A Simple Immunofluorescence Method to Characterize Neurodegeneration and Tyrosine Hydroxylase Reduction in Whole Brain of a Drosophila Model of Parkinson's Disease.

Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson's disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories. Key features • Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body. • Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.

Prevention strategies for vasovagal reaction in whole blood donors: A quadri-armed randomised control trial.

INTRODUCTION: Vasovagal reaction (VVR) is a frequently encountered generalised donor adverse reaction, associated with donor deterrence towards future donation. Several mitigation strategies for prevention of VVR were tried but still not standardised. This quadri-armed randomised study evaluated the utility of water ingestion, applied muscle tension (AMT) and combination of both in preventing the VVR among blood donors. METHODS: A quadri-armed randomised controlled trial was performed on 4320 whole blood donors. Blood donors of 18-65 years of age were randomised into four groups based on the interventions performed i.e., control with no intervention (Group 1, n = 1081), water ingestion (Group 2, n = 1082), AMT (Group 3, n = 1070) and combined intervention (Group 4, n = 1087). VVR during and immediately after blood donation were observed along with assessment of risk factors in blood donors and the effectiveness of interventions were analysed. RESULTS: The incidence of VVR observed 1.6% in our study, with the highest occurrence in the control group (2.5%) and the lowest in the combined intervention group (0.9%). Multivariable logistic regression revealed that the control group donors faced a 1.38-fold greater risk of VVR compared to those receiving interventions (OR: 1.38, 95% CI: 1.10-1.75). Other risk factors included younger age (OR: 1.5, 95% CI: 1.05-2.17), first-time donation (OR: 5.7, 95% CI: 1.66-5.74), prior history of VVR (OR: 2.5, 95% CI: 10.4-101.52). DISCUSSION/CONCLUSION: The combined approach of water ingestion and AMT proved significantly more effective in VVR prevention compared to individual interventions.

Notification and follow-up of blood donors reactive for transfusion-transmitted infections: A narrative review of the literature from India.

BACKGROUND AND OBJECTIVES: Notifying blood donors of their reactive status for transfusion-transmitted infections (TTIs) plays a vital role in enabling early diagnosis and management while also preventing these donors from making future donation and transmission of the infectious agent. Given the limited data on donor notification processes in India, a narrative review was conducted to assess the existing notification process and identify areas requiring enhancement. MATERIALS AND METHODS: We conducted literature searches using PubMed, Google Scholar and Scopus, employing various keywords. The review included data on the year of the study, study design, donor numbers, TTI screening methods, sero-reactive donor confirmation, notification frequency and methods, donor responses, post-test counselling and risk factor assessment. RESULTS: Out of the 29 identified articles, 16 studies were included in the analysis. Repeat testing for initially reactive results was conducted in nine studies for 24.3% reactive donors. Phone calls were the primary notification method in most studies (8; 50%), with letters sent in cases of no response. Only 12 studies provided data on notified donors, revealing a notification rate of 71.2%. Of all initially reactive donors, 33.3% sought post-test counselling. Data from six studies indicated that 74.3% of responsive donors had identifiable TTI risk factors. CONCLUSION: Our review revealed significant variability in the notification processes across different studies. To enhance the management of TTI-reactive donor notifications and responses, we recommend the establishment of universal protocols encompassing pre-donation counselling, repeat/confirmatory testing, notification methods and comprehensive follow-up and treatment.

Fluorescence microscopy-based sensitive method to quantify dopaminergic neurodegeneration in a Drosophila model of Parkinson's disease.

Death of dopaminergic (DAergic) neurons in the substantia nigra pars compacta of the human brain is the characteristic pathological feature of Parkinson's disease (PD). On exposure to neurotoxicants, Drosophila too exhibits mobility defects and diminished levels of brain dopamine. In the fly model of sporadic PD, our laboratory has demonstrated that there is no loss of DAergic neuronal number, however, a significant reduction in fluorescence intensity (FI) of secondary antibodies that target the primary antibody-anti-tyrosine hydroxylase (TH). Here, we present a sensitive, economical, and repeatable assay to characterize neurodegeneration based on the quantification of FI of the secondary antibody. As the intensity of fluorescence correlates with the amount of TH synthesis, its reduction under PD conditions denotes the depletion in the TH synthesis, suggesting DAergic neuronal dysfunction. Reduction in TH protein synthesis is further confirmed through Bio-Rad Stain-Free Western Blotting. Quantification of brain DA and its metabolites (DOPAC and HVA) using HPLC-ECD further demonstrated the depleted DA level and altered DA metabolism as evident from enhanced DA turnover rate. Together all these PD marker studies suggest that FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using ZEN 2012 SP2, a licensed software from Carl Zeiss, Germany. This method will be of good use to biologists, as it with few modifications, can also be implemented to characterize the extent of degeneration of different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method using fluorescence microscopy will be a feasible option for fund-constrained neurobiology laboratories in developing countries.

Sexual dysfunction precedes motor defects, dopaminergic neuronal degeneration, and impaired dopamine metabolism: Insights from Drosophila model of Parkinson's disease.

Sexual dysfunction (SD) is one of the most common non-motor symptoms of Parkinson's disease (PD) and remains the most neglected, under-reported, and under-recognized aspect of PD. Studies have shown that Dopamine (DA) in the hypothalamus plays a role in regulating sexual behavior. But the detailed mechanism of SD in PD is not known. Drosophila melanogaster shares several genes and signaling pathways with humans which makes it an ideal model for the study of a neurodegenerative disorder such as PD. Courtship behavior of Drosophila is one such behavior that is closely related to human sexual behavior and so plays an important role in understanding sexual behavior in diseased conditions as well. In the present study, a sporadic SD model of PD using Drosophila was developed and SD phenotype was observed based on abnormalities in courtship behavior markers. The Drosophila SD model was developed in such a way that at the window of neurotoxin paraquat (PQ) treatment [PQ is considered a crucial risk factor for PD due to its structural similarity with 1-methyl-4-phenyl pyridinium (MPP+), the active form of PD-inducing agent, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)], it does not exhibit mobility defects but shows SD. The whole brain tyrosine hydroxylase immunostaining showed no observable dopaminergic (DAergic) degeneration (number of DA neurons and fluorescence intensity of fluorescently labeled secondary antibodies that target anti-TH primary antibody) of the SD model. Similarly, there was no significant depletion of brain DA and its metabolite levels (HVA and DOPAC) as determined using HPLC-ECD (High-Performance Liquid Chromatography using Electrochemical Detector). The present study illustrates that the traits associated with courtship and sexual activity provide sensitive markers at the earlier stage of PD onset. This PQ-induced SD fly model throws an opportunity to decipher the molecular basis of SD under PD conditions and to screen nutraceuticals/potential therapeutic molecules to rescue SD phenotype and further to DAergic neuroprotection.

Critical appraisal of knowledge, attitude and practice studies for blood donation in India.

BACKGROUND AND OBJECTIVES: A critical appraisal of the literature helps to assess the strength and weakness of the research and suggests ways to improve future research. Our aim was to critically appraise the knowledge, attitude, and practice (KAP) studies conducted in India for blood donation. MATERIALS AND METHODS: Of 70 articles identified in our search on PubMed, Scopus and Google Scholar, 32 were assessed for quality using an appraisal tool for cross-sectional studies (AXIS) and questionnaire items. RESULTS: Quality assessment revealed that only 6 of 32 studies had acceptable reporting (≥80% score on the AXIS tool). The most frequently identified shortcomings were failure to address the non-responders, lack of justification for sample size, assessment of outcome variables and demographic results for the survey. Our evaluation of questionnaires revealed that knowledge for need for blood donation, its benefits and site/place for blood donation were assessed by very few studies. With this, issues such as parental/family consent, religious beliefs, and indifference to blood donation process were amongst the common reasons for non-donation. Many studies also failed to ask questions related to procedural information/instructions, which are necessary for promoting voluntary blood donations. CONCLUSION: Most published KAP studies for blood donation in India were not appropriately described, especially the methodology and result section. These deficiencies could have led to suboptimal interpretation of the prevalent issues. Use of an open-ended and validated KAP questionnaire with a problem-based approach and inclusion of participants from various socio-cultural backgrounds is required for good quality of evidence.

Assessment of Transfusion Practices Among Doctors During COVID-19 Pandemic Using Questionnaire-Based Survey.

During COVID-19 pandemic, many doctors were redirected from various disciplines for care of COVID-19 patients. A survey was conducted among doctors involved, to assess transfusion practices during the pandemic. To assess the knowledge, attitude, and practices of blood transfusion among doctors involved in care of COVID-19 patients. A cross-sectional survey using an online questionnaire (Google form) was used to assess knowledge and need of transfusion, attitude towards modifications in transfusion process, and practices during pandemic. Analysis was done among subgroups based on experience (designation), user type (speciality) and frequency of blood usage in parent department. Of 1900 invitations, 437 responses were received from resident doctors and faculty members across various disciplines. Of these, 354(81%) participants were included in analysis. Mean knowledge score was 6.2, majority 297(83.9%) had adequate knowledge scores (≥ 5 of total 12). Knowledge levels were higher among frequent blood users. Positive attitude towards changes in transfusion process was observed in 72.9% participants with similar scores in subgroups. Practice was assessed in 222(62.7%) participants. Mean practice score was 6.9, wherein 57.7% participants had optimal scores (≥ 7 of total 14). Positive correlation was observed between attitude and practice, unlike knowledge and practice. Although most participants had demonstrated adequate transfusion knowledge levels and positive attitude, transfusion practices during pandemic were affected mainly due to shortage of blood components and modifications in transfusion requisition process due to stringent COVID-19 containment measures. Thus, indicating need for improvement in the basic understanding of the transfusion process. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-022-01613-2.

Epidemiology of severe acute respiratory syndrome-related coronavirus 2 antibodies in healthy blood donors and their follow-up.

BACKGROUND: Asymptomatic/presymptomatic COVID-19 affected individuals who may appear healthy during blood donor screening can donate blood despite being infective. Most blood donors in India are relatives/friends/acquaintances of patients, who under peer pressure overlook the donor selection process, which can significantly impact the transfusion safety. AIMS: The prevalence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) antibodies among blood donors was assessed, along with the possible transmissibility of SARS-CoV-2 virus in transfusion recipients of blood components prepared from sero-reactive blood donors. SETTINGS AND DESIGN: A prospective cross-sectional study was conducted among eligible blood donors from November-2020 to April 2021. METHODS: 1500 blood donors were tested for SARS-CoV-2 IgG antibodies. Sero-reactive donors were followed-up telephonically to inquire about risk factors prior to donation or appearance of COVID-19 related symptoms postdonation. Patients transfused with blood components from seroreactive donors were also followed up for posttransfusion symptoms suggestive for COVID-19. Descriptive analysis was done for the donor and patient follow-up data. RESULTS: A total of 452 (30.1%) donor were reactive, with median S/CO ratio of 2.8 (interquartile range 1.5-5.5). Risk factors such as travel, contact, or quarantine were significantly higher among reactive donors. History of diabetes and/or hypertension was associated with seroreactivity. Total 516 patients were transfused with blood components from these seroreactive donors. Three patients developed fever after transfusion, one of which was found to be PCR positive after 4 days of transfusion. CONCLUSION: Sero-reactivity rate among blood donors was lower than the general population. Optimum blood donor screening strategies can help decrease the possibility of blood collection from infected blood donors.

Adult health and transition stage-specific rotenone-mediated Drosophila model of Parkinson's disease: Impact on late-onset neurodegenerative disease models.

Parkinson's disease (PD) affects almost 1% of the population worldwide over the age of 50 years. Exposure to environmental toxins like paraquat and rotenone is a risk factor for sporadic PD which constitutes 95% of total cases. Herbicide rotenone has been shown to cause Parkinsonian symptoms in multiple animal models. Drosophila is an excellent model organism for studying neurodegenerative diseases (NDD) including PD. The aging process is characterized by differential expression of genes during different life stages. Hence it is necessary to develop life-stage-matched animal models for late-onset human disease(s) such as PD. Such animal models are critical for understanding the pathophysiology of age-related disease progression and important to understand if a genotropic drug/nutraceutical can be effective during late stages. With this idea, we developed an adult life stage-specific (health and transition phase, during which late-onset NDDs such as PD sets in) rotenone-mediated Drosophila model of idiopathic PD. Drosophila is susceptible to rotenone in dose-time dependent manner. Rotenone-mediated fly model of sporadic PD exhibits mobility defects (independent of mortality), inhibited mitochondrial complex I activity, dopaminergic (DAergic) neuronal dysfunction (no loss of DAergic neuronal number; however, reduction in rate-limiting enzyme tyrosine hydroxylase (TH) synthesis), and alteration in levels of dopamine (DA) and its metabolites; 3,4-Dihydroxyphenylacetic acid (DOPAC) and Homovanilic acid (HVA) in brain-specific fashion. These PD-linked behaviors and brain-specific phenotypes denote the robustness of the present fly model of PD. This novel model will be of great help to decipher life stage-specific genetic targets of small molecule mediated DAergic neuroprotection; understanding of which is critical for formulating therapeutic strategies for PD.

Advances in protein misfolding, amyloidosis and its correlation with human diseases.

Protein aggregation, their mechanisms and trends in the field of neurodegenerative diseases is still far from completely being decoded. It is mainly attributed to the complexity surrounding the interaction between proteins which includes various regulatory mechanisms involved with the presentation of abnormal conditions. Although most proteins are functional in their soluble form, they have also been reported to convert themselves into insoluble aggregates under certain conditions naturally. Misfolded protein forms aggregates which are mostly unwanted by the cellular system and are mostly involved in various pathophysiologies including Alzheimer's, Type II Diabetes mellitus, Kurus's etc. Challenges lie in understanding the complex mechanism of protein misfolding and its correlation with clinical evidence. It is often understood that due to the slowness of the process and its association with ageing, timely intervention with drugs or preventive measures will play an essential role in lowering the rate of dementia causing diseases and associated ailments in the future. Today approximately more than 35 proteins have been identified capable of forming amyloids under defined conditions, and nearly all of them have been associated with disease outcomes. This review incorporates a major understanding from the history of diseases associated with protein misfolding, to the current state of neurodegenerative diseases globally, highlighting challenges in drug development and current state of research in a comprehensive manner in the field of protein misfolding diseases. There is increasing clinical association of protein misfolding with regards to amyloids compelling us to thread questions solved and further helping us design possible solutions by generating a pathway-based research on which future work in this field could be driven.

Discrepancy in Blood Grouping: Subgroups of B-Challenges and Dilemma.

Though not very frequent, blood group discrepancies are of common occurrence which must be promptly resolved for safe transfusion practices. Blood group discrepancy is defined as the non-corroboration between cell grouping and serum grouping. Variable erythrocyte antigenic expressions (qualitative and/or quantitative difference) owing to allelic heterogeneity, give rise to subgroups. Problems during blood grouping due to weakened expression of antigens are a rare cause for ABO discrepancies. Of note, subgroups of 'B' with decreased expression of the B antigen are of very rare occurrence in the general population. Serologically, the variants of 'B' can be classified into B3, Bx, Bm and Bel. Serological confirmation of such subgroups requires special immuno-haematological procedures. The current study reports 3 cases of subgroups of B (2 cases of Bm and 1 case of Bw) detected at our centre. It is important to detect and resolve discrepant ABO grouping results so as to prevent ABO mis-match transfusions. Such individuals should also be pre-emptively informed of their respective blood donor and transfusion recipient status, as well.

Autologous Blood Transfusion as a Life Saving Measure for a Trauma Patient with Fracture Femur and Drug Induced Hemolytic Anemia: A Case Report.

A positive direct antiglobulin test has been reported in 1:1000 to 1:14,000 blood donors and 1-15 % of hospital patients. Drugs may cause a positive direct antiglobulin test result and/or immune-mediated haemolysis with an incidence of approximately 1 in a 1 million population. Our aim is to highlight the importance of following strict transfusion protocols and management insight in a direct antiglobulin test positive patient showing incompatibility with multiple units possibly due to drug induced immune haemolytic anaemia (DIIHA). We also aim to highlight importance of autologous blood transfusion in an orthopaedic procedure in which homologous transfusion may be needed. We are presenting case of a 36 year old male with alleged h/o of road traffic accident with comminuted intra-articular fracture of the distal femur. He had h/o of ankylosing spondylitis since last 20 years on medication with indomethacin and methotrexate. A review of the literature was performed which showed use of drug methotrexate as an uncommon clinical entity for DIIHA; sporadic reports exist in the medical literature to support this view. The review of the literature in combination with our own data showed methotrexate can be a cause of DIIHA. We therefore advocate proper immunohaematological work up and use of autologous blood for management of at-risk-patients of DIIHA.

Low-density lipoprotein apheresis in a pediatric patient of familial hypercholesterolemia: Primi experientia from a tertiary care center in North India.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder due to mutation of apolipoprotein-B receptor gene causing severe dyslipidemia. Lifestyle modification and medical treatment attenuate the disease progression, but as these fail to control the blood cholesterol levels, low-density lipoprotein (LDL) apheresis comes forth as a treatment option. To the best of our knowledge, the following is the very first case of pediatric FH being treated by LDL-apheresis to be reported from India. A severely malnourished female child presented with yellowish skin lesions over different parts of the body, viz., bilateral Achilles tendon, both knees, elbows, both pinnae, and outer canthus of both eyes. She had a strong family history of borderline hypercholesterolemia and was diagnosed as a case of FH. She was maintained on diet modification. LDL-apheresis was planned as the cholesterol levels were not controlled with the diet modificationt. However, unavailability of an appropriate kit in India for LDL-apheresis led to the use of the modified PL1 kit meant for therapeutic plasma exchange procedures. We conducted two sessions of LDL-apheresis. After the first session, the LDL-cholesterol (LDL-C) level fell by 75.9% and the total cholesterol fell by 73.5%. A second procedure led to a decline in total cholesterol level by 18.6% and LDL-C by 19.46%. Subsequently, she was advised diet modification and statin therapy with regular follow-up after every 6 months. Thus, the cascade filtration technique is a safe and effective treatment option for removing the undesired lipoproteins.