Harnessing the Cross-Neutralisation Potential of Existing Antivenoms for Mitigating the Outcomes of Snakebite in Sub-Saharan Africa.

Over 32,000 individuals succumb to snake envenoming in sub-Saharan Africa (sSA) annually. This results from several factors, including a lack of antivenom products capable of neutralising the venoms of diverse snake species in this region. Most manufacturers produce polyvalent antivenoms targeting 3 to 16 clinically important snake species in sSA. However, specific products are unavailable for many others, especially those with a restricted geographic distribution. While next-generation antivenoms, comprising a cocktail of broadly neutralising antibodies, may offer an effective solution to this problem, given the need for their clinical validation, recombinant antivenoms are far from being available to snakebite victims. One of the strategies that could immediately address this issue involves harnessing the cross-neutralisation potential of existing products. Therefore, we assessed the neutralisation potency of PANAF-Premium antivenom towards the venoms of 14 medically important snakes from 13 countries across sSA for which specific antivenom products are unavailable. Preclinical assays in a murine model of snake envenoming revealed that the venoms of most snake species under investigation were effectively neutralised by this antivenom. Thus, this finding highlights the potential use of PANAF-Premium antivenom in treating bites from diverse snakes across sSA and the utility of harnessing the cross-neutralisation potential of antivenoms.

Nuclear architecture and the structural basis of mitotic memory.

The nucleus is a complex organelle that hosts the genome and is essential for vital processes like DNA replication, DNA repair, transcription, and splicing. The genome is non-randomly organized in the three-dimensional space of the nucleus. This functional sub-compartmentalization was thought to be organized on the framework of nuclear matrix (NuMat), a non-chromatin scaffold that functions as a substratum for various molecular processes of the nucleus. More recently, nuclear bodies or membrane-less subcompartments of the nucleus are thought to arise due to phase separation of chromatin, RNA, and proteins. The nuclear architecture is an amalgamation of the relative organization of chromatin, epigenetic landscape, the nuclear bodies, and the nucleoskeleton in the three-dimensional space of the nucleus. During mitosis, the nucleus undergoes drastic changes in morphology to the degree that it ceases to exist as such; various nuclear components, including the envelope that defines the nucleus, disintegrate, and the chromatin acquires mitosis-specific epigenetic marks and condenses to form chromosome. Upon mitotic exit, chromosomes are decondensed, re-establish hierarchical genome organization, and regain epigenetic and transcriptional status similar to that of the mother cell. How this mitotic memory is inherited during cell division remains a puzzle. NuMat components that are a part of the mitotic chromosome in the form of mitotic chromosome scaffold (MiCS) could potentially be the seeds that guide the relative re-establishment of the epigenome, chromosome territories, and the nuclear bodies. Here, we synthesize the advances towards understanding cellular memory of nuclear architecture across mitosis and propose a hypothesis that a subset of NuMat proteome essential for nucleation of various nuclear bodies are retained in MiCS to serve as seeds of mitotic memory, thus ensuring the daughter cells re-establish the complex status of nuclear architecture similar to that of the mother cells, thereby maintaining the pre-mitotic transcriptional status.

In situ Nuclear Matrix preparation in Drosophila melanogaster enabling genetic analysis of the nuclear architecture.

Nuclear Matrix (NuMat) is a biochemically defined entity that provides us with a snapshot of the features of the nuclear architecture. Here, we present a protocol to isolate and visualize NuMat in situ in the intact embryo or tissues of Drosophila melanogaster and its applications. We remove the chromatin to reveal underlying nuclear architectural components in organismal context. This protocol couples the power of Drosophila genetics with cell biological observation of the nuclear architecture. For complete details on the use and execution of this protocol, please refer to Pathak et al. (2022), Sureka et al. (2018), and Pathak et al. (2013).

In situ nuclear matrix preparation in Drosophila melanogaster embryos/tissues and its use in studying the components of nuclear architecture.

The study of nuclear matrix (NuMat) over the last 40 years has been limited to either isolated nuclei from tissues or cells grown in culture. Here, we provide a protocol for NuMat preparation in intact Drosophila melanogaster embryos and its use in dissecting the components of nuclear architecture. The protocol does not require isolation of nuclei and therefore maintains the three-dimensional milieu of an intact embryo, which is biologically more relevant compared to cells in culture. One of the advantages of this protocol is that only a small number of embryos are required. The protocol has been extended to larval tissues like salivary glands with little modification. Taken together, it becomes possible to carry out such studies in parallel to genetic experiments using mutant/transgenic flies. This protocol, therefore, opens the powerful field of fly genetics to cell biology in the study of nuclear architecture.Summary: Nuclear Matrix is a biochemically defined entity and a basic component of the nuclear architecture. Here we present a protocol to isolate and visualize Nuclear Matrix in situ in the Drosophila melanogaster and its potential applications.

Genomic organization of the autonomous regulatory domain of eyeless locus in Drosophila melanogaster.

In Drosophila, expression of eyeless (ey) gene is restricted to the developing eyes and central nervous system. However, the flanking genes, myoglianin (myo), and bent (bt) have different temporal and spatial expression patterns as compared to the ey. How distinct regulation of ey is maintained is mostly unknown. Earlier, we have identified a boundary element intervening myo and ey genes (ME boundary) that prevents the crosstalk between the cis-regulatory elements of myo and ey genes. In the present study, we further searched for the cis-elements that define the domain of ey and maintain its expression pattern. We identify another boundary element between ey and bt, the EB boundary. The EB boundary separates the regulatory landscapes of ey and bt genes. The two boundaries, ME and EB, show a long-range interaction as well as interact with the nuclear architecture. This suggests functional autonomy of the ey locus and its insulation from differentially regulated flanking regions. We also identify a new Polycomb Response Element, the ey-PRE, within the ey domain. The expression state of the ey gene, once established during early development is likely to be maintained with the help of ey-PRE. Our study proposes a general regulatory mechanism by which a gene can be maintained in a functionally independent chromatin domain in gene-rich euchromatin.

Remarkable intrapopulation venom variability in the monocellate cobra (Naja kaouthia) unveils neglected aspects of India's snakebite problem.

Interpopulation venom variation has been widely documented in snakes across large geographical distances. This variability is known to markedly influence the effectiveness of snakebite therapy, as antivenoms manufactured against one population may not be effective against others. In contrast, the extent of intrapopulation venom variability, especially at finer geographical scales, remains largely uninvestigated. Moreover, given the historical focus on the 'big four' Indian snakes, our understanding of venom variation in medically important yet neglected snakes, such as the monocellate cobra (Naja kaouthia), remains unclear. To address this shortcoming, we investigated N. kaouthia venoms sampled across a small spatial scale (<50 km) in Eastern India. An interdisciplinary approach employed in this study unveiled considerable intrapopulation differences in the venom proteomic composition, pharmacological and biochemical activities, and toxicity profiles. Documentation of stark differences in venoms at such a finer geographical scale, despite the influence of similar ecological and environmental conditions, is intriguing. Furthermore, evaluation of in vitro and in vivo venom recognition and neutralisation potential of Indian polyvalent 'big four' antivenoms and Thai monovalent N. kaouthia antivenom revealed concerning deficiencies. These results highlight the negative impact of phylogenetic divergence and intrapopulation snake venom variation on the effectiveness of conventional antivenom therapy. SIGNIFICANCE: In contrast to our understanding of snake venom variation across large distances, which is theorised to be shaped by disparities in ecology and environment, intrapopulation variation at finer geographic scales remains scarcely investigated. Assessment of intrapopulation venom variability in Naja kaouthia at a small spatial scale (<50 km) in Eastern India unravelled considerable differences in venom compositions, activities and potencies. While the influence of subtle differences in prey preference and local adaptations cannot be ruled out, these findings, perhaps, also emphasise the role of accelerated molecular evolutionary regimes that rapidly introduce variations in evolutionarily younger lineages, such as advanced snakes. The inability of 'big four' Indian antivenoms and Thai N. kaouthia monovalent antivenom in countering these variations highlights the importance of phylogenetic considerations for the development of efficacious snakebite therapy. Thus, we provide valuable insights into the venoms of one of the most medically important yet neglected Indian snakes.

Sperm methylome alterations following yoga-based lifestyle intervention in patients of primary male infertility: A pilot study.

A majority of the cases of primary male infertility are idiopathic with the underlying molecular mechanisms contributing to the pathophysiology as yet unknown. Effects of the environment can alter the sperm epigenome thereby impacting male reproductive health. Epigenetic mechanisms are crucial to understanding health and disease, and methylome alterations are now known to have far-reaching clinical implications. Here, we report the results from our pilot study, a first of its kind analysis of the effect of the traditional practice of yoga on human sperm quality. We find marked improvement in sperm characteristics in patients of idiopathic male infertility following a supervised 21-day yoga regimen. Furthermore, next-generation sequencing-based methylome analysis reveals alterations in the sperm epigenome of these patients. We find that the practice of yoga is associated with DNA methylation changes at nearly 400 genes, 147 of which were hypermethylated while 229 were hypomethylated. These included promoters of several genes linked to maintenance of fertility and genomic integrity. This novel piece of work draws a direct link between positive lifestyle practices and male reproductive health.

Comparison of Nuclear Matrix and Mitotic Chromosome Scaffold Proteins in Drosophila S2 Cells-Transmission of Hallmarks of Nuclear Organization Through Mitosis.

Chromatin condenses several folds to form mitotic chromosomes during cell division and decondenses post-mitotically to reoccupy their nuclear territory and regain their specific transcriptional profile in a precisely lineage specific manner. This necessitates that the features of nuclear architecture and DNA topology persist through mitosis. We compared the proteome of nuclease and high salt resistant fraction of interphase nucleus known as nuclear matrix (NuMat) and an equivalent biochemical fraction in the mitotic chromosome known as mitotic chromosome scaffold (MiCS). Our study elucidates that as much as 67% of the NuMat proteins are retained in the MiCS indicating that the features of nuclear architecture in interphase nucleus are retained on the mitotic chromosomes. Proteins of the NuMat/MiCS have large dynamic range of MS signal and were detected in sub-femtomolar amounts. Chromatin/RNA binding proteins with hydrolase and helicase activity are highly enriched in NuMat as well as MiCS. Although several transcription factors involved in functioning of interphase nucleus are present exclusively in NuMat, protein components responsible for assembly of membrane-less nuclear bodies are uniquely retained in MiCS. Our study clearly indicates that the features of nuclear architecture, in the structural context of NuMat, are retained in MiCS and possibly play an important role in maintenance of cell lineage specific transcriptional status during cell division and thereby, serve as components of cellular memory.

O-GlcNAcylation of boundary element associated factor (BEAF 32) in Drosophila melanogaster correlates with active histone marks at the promoters of its target genes.

Boundary Element-Associated Factor 32 (BEAF 32) is a sequence specific DNA binding protein involved in functioning of chromatin domain boundaries in Drosophila. Several studies also show it to be involved in transcriptional regulation of a large number of genes, many of which are annotated to have cell cycle, development and differentiation related function. Since post-translational modifications (PTMs) of proteins add to their functional capacity, we investigated the PTMs on BEAF 32. The protein is known to be phosphorylated and O-GlcNAcylated. We mapped O-GlcNAc site at T91 of BEAF 32 and showed that it is linked to the deposition of active histone (H3K4me3) marks at transcription start site (TSS) of associated genes. Its role as a boundary associated factor, however, does not depend on this modification. Our study shows that by virtue of O-GlcNAcylation, BEAF 32 is linked to epigenetic mechanisms that activate a subset of associated genes.

Long Noncoding RNAs in Mammalian Development and Diseases.

Following analysis of sequenced genomes and transcriptome of many eukaryotes, it is evident that virtually all protein-coding genes have already been discovered. These advances have highlighted an intriguing paradox whereby the relative amount of protein-coding sequences remain constant but nonprotein-coding sequences increase consistently in parallel to increasing evolutionary complexity. It is established that differences between species map to nonprotein-coding regions of the genome that surprisingly is transcribed extensively. These transcripts regulate epigenetic processes and constitute an important layer of regulatory information essential for organismal development and play a causative role in diseases. The noncoding RNA-directed regulatory circuit controls complex characteristics. Sequence variations in noncoding RNAs influence evolution, quantitative traits, and disease susceptibility. This chapter presents an account on a class of such noncoding transcripts that are longer than 200 nucleotides (long noncoding RNA-lncRNA) in mammalian development and diseases.

Knockdown of Broad-Complex Gene Expression of Bombyx mori by Oligopyrrole Carboxamides Enhances Silk Production.

Bombyx mori (B. mori) is important due to its major role in the silk production. Though DNA binding ligands often influence gene expression, no attempt has been made to exploit their use in sericulture. The telomeric heterochromatin of B. mori is enriched with 5'-TTAGG-3' sequences. These sequences were also found to be present in several genes in the euchromatic regions. We examined three synthetic oligopyrrole carboxamides that target 5'-TTAGG-3' sequences in controlling the gene expression in B. mori. The ligands did not show any defect or feeding difference in the larval stage, crucial for silk production. The ligands caused silencing of various isoforms of the broad-complex transcription factor and cuticle proteins which resulted in late pupal developmental defects. Furthermore, treatment with such drugs resulted in statistically enhanced cocoon weight, shell weight, and silk yield. This study shows for the first time use of oligopyrrole carboxamide drugs in controlling gene expression in B. mori and their long term use in enhancing silk production.

Impact of cardiac resynchronization therapy on clinical outcomes in patients with continuous-flow left ventricular assist devices.

BACKGROUND: Implantable cardioverter-defibrillators (ICDs) can improve survival in left ventricular assist device (LVAD) recipients. However, the impact of cardiac resynchronization therapy (CRT-D) on outcomes in continuous-flow left ventricular assist device (CF-LVAD) patients is not known. We sought to determine if CRT-D improved clinical outcomes in CF-LVAD patients compared with ICDs alone. METHODS AND RESULTS: Sixty-one consecutive CF-LVAD patients with an ICD or CRT-D were evaluated. Impacts of CRT-D on mortality, all-cause hospitalization, and incidence of atrial (AA) and ventricular (VA) arrhythmias after LVAD implantation was compared with patients with ICD alone. Of the 61 LVAD patients, 31 (age 59.8 ± 16 years, 84% male) had CRT-D and 30 (age 57.2 ± 13 years, 74% male) had ICD. Before LVAD implantation, no significant differences were noted between the groups in demographic and clinical characteristics, LVAD indications, and incidence of AA and VA. Over 682 ± 45 days of LVAD support, 8 patients (25.8%) died in the CRT-D arm versus 5 (16.7%) in the ICD arm (P = .35). No differences were noted between the CRT-D and ICD groups in all-cause (96.8 vs 93.3%; P = .63) and HF (19.4 vs 26.7%; P = .78) hospitalizations, left ventricular (LV) end-diastolic diameter (6.4 ± 1.5 vs 6.2 ± 1.1 cm, P = .47), and incidence of AA (35.4% vs 33.3%; P = .80), VA (29% vs 26.6%; P = .86), and ICD shocks (22.6% vs 16.7%; P = .93). Beta-blocker and antiarrhythmic drug use after LVAD implantation was similar in both groups. CONCLUSIONS: In patients with refractory HF who received CF-LVADs, CRT-D, compared with ICD, did not significantly improve mortality, all-cause hospitalization, LV dimensions, and incidence of AA and VA.

Genome-wide mapping of matrix attachment regions in Drosophila melanogaster.

BACKGROUND: Eukaryotic genome acquires functionality upon proper packaging within the nucleus. This process is facilitated by the structural framework of Nuclear Matrix, a nucleo-proteinaceous meshwork. Matrix Attachment Regions (MARs) in the genome serve as anchoring sites to this framework. RESULTS: Here we report direct sequencing of the MAR preparation from Drosophila melanogaster embryos and identify >7350 MARs. This amounts to ~2.5% of the fly genome and often coincide with AT rich non-coding regions. We find significant association of MARs with the origins of replication, transcription start sites, paused RNA Polymerase II sites and exons, but not introns, of highly expressed genes. We also identified sequence motifs and repeats that constitute MARs. CONCLUSION: Our data reveal the contact points of genome to the nuclear architecture and provide a link between nuclear functions and genomic packaging.

Inpatient hospitalization and intimate partner violence: who are we treating?

BACKGROUND: The majority of research into health care use of intimate partner violence (IPV) has focused on female victims and primarily their emergency department use. There are limited data on injury-related hospitalization rates for female victims and even less for male victims and perpetrators. The goal of this study was to determine the annualized rates of inpatient injury-related hospitalization among individuals involved as either victims or defendants in IPV. METHODS: This was an observational retrospective study linking data from two Level 1 trauma centers and the county prosecutor's office from 2000 to 2010 in Kalamazoo County, Michigan. (1) Hospital data included injury-related admissions (DRG International Classification of Diseases-9th Rev. codes 800-959.9 excluding 905-909.9). (2) Prosecutor data contained all charging requests for crimes between intimate partners. Annualized rates were calculated for the year before the IPV crime and for the year after, using the following algorithm: (number of hospitalizations) / (total population) × (per 10,000). Confidence intervals and two-sided statistical significance were calculated at the 95% confidence level. RESULTS: During the study period, 21,179 IPV crimes were committed, involving 12,913 individual defendants and 14,797 victims. There were 30,301 injury-related hospitalizations by this group during this period. Compared with national hospitalization rates of 3.2 per 10,000 people for injury/poisoning (DRG International Classification of Diseases-9th Rev. 800-959.9 and 990-995), IPV victim annual admission rates were 31.9, defendants at 90.4, and bidirectional individuals at 339.1 per 10,000 people, in the 2 years surrounding the crime. Males, regardless of crime role, have higher injury-related hospitalization rates in this period compared with females (male, 115.6; female, 41.8). Males (victims or defendants) and bidirectional participants of either sex had rates that were significantly higher the year after than the year before the crime. CONCLUSION: Individuals involved in IPV have a 10-fold higher injury-related hospitalization rate as compared with age-matched national rates. Admission rates vary by sex, crime role, and time frame, with males and bidirectional participants having the highest rates. LEVEL OF EVIDENCE: Epidemiological study, level III.

Catheter ablation of atrial flutter in patients with left ventricular assist device improves symptoms of right heart failure.

Persistent atrial flutter (AFL) in left ventricular assist device (LVAD) recipients can result in loss of AV synchrony, impaired ventricular filling and right heart failure (RHF). The authors report the largest series of HeartMate II (HMII) patients who developed AFL with decompensated RHF, which successfully resolved with AFL ablation. Eight patients with HMII LVAD (mean age, 57±12 years) had medically refractory AFL, with 7 developing de novo AFL after LVAD implant (onset range, 2 days-22 months post-implant). Three patients developed recurrent syncope, 2 had inappropriate implantable cardioverter-defibrillator shocks, and 6 had new or escalating need for inotropes. All had features of decompensated RHF. Seven patients underwent electrophysiology testing where mapping confirmed typical counterclockwise AFL (mean AFL cycle length, 252±49 ms) and radiofrequency ablation of cavotricuspid isthmus restored sinus rhythm in all patients. Complete resolution of symptoms and signs of RHF with improved quality of life were noted in all. No procedural complications were noted. During a mean follow-up of 9±5 months, all patients remained free of atrial flutter. Catheter ablation of AFL in LVAD patients is safe and highly effective, resulting in immediate and significant improvement in symptoms of RHF, and should be considered first-line therapy for AFL in these patients.

AAGAG repeat RNA is an essential component of nuclear matrix in Drosophila.

Eukaryotic nucleus is functionally as well as spatially compartmentalized and maintains dynamic organization of sub-nuclear bodies. This organization is supported by a non-chromatin nuclear structure called the nuclear matrix. Although the precise molecular composition and ultra-structure of the nuclear matrix is not known, proteins and RNA molecules are its major components and several nuclear matrix proteins have been identified. However, the nature of its RNA component is unknown. Here we show that in Drosophila melanogaster, transcripts from AAGAG repeats of several hundred nucleotide in length are critical constituents of the nuclear matrix. While both the strands of this repeat are transcribed and are nuclear matrix associated, the polypurine strand is predominantly detected in situ. We also show that AAGAG RNA is essential for viability. Our results reveal the molecular identity of a critical RNA component of the nuclear architecture and point to one of the utilities of the repetitive part of the genome that has accumulated in higher eukaryotes.

'Inappropriate' sinus tachycardia: does the 100 beats per min cut-off matter?

Sinus tachycardia is commonly encountered in clinical practice and when persistent, can result in significant symptoms and impaired quality of life, warranting further evaluation. On the other hand, a growing body of epidemiological and clinical evidence has shown that high resting heart rate (HR) within the accepted normal range is independently associated with increased risk of all-cause and cardiovascular mortality. However, higher HR as a risk factor for adverse cardiovascular outcomes is frequently underappreciated. In this review, we focus on two challenging problems that span the spectrum of abnormally fast sinus HR. The first section reviews inappropriate sinus tachycardia, a complex disorder characterized by rapid sinus HR without a clear underlying cause, with particular emphasis on current management options. The latter section discusses the prognostic significance of elevated resting HR and reviews clinical evidence aimed at modifying this simple, yet highly important risk factor.

Transposable element 'roo' attaches to nuclear matrix of the Drosophila melanogaster.

The genome of eukaryotes is organized into structural units of chromatin loops. This higher order organization is supported by a nuclear skeleton called the nuclear matrix. The genomic DNA associated with the nuclear matrix is called the matrix associated region (MAR). Only a few genome-wide screens have been attempted, although many studies have characterized locusspecific MAR DNA sequences. In this study, a MAR DNA library was prepared from the Drosophila melanogaster Meigen (Diptera: Drosophilidae) genome. One of the sequences identified as a MAR was from a long terminal repeat region of 'roo' retrotransposon (roo MAR). Sequence analysis of roo MAR showed its distribution across the D. melanogaster genome. roo MAR also showed high sequence similarity with a previously identified MAR in Drosophila, namely the 'gypsy' retrotransposon. Analysis of the genes flanking roo MAR insertions in the Drosophila genome showed that genes were co-ordinately expressed. The results from the present study in D. melanogaster suggest this sequence plays an important role in genome organization and function. The findings point to an evolutionary role of retrotransposons in shaping the genomic architecture of eukaryotes.

Complex I and energy thresholds in the brain.

Mitochondrial electron transport chain (ETC) deficiencies are thought to underlie defects in energy metabolism and have been implicated in the neurodegenerative process. In particular, reductions in complex I activities in Parkinson's disease are thought to cause bioenergetic dysfunction with subsequent degeneration of dopaminergic neurons. In terms of bioenergetics and assessing ETC-related problems in the brain, the presence of heterogeneous mitochondria has complicated matters as isolated non-synaptic mitochondria have different energy thresholds and flux control coefficients compared to isolated mitochondria of synaptic origin. The molecular mechanisms that underlie complex I deficiencies in the parkinsonian brain are unknown and are the source of intensive research. This review explores the relationship between complex I activity and energy metabolism in the brain as well as the nature of the complex I defect.

Reducing delay in seeking treatment by patients with acute coronary syndrome and stroke: a scientific statement from the American Heart Association Council on Cardiovascular Nursing and Stroke Council.

Patient delay in seeking treatment for acute coronary syndrome and stroke symptoms is the major factor limiting delivery of definitive treatment in these conditions. Despite decades of research and public education campaigns aimed at decreasing patient delay times, most patients still do not seek treatment in a timely manner. In this scientific statement, we summarize the evidence that (1) demonstrates the benefits of early treatment, (2) describes the extent of the problem of patient delay, (3) identifies the factors related to patient delay in seeking timely treatment, and (4) reveals the inadequacies of our current approaches to decreasing patient delay. Finally, we offer suggestions for clinical practice and future research.