NADH Distribution in Live Progenitor Stem Cells by Phasor-Fluorescence Lifetime Image Microscopy

AbstractNADH is a naturally fluorescent metabolite associated with cellular respiration. Exploiting the different fluorescence lifetime of free and bound NADH has the potential to quantify the relative amount of bound and free NADH, enhancing understanding of cellular processes including apoptosis, cancer pathology, and enzyme kinetics. We use the phasor- fluorescence lifetime image microscopy approach to spatially map NADH in both the free and bound forms of live undifferentiated and differentiated myoblast cells. The phasor approach graphically depicts the change in lifetime at a pixel level without the requirement for fitting the decay. Comparison of the spatial distribution of NADH in the nucleus of cells induced to differentiate through serum starvation and undifferentiated cells show differing distributions of bound and free NADH. Undifferentiated cells displayed a short lifetime indicative of free NADH in the nucleus and a longer lifetime attributed to the presence of bound NADH outside of the nucleus. Differentiating cells displayed redistribution of free NADH with decreased relative concentration of free NADH within the nucleus whereas the majority of NADH was found in the cytoplasm

ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity

Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoidlike proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B1, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B1 and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network

Diamond membrane production : the critical role of radicals in the non-contact electrochemical etching of sp2 carbon

Sub-micrometre single crystal diamond membranes are of huge importance for next generation optical, quantum and electronic device applications. Electrochemical etching has proven a critical step in the production of such membranes. Etching is used to selectively remove a very thin layer of sub-surface sp2 carbon, prepared by ion implantation in bulk diamond, releasing the diamond membrane. Due to the nanosized dimensions, etching is typically carried out using non-contact electrochemistry in low conductivity solutions (bipolar arrangement) which whilst effective, results in extremely slow etch rates. In this work, a new method of non-contact electrochemical etching is presented which uses high conductivity, high concentration, fully dissociated aqueous electrolytes. Careful choice of the electrolyte anion results in significant improvements in the sp2 carbon etch rate. In particular, we show both chloride and sulfate electrolytes increase etch rates significantly (up to × 40 for sulfate) compared to our measurements using the current state-of-the-art solutions and methodologies. Electron paramagnetic resonance experiments, recorded after the electrode potential has been switched off, reveal sizeable hydroxyl radical concentrations at timescales > 107 longer than their lifetime (≤μs). These measurements highlight the importance of electrochemically initiated, solution chemistry radical generation and regeneration pathways in high concentration sulfate and chloride solutions for nano-etching applications

Motivating students and improving engagement in biology units using online QS modules

MathBench biology modules represent one example of how biology educators can incorporate materials to improve quantitative skills and reasoning into introductory courses. The MathBench- Australia project not only aims to ensure that the science and the maths content of MathBench (USA) modules are accurate, but also appropriate to an Australian context, and further aid to minimise students’ negative attitude towards quantitative skills and increase student engagement. Hence, in this ideas exchange we will explore the strategies to embed the contextualised MathBench modules in first and second year science units to improve student engagement and students’ QS

Guideline for reporting systematic reviews of outcome measurement instruments (OMIs):PRISMA-COSMIN for OMIs 2024

PurposeAlthough comprehensive and widespread guidelines on how to conduct systematic reviews of outcome measurement instruments (OMIs) exist, for example from the COSMIN (COnsensus-based Standards for the selection of health Measure- ment INstruments) initiative, key information is often missing in published reports. This article describes the development of an extension of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guideline: PRISMA-COSMIN for OMIs 2024.MethodsThe development process followed the Enhancing the QUAlity and Transparency Of health Research (EQUATOR) guidelines and included a literature search, expert consultations, a Delphi study, a hybrid workgroup meeting, pilot testing, and an end-of-project meeting, with integrated patient/public involvement.ResultsFrom the literature and expert consultation, 49 potentially relevant reporting items were identified. Round 1 of the Delphi study was completed by 103 panelists, whereas round 2 and 3 were completed by 78 panelists. After 3 rounds, agreement (≥ 67%) on inclusion and wording was reached for 44 items. Eleven items without consensus for inclusion and/or wording were discussed at a workgroup meeting attended by 24 participants. Agreement was reached for the inclusion and wording of 10 items, and the deletion of 1 item. Pilot testing with 65 authors of OMI systematic reviews further improved the guideline through minor changes in wording and structure, finalized during the end-of-project meeting. The final check- list to facilitate the reporting of full systematic review reports contains 54 (sub)items addressing the review’s title, abstract, plain language summary, open science, introduction, methods, results, and discussion. Thirteen items pertaining to the title and abstract are also included in a separate abstract checklist, guiding authors in reporting for example conference abstracts.ConclusionPRISMA-COSMIN for OMIs 2024 consists of two checklists (full reports; abstracts), their corresponding expla- nation and elaboration documents detailing the rationale and examples for each item, and a data flow diagram. PRISMA- COSMIN for OMIs 2024 can improve the reporting of systematic reviews of OMIs, fostering their reproducibility and allowing end-users to appraise the quality of OMIs and select the most appropriate OMI for a specific application

Guideline for reporting systematic reviews of outcome measurement instruments (OMIs):PRISMA-COSMIN for OMIs 2024

PurposeAlthough comprehensive and widespread guidelines on how to conduct systematic reviews of outcome measurement instruments (OMIs) exist, for example from the COSMIN (COnsensus-based Standards for the selection of health Measure- ment INstruments) initiative, key information is often missing in published reports. This article describes the development of an extension of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guideline: PRISMA-COSMIN for OMIs 2024.MethodsThe development process followed the Enhancing the QUAlity and Transparency Of health Research (EQUATOR) guidelines and included a literature search, expert consultations, a Delphi study, a hybrid workgroup meeting, pilot testing, and an end-of-project meeting, with integrated patient/public involvement.ResultsFrom the literature and expert consultation, 49 potentially relevant reporting items were identified. Round 1 of the Delphi study was completed by 103 panelists, whereas round 2 and 3 were completed by 78 panelists. After 3 rounds, agreement (≥ 67%) on inclusion and wording was reached for 44 items. Eleven items without consensus for inclusion and/or wording were discussed at a workgroup meeting attended by 24 participants. Agreement was reached for the inclusion and wording of 10 items, and the deletion of 1 item. Pilot testing with 65 authors of OMI systematic reviews further improved the guideline through minor changes in wording and structure, finalized during the end-of-project meeting. The final check- list to facilitate the reporting of full systematic review reports contains 54 (sub)items addressing the review’s title, abstract, plain language summary, open science, introduction, methods, results, and discussion. Thirteen items pertaining to the title and abstract are also included in a separate abstract checklist, guiding authors in reporting for example conference abstracts.ConclusionPRISMA-COSMIN for OMIs 2024 consists of two checklists (full reports; abstracts), their corresponding expla- nation and elaboration documents detailing the rationale and examples for each item, and a data flow diagram. PRISMA- COSMIN for OMIs 2024 can improve the reporting of systematic reviews of OMIs, fostering their reproducibility and allowing end-users to appraise the quality of OMIs and select the most appropriate OMI for a specific application

Loss-of-function mutations in the X-linked biglycan gene cause a severe syndromic form of thoracic aortic aneurysms and dissections.

Thoracic aortic aneurysm and dissection (TAAD) is typically inherited in an autosomal dominant manner, but rare X-linked families have been described. So far, the only known X-linked gene is FLNA, which is associated with the periventricular nodular heterotopia type of Ehlers-Danlos syndrome. However, mutations in this gene explain only a small number of X-linked TAAD families. We performed targeted resequencing of 368 candidate genes in a cohort of 11 molecularly unexplained Marfan probands. Subsequently, Sanger sequencing of BGN in 360 male and 155 female molecularly unexplained TAAD probands was performed. We found five individuals with loss-of-function mutations in BGN encoding the small leucine-rich proteoglycan biglycan. The clinical phenotype is characterized by early-onset aortic aneurysm and dissection. Other recurrent findings include hypertelorism, pectus deformity, joint hypermobility, contractures, and mild skeletal dysplasia. Fluorescent staining revealed an increase in TGF-β signaling, evidenced by an increase in nuclear pSMAD2 in the aortic wall. Our results are in line with those of prior reports demonstrating that Bgn-deficient male BALB/cA mice die from aortic rupture. In conclusion, BGN gene defects in humans cause an X-linked syndromic form of severe TAAD that is associated with preservation of elastic fibers and increased TGF-β signaling.Genet Med 19 4, 386-395