Frequent down-regulation of ABC transporter genes in prostate cancer

Background: ATP-binding cassette (ABC) transporters are transmembrane proteins responsible for the efflux of a wide variety of substrates, including steroid metabolites, through the cellular membranes. For better characterization of the role of ABC transporters in prostate cancer (PCa) development, the profile of ABC transporter gene expression was analyzed in PCa and noncancerous prostate tissues (NPT). Methods: TaqMan Low Density Array (TLDA) human ABC transporter plates were used for the gene expression profiling in 10 PCa and 6 NPT specimens. ABCB1 transcript level was evaluated in a larger set of PCa cases (N = 78) and NPT (N = 15) by real-time PCR, the same PCa cases were assessed for the gene promoter hypermethylation by methylation-specific PCR. Results: Expression of eight ABC transporter genes (ABCA8, ABCB1, ABCC6, ABCC9, ABCC10, ABCD2, ABCG2, and ABCG4) was significantly down-regulated in PCa as compared to NPT, and only two genes (ABCC4 and ABCG1) were up-regulated. Down-regulation of ABC transporter genes was prevalent in the TMPRSS2-ERG-negative cases. A detailed analysis of ABCB1 expression confirmed TLDA results: a reduced level of the transcript was identified in PCa in comparison to NPT (p = 0.048). Moreover, the TMPRSS2-ERG-negative PCa cases showed significantly lower expression of ABCB1 in comparison to NPT (p = 0.003) or the fusion-positive tumors (p = 0.002). Promoter methylation of ABCB1 predominantly occurred in PCa and was rarely detected in NPT (p < 0.001). Conclusions: The study suggests frequent down-regulation of the ABC transporter genes in PCa, especially in the TMPRSS2-ERG-negative tumors

The Effects of Mindful Meditation on Body Image in Female Collegiate Athletes During the Late Luteal and Early Follicular Phases of the Menstrual Cycle

Body image has been studied in previous research but there is a gap in research with the consideration of body image changes during the phases of the menstrual cycle. The purpose of this study was to examine the effects of a mindful meditation intervention on body image during the late luteal and early follicular phases of the menstrual cycle in collegiate female athletes. Eight female collegiate athletes (age: 20.5 ± 1.8 years, height: 1.7 ± 0.1 m, weight: 62.4 ± 8.9 kg, body fat: 26.4 ± 6.9%) participated in both the control and mindful meditation intervention with each taking three weeks to complete. Body image was measured by using 3 surveys, the Self-Compassion Scale, Rosenberg Self-Esteem Scale and the Body Appreciation Scale to compare the control to the intervention. These surveys were taken prior to each of the three weeks and at the end of the three weeks. Participants made no change to their lifestyle during the control period. During the mindful meditation intervention, participants completed the intervention with each session lasting approximately 20 minutes for five days a week for the three weeks of the intervention protocol. Each week of the control was compared to the corresponding week during the intervention for all of the surveys. There were no significant differences found in any of the surveys between the control and the intervention. Although no significant difference found, there was a trend of body image improving with the addition of mindful meditation. The results of this study along with future research in body image may help to clarify the influence of the menstrual cycle on the body image of females

Effect of Single Vs Accumulated Bouts of Exercise on Body Composition, Fitness, and Resting Metabolic Rate

PURPOSE: To examine how the effects of accumulated exercise compare to continuous exercise on body composition, VO2max, and metabolic rate. METHODS: Mildly active males (n = 4) and females (n = 5) were randomly selected to perform either 1) continuous exercise consisting of one 30-minute bout of Tabata (1-bout), 2) accumulated exercise consisting of two 15-minute bouts of Tabata (2-bout), or 3) no exercise (control). Both exercise groups performed Tabata three times per week for 4 weeks. Each group had three participants (2 males and 1 female) with the exception of the 2-bout group which had 3 female participants. Changes in body composition (i.e., body mass (kg) and percent body fat (BF%)) were assessed using Dual-Energy X-Ray Absorptiometry (DXA) measurements taken at baseline and after 4 weeks of training. VO2max (ml/kg/min) and resting metabolic rate (kcal/day) were assessed at baseline and after 4 weeks of training using open spirometry on a ParvoMedics True Max 2400 Oxygen Uptake system. For the female participants, both the baseline and 4-week measurements were assessed in the mid-follicular phase of the menstrual cycle. Participants maintained their normal diet throughout the study. Participants were provided with Fitbit watches to keep track of their activity (avg. heart rate & steps per day) during the study. Significant differences (pRESULTS: No significant changes in body mass were detected within the groups (p \u3e .05) or between the groups (p =.72). No significant changes in fat mass were detected within the groups (p \u3e .05) or between the groups (p =.22). With that being said, there was a trend towards a decrease in fat mass within the 2-bout group (1.2 ± .45 kg; p=.061; ES= -.22). No significant changes in BF% (p\u3e.05) were detected within the control or 1-bout group. There was a significant reduction in BF% within the 2-bout group (1.43 ± 0.38%; p=.03; ES= -.63) and there was a significant difference in the change in BF% between the 3 groups (p=.048; ES= -1.2 and -1.8). No significant changes in muscle mass muscle (kg) were detected within the groups (p\u3e.05) or between the groups (p = .17). With that being said, there was a trend towards an increase in muscle mass within the 2-bout group (.77 ± .27kg; p=.059; ES= -.14). No significant changes (p\u3e.05) in resting metabolic rate or VO2max were reported within the groups or between the groups. CONCLUSION: Four weeks of accumulated exercise reduced BF% when compared to no exercise and continuous exercise. Despite the absence of improvement in VO2max and metabolic rate, accumulated exercise throughout the day may still be a valuable exercise mode as it could help people with busy schedules achieve minimum exercise recommendations. Future studies should include 1) a larger sample size, 2) a greater volume of exercise, or 3) a longer period of observation. These modifications may lead to a more valid evaluation of how metabolic health and fitness are influenced by accumulated and continuous exercise

The Conserved Coronavirus Macrodomain Promotes Virulence and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome Coronavirus Infection.

ADP-ribosylation is a common posttranslational modification that may have antiviral properties and impact innate immunity. To regulate this activity, macrodomain proteins enzymatically remove covalently attached ADP-ribose from protein targets. All members of the Coronavirinae, a subfamily of positive-sense RNA viruses, contain a highly conserved macrodomain within nonstructural protein 3 (nsp3). However, its function or targets during infection remain unknown. We identified several macrodomain mutations that greatly reduced nsp3&apos;s de-ADP-ribosylation activity in vitro Next, we created recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) strains with these mutations. These mutations led to virus attenuation and a modest reduction of viral loads in infected mice, despite normal replication in cell culture. Further, macrodomain mutant virus elicited an early, enhanced interferon (IFN), interferon-stimulated gene (ISG), and proinflammatory cytokine response in mice and in a human bronchial epithelial cell line. Using a coinfection assay, we found that inclusion of mutant virus in the inoculum protected mice from an otherwise lethal SARS-CoV infection without reducing virus loads, indicating that the changes in innate immune response were physiologically significant. In conclusion, we have established a novel function for the SARS-CoV macrodomain that implicates ADP-ribose in the regulation of the innate immune response and helps to demonstrate why this domain is conserved in CoVs. IMPORTANCE: The macrodomain is a ubiquitous structural domain that removes ADP-ribose from proteins, reversing the activity of ADP-ribosyltransferases. All coronaviruses contain a macrodomain, suggesting that ADP-ribosylation impacts coronavirus infection. However, its function during infection remains unknown. Here, we found that the macrodomain is an important virulence factor for a highly pathogenic human CoV, SARS-CoV. Viruses with macrodomain mutations that abrogate its ability to remove ADP-ribose from protein were unable to cause lethal disease in mice. Importantly, the SARS-CoV macrodomain suppressed the innate immune response during infection. Our data suggest that an early innate immune response can protect mice from lethal disease. Understanding the mechanism used by this enzyme to promote disease will open up novel avenues for coronavirus therapies and give further insight into the role of macrodomains in viral pathogenesis

Treatment of recurrent stress urinary incontinence in women: comparison of treatment results for different surgical techniques

INTRODUCTION: There is still no consensus on which surgical technique is the most effective for female recurrent stress urinary incontinence after the initial surgery. AIM: To compare the long-term treatment outcomes of Burch colposuspension operation, transobturator tape implantation (TOT) and tension-free vaginal tape (TVT) procedures performed for female recurrent stress urinary incontinence after the initial surgery. MATERIAL AND METHODS: A retrospective study was performed on 45 women operated on for recurrent stress urinary incontinence after the initial surgery. Depending on the surgical approach, the patients were divided into three groups: group I (n = 19) – Burch colposuspension operation, group II (n = 16) – TOT, and group III (n = 10) – TVT operation was performed. The treatment results were assessed using the UDI-6 (Urogenital Distress Inventory) and IIQ-7 (Incontinence Impact Questionnaire) short form questionnaires. We included one additional question: Is the patient satisfied with the treatment outcome? We classified the urinary continence results after surgery as good when patients were cured or improved, and as bad when the treatment failed. RESULTS: Good urinary continence results were observed in 84.2% of patients in group I, 93.8% of patients in group II, and 90% of patients in group III. 68.4% of patients in group I, 81.3% of patients in group II, and 90% of patients in group III were satisfied with the treatment outcomes. CONCLUSIONS: Burch colposuspension operation, TOT and TVT procedures performed for the female recurrent stress urinary incontinence treatment are effective and show similar good urinary continence results and similar number of patients satisfied with the treatment outcomes

The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism

The ubiquitin-like, containing PHD and RING finger domains protein 1 (UHRF1) is essential for maintenance DNA methylationby DNA methyltransferase 1 (DNMT1). UHRF1 has been shown to recruit DNMT1 to replicated DNA by the ability of its SET andRING-associated (SRA) domain to bind to hemimethylated DNA. Here, we demonstrate that UHRF1 also increases the activity ofDNMT1 by almost 5-fold. This stimulation is mediated by a direct interaction of both proteins through the SRA domain of UHRF1and the replication focus targeting sequence domain of DNMT1, and it does not require DNA binding by the SRA domain. Disruptionof the interaction between DNMT1 and UHRF1 by replacement of key residues in the replication focus targeting sequence domainled to a strong reduction of DNMT1 stimulation. Additionally, the interaction with UHRF1 increased the specificity of DNMT1for methylation of hemimethylated CpG sites. These findings show that apart from the targeting of DNMT1 to the replicatedDNA UHRF1 increases the activity and specificity of DNMT1, thus exerting a multifaceted influence on the maintenance of DNAmethylatio

Molecular basis for DarT ADP-ribosylation of a DNA base

ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2–4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6–9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT–DarG toxin–antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP–HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thyminelinked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT–DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.Peer reviewedFinal Accepted Versio

Structural basis of NINJ1-mediated plasma membrane rupture in cell death

Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event; 1-7; . Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-1; 8; (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death

Toxoplasma gondii: II. Tachyzoite antigenic characterization of eigth strains

Oito amostras de T. gondii - LIV IV, LIV V e S 11 isoladas de suínos, RH e VPS de seres humanos, AS 28 de camundongo, HV III de cão e CN de gato - foram analisadas com o objetivo de verificar a existência de possíveis diferenças na resposta imune quando inoculadas em coelhos. Através da técnica de ELISA, não foram constatadas diferenças entre as oito amostras estudadas. Todas as amostras reagiram de forma semelhante com soros homólogos e heterólogos. A suspensão antigênica, constituída de extrato celular total, mostrou-se eficiente no ELISA teste indireto, já que os soros positivos reagiram fortemente e os soros negativos não apresentaram reação contra os antígenos testados. A análise das amostras, pela técnica de Western blot, revelou que os isolados de T. gondii compartilham vários antígenos com algumas variações. Dentre as bandas reconhecidas no Western blot, três foram comuns a todas as amostras: a p33 (33-37 kDa), p54 (52-55 kDa) e a p66 (66 kDa). A amostra HV III, isolada recentemente de um cão, foi a que mais diferiu no perfil antigênico. Essa amostra não apresentou três antígenos (50, 70 e 75 kDa) presentes nas demais amostras. Apenas dois antígenos, um de 62 kDa da CN e outro de 67 kDa da LIV IV, foram amostra-específicos.Eight Toxoplasma gondii strains were analyzed using ELISA and Western blot techniques, in order to demonstrate possible immunological differences. The analyzed strains were: LIV IV, LIV V and S 11 isolated from swine, RH and VPS from a human being, AS 28 from a wild mouse, HV III from a dog and CN from a cat. With the ELISA assay the eight strains showed similar reactivity with homologous and heterologous sera. The antigenic suspension, consisting of total cellular extract of tachyzoites, was effective in the indirect ELISA assay, with the positive sera reacting strongly and the negative not reacting with the antigens. The Western blot analysis showed that the T. gondii strains have similar antigenic profiles with a few variations. Three bands were observed in all strains: one of about 33 kDa (p33), another of 54 kDa (p54) and a third one of 66 kDa (p66). The HV III strain, isolated from a dog, did not show three antigens (50, 70 and 75 kDa) that were present in the others. However, this difference was not detected by the ELISA assay. Only two antigens (62 kDa of the CN and 67 kDa of the LIV IV) were strain-specific antigens