K-Ras is the most frequently mutated oncoprotein in human cancers, and G12D is its most prevalent mutation. To understand how G12D mutation impacts K-Ras function, we need to understand how it alters the regulation of its dynamics. Here, we present local changes in K-Ras structure, conformation and dynamics upon G12D mutation, from long-timescale Molecular Dynamics simulations of active (GTP-bound) and inactive (GDP-bound) forms of wild-type and mutant K-Ras, with an integrated investigation of atomistic-level changes, local conformational shifts and correlated residue motions. Our results reveal that the local changes in K-Ras are specific to bound nucleotide (GTP or GDP), and we provide a structural basis for this. Specifically, we show that G12D mutation causes a shift in the population of local conformational states of K-Ras, especially in Switch-II (SII) and α3-helix regions, in favor of a conformation that is associated with a catalytically impaired state through structural changes; it also causes SII motions to anti-correlate with other regions. This detailed picture of G12D mutation effects on the local dynamic characteristics of both active and inactive protein helps enhance our understanding of local K-Ras dynamics, and can inform studies on the development of direct inhibitors towards the treatment of K-RasG12D-driven cancers.

Defining oocyte in vitro maturation (IVM) conditions allows for improved reproducibility and efficiency of bovine embryo production. IVM conditions for bovine oocytes have been extensively studied, but beneficial effects of individual supplements remain controversial. This study compared methods of cumulus oocyte complex (COC) isolation, and culture medium requirements, for IVM in order to define optimal conditions. Antral follicles in ovaries were sliced or aspirated to isolate COCs. Brilliant cresyl blue staining of COCs was used to determine the most effective collection technique and the effect of hormones and groups of amino acids in the culture medium was investigated. Our results showed COCs isolated through aspiration had greater meiotic competency to reach MII. Oocyte maturation was achieved with the addition of 1 µg/mL FSH, while estrogen and human chorionic gonadotrophin did not increase the number of MII oocytes. We also provide novel data, that supplementation of a simple inorganic salt solution with L-proline, L-glutamine and essential amino acids in combination, but not individually, resulted in nuclear maturation comparable to TCM199, a more complex medium containing all 20 common amino acids, vitamins, inorganic salts and FBS. Replacement of FBS with BSA in this simplified medium creates a defined medium which provides conditions for IVM that enable reproducible maturation rates.

Mechanical stress on eukaryotic nucleus has been implicated in a diverse range of diseases including muscular dystrophy and cancer metastasis. Today, there are very few non-perturbative methods to quantify nuclear mechanical properties. Interferometric microscopy, also known as quantitative phase microscopy (QPM), is a powerful tool for studying red blood cell biomechanics. The existing QPM tools, however, have not been utilized to study biomechanics of complex eukaryotic cells either due to lack of depth sectioning, limited phase measurement sensitivity, or both. Here, we present depth-resolved confocal reflectance interferometric microscopy as the next generation QPM to study nuclear and plasma membrane biomechanics. The proposed system features multiple confocal scanning foci, affording 1.5 micron depth-resolution and millisecond frame rate. Furthermore, a near common-path interferometer enables quantifying nanometer-scale membrane fluctuations with better than 200 picometers sensitivity. Our results present accurate quantification of nucleic envelope and plasma membrane fluctuations in embryonic stem cells.

Acute liver failure (ALF) is characterized by loss of liver function in response to sustained augmentation of the acute-phase response (APR) in the liver, which can progress even to death. Although the inflammatory interleukin-6 (IL-6)-axis is a crucial factor that drives the hepatic APR by releasing diverse acute-phase proteins (APPs), therapeutic strategies to block the IL-6-STAT3-mediated APR are not well developed. Here, we show that the nuclear receptor retinoic acid-related orphan receptor α (RORα) limits APR-mediated liver injury by inhibiting the hepatic IL-6-STAT3 signaling pathway. Administration of JC1-40, an RORα activator, diminished diethylnitrosamine-induced acute liver injury and repressed transcriptional expression of APPs such as CXCL1 and LCN2 in mice. IL-6-mediated activation of STAT3 was repressed after RORα activation by either adenoviral infusion of RORα or JC1-40 treatment in primary hepatocytes. Activation of RORα decreased transcriptional expression of IL-6 receptor α, an upstream activator of STAT3, both in vitro and in vivo. This may be one mechanism underlying the RORα-mediated inhibition of STAT3. Taken together, our results suggest that RORα is a regulator of the hepatic IL-6-STAT3 signaling pathway and may be a new therapeutic target for treating APR-associated inflammatory ALF.

Biological entities are key elements of biomedical research. Their definition and their relationships are important in areas such as phylogenetic reconstruction, developmental processes or tumor evolution. Hypotheses about relationships like phenotype order are often postulated based on prior knowledge or belief. Evidence on a molecular level is typically unknown and whether total orders are reflected in the molecular measurements is unclear or not assessed. In this work we propose a method that allows a fast and exhaustive screening for total orders in large datasets. We utilise ordinal classifier cascades to identify discriminable molecular representations of the phenotypes. These classifiers are constrained by an order hypothesis and are highly sensitive to incorrect assumptions. Two new error bounds, which are introduced and theoretically proven, lead to a substantial speed-up and allow the application to large collections of many phenotypes. In our experiments we show that by exhaustively evaluating all possible candidate orders, we are able to identify phenotype orders that best coincide with the high-dimensional molecular profiles.

Retinal photoreceptor outer segments (POS) are renewed daily through phagocytosis by the adjacent retinal pigment epithelial (RPE) monolayer. Phagocytosis is mainly driven by the RPE circadian clock but the underlying molecular mechanisms remain elusive. Using ARPE-19 (human RPE cell-line) dispersed and monolayer cell cultures, we investigated the influence of cellular organization on the RPE clock and phagocytosis genes. PCR analysis revealed rhythmic expression of clock and phagocytosis genes in all ARPE-19 cultures. Monolayers had a tendency for higher amplitudes of clock gene oscillations. In all conditions ARNTL, CRY1, PER1-2, REV-ERBα, ITGB5, LAMP1 and PROS1 were rhythmically expressed with REV-ERBα being among the clock genes whose expression showed most robust rhythms in ARPE-19 cells. Using RPE-choroid explant preparations of the mPer2Luc knock-in mice we found that Rev-Erbα deficiency induced significantly longer periods and earlier phases of PER2-bioluminescence oscillations. Furthermore, early phagocytosis factors β5-Integrin and FAK and the lysosomal marker LAMP1 protein levels are rhythmic. Finally, POS incubation affects clock and clock-controlled phagocytosis gene expression in RPE monolayers in a time-dependent manner suggesting that POS can reset the RPE clock. These results shed some light on the complex interplay between POS, the RPE clock and clock-controlled phagocytosis machinery which is modulated by Rev-Erbα.

The increase in antibiotic resistant bacteria has raised global concern regarding the future effectiveness of antibiotics. Human activities that influence microbial communities and environmental resistomes can generate additional risks to human health. In this work, we characterized aquatic microbial communities and their resistomes in samples collected at three sites along the Bogotá River and from wastewaters at three city hospitals, and investigated community profiles and antibiotic resistance genes (ARGs) as a function of anthropogenic contamination. The presence of antibiotics and other commonly used drugs increased in locations highly impacted by human activities, while the diverse microbial communities varied among sites and sampling times, separating upstream river samples from more contaminated hospital and river samples. Clinically relevant antibiotic resistant pathogens and ARGs were more abundant in contaminated water samples. Tracking of resistant determinants to upstream river waters and city sources suggested that human activities foster the spread of ARGs, some of which were co-localized with mobile genetic elements in assembled metagenomic contigs. Human contamination of this water ecosystem changed both community structure and environmental resistomes that can pose a risk to human health.

Microbial electrochemical technology is emerging as an alternative way of treating waste and converting this directly to electricity. Intensive research on these systems is ongoing but it currently lacks the evaluation of possible environmental transmission of enteric viruses originating from the waste stream. In this study, for the first time we investigated this aspect by assessing the removal efficiency of hepatitis B core and surface antigens in cascades of continuous flow microbial fuel cells. The log-reduction (LR) of surface antigen (HBsAg) reached a maximum value of 1.86 ± 0.20 (98.6% reduction), which was similar to the open circuit control and degraded regardless of the recorded current. Core antigen (HBcAg) was much more resistant to treatment and the maximal LR was equal to 0.229 ± 0.028 (41.0% reduction). The highest LR rate observed for HBsAg was 4.66 ± 0.19 h-1 and for HBcAg 0.10 ± 0.01 h-1. Regression analysis revealed correlation between hydraulic retention time, power and redox potential on inactivation efficiency, also indicating electroactive behaviour of biofilm in open circuit control through the snorkel-effect. The results indicate that microbial electrochemical technologies may be successfully applied to reduce the risk of environmental transmission of hepatitis B virus but also open up the possibility of testing other viruses for wider implementation.

Our purpouse was to identify quantitatively and qualitatively the subgingival flora in different gestational trimesters, compared to non-pregnant women; evaluating the correlations between epidemiological characteristics, clinical diagnosis, microbiological findings and levels of estradiol and progesterone. 52 pregnant women divided into 3 groups, according to the gestational trimester and 15 non-pregnant patients, without hormonal contraceptives, were evaluated. Plaque index (PI), gingival index (GI), probing depth (PD) and clinical attachment level (CAL) were evaluated. Subgingival biofilm samples were processed by the qPCR technique and the serum levels of estradiol and progesterone quantified by chemiluminescence. Clinical diagnosis during gestation was correlated with the total bacterial count. A higher prevalence of Tannerella forsythia (Tf) was identified in first trimester of pregnancy and this periodontopathogen was correlated with the diagnosis of gingivitis among pregnant women. Porphyromonas gingivalis (Pg) showed a positive correlation with progesterone levels in the first trimester. High prevalence of periodontopathogens was noticed in this population. Clinical diagnosis in gestation was positively correlated with the total amount of bacteria, without influence of the hormonal levels or the epidemiological factors evaluated. The presence of Tf favored occurrence of gingivitis during pregnancy and the progesterone levels in the first trimester enhanced the growth of Pg.

Parity is associated with the incidence of problems in pregnancy, delivery and the puerperium. The influence of parity in the postpartum period has been poorly studied and the results are incongruous. The objective of this study was to identify the association between parity and the existence of distinct discomfort and problems during the postpartum period. Cross-sectional study with puerperal women in Spain. Data was collected on demographic and obstetric variables and maternal manifestations of discomfort and problems during the postpartum period. An ad hoc online questionnaire was used. Crude odds ratios (ORs) and adjusted odds ratios (aORs) with 95% confidence intervals (CIs) were estimated by conditional logistic regression. 1503 primiparous and 1487 multiparous participated in the study. 53.4% (803) of the primiparous women affirmed to have feelings of sadness, as opposed to 36.2% (539) of multiparous women (aOR: 1.60; 95% CI: 1.35-1.89). 48.3% (726) of primiparous had lactation problems vs 24.7% (367) of multiparous (aOR: 2.46; 95% CI: 2.05-2.94). 37.2% (559) of primiparous reported anxiety, while the percentage in multiparous was 25.7% (382) (aOR: 1.34; 95% CI: 1.12-1.61). 22.2% (333) of primiparous had depressive symptoms, and 11.6% (172) of multiparous (aOR: 1.65; CI 95%: 1.31-2.06). Faecal incontinence was more present in primiparous than in multiparous, 6.5% (97) and 3.3% (49) respectively (aOR: 1.60; 95% CI: 1.07-2.38). Parity is associated with the presence of certain problems in the postpartum period. Thus, primiparous are more likely to have lactation problems, depressive symptoms, anxiety, sadness, and faecal incontinence.

Proteomic biomarker search requires the greatest analytical reproducibility and detailed information on altered proteoforms. Our protein pre-fractionation applies orthogonal native chromatography and conserves important features of protein variants such as native molecular weight, charge and major glycans. Moreover, we maximized reproducibility of sample pre-fractionation and preparation before mass spectrometry by parallelization and automation. In blood plasma and cerebrospinal fluid (CSF), most proteins, including candidate biomarkers, distribute into a multitude of chromatographic clusters. Plasma albumin, for example, divides into 15-17 clusters. As an example of our technique, we analyzed these albumin clusters from healthy volunteers and from dogs and identified cluster-typical modification patterns. Renal disease further modifies these patterns. In human CSF, we found only a subset of proteoforms with fewer modifications than in plasma. We infer from this example that our method can be used to identify and characterize distinct proteoforms and, optionally, enrich them, thereby yielding the characteristics of proteoform-selective biomarkers.

Neuroblastoma (NB) is a pediatric tumor of the peripheral nervous system. Treatment of the disease represents an unsolved clinical problem, as survival of patients with aggressive form of NB remains below 50%. Despite recent identification of numerous potential therapeutic targets, clinical trials validating them are challenging due to the rarity of the disease and its high patient-to-patient heterogeneity. Hence, there is a need for the accurate preclinical models that would allow testing novel therapeutic approaches and prioritizing the clinical studies, preferentially in personalized way. Here, we propose using conditional reprogramming (CR) technology for rapid development of primary NB cell cultures that could become a new model for such tests. This newly established method allowed for indefinite propagation of normal and tumor cells of epithelial origin in an undifferentiated state by their culture in the presence of Rho-associated kinase (ROCK) inhibitor, Y-27632, and irradiated mouse feeder cells. Using a modification of this approach, we isolated cell lines from tumors arising in the TH-MYCN murine transgenic model of NB (CR-NB). The cells were positive for neuronal markers, including Phox2B and peripherin and consisted of two distinct populations: mesenchymal and adrenergic expressing corresponding markers of their specific lineage. This heterogeneity of the CR-NB cells mimicked the different tumor cell phenotypes in TH-MYCN tumor tissues. The CR-NB cells preserved anchorage-independent growth capability and were successfully passaged, frozen and biobanked. Further studies are required to determine the utility of this method for isolation of human NB cultures, which can become a novel model for basic, translational, and clinical research, including individualized drug testing.

Autophagy is an evolutionarily conserved process that captures aberrant intracellular proteins and/or damaged organelles for delivery to lysosomes, with implications for cellular and organismal homeostasis, aging and diverse pathologies, including cancer. During cancer development, autophagy may play both tumour-supporting and tumour-suppressing roles. Any relationships of autophagy to the established oncogene-induced replication stress (RS) and the ensuing DNA damage response (DDR)-mediated anti-cancer barrier in early tumorigenesis remain to be elucidated. Here, assessing potential links between autophagy, RS and DDR, we found that autophagy is enhanced in both early and advanced stages of human urinary bladder and prostate tumorigenesis. Furthermore, a high-content, single-cell-level microscopy analysis of human cellular models exposed to diverse genotoxic insults showed that autophagy is enhanced in cells that experienced robust DNA damage, independently of the cell-cycle position. Oncogene- and drug-induced RS triggered first DDR and later autophagy. Unexpectedly, genetic inactivation of autophagy resulted in RS, despite cellular retention of functional mitochondria and normal ROS levels. Moreover, recovery from experimentally induced RS required autophagy to support DNA synthesis. Consistently, RS due to the absence of autophagy could be partly alleviated by exogenous supply of deoxynucleosides. Our results highlight the importance of autophagy for DNA synthesis, suggesting that autophagy may support cancer progression, at least in part, by facilitating tumour cell survival and fitness under replication stress, a feature shared by most malignancies. These findings have implications for better understanding of the role of autophagy in tumorigenesis, as well as for attempts to manipulate autophagy as an anti-tumour therapeutic strategy.

Deregulated expression of circular RNAs (circRNAs) is associated with various human diseases, including many types of cancer. Despite their growing links to cancer, there has been limited characterization of circRNAs in metastatic castration-resistant prostate cancer, the major cause of prostate cancer mortality. Here, through the analysis of an exome-capture RNA-seq dataset from 47 metastatic castration-resistant prostate cancer samples and ribodepletion and RNase R RNA-sequencing of patient-derived xenografts (PDXs) and cell models, we identified 13 circRNAs generated from the key prostate cancer driver gene-androgen receptor (AR). We validated and characterized the top four most abundant, clinically relevant AR circRNAs. Expression of these AR circRNAs was upregulated during castration-resistant progression of PDXs. The upregulation was not due to global increase of circRNA formation in these tumors. Instead, the levels of AR circRNAs correlated strongly with that of the linear AR transcripts (both AR and AR variants) in clinical samples and PDXs, indicating a transcriptional mechanism of regulation. In cultured cells, androgen suppressed the expression of these AR circRNAs and the linear AR transcripts, and the suppression was attenuated by an antiandrogen. Using nuclear/cytoplasmic fractionation and RNA in-situ hybridization assays, we demonstrated predominant cytoplasmic localization of these AR circRNAs, indicating likely cytoplasmic functions. Overall, this is the first comprehensive characterization of circRNAs arising from the AR gene. With greater resistance to exoribonuclease compared to the linear AR transcripts and detectability of AR circRNAs in patient plasma, these AR circRNAs may serve as surrogate circulating markers for AR/AR-variant expression and castration-resistant prostate cancer progression.

In the English pilot of primary cervical screening with high-risk human papillomavirus (HR-HPV), we exploited natural viral clearance over 24 months to minimise unnecessary referral of HR-HPV+ women with negative cytology. Three laboratories were permitted to use 16/18 genotyping to select women for referral at 12-month recall. We estimated the clinical impact of this early genotyping referral.

Cervical cancer is the second most common malignant cancer in women worldwide. Evidence indicated that miR-664 was significantly downregulated in cervical cancer. However, the mechanisms by which miR-664 regulates the tumorigenesis of cervical cancer remain unclear. Thus, this study aimed to investigate the role of miR-664 in cervical cancer.

Background: South Africa has been experiencing a growing proportion of elderly population with rapid increases in the burden of non-communicable diseases (NCDs) characteristic of population aging. Rural areas in South Africa represent a far smaller fraction of the population, however, share a relatively higher burden of NCDs. In the current literature, there is limited evidence on rural studies in the context of chronic diseases and activities of daily living (ADLs) among the elderly population (60 years and above) in South Africa. Purpose: In this regard, we undertook the present study with the objective of examining the demographic, behavioral, and socioeconomic predictors of subjective health, depression, and quality of life among elderly men and women living in the rural areas (n=2,627). Methods: Data for this study were collected from the Health and Aging in Africa: A Longitudinal Study of an INDEPTH Community in South Africa (HAALSI). Main explanatory variables were self-reported NCDs and difficulties in ADLs. The predictors of subjective health, depression, and quality of life were assessed using multivariable regression methods. Results: We found that the proportion of participants who reported good health, not having depression, and good quality of life was respectively 44.7%, 81.3%, and 63%. Women in the oldest age group (80+ years) were significantly less likely to report good health (OR=0.577, 95% CI=0.420, 0.793) and quality of life (OR=0.709, 95% CI=0.539, 0.933) compared with those in the youngest group. Having more than one chronic condition and ADL difficulties significantly lowered the odds of good health, having no depression, and quality of life among men and women. Conclusion: The present findings suggest the involvement of sociodemographic factors in health and quality of life outcomes among elderly South Africans, and call for enhanced efforts to address these health limiting conditions such as ADLs and chronic multimorbidity.

Background: Candida albicans as an opportunistic fungus is one of the most important causes of late-onset morbidity and mortality in patients with major burns and severely impaired immune system. In recent years, the emergence of resistance to opportunistic fungi and toxicity of antimicrobial drugs make it necessary to develop new drugs. Methods: In the present study, we investigated anticandidal effects of indolicidin, as a representative of host defense peptide, conjugated with gold nanoparticles in fluconazole-resistant clinical isolates of C. albicans. After characterizing the conjugation of indolicidin using biophysical methodologies, the cytotoxicity and hemolytic activity of the nanocomplex were examined. In addition, the expression level of ERG11, responsible for antifungal resistance, and the immunomodulatory effect of peptide-nanomaterial conjugates were assessed. Results: Our data indicated that the nanocomplex was nontoxic for the fibroblast cells and erythrocytes. Treatment with the nanocomplex significantly reduced the expression levels of the ERG11 gene in fluconazole-resistant C. albicans isolates and the iNOS gene in macrophages. Conclusion: The study data provides a chance to develop innovative therapies for the treatment of C. albicans burn infections. However, further investigation is required to examine the efficiency of the nanocomplex.

We created and evaluated an enhanced topical delivery system featuring a combination of highly skin-permeable growth factors (GFs), quercetin (QCN), and oxygen; these synergistically accelerated re-epithelialization and granulation tissue formation of/in diabetic wounds by increasing the levels of GFs and antioxidants, and the oxygen partial pressure, at the wound site.

Glioma is one of the most aggressive primary brain tumors and is incurable. Surgical resection, radiation, and chemotherapies have been the standard treatments for brain tumors, however, they damage healthy tissue. Therefore, there is a need for safe anticancer drug delivery systems. This is particularly true for natural prodrugs such as thymoquinone (TQ), which has a high therapeutic potential for cancers but has poor water solubility and insufficient targeting capacity. We have tailored novel core-shell nanoformulations for TQ delivery against glioma cells using mesoporous silica nanoparticles (MSNs) as a carrier.