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Rotavirus (RV) is a primary cause of malabsorptive diarrhea in children and is associated with significant morbidity and mortality, especially in developing countries. The use of antibiotics exacerbates intestinal microbial imbalance and results in the persistence of RV-induced diarrhea.

Human rotavirus (HRV) is a leading cause of morbidity and mortality in children, especially in developing countries. Malnutrition is prevalent in these countries, which may contribute to the decreased oral vaccine efficacy, posing a concern for global health. Neonatal gnotobiotic (Gn) pigs closely resemble human infants in their anatomy, physiology, and outbred status and are a unique model to investigate malnutrition, oral live attenuated HRV (AttHRV) vaccination, and subsequent virulent HRV (VirHRV) challenge. We evaluated the impact of malnutrition on AttHRV vaccine efficacy and B cell immune responses in neonatal germfree (GF) or Gn pigs transplanted with human infant fecal microbiota (HIFM). Pigs were fed either deficient or sufficient bovine milk diets. Malnutrition did not significantly affect the serum and intestinal contents total or HRV-specific IgG and IgA antibody titers pre VirHRV challenge. However, HRV-specific IgG and IgA antibody secreting cells (ASCs) were reduced in blood or intestinal tissues following AttHRV vaccination and pre VirHRV challenge in deficient HIFM transplanted pigs. Furthermore, post-VirHRV challenge, deficient HIFM pigs had decreased total Ig and HRV-specific IgG and IgA antibody titers in serum or intestinal contents, in addition to decreased HRV-specific IgG and IgA ASCs in blood and ileum, compared with sufficient HIFM pigs. Our results indicate that deficient diet impairs B cell mucosal, and systemic immune responses following HRV vaccination, and challenge. The impaired immune responses contributed to the decreased protective efficacy of the AttHRV vaccine, suggesting that malnutrition may significantly reduce the effectiveness of oral HRV vaccines in children in developing countries.

Human rotavirus (HRV) infection is a major cause of viral gastroenteritis in young children worldwide. Current oral vaccines perform poorly in developing countries where efficacious vaccines are needed the most. Therefore, an alternative affordable strategy to enhance efficacy of the current RV vaccines is necessary. This study evaluated the effects of colonization of neonatal gnotobiotic (Gn) pigs with Escherichia coli Nissle (EcN) 1917 and Lacticaseibacillus rhamnosus GG (LGG) probiotics on immunogenicity and protective efficacy of oral attenuated (Att) HRV vaccine. EcN-colonized pigs had reduced virulent HRV (VirHRV) shedding and decreased diarrhea severity compared with the LGG-colonized group. They also had enhanced HRV-specific IgA antibody titers in serum and antibody secreting cell numbers in tissues pre/post VirHRV challenge, HRV-specific IgA antibody titers in intestinal contents, and B-cell subpopulations in tissues post VirHRV challenge. EcN colonization also enhanced T-cell immune response, promoted dendritic cells and NK cell function, reduced production of proinflammatory cytokines/Toll like receptor (TLR), and increased production of immunoregulatory cytokines/TLR expression in various tissues pre/post VirHRV challenge. Thus, EcN probiotic adjuvant with AttHRV vaccine enhances the immunogenicity and protective efficacy of AttHRV to a greater extent than LGG and it can be used as a safe and economical oral vaccine adjuvant.

Abstract Background Human rotavirus (HRV) is a major cause of viral gastroenteritis in infants; particularly in developing countries where malnutrition is prevalent. Malnutrition perturbs the infant gut microbiota leading to sub-optimal functioning of the immune system and further predisposing infants to enteric infections. Therefore, we hypothesized that malnutrition exacerbates rotavirus disease severity in infants. Methods In the present study, we used a neonatal germ free (GF) piglets transplanted with a two-month-old human infant’s fecal microbiota (HIFM) on protein deficient and sufficient diets. We report the effects of malnourishment on the HRV infection and the HIFM pig microbiota in feces, intestinal and systemic tissues, using MiSeq 16S gene sequencing (V4-V5 region). Results Microbiota analysis indicated that the HIFM transplantation resulted in a microbial composition in pigs similar to that of the original infant feces. This model was then used to understand the interconnections between microbiota diversity, diet, and HRV infection. Post HRV infection, HIFM pigs on the deficient diet had lower body weights, developed more severe diarrhea and increased virus shedding compared to HIFM pigs on sufficient diet. However, HRV induced diarrhea and shedding was more pronounced in non-colonized GF pigs compared to HIFM pigs on either sufficient or deficient diet, suggesting that the microbiota alone moderated HRV infection. HRV infected pigs on sufficient diet showed increased microbiota diversity in intestinal tissues; whereas, greater diversity was observed in systemic tissues of HRV infected pigs fed with deficient diet. Conclusions These results suggest that proper nourishment improves the microbiota quality in the intestines, alleviates HRV disease and lower probability of systemic translocation of potential opportunistic pathogens/pathobionts. In conclusion, our findings further support the role for microbiota and proper nutrition in limiting enteric diseases.

ABSTRACT Malnutrition affects millions of children in developing countries, compromising immunity and contributing to increased rates of death from infectious diseases. Rotavirus is a major etiological agent of childhood diarrhea in developing countries, where malnutrition is prevalent. However, the interactions between the two and their combined effects on immune and intestinal functions are poorly understood. In this study, we used neonatal gnotobiotic (Gn) pigs transplanted with the fecal microbiota of a healthy 2-month-old infant (HIFM) and fed protein-deficient or -sufficient bovine milk diets. Protein deficiency induced hypoproteinemia, hypoalbuminemia, hypoglycemia, stunting, and generalized edema in Gn pigs, as observed in protein-malnourished children. Irrespective of the diet, human rotavirus (HRV) infection early, at HIFM posttransplantation day 3 (PTD3), resulted in adverse health effects and higher mortality rates (45 to 75%) than later HRV infection (PTD10). Protein malnutrition exacerbated HRV infection and affected the morphology and function of the small intestinal epithelial barrier. In pigs infected with HRV at PTD10, there was a uniform decrease in the function and/or frequencies of natural killer cells, plasmacytoid dendritic cells, and CD103+ and apoptotic mononuclear cells and altered gene expression profiles of intestinal epithelial cells (chromogranin A, mucin 2, proliferating cell nuclear antigen, SRY-Box 9, and villin). Thus, we have established the first HIFM-transplanted neonatal pig model that recapitulates major aspects of protein malnutrition in children and can be used to evaluate physiologically relevant interventions. Our findings provide an explanation of why nutrient-rich diets alone may lack efficacy in malnourished children. IMPORTANCE Malnutrition and rotavirus infection, prevalent in developing countries, individually and in combination, affect the health of millions of children, compromising their immunity and increasing the rates of death from infectious diseases. However, the interactions between the two and their combined effects on immune and intestinal functions are poorly understood. We have established the first human infant microbiota-transplanted neonatal pig model of childhood malnutrition that reproduced the impaired immune, intestinal, and other physiological functions seen in malnourished children. This model can be used to evaluate relevant dietary and other health-promoting interventions. Our findings provide an explanation of why adequate nutrition alone may lack efficacy in malnourished children.

Human rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in low- and middle-income countries (LMICs), where HRV vaccine efficacy is low. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of enteric diseases in humans. However, repeated doses of EcN are required to achieve maximum beneficial effects. Administration of EcN on a microsphere biofilm could increase probiotic stability and persistence, thus maximizing health benefits without repeated administrations. Our aim was to investigate immune enhancement by the probiotic EcN adhered to a dextranomar microsphere biofilm (EcN biofilm) in a neonatal, malnourished piglet model transplanted with human infant fecal microbiota (HIFM) and infected with rotavirus. To create malnourishment, pigs were fed a reduced amount of bovine milk. Decreased HRV fecal shedding and protection from diarrhea were evident in the EcN biofilm treated piglets compared with EcN suspension and control groups. Moreover, EcN biofilm treatment enhanced natural killer cell activity in blood mononuclear cells (MNCs). Increased frequencies of activated plasmacytoid dendritic cells (pDC) in systemic and intestinal tissues and activated conventional dendritic cells (cDC) in blood and duodenum were also observed in EcN biofilm as compared with EcN suspension treated pigs. Furthermore, EcN biofilm treated pigs had increased frequencies of systemic activated and resting/memory antibody forming B cells and IgA+ B cells in the systemic tissues. Similarly, the mean numbers of systemic and intestinal HRV-specific IgA antibody secreting cells (ASCs), as well as HRV-specific IgA antibody titers in serum and small intestinal contents, were increased in the EcN biofilm treated group. In summary EcN biofilm enhanced innate and B cell immune responses after HRV infection and ameliorated diarrhea following HRV challenge in a malnourished, HIFM pig model.

Disorders of consciousness (DoC) resulting from severe acute brain injuries may prompt clinicians and surrogate decision makers to consider withdrawal of life-sustaining treatment (WLST) if the neurologic prognosis is poor. Recent guidelines suggest, however, that clinicians should avoid definitively concluding a poor prognosis prior to 28 days post injury, as patients may demonstrate neurologic recovery outside the acute time period. This practice may increase the frequency with which clinicians consider the option of delayed WLST (D-WLST), namely, WLST that would occur after hospital discharge, if the patient's recovery trajectory ultimately proves inconsistent with an acceptable quality of life. However acute care clinicians are often uncertain about what D-WLST entails and therefore find it difficult to properly counsel surrogates about this option. Here, we describe practical and theoretical considerations relevant to D-WLST. We first identify post-acute-care facilities to which patients with DoC are likely to be discharged and where D-WLST may be considered. Second, we describe how clinicians and surrogates may determine the appropriate timing of D-WLST. Third, we outline how D-WLST is practically implemented. And finally, we discuss psychosocial barriers to D-WLST, including the regret paradox, in which surrogates of patients who do not recover to meet preestablished goals frequently choose not to ultimately pursue D-WLST. Together, these practical, logistic, and psychosocial factors must be considered when potentially deferring WLST to the post-acute-care setting to optimize neurologic recovery for patients, avoid prolonged undue suffering, and promote informed and shared decision-making between clinicians and surrogates., (© 2024. The Author(s).)

Background: Although magnetic resonance imaging, particularly diffusion-weighted imaging, has increasingly been used as part of a multimodal approach to prognostication in patients who are comatose after cardiac arrest, the performance of quantitative analysis of apparent diffusion coefficient (ADC) maps, as compared to standard radiologist impression, has not been well characterized. This retrospective study evaluated quantitative ADC analysis to the identification of anoxic brain injury by diffusion abnormalities on standard clinical magnetic resonance imaging reports., Methods: The cohort included 204 previously described comatose patients after cardiac arrest. Clinical outcome was assessed by (1) 3-6 month post-cardiac-arrest cerebral performance category and (2) coma recovery to following commands. Radiological evaluation was obtained from clinical reports and characterized as diffuse, cortex only, deep gray matter structures only, or no anoxic injury. Quantitative analyses of ADC maps were obtained in specific regions of interest (ROIs), whole cortex, and whole brain. A subgroup analysis of 172 was performed after eliminating images with artifacts and preexisting lesions., Results: Radiological assessment outperformed quantitative assessment over all evaluated regions (area under the curve [AUC] 0.80 for radiological interpretation and 0.70 for the occipital region, the best performing ROI, p = 0.011); agreement was substantial for all regions. Radiological assessment still outperformed quantitative analysis in the subgroup analysis, though by smaller margins and with substantial to near-perfect agreement. When assessing for coma recovery only, the difference was no longer significant (AUC 0.83 vs. 0.81 for the occipital region, p = 0.70)., Conclusions: Although quantitative analysis eliminates interrater differences in the interpretation of abnormal diffusion imaging and avoids bias from other prediction modalities, clinical radiologist interpretation has a higher predictive value for outcome. Agreement between radiological and quantitative analysis improved when using high-quality scans and when assessing for coma recovery using following commands. Quantitative assessment may thus be more subject to variability in both clinical management and scan quality than radiological assessment., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature and Neurocritical Care Society.)

Disorders of consciousness represent altered mental status at its most severe, comprising a continuum between coma, the vegetative state/unresponsive wakefulness syndrome, the minimally conscious state, and emergence from the minimally conscious state. Patients often transition between these levels throughout their recovery, and determining a patient's current level can be challenging, particularly in the acute care setting. Although healthcare providers have classically relied on a bedside neurological exam or the Glasgow Coma Scale to aid with assessment of consciousness, studies have identified multiple limitations of doing so. Neurobehavioral assessment measures, such as the Coma Recovery Scale-Revised, have been developed to address these shortcomings. Each behavioral metric has strengths as well as weaknesses when applied in the acute care setting. In this review, we appraise common assessment approaches, outline alternative measures for fine-tuning these assessments in the acute care setting, and highlight strategies for implementing these practices in an interdisciplinary manner., Competing Interests: None declared., (Thieme. All rights reserved.)

Background: Neuroprognostication for disorders of consciousness (DoC) after severe acute brain injury is a major challenge, and the conventional clinical approach struggles to keep pace with a rapidly evolving literature. Lacking specialization, and fragmented between providers, conventional neuroprognostication is variable, frequently incongruent with guidelines, and prone to error, contributing to avoidable mortality and morbidity., Recent Findings: We review the limitations of the conventional approach to neuroprognostication and DoC care, and propose a paradigm entitled the Recovery of Consciousness Via Evidence-Based Medicine and Research (RECOVER) program to address them. The aim of the RECOVER program is to provide specialized, comprehensive, and longitudinal care that synthesizes interdisciplinary perspectives, provides continuity to patients and families, and improves the future of DoC care through research and education., Implications for Practice: This model, if broadly adopted, may help establish neuroprognostication as a new subspecialty that improves the care of this vulnerable patient population., Competing Interests: The authors report no relevant disclosures. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/cp., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

The advent of neurotechnologies including advanced functional magnetic resonance imaging and electroencephalography to detect states of awareness not detectable by traditional bedside neurobehavioral techniques (i.e., covert consciousness) promises to transform neuroscience research and clinical practice for patients with brain injury. As these interventions progress from research tools into actionable, guideline-endorsed clinical tests, ethical guidance for clinicians on how to responsibly communicate the sensitive results they yield is crucial yet remains underdeveloped. Drawing on insights from empirical and theoretical neuroethics research and our clinical experience with advanced neurotechnologies to detect consciousness in behaviorally unresponsive patients, we critically evaluate ethical promises and perils associated with disclosing the results of clinical covert consciousness assessments and describe a semistructured approach to responsible data sharing to mitigate potential risks., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature and Neurocritical Care Society.)

Importance: Among the most impactful neurologic assessments is that of neuroprognostication, defined here as the prediction of neurologic recovery from disorders of consciousness caused by severe, acute brain injury. Across a range of brain injury etiologies, these determinations often dictate whether life-sustaining treatment is continued or withdrawn; thus, they have major implications for morbidity, mortality, and health care costs. Neuroprognostication relies on a diverse array of tests, including behavioral, radiologic, physiological, and serologic markers, that evaluate the brain's functional and structural integrity., Observations: Prognostic markers, such as the neurologic examination, electroencephalography, and conventional computed tomography and magnetic resonance imaging (MRI), have been foundational in assessing a patient's current level of consciousness and capacity for recovery. Emerging techniques, such as functional MRI, diffusion MRI, and advanced forms of electroencephalography, provide new ways of evaluating the brain, leading to evolving schemes for characterizing neurologic function and novel methods for predicting recovery., Conclusions and Relevance: Neuroprognostic markers are rapidly evolving as new ways of assessing the brain's structural and functional integrity after brain injury are discovered. Many of these techniques remain in development, and further research is needed to optimize their prognostic utility. However, even as such efforts are underway, a series of promising findings coupled with the imperfect predictive value of conventional prognostic markers and the high stakes of these assessments have prompted clinical guidelines to endorse emerging techniques for neuroprognostication. Thus, clinicians have been thrust into an uncertain predicament in which emerging techniques are not yet perfected but too promising to ignore. This review illustrates the current, and likely future, landscapes of prognostic markers. No matter how much prognostic markers evolve and improve, these assessments must be approached with humility and individualized to reflect each patient's values.

There is a clinical need for more accurate diagnosis and prognostication in patients with disorders of consciousness (DoC). There are several neuroimaging modalities that enable detailed, quantitative assessment of structural and functional brain injury, with demonstrated diagnostic and prognostic value. Additionally, longitudinal neuroimaging studies have hinted at quantifiable structural and functional neuroimaging biomarkers of recovery, with potential implications for the management of DoC., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Following severe brain injuries, a subset of patients may remain in an altered state of consciousness; most of these patients require artificial feeding. Currently, a functional oral phase and the presence of exclusive oral feeding may constitute signs of consciousness. Additionally, the presence of pharyngo-laryngeal secretions, saliva aspiration, cough reflex and tracheostomy are related to the level of consciousness. However, the link between swallowing and consciousness is yet to be fully understood. The primary aim of this review is to establish a comprehensive overview of the relationship between an individual's conscious behaviour and swallowing (reflexive and voluntary). Previous studies of brain activation during volitional and non-volitional swallowing tasks in healthy subjects are also reviewed. We demonstrate that the areas activated by voluntary swallowing tasks (primary sensorimotor, cingulate, insula, premotor, supplementary motor, cerebellum, and operculum) are not specific to deglutitive function but are shared with other motor tasks and brain networks involved in consciousness. This review also outlines suitable assessment and treatment methods for dysphagic patients with disorders of consciousness. Finally, we propose that markers of swallowing could contribute to the development of novel diagnostic guidelines for patients with disorders of consciousness.

Marginal populations of animals are highly susceptible to environmental pressures associated with climatic changes. Understanding their distribution and ecological requirements is, thus, essential for the development of efficient conservation strategies. The dice snake, Natrixtessellata , is listed as critically endangered in the Czech Republic. In certain regions (Bohemia and Silesia), its populations are located beyond the northern border of the continuous range of the species, while the south Moravian populations are connected to it. Based on the statewide database of the Czech Nature Conservation Agency, we created a predictive model and determined key factors influencing the species distribution. The most relevant factors were: watercourses and bodies, average annual temperatures, altitude, slope inclination and precipitation seasonality. The model fits the presence records well and is applicable in both theory and practice of the species conservation - for example, focusing faunistic research to certain areas, critical analysis of controversial presence reports and as an input for species management in the form of repatriation and introduction., Competing Interests: No conflict of interest to declare Disclaimer: This article is (co-)authored by any of the Editors-in-Chief, Managing Editors or their deputies in this journal., (Jan Chmelař, Petr Civiš, David Fischer, Daniel Frynta, Lenka Jeřábková, Veronika Rudolfová, Ivan Rehák.)

Sports related head injuries can cause transient neurological events including loss of consciousness and dystonic posturing. However, it is unknown why head impacts that appear similar produce distinct neurological effects. The biomechanical effect of impacts can be estimated using computational models of strain within the brain. Here, we investigate the strain and strain rates produced by professional American football impacts that led to loss of consciousness, posturing or no neurological signs. We reviewed 1280 National Football League American football games and selected cases where the team's medical personnel made a diagnosis of concussion. Videos were then analysed for signs of neurological events. We identified 20 head impacts that showed clear video signs of loss of consciousness and 21 showing clear abnormal posturing. Forty-one control impacts were selected where there was no observable evidence of neurological signs, resulting in 82 videos of impacts for analysis. Video analysis was used to guide physical reconstructions of these impacts, allowing us to estimate the impact kinematics. These were then used as input to a detailed 3D high-fidelity finite element model of brain injury biomechanics to estimate strain and strain rate within the brain. We tested the hypotheses that impacts producing loss of consciousness would be associated with the highest biomechanical forces, that loss of consciousness would be associated with high forces in brainstem nuclei involved in arousal and that dystonic posturing would be associated with high forces in motor regions. Impacts leading to loss of consciousness compared to controls produced higher head acceleration (linear acceleration; 81.5 g ± 39.8 versus 47.9 ± 21.4; P = 0.004, rotational acceleration; 5.9 krad/s2 ± 2.4 versus 3.5 ± 1.6; P < 0.001) and in voxel-wise analysis produced larger brain deformation in many brain regions, including parts of the brainstem and cerebellum. Dystonic posturing was also associated with higher deformation compared to controls, with brain deformation observed in cortical regions that included the motor cortex. Loss of consciousness was specifically associated with higher strain rates in brainstem regions implicated in maintenance of consciousness, including following correction for the overall severity of impact. These included brainstem nuclei including the locus coeruleus, dorsal raphé and parabrachial complex. The results show that in head impacts producing loss of consciousness, brain deformation is disproportionately seen in brainstem regions containing nuclei involved in arousal, suggesting that head impacts produce loss of consciousness through a biomechanical effect on key brainstem nuclei involved in the maintenance of consciousness., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Introduction: Animal experiments recently demonstrated that replacing urinary loses with crystalloid diminishes the therapeutic effect of mannitol by reducing the increase in osmolality. We aimed to investigate whether this effect is similarly seen in in brain-injured patients by studying the association between total body fluid balance (TBB) and the osmolar response to mannitol., Methods: We performed a retrospective cohort study of adult patients with acute brain injury between 2015 and 2021 who received ≥ 2 doses of mannitol within 8 hours and no intercurrent concentrated saline solution. We analyzed the association between the change in TBB (∆ TBB ) and change in osmolality (∆ Osm ) before and after mannitol in a linear model, both as univariate and after adjustment for common confounding factors., Results: Of 6,145 patients who received mannitol, 155 patients met inclusion criteria (mean age 60 ± 17 years, 48% male, 83% white). The mean total mannitol dose was 2 ± 0.5 g/kg and the mean change in plasma osmolality was 7.9 ± 7.1 mOsm/kg. Each 1 L increase in ∆ TBB was associated with a change of -1.1 mOsm/L in ∆ Osm (95% CI [-2.2, -0.02], p = 0.045). The magnitude of association was similar to that of total mannitol dose and remained consistent in an adjusted model and after excluding outliers., Conclusions: In patients with acute brain injury, a positive TBB is associated with a diminished mannitol-induced increase in plasma osmolality. Future prospective studies are needed to confirm these findings and their influence on the therapeutic effect of mannitol., Competing Interests: Declaration of Competing Interest This worked was completed while MEB was employed by Massachusetts General Hospital, MEB is now employed by Marinus Pharmaceuticals, but has no conflicts of interest that relate to this work. The rest of authors have no disclosures or conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Resting-state functional MRI is being used to develop diagnostic, prognostic and therapeutic biomarkers for critically ill patients with severe brain injuries. In studies of healthy volunteers and non-critically ill patients, prospective cardiorespiratory data are routinely collected to remove non-neuronal fluctuations in the resting-state functional MRI signal during analysis. However, the feasibility and utility of collecting cardiorespiratory data in critically ill patients on a clinical MRI scanner are unknown. We concurrently acquired resting-state functional MRI (repetition time = 1250 ms) and cardiac and respiratory data in 23 critically ill patients with acute severe traumatic brain injury and in 12 healthy control subjects. We compared the functional connectivity results from two approaches that are commonly used to correct cardiorespiratory noise: (i) denoising with cardiorespiratory data (i.e. image-based method for retrospective correction of physiological motion effects in functional MRI) and (ii) standard bandpass filtering. Resting-state functional MRI data in 7 patients could not be analysed due to imaging artefacts. In 6 of the remaining 16 patients (37.5%), cardiorespiratory data were either incomplete or corrupted. In patients ( n = 10) and control subjects ( n = 10), the functional connectivity results corrected with the image-based method for retrospective correction of physiological motion effects in functional MRI did not significantly differ from those corrected with bandpass filtering of 0.008-0.125 Hz. Collectively, these findings suggest that, in critically ill patients with severe traumatic brain injury, there is limited feasibility and utility to denoising the resting-state functional MRI signal with prospectively acquired cardiorespiratory data., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Halophilic and halotolerant microorganisms represent a promising source of salt-tolerant enzymes suitable for various biotechnological applications where high salt concentrations would otherwise limit enzymatic activity. Considering the current growing enzyme market and the need for more efficient and new biocatalysts, the present study aimed at the characterization of a high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101 T . The protease gene was cloned and expressed in Bacillus subtilis DB104. The recombinant protease SPAO with 269 amino acids belongs to the subfamily of high-alkaline subtilisins. The biochemical characteristics of purified SPAO were analyzed in comparison with subtilisin Carlsberg, Savinase, and BPN'. SPAO, a monomer with a molecular mass of 27.1 kDa, was active over a wide range of pH 6.0-12.0 and temperature 20-80 °C, optimally at pH 9.0-9.5 and 55 °C. The protease is highly oxidatively stable to hydrogen peroxide and retained 58% of residual activity when incubated at 10 °C with 5% (v/v) H 2 O 2 for 1 h while stimulated at 1% (v/v) H 2 O 2 . Furthermore, SPAO was very stable and active at NaCl concentrations up to 5.0 m. This study demonstrates the potential of SPAO for biotechnological applications in the future., (© 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Purpose: Spinal dural arteriovenous fistulas (SDAVFs) represent the most common indication for a spinal angiography. The diagnostic reference level (DRL) for this specific endovascular procedure is still to be determined. This single-center study provides detailed dosimetrics of diagnostic spinal angiography performed in patients with SDAVFs., Methods: Retrospective analysis of all diagnostic spinal angiographies between December 2011 and January 2021. Only patients with an SDAVF who had baseline magnetic resonance angiography (MRA), diagnostic digital subtraction angiography (DSA), treatment and follow-up at this institution were included. Dose area product (DAP, Gy cm 2 ) and fluoroscopy time were compared between preoperative and postoperative angiographies, according to SDAVF locations (common versus uncommon), MRA results at baseline (positive versus negative) and DSA protocols (low-dose, mixed-dose, normal-dose). The 75th percentile of the DAP distribution was used to define the local DRL., Results: A total of 62 spinal angiographies were performed in 25 patients with SDAVF. Preoperative angiographies (30/62, 48%) yielded a significantly higher DAP and longer fluoroscopy time when compared to postoperative angiographies (32/62, 53%) (p < 0.01). The local DRL was 329.41 Gy cm 2 for a nonspecific (n = 62), 395.59 Gy cm 2 for a preoperative and 138.6 Gy cm 2 for a postoperative spinal angiography. Preoperative angiography of uncommonly located SDAVFs yielded a significantly longer fluoroscopy time (p = 0.02). The MRA-based fistula detection had no significant impact on dosimetrics (p > 0.05). A low-dose protocol yielded a 61% reduction of DAP., Conclusion: The results of the present study suggest novel DRLs for spinal angiography in patients with SDAVF. Dedicated low-dose protocols enable radiation dose optimization in these procedures., (© 2022. The Author(s).)

Neuroprognostication, or the prediction of recovery from disorders of consciousness caused by severe brain injury, is as critical as it is complex. With profound implications for mortality and quality of life, neuroprognostication draws upon an intricate set of biomedical, probabilistic, psychosocial and ethical factors. However, the clinical approach to neuroprognostication is often unsystematic, and consequently, variable among clinicians and prone to error. Here, we offer a stepwise conceptual framework for reasoning through neuroprognostic determinations - including an evaluation of neurological function, estimation of a recovery trajectory, definition of goals of care and consideration of patient values - culminating in a clinically actionable formula for weighing the risks and benefits of life-sustaining treatment. Although the complexity of neuroprognostication might never be fully reducible to arithmetic, this systematic approach provides structure and guidance to supplement clinical judgement and direct future investigation., (© 2022. Springer Nature Limited.)

The frequency of surgeries involving the use of metal implants in orthopedic medicine to replace degenerative or fractured joints is increasing, and it is therefore important to optimize the lifespan and quality of these implants. Advances in additive manufacturing (AM), or 3D printing, are creating new opportunities to personalize implants in ways that reduce mechanical stress at the joint implant interface and improve bone ingrowth and implant stability; however, it is not well understood if and to what degree the AM process alters the corrosion behavior of the materials it produces. In this study, six Ti6Al4V prints manufactured via a selective laser melting (SLM) method were examined regarding their corrosion behavior in both saline and bovine calf serum (BCS) solutions. E corr and I corr values were comparable between the CM-Ti6Al4V control and SLM-EDM surfaces; however, SLM surfaces were found to have more narrow passivation behavior evidenced by significant decreases in E pass values relative to CM-Ti6Al4V. We believe this is a consequence of microstructural differences between CM-Ti6Al4V and SLM-Ti6Al4V. Specifically, the SLM-Ti6Al4V demonstrated a dominant α' martensitic microstructure and decreased vanadium-rich β-phase. BCS solution had a detrimental effect on potential parameters, E corr and OCP, decreasing these values relative to their saline counterparts. Increased surface roughness of the SLM-printed surface seemed to amplify the effects of the BCS solution. Furthermore, modest decreases in E pass and I pass were observed in BCS solution, suggesting that the presence of protein may also interfere with passivation behavior. These findings have implications for how SLM-Ti6Al4V implants will perform in vivo and could possibly influence implant longevity and performance., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest.

Disorder of consciousness (DoC) after severe brain injury presents numerous challenges to clinicians, as the diagnosis, prognosis, and management are often uncertain. Magnetic resonance imaging (MRI) has long been used to evaluate brain structure in patients with DoC. More recently, advances in MRI technology have permitted more detailed investigations of the brain's structural integrity (via diffusion MRI) and function (via functional MRI). A growing literature has begun to show that these advanced forms of MRI may improve our understanding of DoC pathophysiology, facilitate the identification of patient consciousness, and improve the accuracy of clinical prognostication. Here we review the emerging evidence for the application of advanced MRI for patients with DoC., Competing Interests: D.F.-E. reports grants or contracts from Medical Research Council (MRC) grant MR/P02596X/1 and Research Collaboration Agreement with HCA Healthcare., (Thieme. All rights reserved.)

The northernmost population of the Balkan wall lizards, Podarcistauricus (Pallas, 1814) was recently discovered in the Czech Republic. We studied genetic variability in a mitochondrial marker cytochrome b to shed light on the origin of this remote population. We detected three unique haplotypes, close to those occurring in the populations of Podarcistauricus from central/north Balkans and Hungary. Our data exclude the hypothesis of a single founder (a randomly or intentionally introduced pregnant female or her progeny) of the Czech population and indicate a native, autochthonous origin of the population or recent introduction/range expansion., (Ivan Rehák, David Fischer, Lukáš Kratochvíl, Michail Rovatsos.)

Background and Objectives: Disorders of consciousness, EEG background suppression, and epileptic seizures are associated with poor outcome after cardiac arrest. Our objective was to identify the distribution of diffusion MRI-measured anoxic brain injury after cardiac arrest and to define the regional correlates of disorders of consciousness, EEG background suppression, and seizures., Methods: We analyzed patients from a single-center database of unresponsive patients who underwent diffusion MRI after cardiac arrest (n = 204). We classified each patient according to recovery of consciousness (command following) before discharge, the most continuous EEG background (burst suppression vs continuous), and the presence or absence of seizures. Anoxic brain injury was measured with the apparent diffusion coefficient (ADC) signal. We identified ADC abnormalities relative to controls without cardiac arrest (n = 48) and used voxel lesion symptom mapping to identify regional associations with disorders of consciousness, EEG background suppression, and seizures. We then used a bootstrapped lasso regression procedure to identify robust, multivariate regional associations with each outcome variable. Last, using area under receiver operating characteristic curves, we then compared the classification ability of the strongest regional associations to that of brain-wide summary measures., Results: Compared to controls, patients with cardiac arrest demonstrated ADC signal reduction that was most significant in the occipital lobes. Disorders of consciousness were associated with reduced ADC most prominently in the occipital lobes but also in deep structures. Regional injury more accurately classified patients with disorders of consciousness than whole-brain injury. Background suppression mapped to a similar set of brain regions, but regional injury could no better classify patients than whole-brain measures. Seizures were less common in patients with more severe anoxic injury, particularly in those with injury to the lateral temporal white matter., Discussion: Anoxic brain injury was most prevalent in posterior cerebral regions, and this regional pattern of injury was a better predictor of disorders of consciousness than whole-brain injury measures. EEG background suppression lacked a specific regional association, but patients with injury to the temporal lobe were less likely to have seizures. Regional patterns of anoxic brain injury are relevant to the clinical and electrographic sequelae of cardiac arrest and may hold importance for prognosis., Classification of Evidence: This study provides Class IV evidence that disorders of consciousness after cardiac arrest are associated with widely lower ADC values on diffusion MRI and are most strongly associated with reductions in occipital ADC., (© 2022 American Academy of Neurology.)

Spatial omics data are advancing the study of tissue organization and cellular communication at an unprecedented scale. Flexible tools are required to store, integrate and visualize the large diversity of spatial omics data. Here, we present Squidpy, a Python framework that brings together tools from omics and image analysis to enable scalable description of spatial molecular data, such as transcriptome or multivariate proteins. Squidpy provides efficient infrastructure and numerous analysis methods that allow to efficiently store, manipulate and interactively visualize spatial omics data. Squidpy is extensible and can be interfaced with a variety of already existing libraries for the scalable analysis of spatial omics data., (© 2022. The Author(s).)

The antibody-drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above ∼4 typically destroys the biophysical properties and in vivo efficacy for ADCs. Herein, we describe the development of a novel bioconjugation platform combining cysteine-engineered (THIOMAB) antibodies and recombinant XTEN polypeptides for the unprecedented generation of homogeneous, stable "TXCs" with DAR of up to 18. Across three different bioactive payloads, we demonstrated improved AMD to tumors and Staphylococcus aureus bacteria for high-DAR TXCs relative to conventional low-DAR ADCs., Competing Interests: NZ, KK, DL, GDL, ED, MP, KZ, CL, JH, CN, DS, LL, PG, SM, WS, JT, JM, VY, GL, JC, JHM, SP, BZ, HHB, KL, MX, KRK, GLP, BQS, CW, KX, PH, SFY, AK, RR, DR, TP, AP, WH, and JS are or were employees of Genentech, Inc. at the time when this work was conducted. VNP and VS are employees of Amunix Pharmaceuticals, Inc.., (This journal is © The Royal Society of Chemistry.)

Background and Objectives: In patients with severe coronavirus disease 2019 (COVID-19), disorders of consciousness (DoC) have emerged as a serious complication. The prognosis and pathophysiology of COVID-DoC remain unclear, complicating decisions about continuing life-sustaining treatment. We describe the natural history of COVID-DoC and investigate its associated brain connectivity profile., Methods: In a prospective longitudinal study, we screened consecutive patients with COVID-19 at our institution. We enrolled critically ill adult patients with a DoC unexplained by sedation or structural brain injury and who were planned to undergo a brain MRI. We performed resting-state fMRI and diffusion MRI to evaluate functional and structural connectivity compared to healthy controls and patients with DoC resulting from severe traumatic brain injury (TBI). We assessed the recovery of consciousness (command following) and functional outcomes (Glasgow Outcome Scale Extended [GOSE] and the Disability Rating Scale [DRS]) at hospital discharge and 3 and 6 months after discharge. We also explored whether clinical variables were associated with recovery from COVID-DoC., Results: After screening 1,105 patients with COVID-19, we enrolled 12 with COVID-DoC. The median age was 63.5 years (interquartile range 55-76.3 years). After the exclusion of 1 patient who died shortly after enrollment, all of the remaining 11 patients recovered consciousness 0 to 25 days (median 7 [5-14.5] days) after the cessation of continuous IV sedation. At discharge, all surviving patients remained dependent: median GOSE score 3 (1-3) and median DRS score 23 (16-30). Ultimately, however, except for 2 patients with severe polyneuropathy, all returned home with normal cognition and minimal disability: at 3 months, median GOSE score 3 (3-3) and median DRS score 7 (5-13); at 6 months, median GOSE score 4 (4-5), median DRS score 3 (3-5). Ten patients with COVID-DoC underwent advanced neuroimaging; functional and structural brain connectivity in those with COVID-DoC was diminished compared to healthy controls, and structural connectivity was comparable to that in patients with severe TBI., Discussion: Patients who survived invariably recovered consciousness after COVID-DoC. Although disability was common after hospitalization, functional status improved over the ensuing months. While future research is necessary, these prospective findings inform the prognosis and pathophysiology of COVID-DoC., Trial Registration Information: ClinicalTrials.gov identifier: NCT04476589., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Granular multiparticle ensembles are of interest from fundamental statistical viewpoints as well as for the understanding of collective processes in industry and in nature. Extraction of physical data from optical observations of three-dimensional (3D) granular ensembles poses considerable problems. Particle-based tracking is possible only at low volume fractions, not in clusters. We apply shadow-based and feature-tracking methods to analyze the dynamics of granular gases in a container with vibrating side walls under microgravity. In order to validate the reliability of these optical analysis methods, we perform numerical simulations of ensembles similar to the experiment. The simulation output is graphically rendered to mimic the experimentally obtained images. We validate the output of the optical analysis methods on the basis of this ground truth information. This approach provides insight in two interconnected problems: the confirmation of the accuracy of the simulations and the test of the applicability of the visual analysis. The proposed approach can be used for further investigations of dynamical properties of such media, including the granular Leidenfrost effect, granular cooling, and gas-clustering transitions.

JOSA A Editor-in-Chief P. Scott Carney, Feature Editor Johannes Courtial, and members of the 2020 Emerging Researcher Best Paper Prize Committee announce the recipient of the 2020 prize for the best paper published by an emerging researcher in the Journal.

Force networks play an important role in the stability of configurations when granular material is packed into a container. These networks can redirect part of the weight of grains inside a container to the side walls. We employ monodisperse stress-birefringent spheres to visualize the contact forces in a quasi-2D and a nearly-2D configuration of these spheres in a thin cuboid cell. The packing structures are particularly simple: a hexagonal lattice in the ground state when the cell width is equal to the sphere diameter, and a frustrated, slightly distorted lattice in thicker cells. The force redistribution is substantially changed by this geometrical modification. In both cases, we observe an 'inverse' Janssen effect with the pressure decreasing from the top to the bottom of the container when the material is loaded with a weight on top of the vessel.

More than 30 years ago Edwards and co-authors proposed a model to describe the statistics of granular packings by an ensemble of equiprobable jammed states. Experimental tests of this model remained scarce so far. We introduce a simple system to analyze statistical properties of jammed granular ensembles to test Edwards theory. Identical spheres packed in a nearly two-dimensional geometrical confinement were studied in experiments and numerical simulations. When tapped, the system evolves toward a ground state, but due to incompatible domain structures it gets trapped. Analytical calculations reproduce relatively well our simulation results, which allows us to test Edwards theory on a coupled system of two subsystems with different properties. We find that the joint system can only be described by the Edwards theory if considered as a single system due to the constraints in the stresses. The results show counterintuitive effects as in the coupled system the change in the order parameter is opposite to what is expected from the change in the compactivity.