Chemical proteomics reveals the target landscape of 1,000 kinase inhibitors.

Medicinal chemistry has discovered thousands of potent protein and lipid kinase inhibitors. These may be developed into therapeutic drugs or chemical probes to study kinase biology. Because of polypharmacology, a large part of the human kinome currently lacks selective chemical probes. To discover such probes, we profiled 1,183 compounds from drug discovery projects in lysates of cancer cell lines using Kinobeads. The resulting 500,000 compound-target interactions are available in ProteomicsDB and we exemplify how this molecular resource may be used. For instance, the data revealed several hundred reasonably selective compounds for 72 kinases. Cellular assays validated GSK986310C as a candidate SYK (spleen tyrosine kinase) probe and X-ray crystallography uncovered the structural basis for the observed selectivity of the CK2 inhibitor GW869516X. Compounds targeting PKN3 were discovered and phosphoproteomics identified substrates that indicate target engagement in cells. We anticipate that this molecular resource will aid research in drug discovery and chemical biology.

Vibrio cholerae pathogenicity island 2 encodes two distinct types of restriction systems.

In response to predation by bacteriophages and invasion by other mobile genetic elements such as plasmids, bacteria have evolved specialized defense systems that are often clustered together on genomic islands. The O1 El Tor strains of Vibrio cholerae responsible for the ongoing seventh cholera pandemic (7PET) contain a characteristic set of genomic islands involved in host colonization and disease, many of which contain defense systems. Notably, Vibrio pathogenicity island 2 contains several characterized defense systems as well as a putative type I restriction-modification (T1RM) system, which, interestingly, is interrupted by two genes of unknown function. Here, we demonstrate that the T1RM system is active, methylates the host genomes of a representative set of 7PET strains, and identify a specific recognition sequence that targets non-methylated plasmids for restriction. We go on to show that the two genes embedded within the T1RM system encode a novel two-protein modification-dependent restriction system related to the GmrSD family of type IV restriction enzymes. Indeed, we show that this system has potent anti-phage activity against diverse members of the Tevenvirinae, a subfamily of bacteriophages with hypermodified genomes. Taken together, these results expand our understanding of how this highly conserved genomic island contributes to the defense of pandemic V. cholerae against foreign DNA. IMPORTANCE: Defense systems are immunity systems that allow bacteria to counter the threat posed by bacteriophages and other mobile genetic elements. Although these systems are numerous and highly diverse, the most common types are restriction enzymes that can specifically recognize and degrade non-self DNA. Here, we show that the Vibrio pathogenicity island 2, present in the pathogen Vibrio cholerae, encodes two types of restriction systems that use distinct mechanisms to sense non-self DNA. The first system is a classical Type I restriction-modification system, and the second is a novel modification-dependent type IV restriction system that recognizes hypermodified cytosines. Interestingly, these systems are embedded within each other, suggesting that they are complementary to each other by targeting both modified and non-modified phages.

A Multi-Epitope Protein for High-Performance Serodiagnosis of Chronic Chagas Disease in ELISA and Lateral Flow Platforms.

We developed a protein to rapidly and accurately diagnose Chagas disease, a life-threatening illness identified by the WHO as a critical worldwide public health risk. Limitations in present day serological tests are complicating the current health situation and contributing to most infected persons being unaware of their condition and therefore untreated. To improve diagnostic testing, we developed an immunological mimic of the etiological agent, Trypanosoma cruzi, by combining ten pathogen-specific epitopes within the beta-barrel protein structure of Thermal Green Protein. The resulting multi-epitope protein, DxCruziV3, displayed high specificity and sensitivity as the antibody capture reagent in an ELISA platform with an analytical sensitivity that exceeds WHO recommendations. Within an immunochromatographic platform, DxCruziV3 showed excellent performance for the point of application diagnosis in a region endemic for multiple diseases, the municipality of Barcelos in the state of Amazonas, Brazil. In total, 167 individuals were rapidly tested using whole blood from a finger stick. As recommended by the Brazilian Ministry of Health, venous blood samples were laboratory tested by conventional assays for comparison. Test results suggest utilizing DxCruziV3 in different assay platforms can confidently diagnose chronic infections by T. cruzi. Rapid and more accurate results will benefit everyone but will have the most noticeable impact in resource-limited rural areas where the disease is endemic.

Frequency and type of cancers in myotonic dystrophy: A retrospective cross-sectional study.

INTRODUCTION/AIMS: Myotonic dystrophies (DMs) are autosomal dominant diseases in which expression of a mutant expanded repeat mRNA leads to abnormal splicing of downstream effector genes thought to be responsible for their multisystem involvement. Cancer risk and cancer-related deaths are increased in DM patients relative to the general population. We aimed at determining the frequency and type of cancers in both DM1 and DM2 vs a non-DM muscular dystrophy cohort. METHODS: A retrospective, cross-sectional study was carried out on patients with genetically confirmed DM1, DM2, facioscapulohumeral muscular dystrophy (FSHD), and oculopharyngeal muscular dystrophy (OPMD) at our institutions from 2000 to 2020. RESULTS: One hundred eighty-five DM1, 67 DM2, 187 FSHD, and 109 OPMD patients were included. Relative to non-DM, DM patients had an increased cancer risk that was independent of age and sex. Specifically, an increased risk of sex-related (ovarian) and non-sex-related (non-melanoma skin, urological, and hematological) cancers was observed in DM1 and DM2, respectively. The length of CTG repeat expansion was not associated with cancer occurrence in the DM1 group. DISCUSSION: In addition to current consensus-based care recommendations, our findings prompt consideration of screening for skin, urological, and hematological cancers in DM2 patients, and screening of ovarian malignancies in DM1 female patients.

Positioning through epistemic cognition in higher education: conceptualising the ways in which academics in a business school view heutagogy.

Teaching and learning in higher education is informed by a multitude of conditioning factors, not least the values systems and outlook of academics. Understanding the epistemological positioning taken by academics in relation to teaching and learning is therefore important if we are to make judgments about how we educate now, and could do so in the future. Developments in educational theory and digital technology have opened-up new possibilities for the ways in which students learn, and to a degree this has been accelerated by the responses from universities to the COVID-19 pandemic. How then should we conceive the future? Heutagogy is one of a number of theoretical approaches that has attracted interest from those who wish to see greater student control over the learning journey- but how widespread is this view amongst academics? This paper reports on a qualitative study in which 12 academics in an English Business School were asked to describe their views on teaching and learning, which we can encapsulate through the concept of epistemic cognition. The findings infer that there is little epistemological underpinning for heutagogy and that if academics are going to innovate, then additional resource and professional development should be put in place to support epistemic reflexivity, and a shift in their epistemological positioning. The paper conceptualises academics' positioning through a typology of epistemic views.

Longitudinal dysphagia assessment in adult patients with nephropathic cystinosis using the Modified Barium Swallow Impairment Profile.

INTRODUCTION/AIMS: Nephropathic cystinosis is a lysosomal storage disorder with known myopathic features, including dysphagia. Evaluation of oropharyngeal swallowing physiology can be standardized using the Modified Barium Swallow Impairment Profile (MBSImP), a validated assessment tool used to analyze and rate swallowing across 17 distinct physiologic domains. Our objective was to better characterize swallowing impairments in nephropathic cystinosis using MBSImP analysis. METHODS: We retrospectively evaluated 40 video fluoroscopic swallowing studies performed at two time points over 1 y in patients with nephropathic cystinosis with various levels of oral and pharyngeal stage dysphagia. Patients completed two self-administered dysphagia outcome measures (the M. D. Anderson Dysphagia Inventory [MDADI] and the 10-item Eating Assessment Tool [EAT-10]). RESULTS: We demonstrated oral stage and pharyngeal stage dysphagia across domains that impacted bolus control, transit, and clearance through both the oral cavity and pharyngeal lumen. Also captured were deficits related to onset and completeness of laryngeal closure that impact airway protection during swallow. There were significant correlations between pharyngeal total score and EAT-10 (r = 0.5, p < 0.001) and between oral total score and EAT-10 (r = 0.7, p < 0.001), MDADI-e (r = -0.6, p < 0.001), MDADI-p (r = -0.5, p < 0.001) and MDADI-c (r = -0.6, p < 0.001). There were no differences in oral or pharyngeal total scores across the 1-y time span. DISCUSSION: This study identifies oral and pharyngeal stage dysphagia as crucial to patients with nephropathic cystinosis and paves the path for future studies of treatment targets.

Parasitic Effects on Electrical Bioimpedance Systems: Critical Review.

Parasitic capacitance represents the main error source in measurement systems based on electrical impedance spectroscopy. The capacitive nature of electrodes' impedance in tetrapolar configuration can give origin to phase errors when electrodes are coupled to parasitic capacitances. Nevertheless, reactive charges in tissue excitation systems are susceptible to instability. Based on such a scenario, mitigating capacitive effects associated with the electrode is a requirement in order to reduce errors in the measurement system. A literature review about the main compensation techniques for parasitic capacitance was carried out. The selected studies were categorized into three groups: (i) compensation in electronic instrumentation; (ii) compensation in measurement processing, and (iii) compensation by negative impedance converters. The three analyzed methods emerged as effective against fixed capacitance. No method seemed capable of mitigating the effects of electrodes' capacitance, that changes in the frequency spectrum. The analysis has revealed the need for a method to compensate varying capacitances, since electrodes' impedance is unknown.

Piper tectoniifolium Kunth: A New Natural Source of the Bioactive Neolignan (-)-Grandisin.

The Piper species are a recognized botanical source of a broad structural diversity of lignans and its derivatives. For the first time, Piper tectoniifolium Kunth is presented as a promising natural source of the bioactive (-)-grandisin. Phytochemical analyses of extracts from its leaves, branches and inflorescences showed the presence of the target compound in large amounts, with leaf extracts found to contain up to 52.78% in its composition. A new HPLC-DAD-UV method was developed and validated to be selective for the identification of (-)-grandisin being sensitive, linear, precise, exact, robust and with a recovery above 90%. The absolute configuration of the molecule was determined by X-ray diffraction. Despite the identification of several enantiomers in plant extracts, the major isolated substance was characterized to be the (-)-grandisin enantiomer. In vascular reactivity tests, it was shown that the grandisin purified from botanical extracts presented an endothelium-dependent vasorelaxant effect with an IC50 of 9.8 ± 1.22 µM and around 80% relaxation at 30 µM. These results suggest that P. tectoniifolium has the potential to serve as a renewable source of grandisin on a large scale and the potential to serve as template for development of new drugs for vascular diseases with emphasis on disorders related to endothelial disfunction.

Screening for Caregiver Burden in the Community: Validation of the European Portuguese Screening Version of the Zarit Burden Interview (ZBI-4).

Objectives: Brief screening instruments are useful in busy clinical practice to identify those requiring further assessment. This study aims to translate and validate a Portuguese version of the four-item Zarit Burden Interview (ZBI-4) to identify caregiver burden in a community-based sample in Northern Portugal.Methods: We collected data from 203 informal caregivers of community-dwellers aged ≥80 years. Internal consistency and factors were measured using Cronbach's alpha. Pearson's correlation was used to examine construct validity against negative and positive aspects of caregiving from the Caregiving Appraisal Scale. Discriminative ability was evaluated from the area under the receiver operating characteristic curve (AUC). Optimal cutoffs were calculated using Youden´s Index.Results: The internal consistency of the Portuguese version of the ZBI-4 was good (alpha = 0.71). Concurrent validity was acceptable, showing strong correlation with the negative (rho = 0.66) and medium correlation with positive (rho = -0.33) aspects of the Caregiving Appraisal Scale. Discriminative accuracy for caregiver burden was also good (AUC = 0.86). Youden's index produced an optimal cutoff of ≥7 points for burden.Conclusions: The Portuguese version of the ZBI-4 screen demonstrates good psychometric properties.Clinical implications: These results show the utility of the Portuguese version of ZBI-4 as a short screen for caregiver burden for use in the community to facilitate rapid screening for this important and complex stressor.

Severe Neurological Toxicity of Immune Checkpoint Inhibitors: Growing Spectrum.

Expanding use of immune-checkpoint inhibitors (ICIs) underscores the importance of accurate diagnosis and timely management of neurological immune-related adverse events (irAE-N). We evaluate the real-world frequency, phenotypes, co-occurring immune-related adverse events (irAEs), and long-term outcomes of severe, grade III to V irAE-N at a tertiary care center over 6 years. We analyze how our experience supports published literature and professional society guidelines. We also discuss these data with regard to common clinical scenarios, such as combination therapy, ICI rechallenge and risk of relapse of irAE-N, and corticosteroid taper, which are not specifically addressed by current guidelines and/or have limited data. Recommendations for management and future irAE-N reporting are outlined. ANN NEUROL 2020;87:659-669.

Retrospective analysis of safety and outcomes of rituximab for myasthenia gravis in patients ≥65 years old.

INTRODUCTION/AIMS: Optimal management of myasthenia gravis (MG) in individuals ≥65 y old is unknown and patient factors may limit therapeutic choices. Safety and efficacy of rituximab in older patients with MG has not been well-studied. METHODS: This retrospective study examined 40 patients (14 patients ≥65 y old) treated with rituximab for MG. The primary efficacy outcome was the proportion of patients reaching "Improved" or better on Myasthenia Gravis Foundation of America (MGFA) Post-Intervention Status (PIS) at 12 mo, compared between younger and older patients. RESULTS: Ninety-two percent of patients ≥65 y old achieved MGFA PIS Improved or better at 12 mo compared to 69% of those <65 y old (P = .11). Median prednisone dose for the cohort decreased in the year following rituximab initiation (20 mg [interquartile range, 10-35] to 10 mg [0-13], P = .01). Non-refractory MG was predictive of favorable outcome, whereas age was not. Serious adverse events (SAEs) were similar between older and younger patients (21.4% vs. 30.8%, P = .715). No patients ≥65 y old required discontinuation of rituximab due to SAE. One death occurred in a patient <65 y old due to systemic inflammatory response syndrome. DISCUSSION: At 12 mo following initiation of rituximab for MG, patients ≥65 y old experienced similarly high rates of improvement in their myasthenic symptoms as younger patients, without an increased risk of experiencing SAEs. Rituximab should be considered in the treatment paradigm in older patients and in non-refractory MG patients of any age.

Compositional flexibility in Li-N-H materials: implications for ammonia catalysis and hydrogen storage.

Li-N-H materials, particularly lithium amide and lithium imide, have been explored for use in a variety of energy storage applications in recent years. Compositional variation within the parent lithium imide, anti-fluorite crystal structure has been related to both its facile storage of hydrogen and impressive catalytic performance for the decomposition of ammonia. Here, we explore the controlled solid-state synthesis of Li-N-H solid-solution anti-fluorite structures ranging from amide-dominated (Li4/3(NH2)2/3(NH)1/3 or Li1.333NH1.667) through lithium imide to majority incorporation of lithium nitride-hydride (Li3.167(NH)0.416N0.584H0.584 or Li3.167NH). Formation of these solid solutions is demonstrated to cause significant changes to the thermal stability and ammonia reactivity of the samples, highlighting the potential use of compositional variation to control the properties of the material in gas storage and catalytic applications.

Injury incidence and characteristics for elite, male, artistic USA gymnastics competitions from 2008 to 2018.

OBJECTIVES: To determine the injury incidence and characteristics for elite, male, artistic USA gymnasts during gymnastics competitions, held in the USA, from 2008 to 2018. METHODS: Injury documentation performed by lead physician and certified athletic trainers at elite junior and senior USA Gymnastics competitions from 2008 to 2018 were reviewed and compiled into an excel database. Injury incidence was computed per 1000 registered gymnasts by competition setting as well as injury location, type, cause, severity, and setting. RESULTS: From 2008 to 2018, 180 injuries were reported in a total of 2102 gymnasts with injury incidence of 85.6 per 1000 gymnasts (95% CI 73.4 to 97.8). The most common injury site was at the ankle (16.7 per 1000 gymnasts, 95% CI 10.9 to 22.4), and muscle strain/rupture/tear was the most common type of injury (28.5 per 1000 gymnasts, 95% CI 21.2 to 35.9). The most common cause was contact with surface (56.1 per 1000 gymnasts, 95% CI 46.1 to 66.2), and the event where most injuries were sustained was the vault (21.9 per 1000 gymnasts, 95% CI 15.4 to 28.4). Incidence of time loss injuries was 38.5 per 1000 gymnasts (95% CI 30.1 to 47.0). Injury incidence was higher during competition (58.5 per 1000 gymnasts, 95% CI 48.2 to 68.8) than during training (27.1 per 1000 RG, 95% CI 19.9 to 34.3; RR 2.16, 95% CI 1.59 to 2.94, p<0.001); injury incidence was greater at Olympic Trials (RR 3.23, 95% CI 1.24 to 8.47, p=0.017) than at National Qualifier meets. We report concussion incidence in gymnastics (5.7 per 1000 gymnasts, 95% CI 2.3 to 9.2). CONCLUSIONS: This is the largest injury study to date for male artistic gymnasts (180 injuries, 2102 gymnasts, 11 years).

HPTLC and FTIR Fingerprinting of Olive Leaves Extracts and ATR-FTIR Characterisation of Major Flavonoids and Polyphenolics.

The aim of this study was to analyse the effect of spontaneous microbial maceration on the release and extraction of the flavonoids and phenolics from olive leaves. Bioprofiling based on thin-layer chromatography effect-directed detection followed by ATR-FTIR spectroscopy proved to be a reliable and convenient method for simultaneous comparison of the extracts. Results show that fermentation significantly enhances the extraction of phenolic compounds and flavonoids. The polyphenolic content was increased from 6.7 µg GAE (gallic acid equivalents) to 25.5 µg GAE, antioxidants from 10.3 µg GAE to 25.3 µg GAE, and flavonoid content from 42 µg RE (rutin equivalents) to 238 µg RE per 20 µL of extract. Increased antioxidant activity of fermented ethyl acetate extracts was attributed to the higher concentration of extracted flavonoids and phenolic terpenoids, while increased antioxidant activity in fermented ethanol extract was due to increased extraction of flavonoids as extraction of phenolic compounds was not improved. Lactic acid that is released during fermentation and glycine present in the olive leaves form a natural deep eutectic solvent (NADES) with significantly increased solubility for flavonoids.

Small Angle X-ray Scattering, Molecular Modeling, and Chemometric Studies from a Thrombin-Like (Lmr-47) Enzyme of Lachesis m. rhombeata Venom.

INTRODUCTION: Snakebite envenomation is considered a neglected tropical disease, and SVTLEs critical elements are involved in serious coagulopathies that occur on envenoming. Although some enzymes of this group have been structurally investigated, it is essential to characterize other proteins to better understand their unique properties such as the Lachesis muta rhombeata 47 kDa (Lmr-47) venom serine protease. METHODS: The structure of Lmr-47 was studied in solution, using SAXS, DLS, CD, and in silico by homology modeling. Molecular docking experiments simulated 21 competitive inhibitors. RESULTS: At pH 8.0, Lmr-47 has an Rg of 34.5 ± 0.6 Å, Dmax of 130 Å, and SR of 50 Å, according to DLS data. Kratky plot analysis indicates a rigid shape at pH 8.0. Conversely, the pH variation does not change the center of mass's intrinsic fluorescence, possibly indicating the absence of fluorescent amino acids in the regions affected by pH variation. CD experiments show a substantially random coiled secondary structure not affected by pH. The low-resolution model of Lmr-47 presented a prolate elongated shape at pH 8.0. Using the 3D structure obtained by molecular modeling, docking experiments identified five good and three suitable competitive inhibitors. CONCLUSION: Together, our work provided insights into the structure of the Lmr-47 and identified inhibitors that may enhance our understanding of thrombin-like family proteins.

Clinical myopathy in patients with nephropathic cystinosis.

BACKGROUND: Nephropathic cystinosis is a lysosomal storage disorder. Patient survival years after renal transplantation has revealed systemic complications including distal myopathy and dysphagia. METHODS: We evaluated 20 adult patients with nephropathic cystinosis using patient-reported and clinical outcome measures. Standard motor measures, video fluoroscopy swallow studies, and tests of respiratory function were performed. We also used Rasch analysis of an initial survey to design a 16-item survey focused on upper and lower extremity function, which was completed by 31 additional patients. RESULTS: Distal myopathy and dysphagia were common in patients with nephropathic cystinosis. Muscle weakness ranges from mild involvement of intrinsic hand muscles to prominent distal greater than proximal weakness and contractures. CONCLUSIONS: In addition to further characterization of underlying dysphagia and muscle weakness, we propose a new psychometrically devised, disease specific, functional outcome measures for distal myopathy in patients with nephropathic cystinosis.

Clinical trial readiness study of distal myopathy and dysphagia in nephropathic cystinosis.

BACKGROUND: Nephropathic cystinosis is a lysosomal storage disorder with late-onset systemic complications, such as myopathy and dysphagia. Currently employed outcome measures lack sensitivity and responsiveness for dysphagia and myopathy, a limitation to clinical trial readiness. METHODS: We evaluated 20 patients with nephropathic cystinosis in two visits over the course of a year to identify outcomes sensitive to detect changes over time. Patients also underwent an expiratory muscle strength training program to assess any effects on aspiration and dysphagia. RESULTS: There were significant differences in the Timed Up and Go Test (TUG) and Timed 25-Foot Walk (25-FW) between baseline and 1-y follow-up (P < .05). Maximum expiratory pressure (MEP) and peak cough flow (PCF) significantly improved following respiratory training (P < .05). CONCLUSIONS: Improved respiratory outcomes may enhance patients ability to expel aspirated material from the airway, stave off pulmonary sequelae associated with chronic aspiration, and yield an overall improvement in physical health and well-being.

COVID-19 in patients with myasthenia gravis.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) has rapidly become a global pandemic, but little is known about its potential impact on patients with myasthenia gravis (MG). METHODS: We studied the clinical course of COVID-19 in five hospitalized patients with autoimmune MG (four with acetylcholine receptor antibodies, one with muscle-specific tyrosine kinase antibodies) between April 1, 2020-April 30-2020. RESULTS: Two patients required intubation for hypoxemic respiratory failure, whereas one required significant supplemental oxygen. One patient with previously stable MG had myasthenic exacerbation. One patient treated with tocilizumab for COVID-19 was successfully extubated. Two patients were treated for MG with intravenous immunoglobulin without thromboembolic complications. DISCUSSION: Our findings suggest that the clinical course and outcomes in patients with MG and COVID-19 are highly variable. Further large studies are needed to define best practices and determinants of outcomes in this unique population.

Comparison of the Accuracy of Short Cognitive Screens Among Adults With Cognitive Complaints in Turkey.

BACKGROUND: Cutoff values of cognitive screen tests vary according to age and educational levels. OBJECTIVE: The objective of this study was to compare the accuracy and determine cutoffs for 3 short cognitive screening instruments: the Mini-Mental State Examination, Montreal Cognitive Assessment (MoCA), and Quick Mild Cognitive Impairment Screen-Turkish version (Qmci-TR), in older adults with low literacy in Turkey. METHODS: In all 321 patients, 133 with subjective cognitive complaints (SCC), 88 amnestic-type mild cognitive impairment (aMCI), and 100 with probable Alzheimer disease (AD) with a median of 5 years education were included. Education and age-specific cutoffs were determined. RESULTS: For the overall population, the Qmci-TR was more accurate than the MoCA in distinguishing between aMCI and AD (area under the curve=0.83 vs. 0.76, P=0.004) and the Qmci-TR and Mini-Mental State Examination were superior to the MoCA in discriminating SCC from aMCI and AD. All instruments had similar accuracy among those with low literacy (primary school and lower educational level or illiterate). CONCLUSIONS: To distinguish between SCC, aMCI, and AD in a sample of older Turkish adults, the Qmci-TR may be preferable. In very low literacy, the choice of the instrument appears less important.