isolateR: an R package for generating microbial libraries from Sanger sequencing data.

MOTIVATION: Sanger sequencing of taxonomic marker genes (e.g. 16S/18S/ITS/rpoB/cpn60) represents the leading method for identifying a wide range of microorganisms including bacteria, archaea, and fungi. However, the manual processing of sequence data and limitations associated with conventional BLAST searches impede the efficient generation of strain libraries essential for cataloging microbial diversity and discovering novel species. RESULTS: isolateR addresses these challenges by implementing a standardized and scalable three-step pipeline that includes: (1) automated batch processing of Sanger sequence files, (2) taxonomic classification via global alignment to type strain databases in accordance with the latest international nomenclature standards, and (3) straightforward creation of strain libraries and handling of clonal isolates, with the ability to set customizable sequence dereplication thresholds and combine data from multiple sequencing runs into a single library. The tool's user-friendly design also features interactive HTML outputs that simplify data exploration and analysis. Additionally, in silico benchmarking done on two comprehensive human gut genome catalogues (IMGG and Hadza hunter-gather populations) showcase the proficiency of isolateR in uncovering and cataloging the nuanced spectrum of microbial diversity, advocating for a more targeted and granular exploration within individual hosts to achieve the highest strain-level resolution possible when generating culture collections. AVAILABILITY AND IMPLEMENTATION: isolateR is available at: https://github.com/bdaisley/isolateR.

Interrogating direct NLRP3 engagement and functional inflammasome inhibition using cellular assays.

As a key regulator of the innate immune system, the NLRP3 inflammasome responds to a variety of environmental insults through activation of caspase-1 and release of the proinflammatory cytokines IL-1ß and IL-18. Aberrant NLRP3 inflammasome function is implicated in numerous inflammatory diseases, spurring drug discovery efforts at NLRP3 as a therapeutic target. A diverse array of small molecules is undergoing preclinical/clinical evaluation with a reported mode of action involving direct modulation of the NLRP3 pathway. However, for a subset of these ligands the functional link between live-cell target engagement and pathway inhibition has yet to be fully established. Herein we present a cohort of mechanistic assays to both query direct NLRP3 engagement in cells, and functionally interrogate different nodes of NLRP3 pathway activity. This system enabled the stratification of potency for five confirmed NLRP3 inhibitors, and identification of two reported NLRP3 inhibitors that failed to demonstrate direct pathway antagonism.

Global synergistic actions to improve brain health for human development.

The global burden of neurological disorders is substantial and increasing, especially in low-resource settings. The current increased global interest in brain health and its impact on population wellbeing and economic growth, highlighted in the World Health Organization's new Intersectoral Global Action Plan on Epilepsy and other Neurological Disorders 2022-2031, presents an opportunity to rethink the delivery of neurological services. In this Perspective, we highlight the global burden of neurological disorders and propose pragmatic solutions to enhance neurological health, with an emphasis on building global synergies and fostering a 'neurological revolution' across four key pillars - surveillance, prevention, acute care and rehabilitation - termed the neurological quadrangle. Innovative strategies for achieving this transformation include the recognition and promotion of holistic, spiritual and planetary health. These strategies can be deployed through co-design and co-implementation to create equitable and inclusive access to services for the promotion, protection and recovery of neurological health in all human populations across the life course.

Antithymocyte globulin for aplastic anemia secondary to pembrolizumab: a case report and review of literature.

Aplastic anemia is a rare but potentially serious complication of immune checkpoint inhibitor therapy. The authors present a case of pembrolizumab-induced aplastic anemia that was refractory to steroids but had some hematologic response to modified-dosing antithymocyte globulin (ATG). This is the first reported case of hematological response to ATG for immune checkpoint inhibitor-induced aplastic anemia and the first reported case of modified ATG dosing for this indication. Cases of immune checkpoint inhibitor-induced aplastic anemia and management options are also summarized. Given the high morbidity and mortality associated with ICI-induced aplastic anemia, more data is necessary to guide evidence-based management recommendations.

Immune checkpoint inhibitors (ICIs) are a form of anticancer therapy that enlists the body's own immune system to fight cancer cells. Although remarkably effective against some types of cancer, ICIs can also cause the augmented immune system to attack noncancer cells, resulting in unwanted off-target side effects. One rare but potentially serious complication of ICIs is aplastic anemia, where the body stops producing enough new blood cells. There is little known about ICI-induced aplastic anemia. The authors present a case of ICI-induced aplastic anemia that did not improve with standard treatment but had some response to antithymocyte globulin, which has not been previously reported. Previously published cases of ICI-induced aplastic anemia and management options are also summarized.

Expression of TCF3 target genes defines a subclass of diffuse large B-cell lymphoma characterized by up-regulation of MYC target genes and poor clinical outcome following R-CHOP therapy.

TCF3 is a lymphopoietic transcription factor that acquires somatic driver mutations in diffuse large B-cell lymphoma (DLBCL). Hypothesizing that expression patterns of TCF3-regulated genes can inform clinical management, we found that unsupervised clustering analysis with 15 TCF3-regulated genes and eight additional ones resolved local DLBCL cases into two main clusters, denoted Groups A and B, of which Group A manifested inferior overall survival (OS, p = 0.0005). We trained a machine learning model to classify samples into the Groups based on expression of the 23 transcripts in an independent validation cohort of 569 R-CHOP-treated DLBCL cases. Group A overlapped with the ABC cell-of-origin subgroup but its prognostic power was superior. GSEA analysis demonstrated asymmetric expression of 30 gene sets between the Groups, pointing to biological differences. We present, validate and make available a novel method to assign DLBCL cases into biologically-distinct groups with divergent OS following R-CHOP therapy.

An IQ Consortium Perspective on Connecting Dissolution Methods to In Vivo Performance: Analysis of an Industrial Database and Case Studies to Propose a Workflow.

Assessment of bioperformance to inform formulation selection and development decisions is an important aspect of drug development. There is high demand in the pharmaceutical industry to develop an efficient and streamlined approach for better understanding and predicting drug product performance to support acceleration of clinical timelines. This manuscript presents an effort from the IQ Formulation Bioperformance Prediction Working Group composed of members from 12 pharmaceutical companies under the IQ Consortium to develop a database around the topic of formulation bioperformance prediction and report findings from the database analysis. Six case studies described in the manuscript demonstrate how bioperformance models were used to predict in vivo performance and to provide guidance addressing questions encountered during oral solid dosage form development. The case studies also described findings of a correlation between in vitro dissolution and in vivo performance and how dissolution data can be incorporated into physiologically based biopharmaceutical modeling. Finally, a workflow for how in vitro dissolution data can be utilized to predict clinical bioperformance of oral solid dosage forms is proposed.

High-sensitivity workflow for LC-MS-based analysis of GalNAc-conjugated oligonucleotides: a case study.

Aim: Mass-selective quantitation is a powerful attribute of LC-MS as a platform for bioanalysis. Here, a sensitive LC-MS approach has been validated for an oligonucleotide having chemical modifications (e.g., N-acetylgalactosamine [GalNAc] conjugated), to distinguish between the conjugated and unconjugated forms of the oligonucleotide, thereby enabling a nuanced view of the pharmacokinetic profile. Results: A high-sensitivity methodology for mass-specific measurement of AZD8233, a GalNAc-conjugated 16-mer oligonucleotide, using LLE-SPE with optimized LC conditions and detection of a low-mass fragment ion was successfully validated in the range of 0.20-100 ng/ml in human plasma. Conclusion: The AZD8233 LC-MS methodology adds valuable insight on the GalNAc linker's in vivo stability to the program and should be broadly applicable to oligonucleotides requiring high sensitivity and mass-selective measurement for quantitative discrimination from metabolites and endogenous interferences.

Validation, Implementation, and Clinical Impact of the Oncomine Myeloid Targeted-Amplicon DNA and RNA Ion Semiconductor Sequencing Assay.

The identification of clinically significant genes recurrently mutated in myeloid malignancies necessitates expanding diagnostic testing with higher throughput, such as targeted next-generation sequencing. We present validation of the Thermo Fisher Oncomine Myeloid Next-Generation Sequencing Panel (OMP), targeting 40 genes and 29 fusion drivers recurrently mutated in myeloid malignancies. The study includes data from a sample exchange between two Canadian hospitals demonstrating high concordance for detection of DNA and RNA aberrations. Clinical validation demonstrates high accuracy, sensitivity, and specificity of the OMP, with a lower limit of detection of 5% for single-nucleotide variants and 10% for insertions/deletions. Prospective sequencing was performed for 187 samples from 168 unique patients presenting with suspected or confirmed myeloid malignancy and other hematological conditions to assess clinical impact of identifying variants. Of detected variants, 48% facilitated or clarified diagnoses, 29% affected prognoses, and 25% had the potential to influence clinical management. Of note, OMP was essential to identifying patients with premalignant clonal states likely contributing to cytopenias. We also found that the detection of even a single variant by the OMP assay, versus 0 variants, was predictive of overall survival, independent of age, sex, or diagnosis (P = 0.03). This study demonstrates that molecular profiling of myeloid malignancies with the OMP represents a promising strategy to advance molecular diagnostics.

Immunotherapy for non-small cell lung cancer (NSCLC), as a stand-alone and in combination therapy.

Non-small cell lung cancer (NSCLC) is of major concern for society as it is associated with high mortality and is one of the most commonly occurring of all cancers. Due to the number of mutational variants and general heterogeneity of this type of cancer, treatment using conventional modalities has been challenging. Therefore, it is important to have improved therapeutic treatments like immunotherapy, that can specifically treat the disease while causing minimal damage to healthy tissue and additionally provide systemic immunity. Cancer vaccines are an important element of cancer immunotherapy and have been approved for treatment of a limited number of cancers, including NSCLC. This article highlights scientific evidence for several therapeutic treatment strategies for NSCLC, alone or in combination, which offers new hope for those suffering. Although cancer vaccines have had some success as a monotherapy, their potential in a combination therapy needs to be critically analyzed for future applications.

Remedial Training of the Less-Impaired Arm in Chronic Stroke Survivors With Moderate to Severe Upper-Extremity Paresis Improves Functional Independence: A Pilot Study.

The ipsilesional arm of stroke patients often has functionally limiting deficits in motor control and dexterity that depend on the side of the brain that is lesioned and that increase with the severity of paretic arm impairment. However, remediation of the ipsilesional arm has yet to be integrated into the usual standard of care for upper limb rehabilitation in stroke, largely due to a lack of translational research examining the effects of ipsilesional-arm intervention. We now ask whether ipsilesional-arm training, tailored to the hemisphere-specific nature of ipsilesional-arm motor deficits in participants with moderate to severe contralesional paresis, improves ipsilesional arm performance and generalizes to improve functional independence. We assessed the effects of this intervention on ipsilesional arm unilateral performance [Jebsen-Taylor Hand Function Test (JHFT)], ipsilesional grip strength, contralesional arm impairment level [Fugl-Meyer Assessment (FM)], and functional independence [Functional independence measure (FIM)] (N = 13). Intervention occurred over a 3 week period for 1.5 h/session, three times each week. All sessions included virtual reality tasks that targeted the specific motor control deficits associated with either left or right hemisphere damage, followed by graded dexterity training in real-world tasks. We also exposed participants to 3 weeks of sham training to control for the non-specific effects of therapy visits and interactions. We conducted five test-sessions: two pre-tests and three post-tests. Our results indicate substantial improvements in the less-impaired arm performance, without detriment to the paretic arm that transferred to improved functional independence in all three posttests, indicating durability of training effects for at least 3 weeks. We provide evidence for establishing the basis of a rehabilitation approach that includes evaluation and remediation of the ipsilesional arm in moderately to severely impaired stroke survivors. This study was originally a crossover design; however, we were unable to complete the second arm of the study due to the COVID-19 pandemic. We report the results from the first arm of the planned design as a longitudinal study.

The Benefits and Costs of Cybersecurity Risk Reduction: A Dynamic Extension of the Gordon and Loeb Model.

This article develops a dynamic extension of the classic model of cybersecurity investment formulated by Gordon and Loeb. In this dynamic model, results are influenced by the rate at which cybersecurity assets depreciate and the rate of return on investment. Depreciation costs are lower in the dynamic model than is implicitly assumed in the classic model, while the rate-of-return threshold is higher. On balance, the user cost of cybersecurity assets is lower in the dynamic model than is implicitly assumed in the classic model. This difference increases the economically efficient size of the cybersecurity system in value terms, increasing the efficient level of risk reduction.

Laboratory practices for manual blood film review: Results of an IQMH patterns of practice survey.

INTRODUCTION: Examination of a blood film is the second most common hematology test, after the complete blood count. Interpretation of a peripheral blood film by trained laboratory professionals provides valuable diagnostic information. The Institute for Quality Management in Healthcare (IQMH) Hematology Scientific Committee developed a questionnaire to gather information regarding current practices for manual blood film review and reporting from laboratories participating in IQMH Morphology proficiency testing (PT) surveys. METHODS: An online survey was distributed to 174 laboratories, 97% submitted results. RESULTS: Of the respondents, the majority (82%) indicated affiliation with small- or medium-sized hospitals (<500 beds). 80% of respondents had core laboratory technologists performing manual blood film reviews, while only 2% utilized dedicated hematology technologists with morphology expertise. All respondents had a policy for manual blood film review by a technologist, 70% did not have blood films reviewed by a senior/charge technologist prior to review by a physician. The majority (88%) of participants included morphological findings in their critical result list; of these, 98% include malaria and 88% include the first-time finding of blasts as critical results. 59% of participants indicated that they have a procedure in place to ensure that interpretation and confirmation of first-time potentially significant morphological findings are available from a physician at all times. CONCLUSION: This survey identified significant variation in blood film review and reporting practices across participating laboratories. The IQMH Hematology Scientific Committee will develop best practice recommendations to guide and standardize practice.

Best Practices in the Development and Validation of Physiologically Based Biopharmaceutics Modeling. A Workshop Summary Report.

This workshop report summarizes the proceedings of Day 2 of a three-day workshop on "Current State and Future Expectations of Translational Modeling Strategies toSupportDrug Product Development, Manufacturing Changes and Controls". From a drug product quality perspective, physiologically based biopharmaceutics modeling (PBBM) is a tool to link variations in the drug product quality attributes to in vivo outcomes enabling the establishment of clinically relevant drug product specifications (CRDPS). Day 2 of the workshop focused on best practices in developing, verifying and validating PBBM. This manuscript gives an overview of podium presentations and summarizes breakout (BO) session discussions related to (1) challenges and opportunities for using PBBM to assess the clinical impact of formulation and manufacturing changes on the in vivo performance of a drug product, (2) best practices to account for parameter uncertainty and variability during model development, (3) best practices in the development, verification and validation of PBBM and (4) opportunities and knowledge gaps related to leveraging PBBM for virtual bioequivalence simulations.

Deficits in Performance on a Mechanically Coupled Asymmetrical Bilateral Task in Chronic Stroke Survivors with Mild Unilateral Paresis.

Typical upper limb-mediated activities of daily living involve coordination of both arms, often requiring distributed contributions to mechanically coupled tasks, such as stabilizing a loaf of bread with one hand while slicing with the other. We sought to examine whether mild paresis in one arm results in deficits in performance on a bilateral mechanically coupled task. We designed a virtual reality-based task requiring one hand to stabilize against a spring load that varies with displacement of the other arm. We recruited 15 chronic stroke survivors with mild hemiparesis and 7 age-matched neurologically intact adults. We found that stroke survivors produced less linear reaching movements and larger initial direction errors compared to controls (p < 0.05), and that contralesional hand performance was less linear than that of ipsilesional hand. We found a hand × group interaction (p < 0.05) for peak acceleration of the stabilizing hand, such that the dominant right hand of controls stabilized less effectively than the nondominant left hand while stroke survivors showed no differences between the hands. Our results indicate that chronic stroke survivors with mild hemiparesis show significant deficits in reaching aspects of bilateral coordination, but no deficits in stabilizing against a movement-dependent spring load in this task.

Motor Deficits in the Ipsilesional Arm of Severely Paretic Stroke Survivors Correlate With Functional Independence in Left, but Not Right Hemisphere Damage.

Chronic stroke survivors with severe contralesional arm paresis face numerous challenges to performing activities of daily living, which largely rely on the use of the less-affected ipsilesional arm. While use of the ipsilesional arm is often encouraged as a compensatory strategy in rehabilitation, substantial evidence indicates that motor control deficits in this arm can be functionally limiting, suggesting a role for remediation of this arm. Previous research has indicated that the nature of ipsilesional motor control deficits vary with hemisphere of damage and with the severity of contralesional paresis. Thus, in order to design rehabilitation that accounts for these deficits in promoting function, it is critical to understand the relative contributions of both ipsilesional and contralesional arm motor deficits to functional independence in stroke survivors with severe contralesional paresis. We now examine motor deficits in each arm of severely paretic chronic stroke survivors with unilateral damage (10 left-, 10 right-hemisphere damaged individuals) to determine whether hemisphere-dependent deficits are correlated with functional independence. Clinical evaluation of contralesional, paretic arm impairment was conducted with the upper extremity portion of the Fugl-Meyer assessment (UEFM). Ipsilesional arm motor performance was evaluated using the Jebsen-Taylor Hand Function Test (JTHFT), grip strength, and ipsilesional high-resolution kinematic analysis during a visually targeted reaching task. Functional independence was measured with the Barthel Index. Functional independence was better correlated with ipsilesional than contralesional arm motor performance in the left hemisphere damage group [JTHFT: [r (10) = -0.73, p = 0.017]; grip strength: [r (10) = 0.64, p = 0.047]], and by contralesional arm impairment in the right hemisphere damage group [UEFM: [r (10) = 0.66, p = 0.040]]. Ipsilesional arm kinematics were correlated with functional independence in the left hemisphere damage group only. Examination of hemisphere-dependent motor correlates of functional independence showed that ipsilesional arm deficits were important in determining functional outcomes in individuals with left hemisphere damage only, suggesting that functional independence in right hemisphere damaged participants was affected by other factors.

Left hemisphere damage produces deficits in predictive control of bilateral coordination.

Previous research has demonstrated hemisphere-specific motor deficits in ipsilesional and contralesional unimanual movements in patients with hemiparetic stroke due to MCA infarct. Due to the importance of bilateral motor actions on activities of daily living, we now examine how bilateral coordination may be differentially affected by right or left hemisphere stroke. To avoid the caveat of simply adding unimanual deficits in assessing bimanual coordination, we designed a unique task that requires spatiotemporal coordination features that do not exist in unimanual movements. Participants with unilateral left (LHD) or right hemisphere damage (RHD) and age-matched controls moved a virtual rectangle (bar) from a midline start position to a midline target. Movement along the long axis of the bar was redundant to the task, such that the bar remained in the center of and parallel to an imaginary line connecting each hand. Thus, to maintain midline position of the bar, movements of one hand closer to or further away from the bar midline required simultaneous, but oppositely directed displacements with the other hand. Our findings indicate that left (LHD), but not right (RHD) hemisphere-damaged patients showed poor interlimb coordination, reflected by significantly lower correlations between displacements of each hand along the bar axis. These left hemisphere-specific deficits were only apparent prior to peak velocity, likely reflecting predictive control of interlimb coordination. In contrast, the RHD group bilateral coordination was not significantly different than that of the control group. We conclude that predictive mechanisms that govern bilateral coordination are dependent on left hemisphere mechanisms. These findings indicate that assessment and training in cooperative bimanual tasks should be considered as part of an intervention framework for post-stroke physical rehabilitation.

IMI - Oral biopharmaceutics tools project - Evaluation of bottom-up PBPK prediction success part 4: Prediction accuracy and software comparisons with improved data and modelling strategies.

Oral drug absorption is a complex process depending on many factors, including the physicochemical properties of the drug, formulation characteristics and their interplay with gastrointestinal physiology and biology. Physiological-based pharmacokinetic (PBPK) models integrate all available information on gastro-intestinal system with drug and formulation data to predict oral drug absorption. The latter together with in vitro-in vivo extrapolation and other preclinical data on drug disposition can be used to predict plasma concentration-time profiles in silico. Despite recent successes of PBPK in many areas of drug development, an improvement in their utility for evaluating oral absorption is much needed. Current status of predictive performance, within the confinement of commonly available in vitro data on drugs and formulations alongside systems information, were tested using 3 PBPK software packages (GI-Sim (ver.4.1), Simcyp® Simulator (ver.15.0.86.0), and GastroPlus™ (ver.9.0.00xx)). This was part of the Innovative Medicines Initiative (IMI) Oral Biopharmaceutics Tools (OrBiTo) project. Fifty eight active pharmaceutical ingredients (APIs) were qualified from the OrBiTo database to be part of the investigation based on a priori set criteria on availability of minimum necessary information to allow modelling exercise. The set entailed over 200 human clinical studies with over 700 study arms. These were simulated using input parameters which had been harmonised by a panel of experts across different software packages prior to conduct of any simulation. Overall prediction performance and software packages comparison were evaluated based on performance indicators (Fold error (FE), Average fold error (AFE) and absolute average fold error (AAFE)) of pharmacokinetic (PK) parameters. On average, PK parameters (Area Under the Concentration-time curve (AUC0-tlast), Maximal concentration (Cmax), half-life (t1/2)) were predicted with AFE values between 1.11 and 1.97. Variability in FEs of these PK parameters was relatively high with AAFE values ranging from 2.08 to 2.74. Around half of the simulations were within the 2-fold error for AUC0-tlast and around 90% of the simulations were within 10-fold error for AUC0-tlast. Oral bioavailability (Foral) predictions, which were limited to 19 APIs having intravenous (i.v.) human data, showed AFE and AAFE of values 1.37 and 1.75 respectively. Across different APIs, AFE of AUC0-tlast predictions were between 0.22 and 22.76 with 70% of the APIs showing an AFE > 1. When compared across different formulations and routes of administration, AUC0-tlast for oral controlled release and i.v. administration were better predicted than that for oral immediate release formulations. Average predictive performance did not clearly differ between software packages but some APIs showed a high level of variability in predictive performance across different software packages. This variability could be related to several factors such as compound specific properties, the quality and availability of information, and errors in scaling from in vitro and preclinical in vivo data to human in vivo behaviour which will be explored further. Results were compared with previous similar exercise when the input data selection was carried by the modeller rather than a panel of experts on each in vitro test. Overall, average predictive performance was increased as reflected in smaller AAFE value of 2.8 as compared to AAFE value of 3.8 in case of previous exercise.