Ubiquitin-assisted phase separation of dishevelled-2 promotes Wnt signalling.

Dishvelled-2 (Dvl2) is an essential component of Wnt pathway, which controls several cell fate decisions during development, such as proliferation, survival and differentiation. Dvl2 forms higher-order protein assemblies in the cell that are critical for relaying the signal from upstream Wnt ligand-frizzled receptor binding to downstream effector ß-catenin activation. However, the precise molecular nature and contribution of Dvl2 protein assemblies during Wnt signalling is unknown. Here, we show that Dvl2 forms protein condensates driven by liquid-liquid phase separation. An intrinsically disordered region (IDR) at the N-terminus is essential for Dvl2 phase separation. Importantly, we identified the HECT-E3 ligase WWP2 as an essential driver of Dvl2 phase separation in vitro and in cells. We demonstrated that ubiquitylation of Dvl2 through K63 linkage by WWP2 is required for formation of Dvl2 condensates. Phase-separated Dvl2 activates Wnt signaling by sequestering the components of destruction complex and thus relieving ß-catenin. Together, our results reveal a ubiquitylation-dependent liquid-liquid phase separation as a new process through which Dvl2 forms condensates, which is necessary for transduction of Wnt signalling. This article has an associated First Person interview with the first author of the paper.

Ascorbic acid antagonizes the sedative effect of diazepam possibly through inhibition of GABA(Aρ1) and GABA(B1) receptors.

Gamma-aminobutyric acid (GABA) receptors belong to a ligand-gated ion channels family and are markedly expressed at the axon terminals of retinal bipolar cells. Ascorbic acid (AA), a known and vital antioxidant in the brain can modulate GABA receptors. We postulate that AA would antagonize benzodiazepines' effect via GABA receptor(s) interacting pathway. Here, we evaluated the modulatory sedative effect of AA on diazepam (DZP)'s anxiolytic effects in Swiss albino mice. The anxiolytic study was accomplished by using open-field, hole-board, and by swing and light-dark tests taking DZP as a standard anxiolytic drug. To understand the possible modulatory effects of AA, animals were co-administered with AA and DZP and/or its antagonist flumazenil (FLU). Additionally, an in-silico study was undertaken against GABA(A1), GABA(B1), and GABA(Aρ1) receptors. Data suggest that AA at 25 mg/kg (i.p.) increased (p<0.05) the number of field cross, rearing, number of hole cross, and swing and residence, while decreased grooming and dark residence parameters as compared to the control and DZP groups. In addition, AA and/or FLU combined with DZP (2 mg/kg, i.p.) reversed DZP-mediated sedative effects in mice. Results from in silico study suggest that AA has good interactions with GABA(Aρ1) and GABA(B1) receptors. In conclusion, DZP is a GABA receptor agonist and AA may reverse DZP-mediated sedative effects in a non-competitive binding fashion in mice through inhibition of GABA(Aρ1) and GABA(B1) receptors.

Antibiotics, Acid and Heat Tolerance of Honey adapted Escherichia coli, Salmonella Typhi and Klebsiella pneumoniae.

The medicinal importance of honey has been known for many decades due to its antimicrobial properties against life-threatening bacteria. However, previous studies revealed that microorganisms are able to develop adaptations after continuous exposure to antimicrobial compounds. The present study was conducted to explore the impact of subinhibitory concentrations of branded honey (Marhaba) and unbranded honey (extracted from Ziziphus mauritiana plant) locally available in Pakistan on Escherichia coli ATCC 10536, Salmonella Typhi and Klebsiella pneumoniae by investigating the development of self- or cross-resistance to antibiotics (gentamicin, kanamycin and imipenem). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of autoclaved honeys were determined. The bacterial cells of E. coli ATCC 10536, S. Typhi and K. pneumoniae were subjected to honey adaptation by exposing to » × MIC (4 passages) and ½ × MIC (4 passages) of both honeys. Moreover, tolerance to low pH and high temperature was also studied in adapted and unadapted cells. The decreasing trend in growth pattern (OD600nm) of E. coli ATCC 10536, S. Typhi and K. pneumoniae was observed with increases in the concentration of honeys (6.25-50% v/v) respectively. Our results showed that continuous exposure of both honeys did not lead to the development of any self- or cross-resistance in tested bacteria. However, percent survival to low pH was found to be significantly higher in adapted cells as compared to unadapted cells. The results indicate that both branded honey (Marhaba) and unbranded honey (extracted from Ziziphus mauritiana plant) were effective in controlling the growth of tested pathogenic bacteria. However, the emergence of tolerance to adverse conditions (pH 2.5, temperature 60 °C) deserves further investigation before proposing honey as a better antibacterial agent in food fabrication/processing, where low pH and high temperatures are usually implemented.

Controlled hypotension for FESS: A randomised double-blinded comparison of magnesium sulphate and dexmedetomidine.

BACKGROUND AND AIMS: Intense bleeding during general anaesthesia (GA) is the major limitation during functional endoscopic sinus surgery (FESS). This study was aimed to compare the efficacy of dexmedetomidine and magnesium sulphate (MgSO4) for controlled hypotension in FESS. METHODS: Sixty eight patients undergoing FESS were randomised to receive either dexmedetomidine 1 µg/kg over 10 min followed by infusion at 0.2 to 0.7 µg/kg/h (Group D) or MgSO4 40 mg/kg over 10 min followed by an infusion at 10 to 15 mg/kg/h (Group M). Anaesthesia and infusion rates for study drugs were maintained with sevoflurane to keep MAP between 60-70 mmHg throughout the surgery. The time to reach the target MAP, the number of patients requiring a minimum and maximum infusion doses of study drugs were noted. RESULTS: The mean time to achieve target mean arterial pressure (MAP) was less in group D (10.59 ± 2.04) as compared with (21.32 ± 4.65 min) group M (P < 0.001). The target MAP was achieved between 5-15 min in 73.52% patients (Group D) with an infusion dose of 0.2-0.4 µg/kg/h of dexmedetomidine without the use of sevoflurane, while 82.35% patients in group M required 4% sevoflurane along with >12-15 mg/kg/hr infusion of MgSO4 to achieve target MAP in 10-20 min. CONCLUSION: Dexmedetomidine is superior to MgSO4 in achieving target MAP in lesser time with minimum infusion dose.

PPM1G forms a PPP-type phosphatase holoenzyme with B56δ that maintains adherens junction integrity.

Serine/threonine phosphatases achieve substrate diversity by forming distinct holoenzyme complexes in cells. Although the PPP family of serine/threonine phosphatase family members such as PP1 and PP2A are well known to assemble and function as holoenzymes, none of the PPM family members were so far shown to act as holoenzymes. Here, we provide evidence that PPM1G, a member of PPM family of serine/threonine phosphatases, forms a distinct holoenzyme complex with the PP2A regulatory subunit B56δ. B56δ promotes the re-localization of PPM1G to the cytoplasm where the phosphatase can access a discrete set of substrates. Further, we unveil α-catenin, a component of adherens junction, as a new substrate for the PPM1G-B56 phosphatase complex in the cytoplasm. B56δ-PPM1G dephosphorylates α-catenin at serine 641, which is necessary for the appropriate assembly of adherens junctions and the prevention of aberrant cell migration. Collectively, we reveal a new holoenzyme with PPM1G-B56δ as integral components, in which the regulatory subunit provides accessibility to distinct substrates for the phosphatase by defining its cellular localization.

An approach to suppress the evolution of resistance in BRAFV600E-mutant cancer.

The principles that govern the evolution of tumors exposed to targeted therapy are poorly understood. Here we modeled the selection and propagation of an amplification in the BRAF oncogene (BRAFamp) in patient-derived tumor xenografts (PDXs) that were treated with a direct inhibitor of the kinase ERK, either alone or in combination with other ERK signaling inhibitors. Single-cell sequencing and multiplex fluorescence in situ hybridization analyses mapped the emergence of extra-chromosomal amplification in parallel evolutionary trajectories that arose in the same tumor shortly after treatment. The evolutionary selection of BRAFamp was determined by the fitness threshold, the barrier that subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for inhibitors of ERK signaling, suggesting that sequential monotherapy is ineffective and selects for a progressively higher BRAF copy number. Concurrent targeting of the RAF, MEK and ERK kinases, however, imposed a sufficiently high fitness threshold to prevent the propagation of subclones with high-level BRAFamp. When administered on an intermittent schedule, this treatment inhibited tumor growth in 11/11 PDXs of lung cancer or melanoma without apparent toxicity in mice. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose the highest fitness threshold will likely prevent the evolution of resistance-causing alterations and, thus, merit testing in patients.

14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein.

Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ(-/-) cells. 14-3-3σ(-/-) cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene.

Obstetric Complications and Management in Chronic Myeloid Leukemia.

Chronic myeloid leukaemia (CML) is amongst the most common haematological malignancies encountered in adults. The younger age of onset and increased incidence of CML in Indians leads to higher chances of encountering it in pregnancy. Pregnancy in CML is a complex situation as first line therapy with tyrosine kinase inhibitors (TKI), is fraught with multiple fetal safety issues. The fetal aspects have been elucidated in literature, but there is scarcity of information on the obstetric outcome per se in presence of CML, excluding the influence of TKI. Obstetric outcomes of 5 pregnancies in four patients with CML are being reported. Literature on interplay of CML and bleeding or thrombotic manifestations is reviewed. The major complications encountered were antepartum (APH) and postpartum haemorrhage (PPH), preterm labour, intrauterine growth retardation and intrauterine fetal death. Patients in the reproductive age group with diagnosis of CML should be carefully counseled regarding the effect of disease and TKI on the maternal-fetal health. Bleeding complications, particularly APH and PPH may be encountered in CML patients. Close coordination of the obstetrician, haematologist, and neonatologist is required in managing these cases successfully. The need for absolute contraception till the remission of disease needs to be emphasized for further pregnancies.

Using Virtual Patients to Teach Empathy: A Randomized Controlled Study to Enhance Medical Students' Empathic Communication.

INTRODUCTION: Physician empathy is a complex phenomenon known to improve illness outcomes; however, few tools are available for deliberate practice of empathy. We used a virtual patient (VP) to teach empathic communication to first-year medical students. We then evaluated students' verbal empathy in a standardized patient (SP) interaction. METHODS: Seventy medical students, randomly assigned to 3 separate study groups, interacted with (1) a control VP portraying depression, (2) a VP with a backstory simulating patient shadowing, or (3) a VP able to give immediate feedback about empathic communication (empathy-feedback VP). Subsequently, the students interviewed an SP portraying a scenario that included opportunities to express empathy. All SP interviews were recorded and transcribed. The study outcomes were (1) the students' verbal response to the empathic opportunities presented by the SP, as coded by reliable assessors using the Empathic Communication Coding System, and (2) the students' responses as coded by the SPs, using a communication checklist. RESULTS: There were no significant differences in student demographics between groups. The students who interacted with the empathy-feedback VP showed higher empathy in the SP interview than did the students in the backstory VP and the control VP groups [mean (SD) empathy scores coded on a 0-6 scale were 2.91 (0.16) vs. 2.20 (0.22) and 2.27 (0.21), respectively). The difference in scores was significant only for the empathy-feedback VP versus the backstory VP group (P = 0.027). The SPs rated the empathy-feedback and the backstory VP groups significantly higher than the control VP group on offering empathic statements (P < 0.0001), appearing warm and caring (P = 0.015), and forming rapport (P = 0.004). CONCLUSIONS: Feedback on empathy in a VP interaction increased students' empathy in encounters with SPs, as rated by trained assessors, whereas a simulation of patient shadowing did not. Both VP interventions increased students' empathy as rated by SPs, compared with the control VP group.

The Use of Simulation to Teach Suicide Risk Assessment to Health Profession Trainees-Rationale, Methodology, and a Proof of Concept Demonstration with a Virtual Patient.

OBJECTIVE: There is increasing use of educational technologies in medical and surgical specialties. Described herein is the development and application of an interactive virtual patient (VP) to teach suicide risk assessment to health profession trainees. We studied the effect of the following: (1) an interaction with a bipolar VP who attempts suicide or (2) completion of a video-teaching module on interviewing a bipolar patient, on medical students' proficiency in assessing suicide risk in standardized patients. We hypothesized that students who interact with a bipolar VP will be at least as likely to assess suicide risk, as their peers who completed a video module. METHODS: In a randomized, controlled study, we compared the frequency with which second-year students at the Medical College of Georgia asked suicide risk and bipolar symptoms questions by VP/video group. RESULTS: We recruited 67 students. The VP group inquired more frequently than the video group in 4 of 5 suicide risk areas and 11 of 14 other bipolar symptomatology areas. There were minimal to small effect sizes in favor of the VP technology. The students preferred the video over the VP as an educational tool (p = 0.007). CONCLUSIONS: Our study provides proof of concept that both VP and video module approaches are feasible for teaching students to assess suicide risk, and we present evidence about the role of active learning to improve communication skills. Depending on the learning context, interviewing a VP or observation of a videotaped interview can enhance the students' suicide risk assessment proficiency in an interview with a standardized patient. An interactive VP is a plausible modality to deliver basic concepts of suicide risk assessment to medical students, can facilitate individual preferences by providing easy access and portability, and has potential generalizability to other aspects of psychiatric training.

WWP2-WWP1 ubiquitin ligase complex coordinated by PPM1G maintains the balance between cellular p73 and ΔNp73 levels.

The balance between transcription factor p73 and its functionally opposing N-terminally truncated ΔNp73 isoform is critical for cell survival, but the precise mechanism that regulates their levels is not clear. In our study, we identified WWP2, an E3 ligase, as a novel p73-associated protein that ubiquitinates and degrades p73. In contrast, WWP2 heterodimerizes with another E3 ligase, WWP1, which specifically ubiquitinates and degrades ΔNp73. Further, we identified phosphatase PPM1G as a functional switch that controls the balance between monomeric WWP2 and a WWP2/WWP1 heterodimeric state in the cell. During cellular stress, WWP2 is inactivated, leading to upregulation of p73, whereas WWP2-WWP1 complex is intact to degrade ΔNp73, thus playing an important role in shifting the balance between p73 and ΔNp73. Collectively, our results reveal a new functional E3 ligase complex controlled by PPM1G that differentially regulates cellular p73 and ΔNp73.

Safety of intrauterine contraceptive device (copper T 200 B) in women with cardiac disease.

BACKGROUND: Women with cardiac disease have need for effective long-lasting reversible contraception. Women with cardiac disease are at increased risk for bacterial endocarditis. There is limited research regarding the use of intrauterine contraceptive devices (IUD) in women with cardiac disease. STUDY DESIGN: In a prospective study, the IUD copper (Cu T200B) was inserted in 40 women with cardiac disease. Infective endocarditis prophylaxis was given 1 h before IUD insertion. The IUD was inserted under aseptic conditions. Ten milliliters of venous blood was obtained for culture of aerobic and anerobic bacteria within 1 h of insertion of the copper T IUD. Women were contacted for follow-up at frequent intervals. RESULTS: There was no incidence of uterine perforation, hemorrhage or spontaneous expulsion of the IUD. All blood cultures were sterile. There were no cases of infective endocarditis. Four women (10%) had menorrhagia at the 6-month follow-up which responded to medical management. One woman had PID for which antibiotics were given. Five women had mild cramps and five had spotting after insertion of the IUD. Patient adherence was excellent as none returned for removal for reasons other than desire for another pregnancy. CONCLUSION: The Cu T200B IUD is a reasonably safe and effective method of temporary contraception in women with cardiac disease who are not receiving anticoagulant therapy.