.5-7.5 mNm) were applied in clockwise and anticlockwise directions to a standard central site on the fingerpads of 34 human subjects (19 females). Torques were superimposed on a 2, 3, or 4 N background normal force. Unitary recordings were made from fast-adapting Type-I (FA-I, n = 39), and slowly-adapting Type-I (SA-I, n = 31) and Type-II (SA-II, n = 13) afferents supplying the fingerpads via microelectrodes inserted into the median nerve. All three afferent types encoded torque magnitude and direction, with torque sensitivity being higher with smaller normal forces. SA-I afferent responses to static torque were inferior to dynamic stimuli in humans, while in monkeys the opposite was true. In humans this might be compensated by the addition of sustained SA-II afferent input, and their capacity to increase or decrease firing rates with direction of rotation. We conclude that the discrimination capacity of individual afferents of each type was inferior in humans than monkeys which could be because of differences in fingertip tissue compliance and skin friction.SIGNIFICANCE STATEMENT We investigated how individual human tactile nerve fibers encode rotational forces (torques) and compared them to their monkey counterparts. Human hands, but not monkey hands, are innervated by a tactile neuron type (SA-II afferents) specialized to encode directional skin strain yet, so far, torque encoding has only been studied in monkeys. We find that human SA-I afferents were generally less sensitive and less able to discriminate torque magnitude and direction than their monkey counterparts, especially during the static phase of torque loading. However, this shortfall in humans could be compensated by SA-II afferent input. This indicates that variation in afferent types might complement each other signaling different stimulus features possibly providing computational advantage to discriminate stimuli.

The two paralogous copies of the YoeB-YefM toxin-antitoxin module in Staphylococcus aureus differ in DNA binding and recognition patterns.

Toxin-antitoxin (TA) systems are ubiquitous regulatory modules for bacterial growth and cell survival following stress. YefM-YoeB, the most prevalent type II TA system, is present in a variety of bacterial species. In Staphylococcus aureus, the YefM-YoeB system exists as two independent paralogous copies. Our previous research resolved crystal structures of the two oligomeric states (heterotetramer and heterohexamer-DNA ternary complex) of the first paralog as well as the molecular mechanism of transcriptional autoregulation of this module. However, structural details reflecting molecular diversity in both paralogs have been relatively unexplored. To understand the molecular mechanism of how Sa2YoeB and Sa2YefM regulate their own transcription and how each paralog functions independently, we solved a series of crystal structures of the Sa2YoeB-Sa2YefM. Our structural and biochemical data demonstrated that both paralogous copies adopt similar mechanisms of transcriptional autoregulation. In addition, structural analysis suggested that molecular diversity between the two paralogs might be reflected in the interaction profile of YefM and YoeB and the recognition pattern of promoter DNA by YefM. Interaction analysis revealed unique conformational and activating force effected by the interface between Sa2YoeB and Sa2YefM. In addition, the recognition pattern analysis demonstrated that residues Thr7 and Tyr14 of Sa2YefM specifically recognizes the flanking sequences (G and C) of the promoter DNA. Together, these results provide the structural insights into the molecular diversity and independent function of the paralogous copies of the YoeB-YefM TA system.

An active domain SA-2 derived from cystatin-SA, and its antifungal activity.

Infections induced by fungi, especially the drug-resistant fungi, are difficult clinical problems. Conventional antifungal treatment is effective but due to resistance, treatment failure, and treatment-related toxicity, there is a need for new antifungal drugs. In this study, SA-2 (YYRRLLRVLRRRW) was derived from Cystatin-SA, a saliva protein with a molecular weight of 14 kDa. Meanwhile, the structure-activity of SA-2 and its mutants was also studied. We detected the antimicrobial activity and cytotoxicity of SA-2 and found that SA-2 had a low cytotoxicity toward mammalian cells but a good inhibitory effect on Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans), with MIC values of 16-64 µg/mL and 8-32 µg/mL, respectively. Interestingly, SA-2 effectively killed fluconazole-resistant C. neoformans and C. albicans within 12 h. This antifungal activity against fluconazole-resistant fungi was comparable to that of amphotericin B. In addition, the C. neoformans-infected mice model was established to evaluate the anti-infective activity of SA-2 in vivo. Results showed that SA-2 significantly reduced the counts of fungi in lung and brain tissues to protect fluconazole-resistant C. neoformans-infected mice from death without changing mice body weights. Moreover, the dramatically increased pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß induced by intranasal infection of C. neoformans could be obviously declined due to the treatment of SA-2, which may be attributed to the elimination of C. neoformans in time in the infected tissue. For the mode of actions underlying SA-2 against C. neoformans, we found that the cationic peptide SA-2 could adhere to the negatively charged fungal cell membrane to increase the surface potential of C. neoformans in a dose-dependent manner, and finally disrupted the integrity of fungal cell membrane, reflecting as a 60% positive rate of propidium iodide uptake of C. neoformans cells after SA-2 (4 × MIC) treatment. Our study indicated that SA-2 has the potential to develop as a new therapeutic agent against infection induced by drug-resistant fungi.

Improving FLAIR SAR efficiency at 7T by adaptive tailoring of adiabatic pulse power through deep learning B1+ estimation.

PURPOSE: The purpose of this study is to demonstrate a method for specific absorption rate (SAR) reduction for 2D T2 -FLAIR MRI sequences at 7 T by predicting the required adiabatic radiofrequency (RF) pulse power and scaling the RF amplitude in a slice-wise fashion. METHODS: We used a time-resampled frequency-offset corrected inversion (TR-FOCI) adiabatic pulse for spin inversion in a T2 -FLAIR sequence to improve B1+ homogeneity and calculated the pulse power required for adiabaticity slice-by-slice to minimize the SAR. Drawing on the implicit B1+ inhomogeneity in a standard localizer scan, we acquired 3D AutoAlign localizers and SA2RAGE B1+ maps in 28 volunteers. Then, we trained a convolutional neural network (CNN) to estimate the B1+ profile from the localizers and calculated pulse scale factors for each slice. We assessed the predicted B1+ profiles and the effect of scaled pulse amplitudes on the FLAIR inversion efficiency in oblique transverse, sagittal, and coronal orientations. RESULTS: The predicted B1+ amplitude maps matched the measured ones with a mean difference of 9.5% across all slices and participants. The slice-by-slice scaling of the TR-FOCI inversion pulse was most effective in oblique transverse orientation and resulted in a 1 min and 30 s reduction in SAR induced delay time while delivering identical image quality. CONCLUSION: We propose a SAR reduction technique based on the estimation of B1+ profiles from standard localizer scans using a CNN and show that scaling the inversion pulse power slice-by-slice for FLAIR sequences at 7T reduces SAR and scan time without compromising image quality.

NO● Represses the Oxygenation of Arachidonoyl PE by 15LOX/PEBP1: Mechanism and Role in Ferroptosis.

We recently discovered an anti-ferroptotic mechanism inherent to M1 macrophages whereby high levels of NO● suppressed ferroptosis via inhibition of hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) production by 15-lipoxygenase (15LOX) complexed with PE-binding protein 1 (PEBP1). However, the mechanism of NO● interference with 15LOX/PEBP1 activity remained unclear. Here, we use a biochemical model of recombinant 15LOX-2 complexed with PEBP1, LC-MS redox lipidomics, and structure-based modeling and simulations to uncover the mechanism through which NO● suppresses ETE-PE oxidation. Our study reveals that O2 and NO● use the same entry pores and channels connecting to 15LOX-2 catalytic site, resulting in a competition for the catalytic site. We identified residues that direct O2 and NO● to the catalytic site, as well as those stabilizing the esterified ETE-PE phospholipid tail. The functional significance of these residues is supported by in silico saturation mutagenesis. We detected nitrosylated PE species in a biochemical system consisting of 15LOX-2/PEBP1 and NO● donor and in RAW264.7 M2 macrophages treated with ferroptosis-inducer RSL3 in the presence of NO●, in further support of the ability of NO● to diffuse to, and react at, the 15LOX-2 catalytic site. The results provide first insights into the molecular mechanism of repression of the ferroptotic Hp-ETE-PE production by NO●.

Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates.

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown. Here, using single-molecule atomic force and fluorescence microscopy imaging as well as fluorescence anisotropy measurements, we established that SA2 binds to both dsDNA and ssDNA, albeit with a higher binding affinity for ssDNA. We observed that SA2 can switch between the 1D diffusing (search) mode on dsDNA and stable binding (recognition) mode at ssDNA gaps. Although SA2 does not specifically bind to centromeric or telomeric sequences, it does recognize DNA structures often associated with DNA replication and double-strand break repair, such as a double-stranded end, single-stranded overhang, flap, fork, and ssDNA gap. SA2 loss leads to a defect in homologous recombination-mediated DNA double-strand break repair. These results suggest that SA2 functions at intermediate DNA structures during DNA transactions in genome maintenance pathways. These findings have important implications for understanding the function of cohesin in these pathways.

Therapeutic Potential of Antioxidants and Hybrid TEMPOL Derivatives in Ocular Neurodegenerative Diseases: A Glimpse into the Future.

Reactive oxygen species play a significant role in the pathogenesis of various ocular neurodegenerative diseases especially glaucoma, age-related macular degeneration (AMD), and ocular ischemic stroke. Increased oxidative stress and the accumulation of ROS have been implicated in the progression of these diseases. As a result, there has been growing interest in exploring potential therapeutic and prophylactic strategies involving exogenous antioxidants. In recent years, there have been significant advancements in the development of synthetic therapeutic antioxidants for targeting reactive oxygen species (ROS) in neurodegenerative diseases. One area of focus has been the development of hybrid TEMPOL derivatives. In the context of ocular diseases, the application of next-generation hybrid TEMPOL antioxidants may offer new avenues for neuroprotection. By targeting ROS and reducing oxidative stress in the retina and optic nerve, these compounds have the potential to preserve retinal ganglion cells and trabecular meshwork and protect against optic nerve damage, mitigating irreversible blindness associated with these diseases. This review seeks to highlight the potential impact of hybrid TEMPOL antioxidants and their derivatives on ocular neurodegenerative disorders.

Virus isolation and full-length genome sequencing of a representative canine distemper virus wild type strain of the South America 2 clade.

Canine Distemper Virus (CDV) is a highly contagious pathogen of dogs that causes severe respiratory, gastrointestinal and nervous signs. Although vaccines have been used to prevent infections, CDV has been reported worldwide, even in vaccinated animals. In the present study, a representative wild type CDV strain (Arg24) was isolated from a sick vaccinated dog and its genome was completely sequenced using Illumina technology. This strain produced a strong cytopathic effect in Vero SLAM (Signaling Lymphocyte Activation Molecule) cells with a higher titer of 1.1 × 105 Median Tissue Culture Infectious Dose (TCID50/mL) at 32 h post infection, in cell-associated virus. The Arg24 strain genome, showed values of 97.1, 90.3, 96.7, 90.6, 89.8 and 97.3 % of amino acid identity with respect to the Onderstepoort vaccine strain (Nucleoprotein, Phosphoprotein, Matrix, Fusion, Hemagglutinin and Large polymerase, respectively). Focusing on the Hemagglutinin gene, which is the target for genetic characterization, Arg24 showed four additional potential glycosylation sites, with respect to the Onderstepoort. The availability of Arg24 strain, which can be easily grown in Vero SLAM cells, is an important tool to perform immunological and antigenic comparative studies, between wild type and vaccine CDV strains.

Development of Novel Magneto-Biosensor for Sulfapyridine Detection.

In this work, we report the development of a highly sensitive biosensor for sulfapyridine detection based on an integrated bio micro-electromechanical system (Bio-MEMS) containing four gold working electrodes (WEs), a platinum counter electrode (CE), and a reference electrode (RE). Firstly, the cleaned WEs were modified with 4-aminophenylacetic acid (CMA). Then, (5-[4-(amino)phenylsulfonamide]-5-oxopentanoic acid (SA2BSA) was immobilized onto the transducers surface by carbodiimide chemistry. The analyte was quantified by competitive detection with SA2BSA immobilized on the WE toward a mixture of Ab155 antibody (with fixed concentration) and sulfapyridine. In order to obtain a highly sensitive biosensor, Ab155 was immobilized onto magnetic latex nanoparticles surface to create a 3D architecture (Ab-MLNp). Using electrochemical impedance spectroscopy (EIS), we investigated the influence of the Ab-MLNp on the sensitivity of our approach. The optimized system was analyzed, as competitive assay, with different concentrations of sulfapyridine (40 µM, 4 µM, and 2 nM) and with phosphate buffer solution. From data fitting calculations and graphs, it was observed that the EIS showed more linearity when Ab-MLNp was used. This result indicates that the magnetic latex nanoparticles increased the sensitivity of the biosensor.

Isolation and characterization of the Staphylococcus aureus bacteriophage vB_SauS_SA2.

A novel bacteriophage vB_SauS_SA2 (hereafter designated SA2) that infects Staphylococcus aureus was isolated. At a multiplicity of infection (MOI) of 0.1, phage SA2 had a latent period of about 10 min with a burst size of 293 PFUs/infected cell (PFU, plaque forming unit). Phage SA2 had a double-stranded DNA genome with a length of 89,055 bp and a G + C content of 31.9%. The genome contained 130 open reading frames (ORFs), 28 of which had assigned functions, and 18 were unique. One tRNA gene (tRNAAsn ) was discovered, and no virulence genes were identified. Its genome showed very low similarity with phage genomes deposited in public databases (75% nucleotide identity and 7% query coverage). The unique characteristics of phage SA2 led to the proposal of a new Siphoviridae genus named 'SA2likevirus'.

The Use of Laser Microirradiation to Investigate the Roles of Cohesins in DNA Repair.

In addition to their mitotic and transcriptional functions, cohesin plays critical roles in DNA damage response (DDR) and repair. Specifically, cohesin promotes homologous recombination (HR) repair of DNA double-strand breaks (DSBs), which is conserved from yeast to humans, and is a critical effector of ATM/ATR DDR kinase-mediated checkpoint control in mammalian cells. Optical laser microirradiation has been instrumental in revealing the damage site-specific functions of cohesin and, more recently, uncovering the unique role of cohesin-SA2, one of the two cohesin complexes uniquely present in higher eukaryotes, in DNA repair in human cells. In this review, we briefly describe what we know about cohesin function and regulation in response to DNA damage, and discuss the optimized laser microirradiation conditions used to analyze cohesin responses to DNA damage in vivo.

First report of Panton-Valentine leukocidin-positive methicillin-susceptible Staphylococcus aureus ST88 harbouring ΦSa2usa isolated from refractory breast abscesses in Japan.

A methicillin-susceptible Staphylococcus aureus with Panton-Valentine leukocidin (PVL) genes was isolated from refractory breast abscesses of 12-year-old girl in Japan, and classified into ST88, spa-t1245 and coa-IIIa. This strain harboured PVL phage ΦSa2usa, which is usually found in ST8 community-acquired methicillin-resistant S. aureus clone USA300.

C-terminus of Sororin interacts with SA2 and regulates sister chromatid cohesion.

Sororin is a conserved protein required for accurate separation of sister chromatids in each cell cycle. Sororin is recruited to chromatin during DNA replication, protects sister chromatid cohesion in S and G2 phase, and regulates the resolution of sister chromatid cohesion in mitosis. Sororin binds to cohesin complex, but how Sororin and cohesin subunits interact remains unclear. Here we report that the C-terminus of Sororin, especially the last 12 amino acid (aa) residues, is important for Sororin to bind cohesin core subunit SA2. Deletion of the last 12aa residues not only inhibits the interactions between Sororin and SA2 but also causes precocious chromosome separation. Our data suggest that the C-terminus of Sororin functions as an anchor binding to SA2, which facilitates other conserved motifs on Sororin to interact with other proteins to regulate sister chromatid cohesion and separation.

Experimental infection of dogs with Bartonella henselae and Bartonella vinsonii subsp. berkhoffii.

The lack of a suitable infection model remains an important obstacle for the pathophysiological understanding of Bartonella spp. The following pilot study was designed to determine whether cell culture-grown Bartonella henselae SA2 and Bartonella vinsonii subsp. berkhoffii genotype III would cause persistent bacteremia in dogs. Pre-inoculation screening established that two laboratory-raised Golden retrievers were naturally-infected with Bartonella koehlerae. Despite prior infection, one dog each was inoculated subcutaneously with 5 × 10(4)B. henselae (SA2 strain) or 3 × 10(4)B. vinsonii subsp. berkhoffii genotype III. Dogs were bled weekly for serological testing and culture using Bartonella alpha proteobacteria growth medium (BAPGM) diagnostic platform. Dog 1 seroconverted to B. henselae and Dog 2 seroconverted to B. vinsonii subsp. berkhoffii genotype III. Throughout the study period, Bartonella spp. DNA was neither amplified nor isolated in ante-mortem BAPGM enrichment blood cultures. B. henselae SA2 was isolated from a postmortem bone marrow from Dog 1 and B. koehlerae DNA was amplified from postmortem lung from Dog 2 following BAPGM enrichment culture. Limitations include lack of uninfected controls, a potentially suboptimal B. vinsonii subsp. berkhoffii inoculum and a relatively short duration of study. We conclude that following intradermal infection, sequestration of Bartonella spp. in tissues may limit diagnostic detection of these bacteria in dog blood samples.

Indicador de saúde entre idosos ativos e insuficientemente ativos residentes em ¨¢reas rurais / Health indicators among elderly active and insufficiently active residents in rural areas

Modelo do estudo: Estudo transversal. Objetivo do estudo: Analisar a associação entre indicadores de sa¨²de em idosos ativos e insuficientemente ativos. Metodologia: Estudo realizado em indiv¨ªduos com 60 anos ou mais, residentes na ¨¢rea rural de Jequi¨¦ ¨C BA, Brasil, cadastrados no programa Estrat¨¦gia Sa¨²de da Fam¨ªlia do distrito de Itajur¨². Foram analisadas as caracter¨ªsticas sociodemogr¨¢ficas, comportamentais, estado de sa¨²de, hist¨®rico de quedas, estado cognitivo e Índice de Massa Corporal (IMC).Para an¨¢lise dos dados, utilizaram-se procedimentos da estat¨ªstica descritiva, testes Qui-quadrado, teste¡°t¡± para amostras independentes e U de Mann-Whitney (n¨ªvel de significância utilizado pd¡±0,05). Resultados: Foram entrevistados 95 idosos, sendo 55 mulheres e 40 homens com idade entre 60 e 96 anos(73,5 ¡À 9,4). A preval¨ºncia de inatividade f¨ªsica foi de 40%, mostrando-se superior entre os idosos que relataram viver sozinho, que apresentaram d¨¦ficit cognitivo, que não sabiam ler e escrever, e com hist¨®rico de quedas no ¨²ltimo ano. Os idosos mais jovens e com menor IMC eram mais ativos, quando comparados com seus pares. Conclusão: Os idosos que não alcançaram as recomendações de n¨ªveis adequados de atividade f¨ªsica apresentaram condições de sa¨²de mais desfavor¨¢veis. Recomenda-se a implementação de pol¨ªticas p¨²blicas para promoção da atividade f¨ªsica no intuito de melhorar as condições de sa¨²de, em especial os idosos residentes de ¨¢reas rurais...

Study design: cross-sectional study. Study objective: To examine the association between health indicators in active and insufficiently active older adults. Methodology: The study included individuals 60 years or older residing in rural Jequie - BA who were registered in the Family Health Strategy program in the district Itajur¨². Sociodemographic data, behavioral characteristics, health status, history of falls, cognitive status and body mass index (BMI) were analyzed. For data analysis, we used the procedures of descriptive statistics, chi-square tests, ¡°t¡± test for independent samples and the Mann-Whitney test (significance level p ¡Ü0.05). Results: Ninety-five individuals were interviewed, including 55 women and 40 men between the ages of 60 and 96 years (73.5 ¡À 9.4). The prevalence of physical inactivity was 40%, being higher among the elderly who live alone reported that patients with cognitive impairment, who could not read and write with a history of falls in the last year. The older people with lower BMI were more active compared to their peers. Conclusion: The elderly who not reached the recommended adequate levels of physical activity had health conditions more unfavorable. We recommend the implementation of public policies to promote physical activity in order to improve health conditions, especially the elderly residents of rural areas...