Expression Levels of a Kinesin-13 Microtubule Depolymerase Modulates the Effectiveness of Anti-Microtubule Agents

Chemotherapeutic drugs often target the microtubule cytoskeleton as a means to disrupt cancer cell mitosis and proliferation. Anti-microtubule drugs inhibit microtubule dynamics, thereby triggering apoptosis when dividing cells activate the mitotic checkpoint. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs); however, we lack a comprehensive understanding about how anti-microtubule agents functionally interact with MAPs. In this report, we test the hypothesis that the cellular levels of microtubule depolymerases, in this case kinesin-13 s, modulate the effectiveness of the microtubule disrupting drug colchicine.We used a combination of RNA interference (RNAi), high-throughput microscopy, and time-lapse video microscopy in Drosophila S2 cells to identify a specific MAP, kinesin-like protein 10A (KLP10A), that contributes to the efficacy of the anti-microtubule drug colchicine. KLP10A is an essential microtubule depolymerase throughout the cell cycle. We find that depletion of KLP10A in S2 cells confers resistance to colchicine-induced microtubule depolymerization to a much greater extent than depletion of several other destabilizing MAPs. Using image-based assays, we determined that control cells retained 58% (+/-2%SEM) of microtubule polymer when after treatment with 2 microM colchicine for 1 hour, while cells depleted of KLP10A by RNAi retained 74% (+/-1%SEM). Likewise, overexpression of KLP10A-GFP results in increased susceptibility to microtubule depolymerization by colchicine.Our results demonstrate that the efficacy of microtubule destabilization by a pharmacological agent is dependent upon the cellular expression of a microtubule depolymerase. These findings suggest that expression levels of Kif2A, the human kinesin-13 family member, may be an attractive biomarker to assess the effectiveness of anti-microtubule chemotherapies. Knowledge of how MAP expression levels affect the action of anti-microtubule drugs may prove useful for evaluating possible modes of cancer treatment

Effects of Short-Term Continuous Montmorency Tart Cherry Juice Supplementation in Participants with Metabolic Syndrome

© 2020 Springer-Verlag. The final publication is available at Springer via https://doi.org/10.1007/s00394-020-02355-5Purpose: Metabolic Syndrome (MetS) augments the incidence of cardiovascular disease by two-fold and type II diabetes mellitus by five-fold. Montmorency tart cherries are rich in phytochemicals shown to improve biomarkers related to cardio-metabolic health in humans. This study aimed to examine cardio-metabolic responses after 7-days Montmorency tart cherry juice (MTCJ) supplementation and also acute on short-term supplementation responses to a single bolus, in humans with MetS. Methods: In a randomised, single-blind, placebo-controlled, crossover trial, 12 participants with MetS (50 ± 10 years; 6M/6F), consumed MTCJ or placebo (PLA) for 7 days. Blood-based and functional cardio-metabolic biomarkers were measured pre- and post-supplementation, and acute responses measured pre-bolus and up to 5 h post-bolus on the 7th day. Results: 24-h ambulatory systolic (P = 0.016), diastolic (P = 0.009) blood pressure and mean arterial pressure (P = 0.041) were significantly lower after 7-days MTCJ supplementation compared to PLA. Glucose (P = 0.038), total cholesterol (P = 0.036), LDL (P = 0.023) concentrations, total cholesterol:HDL ratio (P = 0.004) and respiratory exchange ratio values (P = 0.009) were significantly lower after 6-days MTCJ consumption compared to PLA. Conclusions: This study revealed for the first time in humans that MTCJ significantly improved 24-h BP, fasting glucose, total cholesterol and total cholesterol:HDL ratio, and also lowered resting respiratory exchange ratio compared to a control group. Responses demonstrated clinically relevant improvements on aspects of cardio-metabolic function, emphasising the potential efficacy of MTCJ in preventing further cardio-metabolic dysregulation in participants with MetS. Registered at clinicaltrials.gov (NCT03619941).Peer reviewedFinal Accepted Versio

Homeostatic Plasticity Studied Using In Vivo Hippocampal Activity-Blockade: Synaptic Scaling, Intrinsic Plasticity and Age-Dependence

Homeostatic plasticity is thought to be important in preventing neuronal circuits from becoming hyper- or hypoactive. However, there is little information concerning homeostatic mechanisms following in vivo manipulations of activity levels. We investigated synaptic scaling and intrinsic plasticity in CA1 pyramidal cells following 2 days of activity-blockade in vivo in adult (postnatal day 30; P30) and juvenile (P15) rats. Chronic activity-blockade in vivo was achieved using the sustained release of the sodium channel blocker tetrodotoxin (TTX) from the plastic polymer Elvax 40W implanted directly above the hippocampus, followed by electrophysiological assessment in slices in vitro. Three sets of results were in general agreement with previous studies on homeostatic responses to in vitro manipulations of activity. First, Schaffer collateral stimulation-evoked field responses were enhanced after 2 days of in vivo TTX application. Second, miniature excitatory postsynaptic current (mEPSC) amplitudes were potentiated. However, the increase in mEPSC amplitudes occurred only in juveniles, and not in adults, indicating age-dependent effects. Third, intrinsic neuronal excitability increased. In contrast, three sets of results sharply differed from previous reports on homeostatic responses to in vitro manipulations of activity. First, miniature inhibitory postsynaptic current (mIPSC) amplitudes were invariably enhanced. Second, multiplicative scaling of mEPSC and mIPSC amplitudes was absent. Third, the frequencies of adult and juvenile mEPSCs and adult mIPSCs were increased, indicating presynaptic alterations. These results provide new insights into in vivo homeostatic plasticity mechanisms with relevance to memory storage, activity-dependent development and neurological diseases

Biochemical Properties of a Novel Cysteine Protease of Plasmodium vivax, Vivapain-4

Plasmodium vivax affects hundreds of millions each year and results in severe morbidity and mortality. Plasmodial cysteine proteases (CPs) play crucial roles during the progression of malaria since inhibition of these molecules impairs parasite growth. These CPs might be targeted for new antimalarial drugs. We characterized a novel P. vivax CP, vivapain-4 (VX-4), which appeared to evolve differentially among primate Plasmodium species. VX-4 showed highly unique substrate preference depending on surrounding micro-environmental pH. It effectively hydrolyzed benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA at acidic pH and Z-Arg-Arg-MCA at neutral pH. Three amino acids (Ala90, Gly157 and Glu180) that delineate the S2 pocket were found to be substituted in VX-4. Alteration of Glu180 abolished hydrolytic activity against Z-Arg-Arg-MCA at neutral pH, indicating Glu180 is intimately involved in the pH-dependent substrate preference. VX-4 hydrolyzed actin at neutral pH and hemoglobin at acidic pH, and participated in plasmepsin 4 activation at neutral/acidic pH. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. The differential substrate preferences depending on pH suggested a highly efficient mechanism to enlarge biological implications of VX-4, including hemoglobin degradation, maturation of plasmepsin, and remodeling of the parasite architecture during growth and development of P. vivax

Evaluation of cell-free DNA approaches for multi-cancer early detection

In the Circulating Cell-free Genome Atlas (NCT02889978) substudy 1, we evaluate several approaches for a circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test by defining clinical limit of detection (LOD) based on circulating tumor allele fraction (cTAF), enabling performance comparisons. Among 10 machine-learning classifiers trained on the same samples and independently validated, when evaluated at 98% specificity, those using whole-genome (WG) methylation, single nucleotide variants with paired white blood cell background removal, and combined scores from classifiers evaluated in this study show the highest cancer signal detection sensitivities. Compared with clinical stage and tumor type, cTAF is a more significant predictor of classifier performance and may more closely reflect tumor biology. Clinical LODs mirror relative sensitivities for all approaches. The WG methylation feature best predicts cancer signal origin. WG methylation is the most promising technology for MCED and informs development of a targeted methylation MCED test

MHO1, an Evolutionarily Conserved Gene, Is Synthetic Lethal with PLC1; Mho1p Has a Role in Invasive Growth

The novel protein Memo (Mediator of ErbB2 driven cell motility) was identified in a screen for ErbB2 interacting proteins and found to have an essential function in cell motility. Memo is evolutionarily conserved with homologs found in all branches of life; the human and yeast proteins have a similarity of >50%. In the present study we used the model organism S. cerevisiae to characterize the Memo-homologue Mho1 (Yjr008wp) and to investigate its function in yeast. In a synthetic lethal screen we found MHO1 as a novel synthetic lethal partner of PLC1, which encodes the single phospholipase C in yeast. Double-deleted cells lacking MHO1 and PLC1, proliferate for up to ten generations. Introduction of human Memo into the memoΔplc1Δ strain rescued the synthetic lethal phenotype suggesting that yeast and human proteins have similar functions. Mho1 is present in the cytoplasm and the nucleus of yeast cells; the same distribution of Memo was found in mammalian cells. None of the Memo homologues have a characteristic nuclear localization sequence, however, a conserved nuclear export sequence is found in all. In mammalian cells, blocking nuclear export with Leptomycin B led to nuclear Memo accumulation, suggesting that it is actively exported from the nucleus. In yeast MHO1 expression is induced by stress conditions. Since invasive growth in S. cerevisiea is also stress-induced, we tested Mho1's role in this response. MHO1 deletion had no effect on invasion induced by nutrient deprivation, however, Mho1 overexpression blocked the invasive ability of yeast cells, suggesting that Mho1 might be acting in a dominant negative manner. Taken together, our results show that MHO1 is a novel synthetic lethal interactor with PLC1, and that both gene products are required for proliferation. Moreover, a role for Memo in cell motility/invasion appears to be conserved across species

A promising method for identifying cross-cultural differences in patient perspective: the use of Internet-based focus groups for content validation of new Patient Reported Outcome assessments

OBJECTIVES: This proof of concept (POC) study was designed to evaluate the use of an Internet-based bulletin board technology to aid parallel cross-cultural development of thematic content for a new set of patient-reported outcome measures (PROs). METHODS: The POC study, conducted in Germany and the United States, utilized Internet Focus Groups (IFGs) to assure the validity of new PRO items across the two cultures – all items were designed to assess the impact of excess facial oil on individuals' lives. The on-line IFG activities were modeled after traditional face-to-face focus groups and organized by a common 'Topic' Guide designed with input from thought leaders in dermatology and health outcomes research. The two sets of IFGs were professionally moderated in the native language of each country. IFG moderators coded the thematic content of transcripts, and a frequency analysis of code endorsement was used to identify areas of content similarity and difference between the two countries. Based on this information, draft PRO items were designed and a majority (80%) of the original participants returned to rate the relative importance of the newly designed questions. FINDINGS: The use of parallel cross-cultural content analysis of IFG transcripts permitted identification of the major content themes in each country as well as exploration of the possible reasons for any observed differences between the countries. Results from coded frequency counts and transcript reviews informed the design and wording of the test questions for the future PRO instrument(s). Subsequent ratings of item importance also deepened our understanding of potential areas of cross-cultural difference, differences that would be explored over the course of future validation studies involving these PROs. CONCLUSION: The use of IFGs for cross-cultural content development received positive reviews from participants and was found to be both cost and time effective. The novel thematic coding methodology provided an empirical platform on which to develop culturally sensitive questionnaire content using the natural language of participants. Overall, the IFG responses and thematic analyses provided a thorough evaluation of similarities and differences in cross-cultural themes, which in turn acted as a sound base for the development of new PRO questionnaires

A DECam Search for Explosive Optical Transients Associated with IceCube Neutrino Alerts

In this work, we investigate the likelihood of association between real-time, neutrino alerts with teraelectronvolt to petaelectronvolt energy from IceCube and optical counterparts in the form of core-collapse supernovae (CC SNe). The optical follow-up of IceCube alerts requires two main instrumental capabilities: (1) deep imaging, since 73% of neutrinos would come from CC SNe at redshifts z > 0.3, and (2) a large field of view (FoV), since typical IceCube muon neutrino pointing accuracy is on the order of ~1 deg. With Blanco/DECam (gri to 24th magnitude and 2.2 deg diameter FoV), we performed a triggered optical follow-up observation of two IceCube alerts, IC170922A and IC171106A, on six nights during the three weeks following each alert. For the IC170922A (IC171106A) follow-up observations, we expect that 12.1% (9.5%) of coincident CC SNe at z lesssim 0.3 are detectable, and that, on average, 0.23 (0.07) unassociated SNe in the neutrino 90% containment regions also pass our selection criteria. We find two candidate CC SNe that are temporally coincident with the neutrino alerts in the FoV, but none in the 90% containment regions, a result that is statistically consistent with expected rates of background CC SNe for these observations. If CC SNe are the dominant source of teraelectronvolt to petaelectronvolt neutrinos, we would expect an excess of coincident CC SNe to be detectable at the 3σ confidence level using DECam observations similar to those of this work for ~60 (~200) neutrino alerts with (without) redshift information for all candidates

ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation

The LIM homeodomain gene Islet-1 (ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations

Heavy Ion Carcinogenesis and Human Space Exploration

Prior to the human exploration of Mars or long duration stays on the Earth s moon, the risk of cancer and other diseases from space radiation must be accurately estimated and mitigated. Space radiation, comprised of energetic protons and heavy nuclei, has been show to produce distinct biological damage compared to radiation on Earth, leading to large uncertainties in the projection of cancer and other health risks, while obscuring evaluation of the effectiveness of possible countermeasures. Here, we describe how research in cancer radiobiology can support human missions to Mars and other planets