RNA-binding proteins direct myogenic cell fate decisions.

RNA-binding proteins (RBPs), essential for skeletal muscle regeneration, cause muscle degeneration and neuromuscular disease when mutated. Why mutations in these ubiquitously expressed RBPs orchestrate complex tissue regeneration and direct cell fate decisions in skeletal muscle remains poorly understood. Single-cell RNA-sequencing of regenerating Mus musculus skeletal muscle reveals that RBP expression, including the expression of many neuromuscular disease-associated RBPs, is temporally regulated in skeletal muscle stem cells and correlates with specific stages of myogenic differentiation. By combining machine learning with RBP engagement scoring, we discovered that the neuromuscular disease-associated RBP Hnrnpa2b1 is a differentiation-specifying regulator of myogenesis that controls myogenic cell fate transitions during terminal differentiation in mice. The timing of RBP expression specifies cell fate transitions by providing post-transcriptional regulation of messenger RNAs that coordinate stem cell fate decisions during tissue regeneration.

Early ventilator liberation and decreased sedation needs after tracheostomy in patients with COVID-19 infection.

BACKGROUND: Since the outset of the coronavirus disease 2019 (COVID-19) pandemic, published tracheostomy guidelines have generally recommended deferral of the procedure beyond the initial weeks of intubation given high mortality as well as concerns about transmission of the infection to providers. It is unclear whether tracheostomy in patients with COVID-19 infection facilitates ventilator weaning, and long-term outcomes are not yet reported in the literature. METHODS: This is a retrospective study of tracheostomy outcomes in patients with COVID-19 infection at a single-center academic tertiary referral intensive care unit. Patients underwent percutaneous tracheostomy at the bedside; the procedure was performed with limited staffing to reduce risk of disease transmission. RESULTS: Between March 1 and June 30, 2020, a total of 206 patients with COVID-19 infection required mechanical ventilation and 26 underwent tracheostomy at a mean of 25±5 days after initial intubation. Overall, 81% of tracheostomy patients were liberated from the ventilator at a mean of 9±6 days postprocedure, and 54% were decannulated prior to hospital discharge at a mean of 21±10 days postprocedure. Sedation and pain medication requirements decreased significantly in the week after the procedure. In-hospital mortality was 15%. Among tracheostomy survivors, 68% were discharged to a facility. DISCUSSION: The management of patients with COVID-19 related respiratory failure can be challenging due to prolonged ventilator dependency. In our initial experience, outcomes post-tracheostomy in this population are encouraging, with short time to liberation from the ventilator, a high rate of decannulation prior to hospital discharge, and similar mortality to tracheostomy performed for other indications. Barriers to weaning ventilation in this cohort may be high sedation needs and ventilator dyssynchrony. LEVEL OF EVIDENCE: Level V-Therapeutic/care management.

Injury-mediated stiffening persistently activates muscle stem cells through YAP and TAZ mechanotransduction.

The skeletal muscle microenvironment transiently remodels and stiffens after exercise and injury, as muscle ages, and in myopathic muscle; however, how these changes in stiffness affect resident muscle stem cells (MuSCs) remains understudied. Following muscle injury, muscle stiffness remained elevated after morphological regeneration was complete, accompanied by activated and proliferative MuSCs. To isolate the role of stiffness on MuSC behavior and determine the underlying mechanotransduction pathways, we cultured MuSCs on strain-promoted azide-alkyne cycloaddition hydrogels capable of in situ stiffening by secondary photocrosslinking of excess cyclooctynes. Using pre- to post-injury stiffness hydrogels, we found that elevated stiffness enhances migration and MuSC proliferation by localizing yes-associated protein 1 (YAP) and WW domain-containing transcription regulator 1 (WWTR1; TAZ) to the nucleus. Ablating YAP and TAZ in vivo promotes MuSC quiescence in postinjury muscle and prevents myofiber hypertrophy, demonstrating that persistent exposure to elevated stiffness activates mechanotransduction signaling maintaining activated and proliferating MuSCs.

"And if you gaze long into an abyss, the abyss gazes also into thee": four morphs of Arctic charr adapting to a depth gradient in Lake Tinnsjøen.

The origin of species is a central topic in biology. Ecological speciation might be a driver in adaptive radiation, providing a framework for understanding mechanisms, level, and rate of diversification. The Arctic charr Salvelinus alpinus L. is a polymorphic species with huge morphological and life-history diversity in Holarctic water systems. We studied adaptive radiation of Arctic charr in the 460-m-deep Lake Tinnsjøen to (a) document eco-morphology and life-history traits of morphs, (b) estimate reproductive isolation of morphs, and (c) illuminate Holarctic phylogeography and lineages colonizing Lake Tinnsjøen. We compared Lake Tinnsjøen with four Norwegian outgroup populations. Four field-assigned morphs were identified in Lake Tinnsjøen: the planktivore morph in all habitats except deep profundal, the dwarf morph in shallow-moderate profundal, the piscivore morph mainly in shallow-moderate profundal, and a new undescribed abyssal morph in the deep profundal. Morphs displayed extensive life-history variation in age and size. A moderate-to-high concordance was observed among morphs and four genetic clusters from microsatellites. mtDNA suggested two minor endemic clades in Lake Tinnsjøen originating from one widespread colonizing clade in the Holarctic. All morphs were genetically differentiated at microsatellites (F ST: 0.12-0.20), associated with different mtDNA clade frequencies. Analyses of outgroup lakes implied colonization from a river below Lake Tinnsjøen. Our findings suggest postglacial adaptive radiation of one colonizing mtDNA lineage with niche specialization along a depth-temperature-productivity-pressure gradient. Concordance between reproductive isolation and habitats of morphs implies ecological speciation as a mechanism. Particularly novel is the extensive morph diversification with depth into the often unexplored deepwater profundal habitat, suggesting we may have systematically underestimated biodiversity in lakes. In a biological conservation framework, it is imperative to protect endemic below-species-level biodiversity, particularly so since within-species variation comprises an extremely important component of the generally low total biodiversity observed in the northern freshwater systems.

Fibrinolysis Shutdown Correlation with Thromboembolic Events in Severe COVID-19 Infection.

BACKGROUND: COVID-19 predisposes patients to a prothrombotic state with demonstrated microvascular involvement. The degree of hypercoagulability appears to correlate with outcomes; however, optimal criteria to assess for the highest-risk patients for thrombotic events remain unclear; we hypothesized that deranged thromboelastography measurements of coagulation would correlate with thromboembolic events. STUDY DESIGN: Patients admitted to an ICU with COVID-19 diagnoses who had thromboelastography analyses performed were studied. Conventional coagulation assays, d-dimer levels, and viscoelastic measurements were analyzed using a receiver operating characteristic curve to predict thromboembolic outcomes and new-onset renal failure. RESULTS: Forty-four patients with COVID-19 were included in the analysis. Derangements in coagulation laboratory values, including elevated d-dimer, fibrinogen, prothrombin time, and partial thromboplastin time, were confirmed; viscoelastic measurements showed an elevated maximum amplitude and low lysis of clot at 30 minutes. A complete lack of lysis of clot at 30 minutes was seen in 57% of patients and predicted venous thromboembolic events with an area under the receiver operating characteristic curve of 0.742 (p = 0.021). A d-dimer cutoff of 2,600 ng/mL predicted need for dialysis with an area under the receiver operating characteristic curve of 0.779 (p = 0.005). Overall, patients with no lysis of clot at 30 minutes and a d-dimer > 2,600 ng/mL had a venous thromboembolic event rate of 50% compared with 0% for patients with neither risk factor (p = 0.008), and had a hemodialysis rate of 80% compared with 14% (p = 0.004). CONCLUSIONS: Fibrinolysis shutdown, as evidenced by elevated d-dimer and complete failure of clot lysis at 30 minutes on thromboelastography predicts thromboembolic events and need for hemodialysis in critically ill patients with COVID-19. Additional clinical trials are required to ascertain the need for early therapeutic anticoagulation or fibrinolytic therapy to address this state of fibrinolysis shutdown.

TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle.

A dominant histopathological feature in neuromuscular diseases, including amyotrophic lateral sclerosis and inclusion body myopathy, is cytoplasmic aggregation of the RNA-binding protein TDP-43. Although rare mutations in TARDBP-the gene that encodes TDP-43-that lead to protein misfolding often cause protein aggregation, most patients do not have any mutations in TARDBP. Therefore, aggregates of wild-type TDP-43 arise in most patients by an unknown mechanism. Here we show that TDP-43 is an essential protein for normal skeletal muscle formation that unexpectedly forms cytoplasmic, amyloid-like oligomeric assemblies, which we call myo-granules, during regeneration of skeletal muscle in mice and humans. Myo-granules bind to mRNAs that encode sarcomeric proteins and are cleared as myofibres mature. Although myo-granules occur during normal skeletal-muscle regeneration, myo-granules can seed TDP-43 amyloid fibrils in vitro and are increased in a mouse model of inclusion body myopathy. Therefore, increased assembly or decreased clearance of functionally normal myo-granules could be the source of cytoplasmic TDP-43 aggregates that commonly occur in neuromuscular disease.

Regulation of skeletal muscle stem cells by fibroblast growth factors.

Fibroblast growth factors (FGFs) are essential for self-renewal of skeletal muscle stem cells (satellite cells) and required for maintenance and repair of skeletal muscle. Satellite cells express high levels of FGF receptors 1 and 4, low levels of FGF receptor 3, and little or no detectable FGF receptor 2. Of the multiple FGFs that influence satellite cell function in culture, FGF2 and FGF6 are the only members that regulate satellite cell function in vivo by activating ERK MAPK, p38α/ß MAPKs, PI3 kinase, PLCγ and STATs. Regulation of FGF signaling is complex in satellite cells, requiring Syndecan-4, a heparan sulfate proteoglycan, as well as ß1-integrin and fibronectin. During aging, reduced responsiveness to FGF diminishes satellite cell self-renewal, leading to impaired skeletal muscle regeneration and depletion of satellite cells. Mislocalization of ß1-integrin, reductions in fibronectin, and alterations in heparan sulfate content all contribute to reduced FGF responsiveness in satellite cells. How these cell surface proteins regulate satellite cell self-renewal is incompletely understood. Here we summarize the current knowledge, highlighting the role(s) for FGF signaling in skeletal muscle regeneration, satellite cell behavior, and age-induced muscle wasting. Developmental Dynamics 246:359-367, 2017. © 2017 Wiley Periodicals, Inc.

Isolation, Culture, Functional Assays, and Immunofluorescence of Myofiber-Associated Satellite Cells.

Adult skeletal muscle stem cells, termed satellite cells, regenerate and repair the functional contractile cells in adult skeletal muscle called myofibers. Satellite cells reside in a niche between the basal lamina and sarcolemma of myofibers. Isolating single myofibers and their associated satellite cells provides a culture system that partially mimics the in vivo environment. We describe methods for isolating and culturing intact individual myofibers and their associated satellite cells from the mouse extensor digitorum longus muscle. Following dissection and isolation of individual myofibers we provide protocols for myofiber transplantation, satellite cell transfection, immune detection of satellite cell antigens, and assays to examine satellite cell self-renewal and proliferation.

Minimal PU.1 reduction induces a preleukemic state and promotes development of acute myeloid leukemia.

Modest transcriptional changes caused by genetic or epigenetic mechanisms are frequent in human cancer. Although loss or near-complete loss of the hematopoietic transcription factor PU.1 induces acute myeloid leukemia (AML) in mice, a similar degree of PU.1 impairment is exceedingly rare in human AML; yet, moderate PU.1 inhibition is common in AML patients. We assessed functional consequences of modest reductions in PU.1 expression on leukemia development in mice harboring DNA lesions resembling those acquired during human stem cell aging. Heterozygous deletion of an enhancer of PU.1, which resulted in a 35% reduction of PU.1 expression, was sufficient to induce myeloid-biased preleukemic stem cells and their subsequent transformation to AML in a DNA mismatch repair-deficient background. AML progression was mediated by inhibition of expression of a PU.1-cooperating transcription factor, Irf8. Notably, we found marked molecular similarities between the disease in these mice and human myelodysplastic syndrome and AML. This study demonstrates that minimal reduction of a key lineage-specific transcription factor, which commonly occurs in human disease, is sufficient to initiate cancer development, and it provides mechanistic insight into the formation and progression of preleukemic stem cells in AML.

Structured inquiry-based learning: Drosophila GAL4 enhancer trap characterization in an undergraduate laboratory course.

We have developed and tested two linked but separable structured inquiry exercises using a set of Drosophila melanogaster GAL4 enhancer trap strains for an upper-level undergraduate laboratory methods course at Bucknell University. In the first, students learn to perform inverse PCR to identify the genomic location of the GAL4 insertion, using FlyBase to identify flanking sequences and the primary literature to synthesize current knowledge regarding the nearest gene. In the second, we cross each GAL4 strain to a UAS-CD8-GFP reporter strain, and students perform whole mount CNS dissection, immunohistochemistry, confocal imaging, and analysis of developmental expression patterns. We have found these exercises to be very effective in teaching the uses and limitations of PCR and antibody-based techniques as well as critical reading of the primary literature and scientific writing. Students appreciate the opportunity to apply what they learn by generating novel data of use to the wider research community.

Satb1 regulates the self-renewal of hematopoietic stem cells by promoting quiescence and repressing differentiation commitment.

How hematopoietic stem cells (HSCs) coordinate the regulation of opposing cellular mechanisms such as self-renewal and differentiation commitment remains unclear. Here we identified the transcription factor and chromatin remodeler Satb1 as a critical regulator of HSC fate. HSCs lacking Satb1 had defective self-renewal, were less quiescent and showed accelerated lineage commitment, which resulted in progressive depletion of functional HSCs. The enhanced commitment was caused by less symmetric self-renewal and more symmetric differentiation divisions of Satb1-deficient HSCs. Satb1 simultaneously repressed sets of genes encoding molecules involved in HSC activation and cellular polarity, including Numb and Myc, which encode two key factors for the specification of stem-cell fate. Thus, Satb1 is a regulator that promotes HSC quiescence and represses lineage commitment.

α-Aminoxylation of ketones and ß-chloro-α-aminoxylation of enones with TEMPO and chlorocatecholborane.

Oxidation of various cyclic and acyclic ketones under mild conditions with chlorocatecholborane, a bulky pyridine base, and TEMPO to the corresponding α-aminoxylated products in good to excellent yields (52-99%) is described. For enones as substrates, products of a ß-chloro-α-aminoxylation are obtained (70-89%).

Stem and progenitor cells in myelodysplastic syndromes show aberrant stage-specific expansion and harbor genetic and epigenetic alterations.

Even though hematopoietic stem cell (HSC) dysfunction is presumed in myelodysplastic syndrome (MDS), the exact nature of quantitative and qualitative alterations is unknown. We conducted a study of phenotypic and molecular alterations in highly fractionated stem and progenitor populations in a variety of MDS subtypes. We observed an expansion of the phenotypically primitive long-term HSCs (lineage(-)/CD34(+)/CD38(-)/CD90(+)) in MDS, which was most pronounced in higher-risk cases. These MDS HSCs demonstrated dysplastic clonogenic activity. Examination of progenitors revealed that lower-risk MDS is characterized by expansion of phenotypic common myeloid progenitors, whereas higher-risk cases revealed expansion of granulocyte-monocyte progenitors. Genome-wide analysis of sorted MDS HSCs revealed widespread methylomic and transcriptomic alterations. STAT3 was an aberrantly hypomethylated and overexpressed target that was validated in an independent cohort and found to be functionally relevant in MDS HSCs. FISH analysis demonstrated that a very high percentage of MDS HSC (92% ± 4%) carry cytogenetic abnormalities. Longitudinal analysis in a patient treated with 5-azacytidine revealed that karyotypically abnormal HSCs persist even during complete morphologic remission and that expansion of clonotypic HSCs precedes clinical relapse. This study demonstrates that stem and progenitor cells in MDS are characterized by stage-specific expansions and contain epigenetic and genetic alterations.

Catalytic asymmetric thiofunctionalization of unactivated alkenes.

Catalytic asymmetric sulfenylation of double bonds has been achieved using a BINAM-based phosphoramide catalyst and an electrophilic sulfur source. Simple alkenes as well as styrenes afforded sulfenylated tetrahydrofurans and tetrahydropyrans by closure with pendant hydroxyl or carboxyl groups. Intermolecular thiofunctionalizations were also achieved with simple alcohols or carboxylic acids as the nucleophiles.

Synthesis and reactivity of enantiomerically enriched thiiranium ions.

Enantiomerically enriched thiiranium ion 5 has been prepared by silver-assisted ionization of chloro sulfide 4 at -20 degrees C. This thiiranium ion is configurationally stable in solution up to room temperature as demonstrated by the stereospecific capture of the ion by various oxygen- and nitrogen-based nucleophiles. Both isolated olefins and weak Lewis bases can promote the racemization of 5 but these processes can also be suppressed at low temperature. Capture of 5 by methanol is faster than the racemization processes.

Oxidative coupling of arylboronic acids with arenes via Rh-catalyzed direct C-H arylation.

Oxidative coupling of three different arenes and a thiophene derivative with various arylboronic acids was achieved with a [RhCl(C2H4)2]2/P[p-(CF3)C6H4]3 catalyst system. Commercially available 2,2,6,6-tetramethylpiperidine-N-oxyl radical (TEMPO) was used as a stoichiometric oxidant. A 2-pyridyl group and an imine functional group served as ortho-directing groups to mediate the direct C-H arylation by a Rh complex. Moderate to excellent yields were obtained for the coupling reactions.

A simple cobalt catalyst system for the efficient and regioselective cyclotrimerisation of alkynes.

The intermolecular cyclotrimerisation of terminal and internal alkynes can be catalysed by simple cobalt complexes such as a CoBr2(diimine) under mild reaction conditions when treated with zinc and zinc iodide with high regioselectivity in excellent yields.

Stereoselective synthesis of (+)-nephrosteranic acid, (+)-trans-cognac lactone, and (+)-trans-whisky lactone using a chiral cyclohexadienyl Ti compound.

We present the stereoselective transfer of cyclohexadienyl from 3-metalated 1,4-cyclohexadienes to various aldehydes. Lewis-acid-mediated "allylation" of aldehydes by treatment with 3-silylated and 3-stannylated 1,4-cyclohexadienes could not be achieved with high diastereoselectivity. In contrast, cyclohexadienyl titanium compounds reacted with both aliphatic and aromatic aldehydes with good-to-excellent diastereoselectivities. Reaction of a chiral TADDOL-derived (TADDOL, 2,2-dimethyl-alpha,alpha,alpha',alpha'-tetraphenyl-1,3-dioxolandimethanol) cyclohexadienyl Ti derivative with various aldehydes led to the corresponding homoallylic alcohols with excellent diastereo- and enantioselectivities. Lower selectivities were obtained with chiral B-cyclohexadienyldiisopinocampheylborane. The 1,3-cyclohexadienes are very useful building blocks for the preparation of biologically important gamma-butyrolactones. Short efficient syntheses of (+)-nephrosteranic acid, (+)-trans-whisky lactone, and (+)-trans-cognac lactone by desymmetrization of 1,4-cyclohexadiene are described.