Analysis of Nedd8-associated polypeptides: a model for deciphering the pathway for ubiquitin-like modifications

Ubiquitin-like proteins modify target proteins, altering their activities or causing them to be slated for degradation. These modifications are used to efficiently regulate key events in the cell. To explore the set of proteins modified by a small ubiquitin-like protein, we have developed a proteomic approach. Affinity purification of an epitope-tagged Nedd8 allowed the identification of the majority of proteins known to be involved with the neddylation pathway. This purification not only isolated the known targets of neddylation but also the constellation of enzymes and complexes known to regulate neddylation and deneddylation, including the COP9 signalosome, Nub1, and enzymes in the neddylation cascade. This purification scheme can be applied to other small ubiquitin-like proteins, especially those with limited protein targets such as the SUMOs (1, 2, and 3), Isg15, or FAT10.     

Effect of pregnancy on joint contracture in the rat knee.

As there is evidence that ligamentous laxity is affected by the female hormones, we hypothesized that hormonal changes occurring during pregnancy could have a therapeutic role in preventing the development of a joint contracture. Knee joint contractures were created in pregnant and nonpregnant rats. After 2 wk of immobilization, the degree of contracture was measured with structural properties of the medial collateral and anterior cruciate ligaments and the pubic symphysis. Although not statistically significant, there was a general trend toward reduced contracture in pregnant compared with nonpregnant rats. Cutting the posterior capsule significantly decreased contracture for both the pregnant and nonpregnant groups, confirming the contribution of capsular structures to contracture. Ultimate loads of the medial collateral and anterior cruciate ligaments significantly decreased after immobilization compared with control, but there was no significant effect due to pregnancy. Stiffness and ultimate load of the pubic symphysis were not significantly different between pregnant and nonpregnant groups. The trend toward reduced contracture with pregnancy points toward a possible therapeutic role for female hormones in the prevention of postoperative and/or posttraumatic joint contracture.     

Proteomic‐based investigations on the mode of action of the marine anticancer compound rhizochalinin

Rhizochalinin (Rhiz) is a novel marine natural sphingolipid‐like compound, which shows promising in vitro and in vivo activity in human castration‐resistant prostate cancer. In the present study, a global proteome screening approach was applied to investigate molecular targets and biological processes affected by Rhiz in castration‐resistant prostate cancer. Bioinformatical analysis of the data predicted an antimigratory effect of Rhiz on cancer cells. Validation of proteins involved in the cancer‐associated processes, including cell migration and invasion, revealed downregulation of specific isoforms of stathmin and LASP1, as well as upregulation of Grp75, keratin 81, and precursor IL‐1β by Rhiz. Functional analyses confirmed an antimigratory effect of Rhiz in PC‐3 cells. Additionally, predicted ERK1/2 activation was confirmed by Western blotting analysis, and revealed prosurvival effects in Rhiz‐treated prostate cancer cells indicating a potential mechanism of resistance. A combination of Rhiz with MEK/ERK inhibitors PD98059 (non‐ATP competitive MEK1 inhibitor) and FR180204 (ATP‐competitive ERK1/2 inhibitor) resulted in synergistic effects. This work provides further insights into the molecular mechanisms underlying Rhiz bioactivity. Furthermore, our research is exemplary for the ability of proteomics to predict drug targets and mode of action of natural anticancer agents.     

Heavy metal contamination and health risk assessment in drinking water of Sistan and Baluchistan,Southeastern Iran

Access to safe and clean drinking water is an essential element of healthy life also known as the primary human needs. The present study was conducted to investigate heavy metal (HM) concentrations of drinking water. Excess health risk of HM (Cr, Pb, and Cd) intake is related to the drinking water consumption in local population. HMs concentrations were analyzed by using graphite furnace atomic absorption spectrometer and were compared with permissible limits regulated by country and World Health Organization (WHO). The hazard quotient (HQ) and Excess Lifetime Cancer Risk (ELCR) were determined to show the carcinogenic and non-carcinogenic effects of HMs, respectively. HQs were found in the order of Pb > Cd > Cr and subsequently HI index was also estimated for all HM in two age groups (children and adults). The comparisons indicate no possibility of non-carcinogenic effects to the local population. The values for ELCR were found in the order of Cr > Cd > Pb. The ELCR index was found above acceptable risk levels for chromium and cadmium in both children and adults groups. Furthermore, intermetal correlation results revealed that heavy metals have common sources resulting from geogenic and anthropogenic activities and these are major sources of water contamination in Sistan and Baluchestan province.     

Virtual screening of small molecules databases for discovery of novel PARP-1 inhibitors: combination of in silico and in vitro studies

Poly(ADP-ribose) polymerase-1 (PARP-1) enzyme has critical roles in DNA replication repair and recombination. Thus, PARP-1 inhibitors play an important role in the cancer therapy. In the current study, we have performed combination of in silico and in vitro studies in order to discover novel inhibitors against PARP-1 target. Structure-based virtual screening was carried out for an available small molecules database. A total of 257,951 ligands from Otava database were screened at the binding pocket of PARP-1 using high-throughput virtual screening techniques. Filtered structures based on predicted binding energy results were then used in more sophisticated molecular docking simulations (i.e. Glide/standard precision, Glide/XP, induced fit docking – IFD, and quantum mechanics polarized ligand docking – QPLD). Potential high binding affinity compounds that are predicted by molecular simulations were then tested by in vitro methods. Computationally proposed compounds as PARP-1 inhibitors (Otava Compound Codes: 7111620047 and 7119980926) were confirmed by in vitro studies. In vitro results showed that compounds 7111620047 and 7119980926 have IC₅₀ values of 0.56 and 63 μM against PARP-1 target, respectively. The molecular mechanism analysis, free energy perturbation calculations using long multiple molecular dynamics simulations for the discovered compounds which showed high binding affinity against PARP-1 enzyme, as well as structure-based pharmacophore development (E-pharmacophore) studies were also studied.     

Over-expression of tobacco <Emphasis Type="Italic">UBC1</Emphasis> encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>)

Ubiquitin (Ub)-conjugating enzyme (UBC, E2) receives Ub from Ub-activating enzyme (E1) and transfers it to target proteins, thereby playing a key role in Ub/26S proteasome-dependent proteolysis. UBC has been reported to be involved in tolerating abiotic stress in plants, including drought, salt, osmotic and water stresses. To isolate the genes involved in Cd tolerance, we transformed WT (wild-type) yeast Y800 with a tobacco cDNA expression library and isolated a tobacco cDNA, NtUBC1 (Ub-conjugating enzyme), that enhances cadmium tolerance. When NtUBC1 was over-expressed in tobacco, cadmium tolerance was enhanced, but the Cd level was decreased. Interestingly, 20S proteasome activity was increased and ubiquitinated protein levels were diminished in response to cadmium in NtUBC1 tobacco. By contrast, proteasome activity was decreased and ubiquitinated protein levels were slightly enhanced by Cd treatment in control tobacco, which is sensitive to Cd. Moreover, the oxidative stress level was induced to a lesser extent by Cd in NtUBC1 tobacco compared with control plants, which is ascribed to the higher activity of antioxidant enzymes in NtUBC1 tobacco. In addition, NtUBC1 tobacco displayed a reduced accumulation of Cd compared with the control, likely due to the higher expression of CAX3 (Ca²⁺/H⁺ exchanger) and the lower expression of IRT1 (iron-responsive transporter 1) and HMA-A and -B (heavy metal ATPase). In contrast, atubc1 and atubc1atubc2 Arabidopsis exhibited lower Cd tolerance and proteasome activity than WT. In conclusion, NtUBC1 expression promotes cadmium tolerance likely by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or the Ub-independent 20S proteasome and by diminishing oxidative stress through the activation of antioxidant enzymes and decreasing Cd accumulation due to higher CAX3 and lower IRT1 and HMA-A/B expression in response to 50 µM Cd challenge for 3 weeks.     

Has increased provision of treatment reduced the prevalence of common mental disorders? Review of the evidence from four countries

Many people identified as having common mental disorders in community surveys do not receive treatment. Modelling has suggested that closing this “treatment gap” should reduce the population prevalence of those disorders. To evaluate the effects of reducing the treatment gap in industrialized countries, data from 1990 to 2015 were reviewed from four English‐speaking countries: Australia, Canada, England and the US. These data show that the prevalence of mood and anxiety disorders and symptoms has not decreased, despite substantial increases in the provision of treatment, particularly antidepressants. Several hypotheses for this lack of improvement were considered. There was no support for the hypothesis that reductions in prevalence due to treatment have been masked by increases in risk factors. However, there was little evidence relevant to the hypothesis that improvements have been masked by increased reporting of symptoms because of greater public awareness of common mental disorders or willingness to disclose. A more strongly supported hypothesis for the lack of improvement is that much of the treatment provided does not meet the minimal standards of clinical practice guidelines and is not targeted optimally to those in greatest need. Lack of attention to prevention of common mental disorders may also be a factor. Reducing the prevalence of common mental disorders remains an unsolved challenge for health systems globally, which may require greater attention to the “quality gap” and “prevention gap”. There is also a need for nations to monitor outcomes by using standardized measures of service provision and mental disorders over time.     

MAP3K4 Controls the Chromatin Modifier HDAC6 during Trophoblast Stem Cell Epithelial-to-Mesenchymal Transition

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Association of Hypertension,Body Mass Index,and Waist Circumference with Fluoride Intake; Water Drinking in Residents of Fluoride Endemic Areas,Iran

Hypertension is becoming a global epidemic for both rural and urban populations; it is a major public health challenge in Iran. Fluoride can be a risk factor for hypertension. Cross-sectional analysis was conducted in two study areas to assess the relation of fluoride with blood pressure prevalence, BMI, waist circumference, and waist-to-hip ratio (WHR) among different age groups in both sexes. The mean value of fluoride concentration in the drinking water from the four study villages varied from 0.68 to 10.30 mg/L. The overall prevalence of HTN and prehypertension in all subjects was 40.7%. The prevalence of isolated systolic hypertension, isolated diastolic hypertension, systolic-diastolic hypertension, and prehypertension in the total sample population was 1.15, 0.28, 9.53, and 29.76%, respectively. The odd ratio of hypertension in residents who drank water with high fluoride levels was higher than that in residents who drank water with lower level of fluoride (OR 2.3, 1.03–5.14). Logistic regression results showed that age (P?<?0.001), sex (P?=?0.018), BMI (P?=?0.015), and the fluoride level in drinking water (P?=?0.041) had a significant relationship with increased blood pressure. There were no statistically significant correlations between fluoride and BMI, hip circumference, and waist to hip ratio (WHR). The findings of this study are important for health care personnel and policymakers.     

An in vitro evaluation of browser and grazer fermentation efficiency and microbiota using European moose spring and summer foods

Evolutionary morphological and physiological differences between browsers and grazers contribute to species‐specific digestion efficiency of food resources. Rumen microbial community structure of browsers is supposedly adapted to characteristic nutrient composition of the diet source. If this assumption is correct, domesticated ruminants, or grazers, are poor model animals for assessing the nutritional value of food consumed by browsing game species. In this study, typical spring and summer foods of the European moose (Alces alces) were combined with rumen fluid collected from both dairy cows (Bos taurus) and from moose, with the aim of comparing fermentation efficiency and microbial community composition. The nutritional value of the food resources was characterized by chemical analysis and advanced in vitro measurements. The study also addressed whether or not feed evaluation based on in vitro techniques with cattle rumen fluid as inoculum could be a practical alternative when evaluating the nutritional value of plants consumed by wild browsers. Our results suggest that the fermentation characteristics of moose spring and summer food are partly host‐specific and related to the contribution of the bacterial phyla Firmicutes and Bacteriodetes to the rumen microbial community. Host‐specific adaptations of the ruminal microbial community structure could be explained from the evolutionary adaptations related to feeding habitats and morphophysiological differences between browsers and grazers. However, the observed overall differences in microbial community structure could not be related to ruminal digestion parameters measured in vitro. The in vitro evaluation of digestion efficiency reveals that equal amounts of methane were produced across all feed samples regardless of whether the ruminal fluid was from moose or dairy cow. The results of this study suggested that the nutritional value of browsers' spring and summer food can be predicted using rumen fluid from domesticated grazers as inoculum in in vitro assessments of extent of digestion when excluding samples of the white water lily root, but not of fermentation characteristics as indicated by the proportions of individual fermentation fatty acids to the total of volatile fatty acids.