Assessing organophosphate insecticide retention in muscle tissues of juvenile common carp fish under acute toxicity tests.

Organophosphate insecticide spray poses potential threat of contamination of environmental components their accumulation in aquatic organisms. Although various physiological deficits associated with their exposure in fishes are documented, yet their retention in their edible muscle tissues has been poorly studied. In this context, the study was undertaken to ascertain the bioaccumulation of two organophosphate insecticide compounds (dimethoate and chlorpyrifos) in the muscles of juvenile Cyprinus carpio. The study could provide insight into the risks to human health associated with consuming contaminated fish flesh. The fishes exposed to various concentrations of dimethoate and chlorpyrifos in-vivo for 96 to ascertain the uptake and retention of these insecticides in the muscle. Results indicated that fish muscles accumulated the residues at all the concentrations with the recovery of 2.99% (0.032 ppm) of dimethoate exposed to LC50 concentrations. In contrast, the chlorpyrifos residues were found Below the Detection Level (BDL) in the fishes exposed to LC50 concentrations. The percentage bioaccumulation of dimethoate in fish muscle was 88.10%, and that of chlorpyrifos was BDL. The bio-concentration factor was dose-dependent and increased with increasing doses of both insecticides. The study invites attention to human health risk assessment in the regions where contaminated fish are consumed without scientific supervision.

Development of a machine learning model to predict risk of development of COVID-19-associated mucormycosis.

Aim: The study aimed to identify quantitative parameters that increase the risk of rhino-orbito-cerebral mucormycosis, and subsequently developed a machine learning model that can anticipate susceptibility to developing this condition. Methods: Clinicopathological data from 124 patients were used to quantify their association with COVID-19-associated mucormycosis (CAM) and subsequently develop a machine learning model to predict its likelihood. Results: Diabetes mellitus, noninvasive ventilation and hypertension were found to have statistically significant associations with radiologically confirmed CAM cases. Conclusion: Machine learning models can be used to accurately predict the likelihood of development of CAM, and this methodology can be used in creating prediction algorithms of a wide variety of infections and complications.

Fungal infections caused by the Mucorales order of fungi usually target patients with a weakened immune system. They are usually also associated with abnormal blood sugar states, such as in diabetic patients. Recent work during the COVID-19 outbreak suggested that excessive steroid use and diabetes may be behind the rise in fungal infections caused by Mucorales, known as mucormycosis, in India, but little work has been done to see whether we can predict the risk of mucormycosis. This study found that these fungal infections need not necessarily be caused by Mucorales' species, but by a wide variety of fungi that target patients with weak immune systems. Secondly, we found that diabetes, breathing-assisting devices and high blood pressure states had associations with COVID-19-associated fungal infections. Finally, we were able to develop a machine learning model that showed high accuracy when predicting the risk of development of these fungal infections.

Rhino-orbital cerebral mycosis: a case series of non-mucorales in COVID patients.

Introduction: Rhino-Orbito-cerebral mycoses are not only caused by Aspergillus spp. and Zygomycetes spp. but also can be associated with other rare species such as Neurospora spp., Cladosporium spp. and Fusarium spp. Mucormycosis is associated causatively with immunocompromised states, for example patients with comorbidities such as diabetes mellitus. Clinical symptoms of coronavirus disease (COVID) and mucormycosis in tandem are critical and relentless, frequently with no life-saving treatment. Case series: We report three patients with COVID-19 infection, who during the course of treatment developed rhino-orbital-cerebral mycosis including oral cavity involvement. Rhinocerebral mycosis along with oral cavity involvement was diagnosed by radiological investigations and preliminary screening for fungal infection (KOH mount) in all three cases. Empirical treatment was started but patients did not respond to treatment. All patients died even after debridement and maxillectomy. On culture, rare species of fungi were isolated in all three cases which, with the help of a reference laboratory, were identified as Neurospora, Cladosporium and Fusarium. Neurospora is considered nonpathogenic to humans. Cladosporium is a dematiaceous fungus found in soil in all climates, associated with disseminated or cerebral infections; and Fusarium, though considered a saprophytic colonizer of skin and respiratory mucosa along with other bacteria, is a common cause of mycotic keratitis worldwide. Conclusion: Immune system modifications due to COVID-19 with/without other risk factors can result in fungal co-infections that prove to be fatal for the patients. It is vital to be aware that COVID-19 patients, particularly those who are critically ill, may acquire secondary fungal infections and early detection is critical.

Lest we forget spinal tuberculosis (Potts's spine): Case series with unusual presentation.

Pott's disease, also known as TB spondylitis, is a very uncommon extrapulmonary infection caused by Mycobacterium tuberculosis. As its prevalence is not high it can easily be underdiagnosed. Magnetic resonance imaging (MRI), computed tomographic (CT) guided needle aspiration, or biopsy are known to be the best techniques for early histopathological diagnosis along with confirmation by microbiological results. Ziehl Neelson stain (ZN) can detect Mycobacterium infections when clinically suspected samples are adequate and optimally stained. No single method or simple guideline can diagnose spinal tuberculosis. Early diagnosis and prompt treatment are necessary to prevent permanent neurological disability and to minimize spinal deformity. We are reporting three cases of Potts disease which could have been easily missed if we would have relied on one single investigation.

Establishment of a three­dimensional triculture model on the novel AXTEX­4D™ platform.

Cancer can be fatal if it is not treated in a timely manner; therefore, there is a high demand for more specific oncology drugs. Unfortunately, drugs showing positive responses on a two­dimensional (2D) culture platform do not often show the same effect in clinical trials. Therefore, three­dimensional (3D) culture platforms are garnering attention since they more closely mimic the tumor microenvironment (TME). The TME stimulates metastasis and drug resistance, and serves an essential role in tumor formation. An accurate understanding of tumor­stroma interactions is undoubtedly required to improve the response of patients to therapeutic strategies, and cancer therapeutic strategies that do not account for the stroma are considered inadequate. It should be noted that 3D monoculture systems do not completely mimic the TME since other cells in the 3D culture are missing, such as fibroblast or endothelial cells, which are essential components of the stroma; therefore, it is essential to develop advanced 3D culture systems. The present study aimed to develop a versatile triculture model that mimics the native TME; therefore, it could aid in high­throughput screening of chemotherapeutic drugs against cancer by evaluating their effects on tumor progression and cell cytotoxicity. The present study demonstrated the use of the AXTEX­4D™ platform in developing triculture tissueoids composed of MCF­7, human umbilical vein endothelial cells and MRC5 cells, and compared it with a 3D monoculture model (MCF­7) and a 2D culture model. The triculture model was validated for proliferation, ECM markers and T­cell infiltration by confocal microscopy. Alamar Blue assay demonstrated that triculture tissueoids exhibited higher drug resistance than the other two models, thus demonstrating their use in the screening of oncology drugs.

Role of GeneXpert MTB/RIF assay for the diagnosis of cervical lymph node tuberculosis and rifampicin resistance.

Objectives: Tuberculosis (TB) of lymph node (TB lymphadenitis) is one of the most common forms of extrapulmonary TB (EPTB) whose diagnosis is critically challenging. Although new diagnostic methods have been developed, especially in patients without a history of TB, the cervical tuberculous lymphadenitis diagnosis is still elusive. This study assessed the applicability of GeneXpert in early diagnosis of EPTB, especially cervical lymphadenopathy. Materials and Methods: The study was conducted in a tertiary care hospital from January 2018 to December 2020 at the department of microbiology. All the samples of cervical lymph node tissue and lymph node aspirate were followed as per the routine protocol for mycobacterial identification. The sample was divided into two parts: one was used for the new molecular-based GeneXpert MTB/RIF assay and the second one was tested by direct and concentrated acid-fast bacilli microscopy by Z-N staining and culture for the detection of MTB. Results: Among the 145 samples tested, the GeneXpert detected the DNA of MTB in 89 samples (61.37%), whereas the culture test was positive in 42 (28.93%) specimens. GeneXpert also detected 7 rifampicin resistance cases. GeneXpert sensitivity and specificity results were assessed according to culture results. The sensitivity and specificity of the GeneXpert assay were 85.71% and 48.54%, respectively. Conclusion: GeneXpert MTB/RIF should be used in conjunction with clinical presentation and other molecular investigation in nonrespiratory specimens.

Behavioural incongruities in juvenile Cyprinus carpio exposed to organophosphate compounds.

For the ever increasing human population, the necessity to produce the food in large quantities has become the main goal internationally which has led to increase the practice of pesticides globally. Presence of pesticides in aquatic water bodies is largely due to the runoff from agricultural fields causing to deteriorate the healthy characteristics of an aquatic environment system leading to the toxic impact on non-target aquatic organism such as fish. Approach: In fish, there are various portal of entry through which the contaminants enter. Via various routes, the contaminants reach into the blood and subsequently to different organs or systems. Since Pesticides are known to modify the behavior of animals when exposed to toxic levels. The behavioral changes may be caused by the changes in the nervous system triggered directly or through metabolic or physiological activities. However, the effects have been found to be multifarious and known to differ at different concentrations. Also, Blood is the most accessible component of the vertebrate body fluid system and consequences of direct and indirect damage to blood cells and their precursors are predictable and potentially life threatening. Therefore, behavioural and genotoxicological studies have been considered and used as diagnostic tool in order to investigate behavioural and genotoxicological alterations. This study was undertaken to investigate behavioural changes in Cyprinus carpio exposed to two organophosphate compounds, chlorpyrifos (cpf) and dimethoate (dim). Fishes weighing 10 ± 2 g were exposed to sub-lethal concentrations of cpf (0.76 ppb, 1.52 ppb, 2.28 ppb, 3.04 ppb and 3.8 ppb) and dimethoate (0.22 ppm, 0.44 ppm, 0.66 ppm, 0.88 ppm and 1.1 ppm) for the period of 96 h and various behavioural indices were evaluated during that period. Both the pesticides were found to induce behavioral toxicity in fishes such as drop in swimming velocity, reduced swimming activity and retarded opercular movements. Cpf was found more detrimental as compared to dimethoate in all cases. Fishes also elicited a genotoxic response which was evaluated by calculating the frequency of micronuclei formation in their hematocytes after 21 days of exposure. Chlorpyrifos induced more genotoxicity than dimethoate which was found dose and time dependent. Conclusion: It was concluded that the behavioural and genotoxic alterations in common carp could be applied as possible biomarkers in risk assessment and monitoring programs for pesticide contamination of aquatic ecosystems. Contribution: This study is one of its kind and will help to form baseline data.

Recapitulating Tumor Microenvironment Using AXTEX-4DTM for Accelerating Cancer Research and Drug Screening.

OBJECTIVE: The formation of three-dimensional spheroid tumor model using the scaffold-based platforms has been demonstrated over many years now. 3D tumor models are generated mainly in non-scalable culture systems, using synthetic and biological scaffolds. Many of these models fail to reflect the complex tumor microenvironment and do not allow long-term monitoring of tumor progression. This has resulted in inconsistent data in drug testing assays during preclinical and clinical studies. METHODS: To overcome these limitations, we have developed 3D tissueoids model by using novel AXTEX-4D platform. RESULTS: Cancer 3D tissueoids demonstrated the basic features of 3D cell culture with rapid attachment, proliferation, and longevity with contiguous cytoskeleton and hypoxic core. This study also demonstrated greater drug resistance in 3D-MCF-7 tissueoids in comparison to 2D monolayer cell culture. CONCLUSION: In conclusion, 3D-tissueoids are more responsive than 2D-cultured cells in simulating important tumor characteristics, anti-apoptotic features, and their resulting drug resistance.

Clinico-radiological features, molecular spectrum, and identification of prognostic factors in developmental and epileptic encephalopathy due to inosine triphosphate pyrophosphatase (ITPase) deficiency.

Developmental and epileptic encephalopathy 35 (DEE 35) is a severe neurological condition caused by biallelic variants in ITPA, encoding inosine triphosphate pyrophosphatase, an essential enzyme in purine metabolism. We delineate the genotypic and phenotypic spectrum of DEE 35, analyzing possible predictors for adverse clinical outcomes. We investigated a cohort of 28 new patients and reviewed previously described cases, providing a comprehensive characterization of 40 subjects. Exome sequencing was performed to identify underlying ITPA pathogenic variants. Brain MRI (magnetic resonance imaging) scans were systematically analyzed to delineate the neuroradiological spectrum. Survival curves according to the Kaplan-Meier method and log-rank test were used to investigate outcome predictors in different subgroups of patients. We identified 18 distinct ITPA pathogenic variants, including 14 novel variants, and two deletions. All subjects showed profound developmental delay, microcephaly, and refractory epilepsy followed by neurodevelopmental regression. Brain MRI revision revealed a recurrent pattern of delayed myelination and restricted diffusion of early myelinating structures. Congenital microcephaly and cardiac involvement were statistically significant novel clinical predictors of adverse outcomes. We refined the molecular, clinical, and neuroradiological characterization of ITPase deficiency, and identified new clinical predictors which may have a potentially important impact on diagnosis, counseling, and follow-up of affected individuals.

AXTEX-4D: A Three-Dimensional Ex Vivo Platform for Preclinical Investigations of Immunotherapy Agents.

The latest advancements in oncology are majorly focused on immuno-oncology (I-O) therapies. However, only ∼7% of drugs are being approved from the preclinical discovery phase to phase 1. The most challenging issues in I-O are the development of active and efficient drugs in an economically feasible way and in a comparatively short time for testing and validation. This mandates an urgent need for the upgradation of preclinical screening models that closely mimic the in vivo tumor microenvironment (TME). The established and most common methods for investigating the tumoricidal activity of I-O drugs are either two-dimensional systems or primary tumor cells in standard tissue culture vessels. Unfortunately, they do not mimic the TME. Consequently, the more in vivo-like three-dimensional (3D) multicellular tumor spheroids are quickly becoming the favored model to examine immune cell-mediated responses in reaction to the administration of I-O drugs. Despite many advantages of multicellular spheroids, challenges (e.g., incompatibility of quantitative assays with spheroid platforms) are still involved in the tedious procedures required for the spheroid culture that is holding back the biological community from adapting the well-recognized spheroid tissue models for studying drug delivery more widely. To this end, we have demonstrated the utility of the 3D ex vivo oncology model, developed on our novel AXTEX-4D™ platform to assess therapeutic efficacies of I-O drugs by investigating immune cell proliferation, migration, infiltration, cytokine profiling, and cytotoxicity of tumor tissueoids. The platform eliminates the need for additional biomolecules such as hydrogels and instead relies on the cancer cells themselves to create their own gradients and microenvironmental factors. In effect, the more comprehensive and ex vivo-like immune-oncology model developed on AXTEX-4D platform can be utilized for high-throughput screening of immunotherapeutic drugs.