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Integrative and conjugative elements (ICEs) are major drivers of horizontal gene transfer in bacteria. They mediate their own transfer from host cells (donors) to recipients and allow bacteria to acquire new phenotypes, including pathogenic and metabolic capabilities and drug resistances. Streptococcus mutans, a major causative agent of dental caries, contains a putative ICE, TnSmu1, integrated at the 3' end of a leucyl tRNA gene. We found that TnSmu1 is a functional ICE, containing all the genes necessary for ICE function. It excised from the chromosome and excision was stimulated by DNA damage. We identified the DNA junctions generated by excision of TnSmu1, defined the ends of the element, and detected the extrachromosomal circle. We found that TnSmu1 can transfer from S. mutans donors to recipients when co-cultured on solid medium. The presence of TnSmu1 in recipients inhibited successful acquisition of another copy and this inhibition was mediated, at least in part, by the likely transcriptional repressor encoded by the element. Using microscopy to track individual cells, we found that activation of TnSmu1 caused an arrest of cell growth. Our results demonstrate that TnSmu1 is a functional ICE that affects the biology of its host cells.

DNA replication is highly regulated and primarily controlled at the step of initiation. In bacteria, the replication initiator DnaA and the origin of replication oriC are the primary targets of regulation. Perturbations that increase or decrease replication initiation can cause a decrease in cell fitness. We found that multiple mechanisms, including an increase in replication elongation and a decrease in replication initiation, can compensate for lethal over-initiation. We found that in Bacillus subtilis, under conditions of rapid growth, loss of yabA, a negative regulator of replication initiation, caused a synthetic lethal phenotype when combined with the dnaA1 mutation that also causes replication over-initiation. We isolated several classes of suppressors that restored viability to dnaA1 ∆yabA double mutants. Some suppressors (relA, nrdR) stimulated replication elongation. Others (dnaC, cshA) caused a decrease in replication initiation. One class of suppressors decreased replication initiation in the dnaA1 ∆yabA mutant by causing a decrease in the amount of the replicative helicase, DnaC. We found that decreased levels of helicase in otherwise wild-type cells were sufficient to decrease replication initiation during rapid growth, indicating that the replicative helicase is limiting for replication initiation. Our results highlight the multiple mechanisms cells use to regulate DNA replication.

Dystroglycan (DG) requires extensive post-translational processing and O-glycosylation to function as a receptor for extracellular matrix (ECM) proteins containing laminin-G (LG) domains. Matriglycan is an elongated polysaccharide of alternating xylose (Xyl) and glucuronic acid (GlcA) that binds with high affinity to ECM proteins with LG domains and is uniquely synthesized on α-dystroglycan (α-DG) by like-acetylglucosaminyltransferase-1 (LARGE1). Defects in the post-translational processing or O-glycosylation of α-DG that result in a shorter form of matriglycan reduce the size of α-DG and decrease laminin binding, leading to various forms of muscular dystrophy. Previously, we demonstrated that protein O-mannose kinase (POMK) is required for LARGE1 to generate full-length matriglycan on α-DG (~150–250 kDa) (Walimbe et al., 2020). Here, we show that LARGE1 can only synthesize a short, non-elongated form of matriglycan in mouse skeletal muscle that lacks the DG N-terminus (α-DGN), resulting in an ~100–125 kDa α-DG. This smaller form of α-DG binds laminin and maintains specific force but does not prevent muscle pathophysiology, including reduced force production after eccentric contractions (ECs) or abnormalities in the neuromuscular junctions. Collectively, our study demonstrates that α-DGN, like POMK, is required for LARGE1 to extend matriglycan to its full mature length on α-DG and thus prevent muscle pathophysiology.

Alpha-dystroglycan (αDG) is a highly glycosylated cell surface protein with a significant role in cell-to-extracellular matrix interactions in muscle. αDG interaction with extracellular ligands relies on the activity of the LARGE1 glycosyltransferase that synthesizes and extends the heteropolysaccharide matriglycan. Abnormalities in αDG glycosylation and formation of matriglycan are the pathogenic mechanisms for the dystroglycanopathies, a group of congenital muscular dystrophies. Muscle biopsies were evaluated from related 6-week-old Labrador retriever puppies with poor suckling, small stature compared to normal litter mates, bow-legged stance and markedly elevated creatine kinase activities. A dystrophic phenotype with marked degeneration and regeneration, multifocal mononuclear cell infiltration and endomysial fibrosis was identified on muscle cryosections. Single nucleotide polymorphism (SNP) array genotyping data on the family members identified three regions of homozygosity in 4 cases relative to 8 controls. Analysis of whole genome sequence data from one of the cases identified a stop codon mutation in the LARGE1 gene that truncates 40% of the protein. Immunofluorescent staining and western blotting demonstrated the absence of matriglycan in skeletal muscle and heart from affected dogs. Compared to control, LARGE enzyme activity was not detected. This is the first report of a dystroglycanopathy in dogs.

Integrative and conjugative elements (ICEs) are major drivers of horizontal gene transfer in bacteria. They mediate their own transfer from host cells (donors) to recipients and allow bacteria to acquire new phenotypes, including pathogenic and metabolic capabilities and drug resistances. Streptococcus mutans, a major causative agent of dental caries, contains a putative ICE, TnSmu1, integrated at the 3’ end of a leucyl tRNA gene. We found that TnSmu1 is a functional ICE, containing all the genes necessary for ICE function. It excised from the chromosome and excision was stimulated by DNA damage. We identified the DNA junctions generated by excision of TnSmu1, defined the ends of the element, and detected the extrachromosomal circle. We found that TnSmu1 can transfer from S. mutans donors to recipients when co-cultured on solid medium. The presence of TnSmu1 in recipients inhibited successful acquisition of another copy and this inhibition was mediated, at least in part, by the likely transcriptional repressor encoded by the element. Using microscopy to track individual cells, we found that activation TnSmu1 caused an arrest of cell growth. Our results demonstrate that TnSmu1 is a functional ICE that affects the biology of its host cells.

Dystroglycan (DG) requires extensive post-translational processing to function as a receptor for extracellular matrix proteins containing laminin-G-like (LG) domains. Matriglycan is an elongated polysaccharide of alternating xylose and glucuronic acid that is uniquely synthesized on α-dystroglycan (α-DG) by like-acetylglucosaminyltransferase-1 (LARGE1) and binds with high affinity to matrix proteins like laminin. Defects in the post-translational processing of α-DG that result in a shorter form of matriglycan reduce the size of α-DG and decrease laminin binding, leading to various forms of muscular dystrophy. However, little is known regarding mechanisms that generate full-length matriglycan on α-DG (~150-250 kDa). Here, we show that LARGE1 can only synthesize a short, non-elongated form of matriglycan in mouse skeletal muscle that lacks the DG N-terminus (α-DGN), resulting in a ~100-125 kDa α-DG. This smaller form of α-DG binds laminin and maintains specific force but does not prevent muscle pathophysiology, including reduced force induced by eccentric contractions and abnormalities in neuromuscular junctions. Collectively, our study demonstrates that α-DGN is required for LARGE1 to extend matriglycan to its full mature length on α-DG and thus prevent muscle pathophysiology.

Muscular dystrophy is a progressive and ultimately lethal neuromuscular disease due to lack of therapeutic options that restore muscle function. Gene editing and gene transfer hold great promise as therapies for various neuromuscular diseases when administered prior to the onset of severe clinical symptoms. However, the efficacy of these strategies for restoring neuromuscular function and improving survival in the late stages of muscular dystrophy with severe muscle pathophysiology is unknown. Dystroglycanopathies are muscular dystrophies characterized by extensive skeletal muscle degeneration and, in many cases, are accompanied by eye and brain abnormalities. Thus far, mutations in at least eighteen human genes are known to cause dystroglycanopathies, including those in the like-acetylglucosaminyltransferase-1 (LARGE1) gene. LARGE1 encodes a xylosyl- and glucuronosyltransferase that modifies α-dystroglycan (α-DG) with matriglycan, a linear repeating disaccharide of alternating xylose and glucuronic acid that binds to the laminin G-like domains of extracellular matrix proteins with high affinity. Largemyd/Largemyd (myd) mice lack expression of Large1, and exhibit severe skeletal muscle pathophysiology, impaired mobility, and a drastically reduced lifespan (50% survivorship at 35 weeks of age). Here, we show that systemic delivery of AAV2/9 CMV Large1 (AAVLarge1) in >34-week-old myd mice with advanced disease restores matriglycan expression, attenuates skeletal muscle pathophysiology, improves motor and respiratory function, and normalizes systemic metabolism, which collectively and dramatically extends survival. Our results demonstrate that in a mouse model of muscular dystrophy, skeletal muscle function can be restored, illustrating its remarkable plasticity, and that survival can be greatly improved even after the onset of severe skeletal muscle pathophysiology.

Matriglycan is a linear polysaccharide of alternating xylose and glucuronate that binds extracellular matrix proteins and acts as a receptor for Lassa fever virus. LARGE1 synthesizes matriglycan on dystroglycan and mutations in LARGE1 cause muscular dystrophy with abnormal brain development. However, the mechanism of matriglycan polymerization by LARGE1 is unknown. Here, we report the cryo-EM structure of LARGE1. We show that LARGE1 functions as a dimer to polymerize matriglycan by alternating activities between the xylose transferase domain on one protomer and the glucuronate transferase domain on the other protomer. Biochemical analyses using a recombinant Golgi form of dystroglycan reveal that LARGE1 polymerizes matriglycan processively. Our results provide mechanistic insights into LARGE1 function and may facilitate novel therapeutic strategies for treating neuromuscular disorders or arenaviral infections.One-Sentence SummaryDimeric LARGE1 processively polymerizes matriglycan on dystroglycan using orthogonal active sites on alternate protomers.

Conjugative elements are widespread in bacteria and include plasmids and integrative and conjugative elements (ICEs). They transfer from donor to recipient cells via an element-encoded type IV secretion system. These elements interact with and utilize host functions for their lifecycles. We sought to identify essential host genes involved in the lifecycle of the integrative and conjugative element ICEBs1 of Bacillus subtilis. We constructed a library of strains for inducible knockdown of essential B. subtilis genes using CRISPR interference. Each strain expressed one guide RNA in ICEBs1. We induced partial interference of essential genes and identified those that caused an acute defect in acquisition of ICEBs1 by recipient cells. This screen revealed that reducing expression of genes needed for synthesis of cell wall teichoic acids caused a decrease in conjugation. Using three different ways to reduce their synthesis, we found that wall teichoic acids were necessary in both donors and recipients for efficient conjugative transfer of ICEBs1. Further, we found that depletion of wall teichoic acids caused cells involved in ICEBs1 conjugation to die, most likely from damage to the cell envelope. Our results indicate that wall teichoic acids help protect against envelope stress caused by active conjugation machines.

Mutations in multiple genes required for proper O-mannosylation of α-dystroglycan are causal for congenital/limb-girdle muscular dystrophies and abnormal brain development in mammals. Previously, we and others further elucidated the functional O-mannose glycan structure that is terminated by matriglycan, [(-GlcA-β3-Xyl-α3-)n]. This repeating disaccharide serves as a receptor for proteins in the extracellular matrix. Here, we demonstrate in vitro that HNK-1 sulfotransferase (HNK-1ST/carbohydrate sulfotransferase) sulfates terminal glucuronyl residues of matriglycan at the 3-hydroxyl and prevents further matriglycan polymerization by the LARGE1 glycosyltransferase. While α-dystroglycan isolated from mouse heart and kidney is susceptible to exoglycosidase digestion of matriglycan, the functional, lower molecular weight α-dystroglycan detected in brain, where HNK-1ST expression is elevated, is resistant. Removal of the sulfate cap by a sulfatase facilitated dual-glycosidase digestion. Our data strongly support a tissue specific mechanism in which HNK-1ST regulates polymer length by competing with LARGE for the 3-position on the nonreducing GlcA of matriglycan.

Integrative and conjugative elements (ICEs) are mobile genetic elements that reside in a bacterial host chromosome and are prominent drivers of bacterial evolution. They are also powerful tools for genetic analyses and engineering. Transfer of an ICE to a new host involves many steps, including excision from the chromosome, DNA processing and replication, transfer across the envelope of the donor and recipient, processing of the DNA, and eventual integration into the chromosome of the new host (now a stable transconjugant). Interactions between an ICE and its host throughout the life cycle likely influence the efficiencies of acquisition by new hosts. Here, we investigated how different functional modules of two ICEs, Tn916 and ICEBs1, affect the transfer efficiencies into different host bacteria. We constructed hybrid elements that utilize the high-efficiency regulatory and excision modules of ICEBs1 and the conjugation genes of Tn916. These elements produced more transconjugants than Tn916, likely due to an increase in the number of cells expressing element genes and a corresponding increase in excision. We also found that several Tn916 and ICEBs1 components can substitute for one another. Using B. subtilis donors and three Enterococcus species as recipients, we found that different hybrid elements were more readily acquired by some species than others, demonstrating species-specific interactions in steps of the ICE life cycle. This work demonstrates that hybrid elements utilizing the efficient regulatory functions of ICEBs1 can be built to enable efficient transfer into and engineering of a variety of other species.

DNA replication is essential for all living organisms. Several events can disrupt replication, including DNA damage (e.g., pyrimidine dimers, crosslinking) and so-called “roadblocks” (e.g., DNA-binding proteins or transcription). Bacteria have several well-characterized mechanisms for repairing damaged DNA and then restoring functional replication forks. However, little is known about the repair of stalled or arrested replication forks in the absence of chemical alterations to DNA. Using a library of random transposon insertions in Bacillus subtilis, we identified 35 genes that affect the ability of cells to survive exposure to an inhibitor that arrests replication elongation, but does not cause chemical alteration of the DNA. Genes identified include those involved in iron-sulfur homeostasis, cell envelope biogenesis, and DNA repair and recombination. In B. subtilis, and many bacteria, two nucleases (AddAB and RecJ) are involved in early steps in repairing replication forks arrested by chemical damage to DNA and loss of either nuclease causes increased sensitivity to DNA damaging agents. These nucleases resect DNA ends, leading to assembly of the recombinase RecA onto the single-stranded DNA. Notably, we found that disruption of recJ increased survival of cells following replication arrest, indicating that in the absence of chemical damage to DNA, RecJ is detrimental to survival. In contrast, and as expected, disruption of addA decreased survival of cells following replication arrest, indicating that AddA promotes survival. The different phenotypes of addA and recJ mutants appeared to be due to differences in assembly of RecA onto DNA. RecJ appeared to promote too much assembly of RecA filaments. Our results indicate that in the absence of chemical damage to DNA, RecA is dispensable for cells to survive replication arrest and that the stable RecA nucleofilaments favored by the RecJ pathway may lead to cell death by preventing proper processing of the arrested replication fork.

The second step in the biosynthesis of the cellular antioxidant glutathione (GSH) is catalyzed by human glutathione synthetase (hGS), a negatively cooperative homodimer. Patients with mutations in hGS have been reported to exhibit a range of symptoms from hemolytic anemia and metabolic acidosis to neurological disorders and premature death. Several patient mutations occur in the S-loop of hGS, a series of residues near the negatively cooperative γ-GC substrate binding site. Experimental point mutations and molecular dynamic simulations show the S-loop not only binds γ-GC through a salt bridge and multiple hydrogen bonds, but the residues also modulate allosteric communication in hGS. By elucidating the role of S-loop residues in active site structure, substrate binding, and allostery, the atomic level sequence of events that leads to the detrimental effects of hGS mutations in patients are more fully understood., Graphical Abstract Unlabelled Image

DNA replication is a highly regulated process that is primarily controlled at the step of initiation. In the gram-positive bacterium Bacillus subtilis the replication initiator DnaA, is regulated by YabA, which inhibits cooperative binding at the origin. Mutants lacking YabA have increased and asynchronous initiation. We found that under conditions of rapid growth, the dnaA1 mutation that causes replication over-initiation, was synthetic lethal with a deletion of yabA. We isolated several classes of suppressors of the lethal phenotype of the ΔyabA dnaA1 double mutant. Some suppressors (dnaC, cshA) caused a decrease in replication initiation. Others (relA, nrdR) stimulate replication elongation. One class of suppressors decreased levels of the replicative helicase, DnaC, thereby limiting replication initiation. We found that decreased levels of helicase were sufficient to decrease replication initiation under fast growth conditions. Our results highlight the multiple mechanisms cells use to regulate DNA replication.

This article is dedicated to the late long-time Editor-in-Chief of Analytical Biochemistry, William Jakoby. As a graduate student, I remember reading many articles in Analytical Biochemistry and Methods in Enzymology, both volumes that Bill edited. I first met him as a graduate student presenting at the American Society of Biochemistry (and Molecular Biology) meetings. My Ph.D. advisor, Alton Meister, would bring over well-known biochemists and introduce me as Dr. Anderson, leaving me a bit tongue-tied being that I was still actually a humble graduate student! I next met Bill at my first Analytical Biochemistry Executive Editors meeting in San Diego when he was Editor-in-Chief Emeritus; I felt honored to be on the same board with him and serving the journal to which he had brought to prominence. His eyes were piercing and he was so sharp; his knowledge was both broad and deep. Since much of the large body of Bill's research was on glutathione S-transferases, my article focuses on the assay of the enzymes that synthesize glutathione, a substrate for glutathione S-transferases.

Matriglycan [-GlcA-β1,3-Xyl-α1,3-]n serves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetyl-glucosaminyltransferase 1 (LARGE1) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O-Mannose Kinase (POMK), which phosphorylates mannose of core M3 (GalNAc-β1,3-GlcNAc-β1,4-Man) preceding matriglycan synthesis, is required for LARGE1-mediated generation of full-length matriglycan on α-DG (~150 kDa). In the absence of Pomk gene expression in mouse skeletal muscle, LARGE1 synthesizes a very short matriglycan resulting in a ~ 90 kDa α-DG which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE1 directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE1 to elongate matriglycan on α-DG, thereby preventing muscular dystrophy.

Matriglycan [-GlcA-β1,3-Xyl-α1,3-]n serves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetylglucosaminyltransferase-1 (LARGE) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O-Mannose Kinase (POMK), which phosphorylates mannose of core M3 (GalNac-β1,3-GlcNac-β1,4-Man) preceding matriglycan synthesis, is required for LARGE-mediated generation of full-length matriglycan on α-DG (∼150 kDa). In the absence of POMK, LARGE synthesizes a very short matriglycan resulting in a ∼90 kDa α-DG in mouse skeletal muscle which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE to elongate matriglycan on α-DG, thereby preventing muscular dystrophy.

Bifunctional methane activation by M(II)-N2S motif.

Density functional theory and ab initio calculations indicate that nucleophiles can significantly reduce enthalpic barriers to methane C-H bond activation. Valence bond analysis suggests the formation of a two-center three-electron bond as the origin for the catalytic nucleophile effect. A predictive model for methane activation catalysis follows, which suggests that strongly electron-attracting and electron-rich radicals, together with both a negatively charged and strongly electron-donating outer sphere nucleophile, result in the lowest reaction barriers. It is corroborated by the sensitivity of the calculated C-H activation barriers to the external nucleophile and to continuum solvent polarity. More generally, from the present studies, one may propose proteins with hydrophobic active sites, available strong nucleophiles, and hydrogen bond donors as attractive targets for engineering novel methane functionalizing enzymes.

Dermatologists are frequently called to evaluate patients with urticaria in the emergency room, urgent care clinics, and hospitals. Many acute urticarial eruptions will resolve without long-term sequelae; however, there are red flags that clinicians must be aware of. First-line treatment for acute urticaria is regular dose H1 antagonists; however, the dose can be increased up to fourfold for refractory disease. Short courses of corticosteroids should be avoided as rebound urticaria is common upon discontinuation. Urticarial vasculitis presents with persistent, atypical urticaria, burning, and residual lesions. The most common extra-cutaneous manifestation of urticarial vasculitis is musculoskeletal involvement. Schnitzler syndrome is a rare, severe condition but new evidence provides promise for use of biologic therapies. Acute spontaneous urticarial eruptions are commonly encountered in hospitalized patients. This review provides readers with the tools needed to delineate benign eruptions from more concerning conditions.

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px 'Helvetica Neue'} Density functional theory and ab initio calculations indicate that nucleophiles can significantly reduce enthalpic barriers to methane C–H bond activation. Different pieces of evidence point to an electrostatic origin for the nucleophile effect such as the sensitivity of the C–H activation barriers to the external nucleophile and to continuum solvent polarity. The data further imply a transition state with significant charge build-up on the active hydrogen of the hydrocarbon substrate. From the present modeling studies, one may propose proteins with hydrophobic active sites, available nucleophiles, and hydrogen bond donors as attractive targets for the engineering of novel methane functionalizing enzymes.

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px 'Helvetica Neue'} Density functional theory and ab initio calculations indicate that nucleophiles can significantly reduce enthalpic barriers to methane C–H bond activation. Different pieces of evidence point to an electrostatic origin for the nucleophile effect such as the sensitivity of the C–H activation barriers to the external nucleophile and to continuum solvent polarity. The data further imply a transition state with significant charge build-up on the active hydrogen of the hydrocarbon substrate. From the present modeling studies, one may propose proteins with hydrophobic active sites, available nucleophiles, and hydrogen bond donors as attractive targets for the engineering of novel methane functionalizing enzymes.

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance is poorly understood. Here, we identified a conserved and essential protein in pneumococci and related Firmicutes named CcrZ (for Cell Cycle Regulator protein interacting with FtsZ) that couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae directly interacts with the cytoskeleton protein FtsZ to place it in the middle of the newborn cell where the DnaA-bound origin is positioned. Together, this work uncovers a new mechanism for the control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.

Background Pleomorphic dermal sarcoma is the cutaneous variant of undifferentiated pleomorphic sarcoma. It is a rare malignancy of unclear histogenesis; it is a diagnosis of exclusion that requires extensive use of immunohistochemistry to rule out other malignancies. Pleomorphic dermal sarcoma typically presents as a solitary tumor in sun-exposed areas and may have unpredictable clinical behavior, with some tumors associated with metastasis and death. Case presentation We present an unusual case of multifocal pleomorphic dermal sarcoma arising in the areas of alpha-1-antitrypsin deficiency panniculitis in a lung transplant patient. Our patient was a 58-year-old white woman whose initial presentation was consistent with alpha-1-antitrypsin deficiency panniculitis. She then developed extensive multifocal, bleeding, and ulcerated nodules in the areas of the panniculitis. A skin biopsy was consistent with a diagnosis of pleomorphic dermal sarcoma. Her immunosuppressive regimen was decreased, and she was treated with liposomal doxorubicin 40 mg/m2 every 3 weeks with some initial improvement in the size of her tumors. However, soon after beginning therapy, she developed pneumonia and septic shock and ultimately died from multi-organ failure. Conclusions We hypothesize that chronic, multifocal inflammation in the skin in the setting of immunosuppression led to simultaneous, malignant transformation in numerous skin lesions. We discuss the challenges of diagnosing pleomorphic dermal sarcoma, therapeutic options, and stress the need for multidisciplinary management of these cases.

Background α-Dystroglycan is the highly glycosylated component of the dystrophin-glycoprotein complex (DGC) that binds with high-affinity to extracellular matrix (ECM) proteins containing laminin-G-like (LG) domains via a unique heteropolysaccharide [-GlcA-beta1,3-Xyl-alpha1,3-]n called matriglycan. Changes in expression of components of the DGC or in the O-glycosylation of α-dystroglycan result in muscular dystrophy but are also observed in certain cancers. In mice, the loss of either of two DGC proteins, dystrophin or α-sarcoglycan, is associated with a high incidence of rhabdomyosarcoma (RMS). In addition, glycosylation of α-dystroglycan is aberrant in a small cohort of human patients with RMS. Since both the glycosylation of α-dystroglycan and its function as an ECM receptor require over 18 post-translational processing enzymes, we hypothesized that understanding its role in the pathogenesis of RMS requires a complete analysis of the expression of dystroglycan-modifying enzymes and the characterization of α-dystroglycan glycosylation in the context of RMS. Methods A series of cell lines and biopsy samples from human and mouse RMS were analyzed for the glycosylation status of α-dystroglycan and for expression of the genes encoding the responsible enzymes, in particular those required for the addition of matriglycan. Furthermore, the glycosyltransferase LARGE1 was ectopically expressed in RMS cells to determine its effects on matriglycan modifications and the ability of α-dystroglycan to function as a laminin receptor. Results Immunohistochemistry and immunoblotting of a collection of primary RMS tumors show that although α-dystroglycan is consistently expressed and glycosylated in these tumors, α-dystroglycan lacks matriglycan and the ability to bind laminin. Similarly, in a series of cell lines derived from human and mouse RMS, α-dystroglycan lacks matriglycan modification and the ability to bind laminin. RNAseq data from RMS cell lines was analyzed for expression of the genes known to be involved in α-dystroglycan glycosylation, which revealed that, for most cell lines, the lack of matriglycan can be attributed to the downregulation of the dystroglycan-modifying enzyme LARGE1. Ectopic expression of LARGE1 in these cell cultures restored matriglycan to levels comparable to those in muscle and restored high-affinity laminin binding to α-dystroglycan. Conclusions Collectively, our findings demonstrate that a lack of matriglycan on α-dystroglycan is a common feature in RMS due to the downregulation of LARGE1, and that ectopic expression of LARGE1 can restore matriglycan modifications and the ability of α-dystroglycan to function as an ECM receptor.

INTRODUCTION UDP N-acetylglucosamine2-epimerase/N-acetylmannosamine-kinase (GNE) gene mutations can cause mostly autosomal-recessive myopathy with juvenile-onset known as hereditary inclusion-body myopathy (HIBM). METHODS We describe a family of a patient showing an unusual HIBM with both vacuolar myopathy and myositis without quadriceps-sparing, hindering diagnosis. We show how genetic testing with functional assays, clinical transcriptome sequencing (RNA-seq) in particular, helped facilitate both the diagnosis and a better understanding of the genotype-phenotype relationship. RESULTS We identified a novel 7.08 kb pathogenic deletion upstream of GNE using array comparative genomic hybridization (aCGH) and a common Val727Met variant. Using RNA-seq, we found only monoallelic (Val727Met-allele) expression, leading to ~50% GNE reduction in muscle. Importantly, α-dystroglycan is hypoglycosylated in the patient muscle, suggesting HIBM could be a "dystroglycanopathy." CONCLUSIONS Our study shows the importance of considering aCGH for GNE-myopathies, and the potential of RNA-seq for faster, definitive molecular diagnosis of unusual myopathies. Muscle Nerve, 2019.

Dystroglycan (DG) is a highly expressed extracellular matrix receptor that is linked to the cytoskeleton in skeletal muscle. DG is critical for the function of skeletal muscle, and muscle with primary defects in the expression and/or function of DG throughout development has many pathological features and a severe muscular dystrophy phenotype. In addition, reduction in DG at the sarcolemma is a common feature in muscle biopsies from patients with various types of muscular dystrophy. However, the consequence of disrupting DG in mature muscle is not known. Here, we investigated muscles of transgenic mice several months after genetic knockdown of DG at maturity. In our study, an increase in susceptibility to contraction-induced injury was the first pathological feature observed after the levels of DG at the sarcolemma were reduced. The contraction-induced injury was not accompanied by increased necrosis, excitation-contraction uncoupling, or fragility of the sarcolemma. Rather, disruption of the sarcomeric cytoskeleton was evident as reduced passive tension and decreased titin immunostaining. These results reveal a role for DG in maintaining the stability of the sarcomeric cytoskeleton during contraction and provide mechanistic insight into the cause of the reduction in strength that occurs in muscular dystrophy after lengthening contractions.

BACKGROUND Medicaid is often associated with longer hospitalizations and higher in-hospital mortality than other insurance types. OBJECTIVE To characterize the impact of state Medicaid expansion status under the Affordable Care Act (ACA) on payer mix, length of stay (LOS), and in-hospital mortality. DESIGN/SETTING/PATIENTS Retrospective cohort study of general medicine patients discharged from academic medical centers (AMCs) within the University HealthSystem Consortium from October 1, 2012 to September 30, 2015. INTERVENTION/MEASUREMENTS Hospitals were stratified according to state Medicaid expansion status. The proportion of discharges by primary payer, LOS index, and mortality index were compared between Medicaid-expansion and nonexpansion hospitals before and after ACA implementation. ACA implementation was defined as January 1, 2014, for all states except Michigan, New Hampshire, Pennsylvania, and Indiana, which had unique dates of Medicaid expansion. RESULTS We identified 3,144,488 discharges from 156 hospitals in 24 Medicaid-expansion states and Washington, DC, and 1,114,464 discharges from 55 hospitals in 14 nonexpansion states during the study period. Hospitals in Medicaid-expansion states experienced a significant 3.7% increase in Medicaid discharges (P = 0.013) and a 2.9% decrease in uninsured discharges (P < 0.001) after ACA implementation, whereas hospitals in nonexpansion states saw no significant change in payer mix. In a difference-in-differences analysis, the changes in LOS and mortality indices pre- to post-ACA implementation did not differ significantly between hospitals in Medicaid-expansion versus nonexpansion states. CONCLUSIONS The differential shift in payer mix between Medicaid-expansion and nonexpansion states under the ACA did not influence LOS or in-hospital mortality for general medicine patients at AMCs in the United States. Journal of Hospital Medicine 2016. © 2016 Society of Hospital Medicine

Unchecked amino acid accumulation in living cells has the potential to cause stress by disrupting normal metabolic processes. Thus, many organisms have evolved degradation strategies that prevent endogenous accumulation of amino acids. L-2,3-diaminopropionate (Dap) is a non-protein amino acid produced in nature where it serves as a precursor to siderophores, neurotoxins and antibiotics. Dap accumulation in Salmonella enterica was previously shown to inhibit growth by unknown mechanisms. The production of diaminopropionate ammonia-lyase (DpaL) alleviated Dap toxicity in S. enterica by catalyzing the degradation of Dap to pyruvate and ammonia. Here, we demonstrate that Dap accumulation in S. enterica elicits a proline requirement for growth and specifically inhibits coenzyme A and isoleucine biosynthesis. Additionally, we establish that the DpaL-dependent degradation of Dap to pyruvate proceeds through an unbound 2-aminoacrylate (2AA) intermediate, thus contributing to 2AA stress inside the cell. The reactive intermediate deaminase, RidA, is shown to prevent 2AA damage caused by DpaL-dependent Dap degradation by enhancing the rate of 2AA hydrolysis. The results presented herein inform our understanding of the effects Dap has on metabolism in S. enterica, and likely other organisms, and highlight the critical role played by RidA in preventing 2AA stress stemming from Dap detoxification.

Targeting patients with prolonged hospitalizations may represent an effective strategy for reducing average hospital length of stay (LOS). We sought to characterize predictors of prolonged hospitalizations among general medicine patients to guide future improvement efforts. We conducted a retrospective cohort study using administrative data of general medicine patients discharged from inpatient status from our academic medical center between 2012 and 2014. Multivariable logistic regression was performed to assess the association between sociodemographic and clinical variables with prolonged LOS, defined as >21 days. Of 18,363 discharges, 416 (2.3%) demonstrated prolonged LOS. Prolonged hospitalizations accounted for 18.6% of total inpatient days and contributed 0.8 days to an average LOS of 4.8 days during the study period. Prolonged hospitalizations were associated with younger age (odds ratio [OR]: 0.80 per 10-year increase in age, 95% confidence interval [CI]: 0.73–0.87) and Medicaid insurance (OR: 1.99, 95% CI: 1.29–3.05, REF = Medicare). Compared to patients without prolonged LOS, prolonged LOS patients were more likely to have methicillin-resistant Staphylococcus aureus septicemia (OR: 8.83, 95% CI: 1.72–45.36); require a palliative care consult (OR: 4.63, 95% CI: 2.86–7.49), ICU stay (OR: 6.66, 95% CI: 5.22–8.50), or surgery (OR: 5.04, 95% CI: 3.90–6.52); and be discharged to a post–acute-care facility (OR: 10.37, 95% CI: 6.92–15.56). Prolonged hospitalizations in a small proportion of patients were an important contributor to overall LOS and particularly affected Medicaid enrollees with complex hospital stays who were not discharged home. Further studies are needed to determine the reasons for discharge delays in this population. Journal of Hospital Medicine 2015;10:623–626. © 2015 Society of Hospital Medicine

Context Fragmentation in geriatric hip fracture care is a growing concern because of the aging population. Patients with hip fractures at our institution historically were admitted to multiple different services and units, leading to unnecessary variation in inpatient care. Such inconsistency contributed to delays in surgery, discharge, and functional recovery; hospital-acquired complications; failure to adhere to best practices in osteoporosis management; and poor coordination with outpatient practitioners. Objective To describe a stepwise approach to systems redesign for this patient population. Design We designed and implemented a comprehensive geriatric hip fracture program for patients aged 65 years and older at our academic Medical Center in October 2014. Key interventions included admission of all ward-status patients to the Orthopedics Service with hospitalist comanagement; geographic placement on the Orthopedics Unit; and standardized, evidence-based electronic order sets bundling geriatric best practices and a streamlined workflow for discharge planning. Main outcome measures Hospital length of stay. Results We identified 271 admissions among 267 patients between January 1, 2012, and March 31, 2016; of those, 154 were before and 117 were after program implementation. Mean hospital length of stay significantly improved from 6.4 to 5.5 days (p = 0.004). The 30-day all-cause readmission rate and discharge disposition remained stable. The percentage of patients receiving osteoporosis evaluation and treatment increased significantly. The rate of completed 30-day outpatient follow-up also improved. Conclusion Our comprehensive geriatric hip fracture program achieved and sustained gains in the quality and efficiency of care by improving fragmentation in the health care system.

BACKGROUND: Care coordination between adult hospitalists and primary care providers (PCPs) is a critical component of successful transitions of care from hospital to home, yet one that is not well understood. OBJECTIVE: The purpose of this study was to understand the challenges in coordination of care, as well as potential solutions, from the perspective of hospitalists and PCPs in North Carolina. DESIGN AND PARTICIPANTS: We conducted an exploratory qualitative study with 58 clinicians in four hospitalist focus groups (n = 32), three PCP focus groups (n = 19), and one hybrid group with both hospitalists and PCPs (n = 7). APPROACH: Interview guides included questions about care coordination, information exchange, follow-up care, accountability, and medication management. Focus group sessions were recorded, transcribed verbatim, and analyzed in ATLAS.ti. The constant comparative method was used to evaluate differences between hospitalists and PCPs. KEY RESULTS: Hospitalists and PCPs were found to encounter similar care coordination challenges, including (1) lack of time, (2) difficulty reaching other clinicians, (3) lack of personal relationships with other clinicians, (4) lack of information feedback loops, (5) medication list discrepancies, and (6) lack of clarity regarding accountability for pending tests and home health. Hospitalists additionally noted difficulty obtaining timely follow-up appointments for after-hours or weekend discharges. PCPs additionally noted (1) not knowing when patients were hospitalized, (2) not having hospital records for post-hospitalization appointments, (3) difficulty locating important information in discharge summaries, and (4) feeling undervalued when hospitalists made medication changes without involving PCPs. Hospitalists and PCPs identified common themes of successful care coordination as (1) greater efforts to coordinate care for "high-risk" patients, (2) improved direct telephone access to each other, (3) improved information exchange through shared electronic medical records, (4) enhanced interpersonal relationships, and (5) clearly defined accountability. CONCLUSIONS: Hospitalists and PCPs encounter similar challenges in care coordination, yet have important experiential differences related to sending and receiving roles for hospital discharges. Efforts to improve coordination of care between hospitalists and PCPs should aim to understand perspectives of clinicians in each setting.

Mutations in the LARGE gene have been identified in congenital muscular dystrophy (CMD) patients with brain abnormalities. Both LARGE and its paralog, LARGE2 (also referred to as GYLTL1B) are bifunctional glycosyltransferases with xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, and are capable of forming polymers consisting of [-3Xyl-α1,3GlcAβ1-] repeats. LARGE-dependent modification of α-dystroglycan (α-DG) with these polysaccharides is essential for the ability of α-DG to act as a receptor for ligands in the extracellular matrix. Here we report on the endogenous enzymatic activities of LARGE and LARGE2 in mice and humans, using a newly developed assay for GlcA-T activity. We show that normal mouse and human cultured cells have endogenous LARGE GlcA-T, and that this activity is absent in cells from the Large(myd) (Large-deficient) mouse model of muscular dystrophy, as well as in cells from CMD patients with mutations in the LARGE gene. We also demonstrate that GlcA-T activity is significant in the brain, heart, and skeletal muscle of wild-type and Large2(-/-) mice, but negligible in the corresponding tissues of the Large(myd) mice. Notably, GlcA-T activity is substantial, though reduced, in the kidneys of both the Large(myd) and Large2(-/-) mice, consistent with the observation of α-DG/laminin binding in these contexts. This study is the first to test LARGE activity in samples as small as cryosections and, moreover, provides the first direct evidence that not only LARGE, but also LARGE2, is vital to effective functional modification of α-DG in vivo.

Wave attenuation by vegetation is a highly dynamic process and its quantification is important for understanding shore protection potential and modeling coastal hydrodynamics. Data documenting the interactions of Spartina alterniflora , represented by polyolefin tubing, and single- and double-peaked irregular waves were collected in a large-scale laboratory flume. The laboratory provided a controlled environment to evaluate wave attenuation, including the parameters of stem density, submergence, wave height, and peak period. Wave attenuation appeared to be most dependent on stem density and the ratio of stem length to water depth. Wave attention increased slightly with wave height while no clear trend with respect to wave period was seen. Treating double-peaked spectra as superimposed wave systems revealed a preferential dissipation of the higher-frequency wave system relative to the lower-frequency wave system under emergent conditions. Wave energy loss occurred at all frequencies of both spectral types, with dissipation increasing with frequency above the spectral peak. Parameterizing the spectral equilibrium range as a function of frequency showed a steepening of the spectral tail compared to the − 4 power law under emergent conditions. An empirical relationship defining the bulk drag coefficient for S. alterniflora as a function of the stem Reynolds number is found to serve as a first estimate for engineering applications.

Dissecting Dystrophies Defects in α-dystroglycan lead to various congenital muscular dystrophies, and its ability to bind to extracellular matrix (ECM) is dependent on formation of a specific O-linked sugar structure. Previous efforts to understand the molecular mechanisms underlying α-dystroglycan's ability to bind to the ECM led to the identification of a phosphorylated O -mannosyl trisaccharide on α-dystroglycan and to the demonstration that addition of this residue is a prerequisite for formation of the ligand-binding motif. However, the biosynthetic pathway that leads to production of the phosphorylated O -mannosyl glycan has not been delineated. Yoshida-Moriguchi et al. (p. 896 , published online 8 August) elucidate the functions of three genes recently found to cause dystroglycan-related disorders and explain the defects in the production of the phosphorylated O -mannosyl glycan that underlie the pathologies of patients with the relevant mutations.

The ‘pseudokinase’ SgK196 is a protein O-mannose kinase (POMK) that catalyzes an essential phosphorylation step during biosynthesis of the laminin-binding glycan on α-dystroglycan. However, the catalytic mechanism underlying this activity remains elusive. Here we present the crystal structure of Danio rerio POMK in complex with Mg2+ ions, ADP, aluminum fluoride, and the GalNAc-β3-GlcNAc-β4-Man trisaccharide substrate, thereby providing a snapshot of the catalytic transition state of this unusual kinase. The active site of POMK is established by residues located in non-canonical positions and is stabilized by a disulfide bridge. GalNAc-β3-GlcNAc-β4-Man is recognized by a surface groove, and the GalNAc-β3-GlcNAc moiety mediates the majority of interactions with POMK. Expression of various POMK mutants in POMK knockout cells further validated the functional requirements of critical residues. Our results provide important insights into the ability of POMK to function specifically as a glycan kinase, and highlight the structural diversity of the human kinome. DOI: http://dx.doi.org/10.7554/eLife.22238.001

International audience; Human glutathione synthetase (hGS) catalyzes the second ATP-dependent step in the biosynthesis of glutathione (GSH) and is negatively cooperative to the γ-glutamyl substrate. The hGS active site is composed of three highly conserved catalytic loops, notably the alanine rich A-loop. Experimental and computational investigations of the impact of mutation of Asp458 are reported, and thus the role of this A-loop residue on hGS structure, activity, negativity cooperativity and stability is defined. Several Asp458 hGS mutants (D458A, D458N and D458R) were constructed using site-directed mutagenesis and their activities determined (10%, 15% and 7% of wild-type hGS, respectively). The Michaelis-Menten constant (K(m)) was determined for all three substrates (glycine, GAB and ATP): glycine K(m) increased by 30-115-fold, GAB K(m) decreased by 8-17-fold, and the ATP K(m) was unchanged. All Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type hGS, as determined by differential scanning calorimetry. The findings indicate that Asp458 is essential for hGS catalysis and that it impacts the allostery of hGS.

Background. To investigate whether human papilloma virus (HPV)-specific immune mechanisms can result in tumor clearance, we have created HPV+ and HPV− tonsil cells that form squamous cancers in immune-competent mice. Here, we determine that an immune-specific response can clear HPV+ tumor cells and the cellular requirements to mediate this tumor clearance. Methods. Through the benefit of this model, we use in vitro and in vivo methods to better understand how HPV+ cells are rejected. Results. The data show that an in vivo antigen-specific antitumor response is generated to HPV+ transformed cells and that this response requires CD4+ and CD8+ cells to mount this antitumor response. Conclusion. The findings from this preclinical model will have implications not only in understanding human disease but also as a valuable model for testing immunotherapeutic strategies for HPV+ head and neck cancer. © 2009 Wiley Periodicals, Inc. Head Neck, 2009

The human papillomavirus (HPV) oncogene E6 has been shown to perform multiple functions (p53 degradation, telomerase activation, etc.) that play a role in oncogenic transformation. Beyond known E6 functions, an undefined mechanism that allows cellular invasion requires the E6 PDZ binding motif (PDZBM). Here, we show that HPV type 16 (HPV16) E6 interacts with and induces loss of a protein tyrosine phosphatase (PTPN13) in a PDZBM-dependent manner. PTPN13 loss induced either by the presence of E6 or by a short hairpin RNA strategy allows for anchorage-independent growth (AIG) and synergy with a known oncogene, Ras v12 , resulting in invasive growth in vivo. Restoring PTPN13 expression reverses AIG in cells lacking PTPN13. A genomic analysis of colorectal carcinoma has identified an association between PTPN13 loss-of-function mutations and aberrant Ras signaling. Our findings support this correlation and provide methods for further evaluation of the mechanisms by which PTPN13 loss/Ras expression leads to invasive growth, the results of which will be important for treatment of HPV-related and non-HPV cancer.