Comprehensive Glycomic and Proteomic Analysis of Mouse Striatum and Lateral Hypothalamus Following Repeated Exposures to Cocaine or Methamphetamine.

Substance use disorder is a major concern, with few therapeutic options. Heparan sulfate (HS) and chondroitin sulfate (CS) interact with a plethora of growth factors and their receptors and have profound effects on cellular signaling. Thus, targeting these dynamic interactions might represent a potential novel therapeutic modality. In the present study, we performed mass spectrometry-based glycomic and proteomic analysis to understand the effects of cocaine and methamphetamine (METH) on HS, CS, and the proteome of two brain regions critically involved in drug addiction: the lateral hypothalamus and the striatum. We observed that cocaine and METH significantly alter HS and CS abundances as well as sulfate contents and composition. In particular, repeated METH or cocaine treatments reduced CS 4-O-sulfation and increased CS 6-O-sulfation. Since C4S and C6S exercise differential effects on axon growth, regeneration, and plasticity, these changes likely contribute to drug-induced neural plasticity in these brain regions. Notably, we observed that restoring these alterations by increasing CS 4-0 levels in the lateral hypothalamus by adeno-associated virus delivery of an shRNA to arylsulfatase B (N-acetylgalactosamine-4-sulfatase) ameliorated anxiety and prevented the expression of preference for cocaine in a novelty induced conditioned place preference test during cocaine withdrawal. Finally, proteomics analyses revealed a number of aberrant proteins in METH- and cocaine-treated versus saline-treated mice, including myelin proteolipid protein, calcium/calmodulin-dependent protein kinase type II subunit alpha, synapsin-2, tenascin-R, calnexin, annexin A7, hepatoma-derived growth factor, neurocan, and CSPG5, and oxidative phosphorylation among the top perturbed pathway. Taken together, these data support the role of HS, CS, and associated proteins in stimulants abuse and suggest that manipulation of HSPGs can represent a novel therapeutic strategy.

Single-cell transcriptomic analysis of human pleura reveals stromal heterogeneity and informs in vitro models of mesothelioma.

The pleural lining of the thorax regulates local immunity, inflammation and repair. A variety of conditions, both benign and malignant, including pleural mesothelioma, can affect this tissue. A lack of knowledge concerning the mesothelial and stromal cells comprising the pleura has hampered the development of targeted therapies. Here, we present the first comprehensive single-cell transcriptomic atlas of the human parietal pleura and demonstrate its utility in elucidating pleural biology. We confirm the presence of known universal fibroblasts and describe novel, potentially pleural-specific, fibroblast subtypes. We also present transcriptomic characterisation of multiple in vitro models of benign and malignant mesothelial cells, and characterise these through comparison with in vivo transcriptomic data. While bulk pleural transcriptomes have been reported previously, this is the first study to provide resolution at the single-cell level. We expect our pleural cell atlas will prove invaluable to those studying pleural biology and disease. It has already enabled us to shed light on the transdifferentiation of mesothelial cells, allowing us to develop a simple method for prolonging mesothelial cell differentiation in vitro.

Remdesivir Resistance in Transplant Recipients With Persistent Coronavirus Disease 2019.

New mutations conferring resistance to SARS-CoV-2 therapeutics have important clinical implications. We describe the first cases of an independently acquired V792I RNA-dependent RNA polymerase mutation developing in renal transplant recipients after remdesivir exposure. Our work underscores the need for augmented efforts to identify concerning mutations and address their clinical implications.

GAGrank: Software for Glycosaminoglycan Sequence Ranking Using a Bipartite Graph Model.

The sulfated glycosaminoglycans (GAGs) are long, linear polysaccharide chains that are typically found as the glycan portion of proteoglycans. These GAGs are characterized by repeating disaccharide units with variable sulfation and acetylation patterns along the chain. GAG length and modification patterns have profound impacts on growth factor signaling mechanisms central to numerous physiological processes. Electron activated dissociation tandem mass spectrometry is a very effective technique for assigning the structures of GAG saccharides; however, manual interpretation of the resulting complex tandem mass spectra is a difficult and time-consuming process that drives the development of computational methods for accurate and efficient sequencing. We have recently published GAGfinder, the first peak picking and elemental composition assignment algorithm specifically designed for GAG tandem mass spectra. Here, we present GAGrank, a novel network-based method for determining GAG structure using information extracted from tandem mass spectra using GAGfinder. GAGrank is based on Google's PageRank algorithm for ranking websites for search engine output. In particular, it is an implementation of BiRank, an extension of PageRank for bipartite networks. In our implementation, the two partitions comprise every possible sequence for a given GAG composition and the tandem MS fragments found using GAGfinder. Sequences are given a higher ranking if they link to many important fragments. Using the simulated annealing probabilistic optimization technique, we optimized GAGrank's parameters on ten training sequences. We then validated GAGrank's performance on three validation sequences. We also demonstrated GAGrank's ability to sequence isomeric mixtures using two mixtures at five different ratios.

Adult heart procurement following circulatory determined death from a donor on venous-venous extracorporeal membrane oxygenation: A case report.

Donation after circulatory death in the context of heart transplants is attracting interest and becoming popular in clinical practice. Activity is growing in the United Kingdom, Australia, and the United States. We believe that a prolonged warm ischemic time (time from asystole to reperfusion of the heart on an ex vivo perfusion system) is a primary indicator of adverse outcomes. However, 1.5 liters of blood must be retrieved from the right atrium following sternotomy prolonging warm ischemic time. The patient in the following case report was supported by veno-venous extra-corporeal membrane oxygenation following drowning, further complicated by aspiration-related lung failure. Following circulatory death and a mandatory five-minute stand-off period, 1.5 liters of blood was drained from the circuit as sternotomy began. Surgeons then proceeded to direct procurement of the heart, aiming for least functional warm ischemic time. Following standard implantation, the patient's postoperative recovery has been unremarkable to date.

Predictors of functional outcomes in patients with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent muscular dystrophies characterized by considerable variability in severity, rates of progression and functional outcomes. Few studies follow FSHD cohorts long enough to understand predictors of disease progression and functional outcomes, creating gaps in our understanding, which impacts clinical care and the design of clinical trials. Efforts to identify molecularly targeted therapies create a need to better understand disease characteristics with predictive value to help refine clinical trial strategies and understand trial outcomes. Here we analysed a prospective cohort from a large, longitudinally followed registry of patients with FSHD in the USA to determine predictors of outcomes such as need for wheelchair use. This study analysed de-identified data from 578 individuals with confirmed FSHD type 1 enrolled in the United States National Registry for FSHD Patients and Family members. Data were collected from January 2002 to September 2019 and included an average of 9 years (range 0-18) of follow-up surveys. Data were analysed using descriptive epidemiological techniques, and risk of wheelchair use was determined using Cox proportional hazards models. Supervised machine learning analysis was completed using Random Forest modelling and included all 189 unique features collected from registry questionnaires. A separate medications-only model was created that included 359 unique medications reported by participants. Here we show that smaller allele sizes were predictive of earlier age at onset, diagnosis and likelihood of wheelchair use. Additionally, we show that females were more likely overall to progress to wheelchair use and at a faster rate as compared to males, independent of genetics. Use of machine learning models that included all reported clinical features showed that the effect of allele size on progression to wheelchair use is small compared to disease duration, which may be important to consider in trial design. Medical comorbidities and medication use add to the risk for need for wheelchair dependence, raising the possibility for better medical management impacting outcomes in FSHD. The findings in this study will require further validation in additional, larger datasets but could have implications for clinical care, and inclusion criteria for future clinical trials in FSHD.

The developmental transcriptome for Lytechinus variegatus exhibits temporally punctuated gene expression changes.

Embryonic development is arguably the most complex process an organism undergoes during its lifetime, and understanding this complexity is best approached with a systems-level perspective. The sea urchin has become a highly valuable model organism for understanding developmental specification, morphogenesis, and evolution. As a non-chordate deuterostome, the sea urchin occupies an important evolutionary niche between protostomes and vertebrates. Lytechinus variegatus (Lv) is an Atlantic species that has been well studied, and which has provided important insights into signal transduction, patterning, and morphogenetic changes during embryonic and larval development. The Pacific species, Strongylocentrotus purpuratus (Sp), is another well-studied sea urchin, particularly for gene regulatory networks (GRNs) and cis-regulatory analyses. A well-annotated genome and transcriptome for Sp are available, but similar resources have not been developed for Lv. Here, we provide an analysis of the Lv transcriptome at 11 timepoints during embryonic and larval development. Temporal analysis suggests that the gene regulatory networks that underlie specification are well-conserved among sea urchin species. We show that the major transitions in variation of embryonic transcription divide the developmental time series into four distinct, temporally sequential phases. Our work shows that sea urchin development occurs via sequential intervals of relatively stable gene expression states that are punctuated by abrupt transitions.

Software for Peak Finding and Elemental Composition Assignment for Glycosaminoglycan Tandem Mass Spectra.

Glycosaminoglycans (GAGs) covalently linked to proteoglycans (PGs) are characterized by repeating disaccharide units and variable sulfation patterns along the chain. GAG length and sulfation patterns impact disease etiology, cellular signaling, and structural support for cells. We and others have demonstrated the usefulness of tandem mass spectrometry (MS2) for assigning the structures of GAG saccharides; however, manual interpretation of tandem mass spectra is time-consuming, so computational methods must be employed. In the proteomics domain, the identification of monoisotopic peaks and charge states relies on algorithms that use averagine, or the average building block of the compound class being analyzed. Although these methods perform well for protein and peptide spectra, they perform poorly on GAG tandem mass spectra, because a single average building block does not characterize the variable sulfation of GAG disaccharide units. In addition, it is necessary to assign product ion isotope patterns to interpret the tandem mass spectra of GAG saccharides. To address these problems, we developed GAGfinder, the first tandem mass spectrum peak finding algorithm developed specifically for GAGs. We define peak finding as assigning experimental isotopic peaks directly to a given product ion composition, as opposed to deconvolution or peak picking, which are terms more accurately describing the existing methods previously mentioned. GAGfinder is a targeted, brute force approach to spectrum analysis that uses precursor composition information to generate all theoretical fragments. GAGfinder also performs peak isotope composition annotation, which is typically a subsequent step for averagine-based methods. Data are available via ProteomeXchange with identifier PXD009101.

Glycomic and Proteomic Changes in Aging Brain Nigrostriatal Pathway.

Parkinson's disease (PD) is a neurological disorder characterized by the progressive loss of functional dopaminergic neurons in the nigrostriatal pathway in the brain. Although current treatments provide only symptomatic relief, gene therapy has the potential to slow or halt the degeneration of nigrostriatal dopamine neurons in PD patients. Adeno-associated viruses (AAV) are vectors of choice in gene therapy because of their well-characterized safety and efficacy profiles; however, although gene therapy has been successful in preclinical models of the disease, clinical trials in humans have failed to demonstrate efficacy. Significantly, all primary AAV receptors of the virus are glycans. We thus hypothesize that age related changes in glycan receptors of heparan sulfate (HS) proteoglycans (receptor for rAAV2), and/or N-glycans with terminal galactose (receptor for rAAV9) results in poor adeno-associated virus binding in either the striatum or substantia nigra, or both, affecting transduction and gene delivery. To test our hypothesis we analyzed the striatum and substantia nigra for changes in HS, N-glycans and proteomic signatures in young versus aged rat brain striatum and substantia nigra. We observed different brain region-specific HS disaccharide profiles in aged compared with young adult rats for brain region-specific profiles in striatum versus substantia nigra. We observed brain region- and age-specific N-glycan compositional profiles with respect to the terminal galactose units that serve as receptors for AAV9. We also observed brain region-specific changes in protein expression in the aging nigrostriatal pathway. These studies provide insight into age- and brain region-specific changes in glycan receptors and proteome that will inform design of improved viral vectors for Parkinson Disease (PD) gene therapy.

Proteomic and biological profiling of extracellular vesicles from Alzheimer's disease human brain tissues.

INTRODUCTION: Extracellular vesicles (EVs) from human Alzheimer's disease (AD) biospecimens contain amyloid beta (Aß) peptide and tau. While AD EVs are known to affect brain disease pathobiology, their biochemical and molecular characterizations remain ill defined. METHODS: EVs were isolated from the cortical gray matter of 20 AD and 18 control brains. Tau and Aß levels were measured by immunoassay. Differentially expressed EV proteins were assessed by quantitative proteomics and machine learning. RESULTS: Levels of pS396 tau and Aß1-42 were significantly elevated in AD EVs. High levels of neuron- and glia-specific factors are detected in control and AD EVs, respectively. Machine learning identified ANXA5, VGF, GPM6A, and ACTZ in AD EV compared to controls. They distinguished AD EVs from controls in the test sets with 88% accuracy. DISCUSSION: In addition to Aß and tau, ANXA5, VGF, GPM6A, and ACTZ are new signature proteins in AD EVs.

Inflammatory and Infectious Syndromes Associated With Cancer Immunotherapies.

Immunotherapy using antibodies to immune checkpoint molecules or targeted chimeric antigen receptor-modified T cells (CAR-T cells) represent dramatic advances in cancer treatment. These therapies mediate immune-related adverse events that may mimic or amplify infectious presentations. Checkpoint inhibitor therapy may be associated with diverse irAEs including mild skin, endocrine, and autoimmune manifestations or severe inflammatory processes including colitis, pneumonitis, myocarditis, and shock. CAR-T-cell therapies may induce toxicities including cytokine-release syndrome with fevers and multiorgan dysfunction, CAR-T-cell-related encephalopathy syndrome with altered mental status and neurologic dysfunction, or hemophagocytic lymphohistiocytosis-macrophage-activation syndrome. Infectious risks may relate to prior cancer therapies or to treatments of inflammatory dysregulation, including corticosteroids and inhibitors of tumor necrosis factor-α and interleukin-6. Immune activation may unmask subclinical infections. Clinical approaches must attempt to identify infections in the face of immunotherapy-associated inflammatory processes. Empirical antimicrobial therapies should not be delayed based on the presumption of noninfectious syndromes.

RNA-Seq identifies SPGs as a ventral skeletal patterning cue in sea urchins.

The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning.

Randomized trial of iReadMore word reading training and brain stimulation in central alexia.

Central alexia is an acquired reading disorder co-occurring with a generalized language deficit (aphasia). We tested the impact of a novel training app, 'iReadMore', and anodal transcranial direct current stimulation of the left inferior frontal gyrus, on word reading ability in central alexia. The trial was registered at www.clinicaltrials.gov (NCT02062619). Twenty-one chronic stroke patients with central alexia participated. A baseline-controlled, repeated-measures, crossover design was used. Participants completed two 4-week blocks of iReadMore training, one with anodal stimulation and one with sham stimulation (order counterbalanced between participants). Each block comprised 34 h of iReadMore training and 11 stimulation sessions. Outcome measures were assessed before, between and after the two blocks. The primary outcome measures were reading ability for trained and untrained words. Secondary outcome measures included semantic word matching, sentence reading, text reading and a self-report measure. iReadMore training resulted in an 8.7% improvement in reading accuracy for trained words (95% confidence interval 6.0 to 11.4; Cohen's d = 1.38) but did not generalize to untrained words. Reaction times also improved. Reading accuracy gains were still significant (but reduced) 3 months after training cessation. Anodal transcranial direct current stimulation (compared to sham), delivered concurrently with iReadMore, resulted in a 2.6% (95% confidence interval -0.1 to 5.3; d = 0.41) facilitation for reading accuracy, both for trained and untrained words. iReadMore also improved performance on the semantic word-matching test. There was a non-significant trend towards improved self-reported reading ability. However, no significant changes were seen at the sentence or text reading level. In summary, iReadMore training in post-stroke central alexia improved reading ability for trained words, with good maintenance of the therapy effect. Anodal stimulation resulted in a small facilitation (d = 0.41) of learning and also generalized to untrained items.10.1093/brain/awy138_video1awy138media15796149281001.

Triazolopyridine ethers as potent, orally active mGlu2 positive allosteric modulators for treating schizophrenia.

Triazolopyridine ethers with mGlu2 positive allosteric modulator (PAM) activity are disclosed. The synthesis, in vitro activity, and metabolic stability data for a series of analogs is provided. The effort resulted in the discovery of a potent, selective, and brain penetrant lead molecule BMT-133218 ((+)-7m). After oral administration at 10mg/kg, BMT-133218 demonstrated full reversal of PCP-stimulated locomotor activity and prevented MK-801-induced working memory deficits in separate mouse models. Also, reversal of impairments in executive function were observed in rat set-shifting studies at 3 and 10mg/kg (p.o.). Extensive plasma protein binding as the result of high lipophilicity likely limited activity at lower doses. Optimized triazolopyridine ethers offer utility as mGlu2 PAMs for the treatment of schizophrenia and merit further preclinical investigation.

Do Inflammatory Indices Play a Role in Distinguishing between Uncomplicated and Complicated Diverticulitis?

BACKGROUND: The usefulness of inflammatory indices in assessment of the severity of acute diverticulitis remains unestablished. The aim of this study was to determine whether inflammatory indices and hematological ratios could be utilised to differentiate between uncomplicated and complicated diverticulitis. METHODS: Hematological and inflammatory indices were recorded for each admission with CT confirmed acute diverticulitis (101 complicated, 127 uncomplicated). Cases were divided into training (n = 57) and test sets (n = 171). A classification and regression tree (CART) analysis was employed in the training set to identify optimal inflammatory marker cut-off points associated with complicated diverticulitis. Samples (test set) were then categorized as (A) greater than and (B) less than CART identified cut-off points. The predictive properties of inflammatory marker cut-off points in distinguishing severity of diverticulitis were assessed using a univariate logistic regression analysis, summary receiver operating characteristic curves and confusion matrix generation. RESULTS: C-reactive protein >109 mg/ml (OR 3.07, 95% CI 1.43-6.61, p = 0.004, area under the curve; AUC = 0.64) and white cell lymphocyte ratio (WLR) >17.72 (OR 4.23, 95% CI 1.95-9.17, p < 0.001, AUC = 0.64) were the most accurate parameters in distinguishing complicated and uncomplicated disease. WCC >21 × 109/l (p = 0.02, AUC = 0.60) and lymphocyte count >0.55 × 109/l (p = 0.009, AUC = 0.60) were less accurate. CONCLUSION: Widely used inflammatory indices are useful in the depiction of complicated diverticulitis. The indices cut-off points highlighted in this study should be considered at the time of diagnosis in combination with radiological features of complicated diverticulitis.