A gene signature for Alzheimer’s disease using RNAi in C. elegans

Alzheimer’s disease (AD) is a complex multifactorial disorder that is responsible for the large majority of the 50 million cases of dementia worldwide. This disease is still incurable, a situation caused at least in part by the fact that its genetics are incompletely known. In our laboratory, we have developed a novel computational approach—Network-based Transcriptome-Wide Association Studies (nTWAS)—that seeks to identify the genes associated with AD by comparing gene expression patterns across tissues in the brain. nTWAS acts as an in silico pre-screen by providing a list of gene candidates, thus enabling us to pursue investigations into each gene candidate with significantly more depth. To that end, we use RNA interference in an AD model in the nematode worm Caenorhabditis elegans to validate the results of this pre-screen. C. elegans is a well-established research tool in biological and biochemical research for its ease of culture, small size, short generation time, and relative simplicity. Furthermore, the worm’s facility for genetic manipulation and remarkably similar cellular characteristics to those of humans have allowed for numerous advances in the study of cancer, neurodegeneration, and ageing. Our approach takes advantage of an automated worm tracking platform, developed in our laboratory, that can simultaneously track hundreds of worms and make precise measurements of their motility, defects of which has been shown to correlate with neurological and muscular toxicity. While standard approaches typically only take data on dozens of worms, the vastly increased population size of our approach greatly improves the statistical power of our screen. We have leveraged these improvements in screening methods to associate the differences in distributions of these parameters with phenotypic changes across various siRNA conditions. Through both motility screening and validation by imaging, we identified ckr-2, skr-21, and Y92H12A.2 as modulators of amyloid beta aggregation. While skr-21 and Y92H12A.2 are both components of the ubiquitin-proteasome system, ckr-2 is an ortholog of a neuronal cholecystokinin receptor which has been suggested to be a biomarker of AD but for which no mechanism is known. The results of this work thus contribute to extending our understanding of the gene signature of AD

Study of serum lactate dehydrogenase level and seasonal variation in preeclampsia and eclampsia with its obstetric outcome

Background: Preeclampsia and eclampsia complicate 6–8% of all pregnancies and lead to various maternal and fetal complications. LDH is an intracellular enzyme and its level is increased in this women due to cellular death. So, serum LDH levels can be used to assess the severity of disease, to improve the maternal and fetal outcome. Studies in several countries have shown higher incidence of the disease in the winter season. This study is being conducted to correlate serum LDH levels and seasonal variation in preeclampsia and eclampsia.Methods: It is a retrospective observational study. Data for 102 cases were collected from the parturition register and patient discharge record from January to December 2016. All singleton pregnant women who came to R L Jalappa Hospital with severe preeclampsia and eclampsia were included in the study.Results: Total of 102 patients were studied. Incidence of the disease was most commonly seen in younger age group, which was statistically significant (p=0.020). Even though most of the cases presented in winter (39), there was no statistically significant association between seasonal variation in occurrence of the disease and serum LDH levels. LDH raised to >800IU/L in the cases was seen more in the younger age group.Conclusions: This study only showed that preeclampsia and eclampsia occurred most commonly in younger women. This study did not show any variation in serum LDH levels in patients presenting in different seasons

Evaluation of vitamin D levels in term pregnancy and its obstetric outcome in Indian women

Background: Vitamin D deficiency is currently a global pandemic affecting all age groups. Vitamin D is considered a fundamental hormone in calcium homeostasis and bone health. Risk of vitamin D deficiency increases during pregnancy due to increased maternal and fetal demands and altered vitamin D metabolism. Recently, maternal vitamin D deficiency has been linked to adverse pregnancy outcomes, including preeclampsia, gestational diabetes, fetal growth restriction and preterm birth. Adequate vitamin D status appears to be relevant to health at all ages, and even in prenatal life.Methods: This is a cross sectional, observational study conducted in the department of obstetrics and gynaecology at R. L. Jalappa Hospital. A total number of 160 subjects were included. 5 ml of venous blood was collected and was centrifuged at 3000 rpm and stored at - 80°C till analysis. Analysis of 25-hydroxy Vitamin D was done using ELISA.Results: Majority of the subjects were vitamin D deficient (81.87%) and 12.5% were vitamin D insufficient and only 5.63% were vitamin D sufficient. The prevalence of vitamin D deficiency was more among primigravidas (85.6%) and was associated with higher rates of caesarean section (92.4%). High prevalence of vitamin D deficiency was seen in lower middle socioeconomic class (62.5%). Maternal vitamin D deficiency was associated low birth weight of neonates (100%).Conclusions: In this study it was concluded that majority of subjects were vitamin D deficient and belonged to lower middle socioeconomic class. Majority of this subjects who underwent caesarean section were vitamin D deficient. Vitamin D deficiency was associated only with low birth weight of neonates and no other adverse obstetric outcome

DNA-Liposome Hybrid Carriers for Triggered Cargo Release

The design of simple and versatile synthetic routes to accomplish triggered-release properties in carriers is of particular interest for drug delivery purposes. In this context, the programmability and adaptability of DNA nanoarchitectures in combination with liposomes have great potential to render biocompatible hybrid carriers for triggered cargo release. We present an approach to form a DNA mesh on large unilamellar liposomes incorporating a stimuli-responsive DNA building block. Upon incubation with a single-stranded DNA trigger sequence, a hairpin closes, and the DNA building block is allowed to self-contract. We demonstrate the actuation of this building block by single-molecule Förster resonance energy transfer (FRET), fluorescence recovery after photobleaching, and fluorescence quenching measurements. By triggering this process, we demonstrate the elevated release of the dye calcein from the DNA-liposome hybrid carriers. Interestingly, the incubation of the doxorubicin-laden active hybrid carrier with HEK293T cells suggests increased cytotoxicity relative to a control carrier without the triggered-release mechanism. In the future, the trigger could be provided by peritumoral nucleic acid sequences and lead to site-selective release of encapsulated chemotherapeutics. © 2022 American Chemical Society. All rights reserved

Widespread Proteome Remodeling and Aggregation in Aging C-elegans

Aging has been associated with a progressive decline of proteostasis, but how this process affects proteome composition remains largely unexplored. Here, we profiled more than 5,000 proteins along the lifespan of the nematode C. elegans. We find that one-third of proteins change in abundance at least 2-fold during aging, resulting in a severe proteome imbalance. These changes are reduced in the long-lived daf-2 mutant but are enhanced in the short-lived daf-16 mutant. While ribosomal proteins decline and lose normal stoichiometry, proteasome complexes increase. Proteome imbalance is accompanied by widespread protein aggregation, with abundant proteins that exceed solubility contributing most to aggregate load. Notably, the properties by which proteins are selected for aggregation differ in the daf-2 mutant, and an increased formation of aggregates associated with small heat-shock proteins is observed. We suggest that sequestering proteins into chaperone-enriched aggregates is a protective strategy to slow proteostasis decline during nematode aging

Supersaturated proteins are enriched at synapses and underlie cell and tissue vulnerability in Alzheimer's disease.

Neurodegenerative disorders progress across the brain in characteristic spatio-temporal patterns. A better understanding of the factors underlying the specific cell and tissue vulnerability responsible for such patterns could help identify the molecular origins of these conditions. To investigate these factors, based on the observation that neurodegenerative disorders are closely associated with the presence of aberrant protein deposits, we made the hypothesis that the vulnerability of cells and tissues is associated to the overall levels of supersaturated proteins, which are those most metastable against aggregation. By analyzing single-cell transcriptomic and subcellular proteomics data on healthy brains of ages much younger than those typical of disease onset, we found that the most supersaturated proteins are enriched in cells and tissues that succumb first to neurodegeneration. Then, by focusing the analysis on a metastable subproteome specific to Alzheimer's disease, we show that it is possible to recapitulate the pattern of disease progression using data from healthy brains. We found that this metastable subproteome is significantly enriched for synaptic processes and mitochondrial energy metabolism, thus rendering the synaptic environment dangerous for aggregation. The present identification of protein supersaturation as a signature of cell and tissue vulnerability in neurodegenerative disorders could facilitate the search for effective treatments by providing clearer points of intervention

A metastable subproteome underlies inclusion formation in muscle proteinopathies

Protein aggregation is a pathological feature of neurodegenerative disorders. We previously demonstrated that protein inclusions in the brain are composed of supersaturated proteins, which are abundant and aggregation-prone, and form a metastable subproteome. It is not yet clear, however, whether this phenomenon is also associated with non-neuronal protein conformational disorders. To respond to this question, we analyzed proteomic datasets from biopsies of patients with genetic and acquired protein aggregate myopathy (PAM) by quantifying the changes in composition, concentration and aggregation propensity of proteins in the fibers containing inclusions and those surrounding them. We found that a metastable subproteome is present in skeletal muscle from healthy patients. The expression of this subproteome escalate as proteomic samples are taken more proximal to the pathologic inclusion, eventually exceeding its solubility limits and aggregating. While most supersaturated proteins decrease or maintain steady abundance across healthy fibers and inclusion-containing fibers, proteins within the metastable subproteome rise in abundance, suggesting that they escape regulation. Taken together, our results show in the context of a human conformational disorder that the supersaturation of a metastable subproteome underlies widespread aggregation and correlates with the histopathological state of the tissue

The H50Q mutation induces a 10-fold decrease in the solubility of α-synuclein.

The conversion of α-synuclein from its intrinsically disordered monomeric state into the fibrillar cross-β aggregates characteristically present in Lewy bodies is largely unknown. The investigation of α-synuclein variants causative of familial forms of Parkinson disease can provide unique insights into the conditions that promote or inhibit aggregate formation. It has been shown recently that a newly identified pathogenic mutation of α-synuclein, H50Q, aggregates faster than the wild-type. We investigate here its aggregation propensity by using a sequence-based prediction algorithm, NMR chemical shift analysis of secondary structure populations in the monomeric state, and determination of thermodynamic stability of the fibrils. Our data show that the H50Q mutation induces only a small increment in polyproline II structure around the site of the mutation and a slight increase in the overall aggregation propensity. We also find, however, that the H50Q mutation strongly stabilizes α-synuclein fibrils by 5.0 ± 1.0 kJ mol(-1), thus increasing the supersaturation of monomeric α-synuclein within the cell, and strongly favors its aggregation process. We further show that wild-type α-synuclein can decelerate the aggregation kinetics of the H50Q variant in a dose-dependent manner when coaggregating with it. These last findings suggest that the precise balance of α-synuclein synthesized from the wild-type and mutant alleles may influence the natural history and heterogeneous clinical phenotype of Parkinson disease

Clinical Utilization of the FilmArray Meningitis/Encephalitis (ME) Multiplex Polymerase Chain Reaction (PCR) Assay

Objective: To assess the clinical utilization and performance of the FilmArray® Meningitis/Encephalitis (ME) multiplex polymerase chain reaction (PCR) panel in a hospital setting.Background: Rapid diagnosis and treatment of central nervous system (CNS) infections are critical to reduce morbidity and mortality. The ME panel is a Food and Drug Administration (FDA) approved rapid multiplex PCR assay that targets 14 bacteria, viruses, and fungi. Previous studies show an overall agreement of 93–99% between the ME panel and conventional diagnostic testing. However, few studies have evaluated the clinical implementation of the ME assay, which is available for routine use at our institution.Methods: We performed a single center retrospective chart review of inpatients who underwent ME panel testing from August 2016 to May 2017. Clinical, radiologic, and laboratory data were reviewed to determine the clinical significance of results. Indication for lumbar puncture (LP), time to results of the ME panel, and duration of antimicrobial therapy were evaluated.Results: Seven hundred and five inpatients underwent ME testing, of whom 480 (68.1%) had clinical suspicion for CNS infection with 416 (59.0%) receiving empiric antimicrobial treatment for CNS infection. The median time-to-result of the ME panel was 1.5 h (IQR, 1.4–1.7). Overall agreement between the ME panel results and clinico-laboratory assessment was 98.2%. Forty-five patients tested positive by ME, of which 12 (26.6%) were determined likely to be clinically insignificant.Conclusions: Routine availability of the ME panel led to overutilization of diagnostic test ordering, as demonstrated by the fact that over one-third of ME panel tests performed were ordered for patients with little or no suspicion for CNS infection. The median time from LP to ME panel result was 1.5 h (IQR, 1.4–1.7). The ME panel's rapid turn-around time contributed to the overuse of the test. Approximately one-quarter of positive ME results were deemed clinically insignificant, though the impact of these positive results requires additional evaluation. Twenty-four and forty-eight hours after the ME panel resulted, 68 and 25% of patients started on empiric therapy remained on antibiotics, respectively. The median time from diagnosis to discontinuation and/or narrowing of antibiotic coverage was 25.6 h (IQR, 3.6–42.5). Further consideration of the appropriate indications for use of the ME panel in clinical settings is required

A metastable subproteome underlies inclusion formation in muscle proteinopathies

Abstract: Protein aggregation is a pathological feature of neurodegenerative disorders. We previously demonstrated that protein inclusions in the brain are composed of supersaturated proteins, which are abundant and aggregation-prone, and form a metastable subproteome. It is not yet clear, however, whether this phenomenon is also associated with non-neuronal protein conformational disorders. To respond to this question, we analyzed proteomic datasets from biopsies of patients with genetic and acquired protein aggregate myopathy (PAM) by quantifying the changes in composition, concentration and aggregation propensity of proteins in the fibers containing inclusions and those surrounding them. We found that a metastable subproteome is present in skeletal muscle from healthy patients. The expression of this subproteome escalate as proteomic samples are taken more proximal to the pathologic inclusion, eventually exceeding its solubility limits and aggregating. While most supersaturated proteins decrease or maintain steady abundance across healthy fibers and inclusion-containing fibers, proteins within the metastable subproteome rise in abundance, suggesting that they escape regulation. Taken together, our results show in the context of a human conformational disorder that the supersaturation of a metastable subproteome underlies widespread aggregation and correlates with the histopathological state of the tissue