Absence of functional deficits in rats following systemic administration of an AAV9 vector despite moderate peripheral nerve and dorsal root ganglia findings: A clinically silent peripheral neuropathy.

Adeno-associated virus (AAV)-based vectors are commonly used for delivering transgenes in gene therapy studies, but they are also known to cause dorsal root ganglia (DRG) and peripheral nerve toxicities in animals. However, the functional implications of these pathologic findings and their time course remain unclear. At 2, 4, 6, and 8 weeks following a single dose of an AAV9 vector carrying human frataxin transgene in rats, non-standard functional assessments, including von Frey filament, electrophysiology, and Rotarod tests, were conducted longitudinally to measure allodynia, nerve conduction velocity, and coordination, respectively. Additionally, DRGs, peripheral nerves, brain and spinal cord were evaluated histologically and circulating neurofilament light chain (NfL) was quantified at 1, 2, 4, and 8 weeks, respectively. At 2 and 4 weeks after dosing, minimal-to-moderate nerve fiber degeneration and neuronal degeneration were observed in the DRGs in some of the AAV9 vector-dosed animals. At 8 weeks, nerve fiber degeneration was observed in DRGs, with or without neuronal degeneration, and in sciatic nerves of all AAV9 vector-dosed animals. NfL values were higher in AAV9 vector-treated animals at weeks 4 and 8 compared with controls. However, there were no significant differences in the three functional endpoints evaluated between the AAV9 vector- and vehicle-dosed animals, or in a longitudinal comparison between baseline (predose), 4, and 8 week values in the AAV9 vector-dose animals. These findings demonstrate that there is no detectable functional consequence to the minimal-to-moderate neurodegeneration observed with our AAV9 vector treatment in rats, suggesting a functional tolerance or reserve for loss of DRG neurons after systemic administration of AAV9 vector.

India Unburdening the Pandemic: Jabs and Talks.

The impact of COVID-19 on the global healthcare system was detrimental, and India was not an exception. A crucial part of India's fight during the pandemic was the nation's astonishing vaccination delivery. From actively curbing the spread of COVID-19 and managing the affected to initiatives for the vaccination of our vast country, India faced numerous challenges in the healthcare delivery system during the pandemic. India's compassionate initiative to supply COVID-19 vaccines across the globe was remarkable. With the rising caseload and increasing case fatality, healthcare workers (HCWs) worked tirelessly to fight the battle against COVID-19. This left gruesome effects on their mental health, leading to various mental health problems. To alleviate such concerns, the government and many renowned institutions in India put forth recommendations, services, and assistance to those suffering. In a nutshell, the healthcare system in India faced countless challenges during the COVID-19 pandemic, but the course of action taken to combat those challenges was truly extraordinary.

Measuring Dipolar Order Parameters in Nondeuterated Proteins Using Solid-State NMR at the Magic-Angle-Spinning Frequency of 100 kHz.

Proteins are dynamic molecules, relying on conformational changes to carry out function. Measurement of these conformational changes can provide insight into how function is achieved. For proteins in the solid state, this can be done by measuring the decrease in the strength of anisotropic interactions due to motion-induced fluctuations. The measurement of one-bond heteronuclear dipole-dipole coupling at magic-angle-spinning (MAS) frequencies >60 kHz is ideal for this purpose. However, rotational-echo double resonance (REDOR), an otherwise gold-standard technique for the quantitative measurement of these couplings, is difficult to implement under these conditions, especially in nondeuterated samples. We present here a combination of strategies based on REDOR variants ϵ-REDOR and DEDOR (deferred REDOR) and simultaneously measure residue-specific 15N-1H and 13Cα-1Hα dipole-dipole couplings in nondeuterated systems at the MAS frequency of 100 kHz. These strategies open up avenues to access dipolar order parameters in a variety of systems at the increasingly fast MAS frequencies that are now available.

Asynchronising five-fold symmetry sequence for better homonuclear polarisation transfer in magic-angle-spinning solid-state NMR.

Recoupling, decoupling, and multidimensional correlation experiments in magic-angle-spinning (MAS) solid-state NMR can be designed by exploiting the symmetry of internal spin interactions. One such scheme, namely, C521, and its supercycled version SPC521, notated as a five-fold symmetry sequence, is widely used for double-quantum dipole-dipole recoupling. Such schemes are generally rotor synchronised by design. We demonstrate an asynchronous implementation of the SPC521 sequence leading to higher double-quantum homonuclear polarisation transfer efficiency compared to the normal synchronous implementation. Rotor-synchronisation is broken in two different ways: lengthening the duration of one of the pulses, denoted as pulse-width variation (PWV), and mismatching the MAS frequency denoted as MAS variation (MASV). The application of this asynchronous sequence is shown on three different samples, namely, U-13C-alanine and 1,4-13C-labelled ammonium phthalate which include 13Cα-13Cß, 13Cα-13Co, and 13Co-13Co spin systems, and adenosine 5'- triphosphate disodium salt trihydrate (ATP⋅3H2O). We show that the asynchronous version performs better for spin pairs with small dipole-dipole couplings and large chemical-shift anisotropies, for example, 13Co-13Co. Simulations and experiments are shown to corroborate the results.

Comparison of Framingham Risk Scores (FRS), Joint British Society (JBS3), and American College of Cardiology/American Heart Association (ACC/AHA) Cardiovascular Risk Scores Among Adults With First Myocardial Infarction.

INTRODUCTION: The prevalence of myocardial infarction (MI) among young Indian adults is on the rise with reports suggesting 32.7% of all deaths in men and 32.6% of all deaths in women between 2010-13 were due to cardiovascular diseases (CVDs). Though various long-term cohort studies have established risk assessment scores none of them are specific to the Indian population. In this study, we look to establish which scoring system among the American College of Cardiology (ACC), Joint British Society (JBS3) and Framingham Risk Scores (FRS) would be reliable for the Indian population. A timely intervention based on the most reliable score can help mitigate cardiovascular diseases. MATERIALS AND METHODS: In this cross-sectional study, we included Indian adults, aged more than 40 years, with first MI. Patients previously on lipid lowering drugs were excluded. Demographic data, history, clinical information, laboratory data and other investigations were noted. Subsequently the predicted cardiovascular risk scores based on JBS3, ACC, and FRS were calculated and divided into low risk, intermediate and high risk based on the categorization of the risk scores individually. RESULTS: There were 102 (79.1%) males and 23 (17.8%) females with a mean age of 51.01 years (standard deviation [SD]=12.82, p value <0.001). There was considerable prevalence of type 2 diabetes mellitus with 56 (47.1%) of the subjects being known diabetics. The mean 10-year risk of MI based on ACC was 12.42% (SD=10.45), mean JBS3 score was 14.45% (SD=12.67) and mean FRS score was 15.75% (SD=14.71). FRS scores when categorized, 48 (40.3%) patients had low risk, 30 (23.3%) had medium risk and 43 (33.3%) had high risk. As for ACC score, 39 (35.8%) patients were in low risk and 29 (26.6%) in intermediate risk, borderline in 18 (16.5%) and high risk in 23 (21.1%). In JBS3 scores, 53 (46.5%) patients were in low risk, 32 (28.1%) were in moderate risk and 29 (25.4%) in high risk. CONCLUSION:  The absolute value of 10-year risk scores was highest for FRS scores. The proportion of patients whose scores were under the category of high risk was highest for FRS.

Study of early warning efficiency of different laboratory markers in predicting progression of disease in COVID-19.

Introduction: COVID 19 pandemic has given rise to several challenges to clinicians and one of the keys in this is to predict the set of patients who progress from mild disease to moderate and severe. Apart from the symptomatology and signs, there are several lab parameters varying from biochemical, hematological to radiological parameters which help us in stratifying the stage of disease and also in deciding on which set of patients need close and vigilant monitoring. This would help us in better stratification of disease and utilize the available infrastructure and resources in an optimum way for better management of the disease. Aim: To analyze the early warning efficiency of laboratory parameters individually or in combination in predicting the progress of disease in patients from mild to moderate/severe disease. Materials and Methods: This was taken up as a retrospective study with 100 cases and 100 controls. The demographic details, inflammatory markers, biochemical markers and hematological markers were analyzed. Test of significance was employed to compare categorical variables while student t-test was employed to test the difference in the mean value such as age between case and control (Mann-Whitney U-test in parameters not having normal distribution). Receiver operating characteristic (ROC) curve was constructed for these parameters using cases and controls and area under the curve (AUC) were estimated which was used as an indicator of sensitivity and specificity of the parameter in their early warning efficiency. The critical values for each of the parameters either individually or in combination was estimated as well. Results: Among the parameters C reactive protein (CRP), d-dimers and eosinopenia have the best early warning efficiency. The area under the ROCs curve for neutrophil lymphocyte ratio (NLR), CRP. Ferritin, lactate dehydrogenase, Eosinopenia was 0.609, 0.947, 0.614, 0.554, 0.617 respectively at triage. However, a combination of eosinopenia with CRP (AUC-0.732) or NLR with CRP (AUC-0.728) have a good sensitivity and specificity in predicting the outcome regarding the progression of the disease. Conclusions: Among the parameters, CRP, d-dimers, Eosinopenia and NLR have the best early warning efficiency. However, a combination of Eosinopenia and CRP at triage should also serve as a red flag sign in patients apart from the well-known NLR and IL6 values.

Résumé Introduction: La pandémie covide 19 a relevé plusieurs défis aux cliniciens et l'une des clés dans ce domaine est de prédire l'ensemble des patients qui passent d'une maladie légère à modérée et sévère. Outre la symptomatologie et les signes, plusieurs paramètres de laboratoire variant des paramètres biochimiques, hématologiques à radiologiques qui nous aident à stratifier le stade de la maladie et également à décider quel ensemble de patients nécessite une surveillance étroite et vigilante. Cela nous aiderait à mieux stratification des maladies et à utiliser l'infrastructure et les ressources disponibles de manière optimale pour une meilleure prise en charge de la maladie. Objectif: Analyser l'efficacité d'alerte précoce des paramètres de laboratoire individuellement ou en combinaison pour prédire les progrès des maladies chez les patients d'une maladie légère à modérée / sévère. Matériaux et méthodes: Ceci a été considéré comme une étude rétrospective avec 100 cas et 100 contrôles. Les détails démographiques, les marqueurs inflammatoires, les marqueurs biochimiques et les marqueurs hématologiques ont été analysés. Le test de signification a été utilisé pour comparer les variables catégorielles tandis que le test T des étudiants a été utilisé pour tester la différence de valeur moyenne telle que l'âge entre le cas et le contrôle (test U Mann - Whitney dans les paramètres n'ayant pas de distribution normale). La courbe des caractéristiques de fonctionnement du récepteur (ROC) a été construite pour ces paramètres en utilisant les cas et les contrôles et la zone sous la courbe (AUC) ont été estimés qui ont été utilisés comme indicateur de sensibilité et de spécificité du paramètre dans leur efficacité d'alerte précoce. Les valeurs critiques pour chacun des paramètres individuellement ou en combinaison ont également été estimées. Résultats: Parmi les paramètres C Protein réactif (CRP), les D - dimères et l'éosinopénie ont la meilleure efficacité d'alerte précoce. La zone sous la courbe ROCS pour le rapport lymphocyte des neutrophiles (NLR), CRP. La ferritine, la lactate déshydrogénase, l'éosinopénie était de 0,609, 0,947, 0,614, 0,554, 0,617 respectivement au triage. Cependant, une combinaison d'éosinopénie avec CRP (AUC - 0,732) ou NLR avec CRP (AUC - 0,728) a une bonne sensibilité et spécificité pour prédire le résultat concernant la progression de la maladie. Conclusions: Parmi les paramètres, le CRP, les D - dimères, l'éosinopénie et le NLR ont la meilleure efficacité d'alerte précoce. Cependant, une combinaison d'éosinopénie et de CRP au triage devrait également servir de signe du drapeau rouge chez les patients en dehors des valeurs NLR et IL6 bien connues. Mots-clés: C Protéine réactive, efficacité d'alerte précoce, éosinopénie, progression de la maladie dans Covid ­ 19.

A study to evaluate the role of eosinophil count as a prognostic marker for assessing the outcome in patients with COVID-19 infection.

Background and Objectives: The triaging of COVID-19 patients is of paramount importance to plan further management. There are several clinical and laboratory parameters that help in categorizing the disease severity, triaging, and prognostication. Little is known about the prognostic significance of eosinopenia in predicting the severity of COVID-19 from large hospital data, especially from low- and middle-income countries. The objective of this study is to evaluate the level of eosinopenia as an early prognostic marker for assessing the outcomes in COVID-19 patients and to assess the superiority of eosinopenia as a prognostic marker for assessing the outcomes in COVID-19 patients compared to lymphopenia and neutrophil-to-lymphocyte ratio (NLR). Methods: The study was carried out in a tertiary care hospital. A retrospective longitudinal approach was adopted wherein the hospital records of COVID-19 patients were analyzed. In our study, two separate groups of patients were included for analysis to describe the association between initial eosinophil counts of the patients and the clinical outcomes. In the first group, the disease severity in terms of clinical and radiological parameters was compared in patients of COVID-19 presenting with and without the presence of initial eosinopenia. Commonly used markers for triage, namely lymphopenia and NLR, were compared with the presence of initial eosinopenia among the patients who progressed to moderate and severe disease. In the second group, an analysis of eosinopenia was made among the patients who succumbed to the illness. Results: It was seen that 29.6% of patients with eosinopenia had moderate and severe disease compared to those without eosinopenia where only 10.8% had moderate disease, none had severe disease. It was seen that 19.7% of patients with eosinopenia but no lymphopenia had more severe disease compared to patients with lymphopenia but no eosinopenia where 10.8% of the patients had moderate disease, none had severe disease. In patients younger than 60 years who died of COVID-19, it was found that initial eosinopenia was found in 86%, whereas a high NLR >17 was seen in only 25.6% of patients who died, thus implying that is eosinopenia is an important marker of disease severity in COVID-19. Conclusions: Eosinopenia is an important parameter in the evaluation of COVID-19 and the presence of it should alert the clinicians regarding the further progression of the disease. It is not only an important marker but also an early marker for severe disease.

Methodologies and Emerging Technologies for the Evaluation of the Hematopoietic System.

Hematology and bone marrow analysis is central to our understanding of the hematopoietic system and how it responds to insults, and this session presented during the 2022 STP symposium provided a review of current and novel approaches for the evaluation of the hematopoietic system in the context of nonclinical investigations. This publication summarizes the information presented on novel approaches for evaluation of the hematopoietic system using automated hematology analyzers, including details around the quantitative assessment of bone marrow cell suspensions as well as introducing several newly available hematology parameters. It was followed by a discussion on intravital microscopy and live cell imaging and how these methods can assist with de-risking hematopoiesis-associated safety concerns, and a review of recent assays using artificial intelligence for the evaluation of bone marrow.

Shimon Vega in the eyes of his students and postdocs.

Professor Shimon Vega (1943-2021) of the Weizmann Institute of Science passed away on the 16-th of November. Shimon Vega established theoretical frameworks to develop and explain solid-state nuclear magnetic resonance (NMR) and dynamic nuclear polarization (DNP) techniques and methodologies. His departure left a profound mark on his many students, postdocs, and colleagues. Shortly after his passing, we all assembled spontaneously for an international online meeting to share our reflections and memories of our experiences in Shimon's lab and how they affected us deeply during that period of timeand throughout our scientific careers. These thoughts and feelings were put here into writing.

Circulating neurofilament light chain as a promising biomarker of AAV-induced dorsal root ganglia toxicity in nonclinical toxicology species.

Adeno-associated virus (AAV)-induced dorsal root ganglia (DRG) toxicity has been observed in several nonclinical species, where lesions are characterized by neuronal degeneration/necrosis, nerve fiber degeneration, and mononuclear cell infiltration. As AAV vectors become an increasingly common platform for novel therapeutics, non-invasive biomarkers are needed to better characterize and manage the risk of DRG neurotoxicity in both nonclinical and clinical studies. Based on biological relevance, reagent availability, antibody cross-reactivity, DRG protein expression, and assay performance, neurofilament light chain (NF-L) emerged as a promising biomarker candidate. Dose- and time-dependent changes in NF-L were evaluated in male Wistar Han rats and cynomolgus monkeys following intravenous or intrathecal AAV injection, respectively. NF-L profiles were then compared against microscopic DRG lesions on day 29 post-dosing. In animals exhibiting DRG toxicity, plasma/serum NF-L was strongly associated with the severity of neuronal degeneration/necrosis and nerve fiber degeneration, with elevations beginning as early as day 8 in rats (≥5 × 1013 vg/kg) and day 14 in monkeys (≥3.3 × 1013 vg/dose). Consistent with the unique positioning of DRGs outside the blood-brain barrier, NF-L in cerebrospinal fluid was only weakly associated with DRG findings. In summary, circulating NF-L is a promising biomarker of AAV-induced DRG toxicity in nonclinical species.

Highly selective and sensitive 'on-off' fluorescent chemosensor for Fe3+ ions crafted by benzofuran moiety in both experimental and theoretical methods.

A benzofuran glycinamide-based chemosensor, 3-(2-([4-fluorobenzyl]amino)acetamido)benzofuran-2-carboxamide (BGA) was developed and synthesized for the selective and sensitive detection of Fe3+ ions. The photophysical properties of the probe BGA were studied using UV-visible light absorption and fluorescence spectrophotometers. The chemosensor BGA showed a marked 'on-off' fluorescence response towards Fe3+ ions in the presence of other metal ions in DMSO/H2 O solution (9/1, v/v). The very low limits of detection (LOD) were calculated to be 10 nM and 43 nM using UV-visible light absorption and fluorescence spectrophotometers, respectively. Job's plot analysis revealed the formation of a BGA-Fe3+ complex with a 1:1 binding stoichiometry ratio using UV-visible light spectroscopy. The sensing mechanism was also demonstrated using density functional theory calculation.

Biodistribution and Tolerability of AAV-PHP.B-CBh-SMN1 in Wistar Han Rats and Cynomolgus Macaques Reveal Different Toxicologic Profiles.

Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high-dose intravenous (IV) administration. To characterize biodistribution, transduction, and toxicity among common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron 1 (SMN1) transgene (AAV-PHP.B-CBh-SMN1) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2 × 1013, 5 × 1013, or 1 × 1014 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5 × 1013 vg/kg was found dead on day 4. Clinical pathology data on days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi, suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while quantitative polymerase chain reaction confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after 1 month in rats, and suggest adding the autonomic system in the watch list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.

Evaluation of the Central Effects of Systemic Lentiviral-Mediated Leptin Delivery in Streptozotocin-Induced Diabetic Rats.

Type 1 diabetes (T1D) is characterized by hyperphagia, hyperglycemia and activation of the hypothalamic-pituitary-adrenal (HPA) axis. We have reported previously that daily leptin injections help to alleviate these symptoms. Therefore, we hypothesized that leptin gene therapy could help to normalize the neuroendocrine dysfunction seen in T1D. Adult male Sprague Dawley rats were injected i.v. with a lentiviral vector containing the leptin gene or green fluorescent protein. Ten days later, they were injected with the vehicle or streptozotocin (STZ). HPA function was assessed by measuring norepinephrine (NE) levels in the paraventricular nucleus (PVN) and serum corticosterone (CS). Treatment with the leptin lentiviral vector (Lepvv) increased leptin and insulin levels in non-diabetic rats, but not in diabetic animals. There was a significant reduction in blood glucose levels in diabetic rats due to Lepvv treatment. Both NE levels in the PVN and serum CS were reduced in diabetic rats treated with Lepvv. Results from this study provide evidence that leptin gene therapy in STZ-induced diabetic rats was able to partially normalize some of the neuroendocrine abnormalities, but studies with higher doses of the Lepvv are needed to develop this into a viable option for treating T1D.

Bacillus-Mediated Silver Nanoparticle Synthesis and Its Antagonistic Activity against Bacterial and Fungal Pathogens.

In this article, the supernatant of the soil-borne pathogen Bacillus mn14 was used as the catalyst for the synthesis of AgNPs. The antibacterial and antifungal activity of Bs-AgNPs was evaluated, in which S. viridans and R. solani showed susceptibility at 70 µL and 100 µL concentrations. Enzyme properties of the isolates, according to minimal inhibitory action and a growth-enhancing hormone-indole acetic acid (IAA) study of the isolates, were expressed in TLC as a purple color with an Rf value of 0.7. UV/Vis spectroscopy revealed the presence of small-sized AgNPs, with a surface plasmon resonance (SPR) peak at 450 nm. The particle size analyzer identified the average diameter of the particles as 40.2 nm. The X-ray diffraction study confirmed the crystalline nature and face-centered cubic type of the silver nanoparticle. Scanning electron microscopy characterized the globular, small, round shape of the silver nanoparticle. AFM revealed the two-dimensional topology of the silver nanoparticle with a characteristic size ranging around 50 nm. Confocal microscopy showed the cell-wall disruption of S. viridans treated with Bs-AgNPs. High-content screening and compound microscopy revealed the destruction of mycelia of R. solani after exposure to Bs-AgNPs. Furthermore, the Bs-AgNPs cured sheath blight disease by reducing lesion length and enhancing root and shoot length in Oryza sativa seeds. This soil-borne pathogen Bacillus-mediated synthesis approach of AgNPs appears to be cost-efficient, ecofriendly, and farmer-friendly, representing an easy way of providing valuable nutritious edibles in the future.