Toll-Like Receptor 11 (TLR11) Interacts with Flagellin and Profilin through Disparate Mechanisms

Toll-like receptors (TLRs) are innate immune receptors that sense a variety of pathogen-associated molecular patterns (PAMPs) by interacting with them and subsequently initiating signal transduction cascades that elicit immune responses. TLR11 has been shown to interact with two known protein PAMPs: Salmonella and E. coli flagellin FliC and Toxoplasma gondii profilin-like protein. Given the highly divergent biology of these pathogens recognized by TLR11, it is unclear whether common mechanisms are used to recognize these distinct protein PAMPs. Here we show that TLR11 interacts with these two PAMPs using different receptor domains. Furthermore, TLR11 binding to flagellin and profilin exhibits differential dependency on pH and receptor ectodomain cleavage

An Essential Role for ECSIT in Mitochondrial Complex I Assembly and Mitophagy in Macrophages

ECSIT is a mitochondrial complex I (CI)-associated protein that has been shown to regulate the production of mitochondrial reactive oxygen species (mROS) following engagement of Toll-like receptors (TLRs). We have generated an Ecsit conditional knockout (CKO) mouse strain to study the in vivo role of ECSIT. ECSIT deletion results in profound alteration of macrophage metabolism, leading to a striking shift to reliance on glycolysis, complete disruption of CI activity, and loss of the CI holoenzyme and multiple subassemblies. An increase in constitutive mROS production in ECSIT-deleted macrophages prevents further TLR-induced mROS production. Surprisingly, ECSIT-deleted cells accumulate damaged mitochondria because of defective mitophagy. ECSIT associates with the mitophagy regulator PINK1 and exhibits Parkin-dependent ubiquitination. However, upon ECSIT deletion, we observed increased mitochondrial Parkin without the expected increase in mitophagy. Taken together, these results demonstrate a key role of ECSIT in CI function, mROS production, and mitophagy-dependent mitochondrial quality control

mTORC1-independent Raptor prevents hepatic steatosis by stabilizing PHLPP2

Mechanistic target of rapamycin complex 1 (mTORC1), defined by the presence of Raptor, is an evolutionarily conserved and nutrient-sensitive regulator of cellular growth and other metabolic processes. To date, all known functions of Raptor involve its scaffolding mTOR kinase with substrate. Here we report that mTORC1-independent (‘free’) Raptor negatively regulates hepatic Akt activity and lipogenesis. Free Raptor levels in liver decline with age and in obesity; restoration of free Raptor levels reduces liver triglyceride content, through reduced β-TrCP-mediated degradation of the Akt phosphatase, PHLPP2. Commensurately, forced PHLPP2 expression ameliorates hepatic steatosis in diet-induced obese mice. These data suggest that the balance of free and mTORC1-associated Raptor governs hepatic lipid accumulation, and uncover the potentially therapeutic role of PHLPP2 activators in non-alcoholic fatty liver disease

Ribonucleoprotein Purification and Characterization Using RNA Mango

The characterization of RNA–protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the S. cerevisiae U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro

Constraints on Type Ia Supernova Progenitor Companions from Early Ultraviolet Observations with Swift

We compare early ultraviolet (UV) observations of Type Ia Supernovae (SNe Ia) with theoretical predictions for the brightness of the shock associated with the collision between SN ejecta and a companion star. Our simple method is independent of the intrinsic flux from the SN and treats the flux observed with the Swift/Ultra-Violet Optical Telescope (UVOT) as conservative upper limits on the shock brightness. Comparing this limit with the predicted flux for various shock models, we constrain the geometry of the SN progenitor-companion system. We find the model of a 1 M_sun red supergiant companion in Roche lobe overflow to be excluded at a 95% confidence level for most individual SNe for all but the most unfavorable viewing angles. For the sample of 12 SNe taken together, the upper limits on the viewing angle are inconsistent with the expected distribution of viewing angles for RG stars as the majority of companions with high confidence. The separation distance constraints do allow MS companions. A better understanding of the UV flux arising from the SN itself as well as continued UV observations of young SNe Ia will further constrain the possible progenitors of SNe Ia.Comment: accepted versio

The Human Gene Mutation Database (HGMD®): optimizing its use in a clinical diagnostic or research setting

The Human Gene Mutation Database (HGMD®) constitutes a comprehensive collection of published germline mutations in nuclear genes that are thought to underlie, or are closely associated with human inherited disease. At the time of writing (June 2020), the database contains in excess of 289,000 different gene lesions identified in over 11,100 genes manually curated from 72,987 articles published in over 3100 peer-reviewed journals. There are primarily two main groups of users who utilise HGMD on a regular basis; research scientists and clinical diagnosticians. This review aims to highlight how to make the most out of HGMD data in each setting

The current state of antifungal stewardship among pediatric antimicrobial stewardship programs

OBJECTIVE: To characterize the current state of antifungal stewardship practices and perceptions of antifungal use among pediatric antimicrobial stewardship programs (ASPs). DESIGN: We developed and distributed an electronic survey, which included 17 closed-ended questions about institutional antifungal stewardship practices and perceptions, among pediatric ASPs. PARTICIPANTS: ASP physicians and pharmacists of 74 hospitals participating in the multicenter Sharing Antimicrobial Reports for Pediatric Stewardship (SHARPS) Collaborative. RESULTS: We sent surveys to 74 hospitals and received 68 unique responses, for a response rate of 92%. Overall, 63 of 68 the respondent ASPs (93%) reported that they conduct 1 or more antifungal stewardship activities. Of these 68 hospital ASPs, 43 (63%) perform prospective audit and feedback (PAF) of antifungals. The most common reasons reported for not performing PAF of antifungals were not enough time or resources (19 of 25, 76%) and minimal institutional antifungal use (6 of 25, 24%). Also, 52 hospitals (76%) require preauthorization for 1 or more antifungal agents. The most commonly restricted antifungals were isavuconazole (42 of 52 hospitals, 80%) and posaconazole (39 of 52 hospitals, 75%). Furthermore, 33 ASPs (48%) agreed or strongly agreed that antifungals are inappropriately used at their institution, and only 25 of 68 (37%) of ASPs felt very confident making recommendations about antifungals. CONCLUSIONS: Most pediatric ASPs steward antifungals, but the strategies employed are highly variable across surveyed institutions. Although nearly half of respondents identified inappropriate antifungal use as a problem at their institution, most ASPs do not feel confident making recommendations about antifungals. Future studies are needed to determine the rate of inappropriate antifungal use and the best antifungal stewardship strategies

Engineered Knottin Peptide Enables Noninvasive Optical Imaging of Intracranial Medulloblastoma

Central nervous system tumors carry grave clinical prognoses due to limited effectiveness of surgical resection, radiation, and chemotherapy. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine knot (knottin) peptide that binds with high affinity to α β , α β , and α β integrin receptors. This integrin-binding knottin peptide, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB. Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared with the normal surrounding brain tissue, as measured by optical imaging. The imaging signal intensity correlated with tumor volume. Due to its unique ability to bind to α β integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared with other engineered integrin-binding knottin peptides and with c(RGDfK), a well-studied integrin-binding peptidomimetic. Next, EETI 2.5F was fused to an antibody fragment crystallizable (Fc) domain (EETI 2.5F-Fc) to determine if a larger integrin-binding protein could also target intracranial brain tumors. EETI 2.5F-Fc, conjugated to a fluorescent dye, illuminated MB following i.v. injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity. These results highlight the potential of using EETI 2.5F and EETI 2.5-Fc as targeted molecular probes for brain tumor imaging

Genomic selection for target traits in the Australian lentil breeding program

Genomic selection (GS) uses associations between markers and phenotypes to predict the breeding values of individuals. It can be applied early in the breeding cycle to reduce the cross-to-cross generation interval and thereby increase genetic gain per unit of time. The development of cost-effective, high-throughput genotyping platforms has revolutionized plant breeding programs by enabling the implementation of GS at the scale required to achieve impact. As a result, GS is becoming routine in plant breeding, even in minor crops such as pulses. Here we examined 2,081 breeding lines from Agriculture Victoria’s national lentil breeding program for a range of target traits including grain yield, ascochyta blight resistance, botrytis grey mould resistance, salinity and boron stress tolerance, 100-grain weight, seed size index and protein content. A broad range of narrow-sense heritabilities was observed across these traits (0.24-0.66). Genomic prediction models were developed based on 64,781 genome-wide SNPs using Bayesian methodology and genomic estimated breeding values (GEBVs) were calculated. Forward cross-validation was applied to examine the prediction accuracy of GS for these targeted traits. The accuracy of GEBVs was consistently higher (0.34-0.83) than BLUP estimated breeding values (EBVs) (0.22-0.54), indicating a higher expected rate of genetic gain with GS. GS-led parental selection using early generation breeding materials also resulted in higher genetic gain compared to BLUP-based selection performed using later generation breeding lines. Our results show that implementing GS in lentil breeding will fast track the development of high-yielding cultivars with increased resistance to biotic and abiotic stresses, as well as improved seed quality traits

Characteristics of antifungal utilization for hospitalized children in the United States

OBJECTIVE: To characterize antifungal prescribing patterns, including the indication for antifungal use, in hospitalized children across the United States. DESIGN: We analyzed antifungal prescribing data from 32 hospitals that participated in the SHARPS Antibiotic Resistance, Prescribing, and Efficacy among Children (SHARPEC) study, a cross-sectional point-prevalence survey conducted between June 2016 and December 2017. METHODS: Inpatients aged \u3c18 years with an active systemic antifungal order were included in the analysis. We classified antifungal prescribing by indication (ie, prophylaxis, empiric, targeted), and we compared the proportion of patients in each category based on patient and antifungal characteristics. RESULTS: Among 34,927 surveyed patients, 2,095 (6%) received at least 1 systemic antifungal and there were 2,207 antifungal prescriptions. Most patients had an underlying oncology or bone marrow transplant diagnosis (57%) or were premature (13%). The most prescribed antifungal was fluconazole (48%) and the most common indication for antifungal use was prophylaxis (64%). Of 2,095 patients receiving antifungals, 79 (4%) were prescribed \u3e1 antifungal, most often as targeted therapy (48%). The antifungal prescribing rate ranged from 13.6 to 131.2 antifungals per 1,000 patients across hospitals ( CONCLUSIONS: Most antifungal use in hospitalized children was for prophylaxis, and the rate of antifungal prescribing varied significantly across hospitals. Potential targets for antifungal stewardship efforts include high-risk, high-utilization populations, such as oncology and bone marrow transplant patients, and specific patterns of utilization, including prophylactic and combination antifungal therapy