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Several nuclear reactor designs rely on passive containment cooling systems. The so-called containment wall condenser relies on natural circulation loops to extract heat from high-temperature steam in the containment to a water tank at ambient pressure. In such passive systems, phase changes can happen and cause flow instabilities in the cooling loop. The flashing-induced instability occurs when the heated fluid in the riser suddenly vaporizes due to a hydrostatic pressure decrease. This instability causes periodic flow peaks, which are of major concern but whose characteristics have not been studied quantitatively. This paper presents two analytical models that predict the flashing frequency and a maximum flow amplitude from geometry and basic operating parameters such as power level and reservoir temperature. The expressions are derived from a physical analysis and do not involve any calibration constants. The flashing frequency appears to be driven by the power level, the inlet temperature and the riser pipe geometry. For the amplitude, the maximum flow rate can be expressed in a Froude number that depends only on the total pressure losses. These models are validated against PASI experiments and system-scale simulations with the CATHARE 3 code, both performed as part of the European Commission funded PASTELS project. Additional data from numerous experimental studies in the literature are used to extend the validity range of the frequency model. Successfully validated against experimental data and additional simulations, these models provide an explicit relationship between oscillations characteristics and design parameters, making them valuable tools for nuclear engineers. • Flashing frequency is proportional to the power-to-volume ratio. • Flashing frequency can be derived from power, inlet temperature and riser pipe geometry. • Flashing amplitude can be derived from riser head and total pressure losses. [ABSTRACT FROM AUTHOR]

Within the Canadian context, the physical activity levels of children and youth in the after school time period has become a source of public health concern. We argue that this concern is informed by broader public health crises, in particular the ‘global obesity epidemic’ and the closely related ‘global pandemic of physical inactivity', and that these so-called ‘crises’ operate as part of a discursive regime that serves to justify after school interventions aimed at increasing the physical activity practices of children and youth. Although the objectives of such interventions are seemingly well intentioned, we suggest that such interventions nonetheless harbor difficult to discern, but potentially pernicious consequences, for the communities in which they are implemented. We focus our attention on the place-specific effects of one Public Health Agency of Canada-funded after school physical activity intervention – After the School Bell Rings (ASBR) – that was implemented in the mostly Indigenous, northern community of Placid, Manitoba. Based on a critical analysis of the ASBR program itself, along with interviews and focus groups with children, parents and recreation providers (n = 10) from the community of Placid, we contend that the ASBR serves to govern Indigenous recreation at a distance. We argue that when implemented in the place-specific context of Placid, the ASBR functions as part of a broader governmental assemblage composed of loosely connected discourses, institutions, socio-structural conditions and individuals that, when assembled, ultimately serve to govern geographically and culturally distinct communities. We conclude by suggesting that the objectives of broad-based health interventions, such as the goal of increasing after school physical activity levels, should not be universally implemented across diverse locales, but need to account for the diverse, place-specific priorities and needs of the communities into which they are implemented. [ABSTRACT FROM PUBLISHER]

Understanding the evolutionary consequences of anthropogenic change is imperative for estimating long‐term species resilience. While contemporary genomic data can provide us with important insights into recent demographic histories, investigating past change using present genomic data alone has limitations. In comparison, temporal genomics studies, defined herein as those that incorporate time series genomic data, utilize museum collections and repeated field sampling to directly examine evolutionary change. As temporal genomics is applied to more systems, species and questions, best practices can be helpful guides to make the most efficient use of limited resources. Here, we conduct a systematic literature review to synthesize the effects of temporal genomics methodology on our ability to detect evolutionary changes. We focus on studies investigating recent change within the past 200 years, highlighting evolutionary processes that have occurred during the past two centuries of accelerated anthropogenic pressure. We first identify the most frequently studied taxa, systems, questions and drivers, before highlighting overlooked areas where further temporal genomic studies may be particularly enlightening. Then, we provide guidelines for future study and sample designs while identifying key considerations that may influence statistical and analytical power. Our aim is to provide recommendations to a broad array of researchers interested in using temporal genomics in their work. [ABSTRACT FROM AUTHOR]

Tumor angiogenesis is critical for the growth and progression of cancer. As such, angiostasis is a treatment modality for cancer with potential utility for multiple types of cancer and fewer side effects. However, clinical success of angiostatic monotherapies has been moderate, at best, causing angiostatic treatments to lose their early luster. Previous studies demonstrated compensatory mechanisms that drive tumor vascularization despite the use of angiostatic monotherapies, as well as the potential for combination angiostatic therapies to overcome these compensatory mechanisms. We screened clinically approved angiostatics to identify specific combinations that confer potent inhibition of tumor-induced angiogenesis. We used a novel modification of the ex ovo chick chorioallantoic membrane (CAM) model that combined confocal and automated analyses to quantify tumor angiogenesis induced by glioblastoma tumor onplants. This model is advantageous due to its low cost and moderate throughput capabilities, while maintaining complex in vivo cellular interactions that are difficult to replicate in vitro. After screening multiple combinations, we determined that glioblastoma-induced angiogenesis was significantly reduced using a combination of bevacizumab (Avastin®) and temsirolimus (Torisel®) at doses below those where neither monotherapy demonstrated activity. These preliminary results were verified extensively, with this combination therapy effective even at concentrations further reduced 10-fold with a CI value of 2.42E-5, demonstrating high levels of synergy. Thus, combining bevacizumab and temsirolimus has great potential to increase the efficacy of angiostatic therapy and lower required dosing for improved clinical success and reduced side effects in glioblastoma patients. [ABSTRACT FROM AUTHOR]

Pan-girder (PG) bridges became popular in Texas, USA, in the 1950s and 1960s as a viable alternative for short-span bridges due to their speed of construction, low-labour requirements and cost–effectiveness. However, these bridges were designed for lighter live loads than the current AASHTO HL-93 loading, resulting in gradual decline in their usage. Existing PG bridges may have various structural and non-structural deteriorations because they are nearing or have crossed their service lives. In this study, the existing condition of four selected PG bridges in the Dallas–Fort Worth metroplex were visually inspected to assess their current condition. The bridges exhibited significant concrete spalling, cracking and crushing, resulting in exposed and corroded steel rebars. The newest bridge, built in 1992, experienced less severe damage than the other three bridges built in 1955–1971. The bridges' safety could be compromised due to deteriorations and lighter design live loads. Therefore, the following recommendations were made: (a) glue a polyvinyl chloride pipe on the form-lowering holes extending beyond the concrete surface of the arch to minimise water infiltration and related damage; (b) conduct load testing and ratings to verify the structural safety and load-carrying capacity of the deteriorated bridges; and (c) retrofit to upgrade the existing bridge condition and safety and mitigate against further damage. [ABSTRACT FROM AUTHOR]

The 5'-leader of the HIV-1 genome regulates multiple functions during viral replication via mechanisms that have yet to be established. We developed a nuclear magnetic resonance approach that enabled direct detection of structural elements within the intact leader (712-nucleotide dimer) that are critical for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the unique-5' region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin and enhances binding by the nucleocapsid (NC) protein, which is the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch. [ABSTRACT FROM AUTHOR]

In case-control single-cell RNA-seq studies, sample-level labels are transferred onto individual cells, labeling all case cells as affected, when in reality only a small fraction of them may actually be perturbed. Here, using simulations, we demonstrate that the standard approach to single cell analysis fails to isolate the subset of affected case cells and their markers when either the affected subset is small, or when the strength of the perturbation is mild. To address this fundamental limitation, we introduce HiDDEN, a computational method that refines the case-control labels to accurately reflect the perturbation status of each cell. We show HiDDEN's superior ability to recover biological signals missed by the standard analysis workflow in simulated ground truth datasets of cell type mixtures. When applied to a dataset of human multiple myeloma precursor conditions, HiDDEN recapitulates the expert manual annotation and discovers malignancy in early stage samples missed in the original analysis. When applied to a mouse model of demyelination, HiDDEN identifies an endothelial subpopulation playing a role in early stage blood-brain barrier dysfunction. We anticipate that HiDDEN should find wide usage in contexts that require the detection of subtle transcriptional changes in cell types across conditions., (© 2024. The Author(s).)

The meningeal lymphatics system plays diverse roles in facilitating neuroimmune function at brain borders, yet its specific contribution toward glial function and homeostasis is not known. In this issue of Immunity, Das Neves et al. (2024) describe a novel role for the meningeal lymphatics in maintaining oligodendrocyte survival and myelination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Molecular chaperones and co-chaperones are highly conserved cellular components that perform variety of duties related to the proper three-dimensional folding of the proteome. The web of factors that carries out this essential task is called the proteostasis network (PN). Ribonucleoproteins (RNPs) represent an underexplored area in terms of the connections they make with the PN. The Survival Motor Neuron (SMN) complex is an RNP assembly chaperone and serves as a paradigm for studying how specific small nuclear (sn)RNAs are identified and paired with their client substrate proteins. SMN protein is the eponymous component of a large complex required for the biogenesis of uridine-rich small nuclear ribonucleoproteins (U-snRNPs) and localizes to distinct membraneless organelles in both the nucleus and cytoplasm of animal cells. SMN forms the oligomeric core of this complex, and missense mutations in its YG box self-interaction domain are known to cause Spinal Muscular Atrophy (SMA). The basic framework for understanding how snRNAs are assembled into U-snRNPs is known, the pathways and mechanisms used by cells to regulate their biogenesis are poorly understood. Given the importance of these processes to normal development as well as neurodegenerative disease, we set out to identify and characterize novel SMN binding partners. Here, we carried out affinity purification mass spectrometry (AP-MS) of SMN using stable fly lines exclusively expressing either wildtype or SMA-causing missense alleles. Bioinformatic analyses of the pulldown data, along with comparisons to proximity labeling studies carried out in human cells, revealed conserved connections to at least two other major chaperone systems including heat shock folding chaperones (HSPs) and histone/nucleosome assembly chaperones. Notably, we found that heat shock cognate protein Hsc70-4 and other HspA family members preferentially interacted with SMA-causing alleles of SMN. Hsc70-4 is particularly interesting because its mRNA is aberrantly sequestered by a mutant form of TDP-43 in mouse and Drosophila ALS (Amyotrophic Lateral Sclerosis) disease models. Most important, a missense allele of Hsc70-4 (HspA8 in mammals) was recently identified as a bypass suppressor of the SMA phenotype in mice. Collectively, these findings suggest that chaperone-related dysfunction lies at the etiological root of both ALS and SMA.

Background: Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by hypomorphic loss of function in the survival motor neuron (SMN) protein. SMA presents across a broad spectrum of disease severity. Unfortunately, genetic models of intermediate SMA have been difficult to generate in vertebrates and are thus unable to address key aspects of disease etiology. To address these issues, we developed a Drosophila model system that recapitulates the full range of SMA severity, allowing studies of pre-onset biology as well as late-stage disease processes., Results: Here, we carried out transcriptomic and proteomic profiling of mild and intermediate Drosophila models of SMA to elucidate molecules and pathways that contribute to the disease. Using this approach, we elaborated a role for the SMN complex in the regulation of innate immune signaling. We find that mutation or tissue-specific depletion of SMN induces hyperactivation of the immune deficiency (IMD) and Toll pathways, leading to overexpression of antimicrobial peptides (AMPs) and ectopic formation of melanotic masses in the absence of an external challenge. Furthermore, the knockdown of downstream targets of these signaling pathways reduced melanotic mass formation caused by SMN loss. Importantly, we identify SMN as a negative regulator of a ubiquitylation complex that includes Traf6, Bendless, and Diap2 and plays a pivotal role in several signaling networks., Conclusions: In alignment with recent research on other neurodegenerative diseases, these findings suggest that hyperactivation of innate immunity contributes to SMA pathology. This work not only provides compelling evidence that hyperactive innate immune signaling is a primary effect of SMN depletion, but it also suggests that the SMN complex plays a regulatory role in this process in vivo. In summary, immune dysfunction in SMA is a consequence of reduced SMN levels and is driven by cellular and molecular mechanisms that are conserved between insects and mammals., (© 2024. The Author(s).)

Background: People receiving hemodialysis experience high symptom burden that contributes to low functional status and poor health-related quality of life. Management of symptoms is a priority for individuals receiving hemodialysis but limited effective treatments exist. There is emerging evidence that exercise programming can improve several common dialysis-related symptoms., Objective: The primary aim of this study is to evaluate the effect of an exercise rehabilitation program on symptom burden in individuals receiving maintenance hemodialysis., Design: Multicenter, randomized controlled, 1:1 parallel, open label, prospective blinded end point trial., Setting: Three facility-based hemodialysis units in Winnipeg, Manitoba, Canada., Participants: Adults aged 18 years or older with end-stage kidney disease receiving facility-based maintenance hemodialysis for more than 3 months, with at least 1 dialysis-related symptom as indicated by the Dialysis Symptom Index (DSI) severity score >0 (n = 150)., Intervention: Supervised 26-week exercise rehabilitation program and 60 minutes of cycling during hemodialysis thrice weekly. Exercise intensity and duration were supervised and individualized by the kinesiologist as per participant baseline physical function with gradual progression over the course of the intervention., Control: Usual hemodialysis care (no exercise program)., Measurements: Our primary outcome is change in symptom burden at 12 weeks as measured by the DSI severity score. Secondary outcomes include change in modified DSI severity score (includes 10 symptoms most plausible to improve with exercise), change in DSI severity score at 26 and 52 weeks; time to recover post-hemodialysis; health-related quality of life measured using EuroQol (EQ)-5D-5L; physical activity behavior measured by self-report (Godin-Shepherd questionnaire) and triaxial accelerometry; exercise capacity (shuttle walk test); frailty (Fried); self-efficacy for exercise; and 1-year hospitalization and mortality., Methods: Change in primary outcome will be compared between groups by independent 2-tailed t test or Mann-Whitney U test depending on data distribution and using generalized linear mixed models, with study time point as a random effect and adjusted for baseline DSI score. Similarly, change in secondary outcomes will be compared between groups over time using appropriate parametric and nonparametric statistical tests depending on data type and distribution., Limitations: The COVID-19 pandemic restrictions on clinical research at our institution delayed completion of target recruitment and prevented collection of accelerometry and physical function outcome data for 15 months until restrictions were lifted., Conclusions: The application of an exercise rehabilitation program to improve symptom burden in individuals on hemodialysis may ameliorate common symptoms observed in individuals on hemodialysis and result in improved quality of life and reduced disability and morbidity over the long term. Importantly, this pragmatic study, with a standardized exercise intervention that is adaptable to baseline physical function, addresses an important gap in both clinical care of hemodialysis patients and our current knowledge., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: C.B. has ownership interest in Precision Advanced Digital Manufacturing Ltd. E.F., K.S., E.G, M.S., R.W., R.G., K.R., T.A.D., M.V., J.Z., P.K., N.T., C.R., J.M.M. report no conflicts of interest., (© The Author(s) 2024.)

Introduction: Molecular chaperones and co-chaperones are highly conserved cellular components that perform a variety of duties related to the proper three-dimensional folding of the proteome. The web of factors that carries out this essential task is called the proteostasis network (PN). Ribonucleoproteins (RNPs) represent an underexplored area in terms of the connections they make with the PN. The Survival Motor Neuron (SMN) complex is an assembly chaperone and serves as a paradigm for studying how specific RNAs are identified and paired with their client substrate proteins to form RNPs. SMN is the eponymous component of a large complex, required for the biogenesis of uridine-rich small nuclear ribonucleoproteins (U-snRNPs), that localizes to distinct membraneless organelles in both the nucleus and cytoplasm of animal cells. SMN protein forms the oligomeric core of this complex, and missense mutations in the human SMN1 gene are known to cause Spinal Muscular Atrophy (SMA). The basic framework for understanding how snRNAs are assembled into U-snRNPs is known. However, the pathways and mechanisms used by cells to regulate their biogenesis are poorly understood., Methods: Given the importance of these processes to normal development as well as neurodegenerative disease, we set out to identify and characterize novel SMN binding partners. We carried out affinity purification mass spectrometry (AP-MS) of Drosophila SMN complexes using fly lines exclusively expressing either wildtype or SMA-causing missense alleles., Results: Bioinformatic analyses of the pulldown data, along with comparisons to proximity labeling studies carried out in human cells, revealed conserved connections to at least two other major chaperone systems including heat shock folding chaperones (HSPs) and histone/nucleosome assembly chaperones. Notably, we found that heat shock cognate protein Hsc70-4 and other HspA family members preferentially associated with SMA-causing alleles of SMN., Discussion: Hsc70-4 is particularly interesting because its mRNA is aberrantly sequestered by a mutant form of TDP-43 in mouse and Drosophila ALS (Amyotrophic Lateral Sclerosis) disease models. Most important, a missense allele of Hsc70-4 (HspA8 in mammals) was recently identified as a bypass suppressor of the SMA phenotype in mice. Collectively, these findings suggest that chaperone-related dysfunction lies at the etiological root of both ALS and SMA., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Background: Spinal Muscular Atrophy (SMA) is a devastating neuromuscular disease caused by hypomorphic loss of function in the Survival Motor Neuron (SMN) protein. SMA presents across broad spectrum of disease severity. Unfortunately, vertebrate models of intermediate SMA have been difficult to generate and are thus unable to address key aspects of disease etiology. To address these issues, we developed a Drosophila model system that recapitulates the full range of SMA severity, allowing studies of pre-onset biology as well as late-stage disease processes., Results: Here, we carried out transcriptomic and proteomic profiling of mild and intermediate Drosophila models of SMA to elucidate molecules and pathways that contribute to the disease. Using this approach, we elaborated a role for the SMN complex in the regulation of innate immune signaling. We find that mutation or tissue-specific depletion of SMN induces hyperactivation of the Immune Deficiency (IMD) and Toll pathways, leading to overexpression of antimicrobial peptides (AMPs) and ectopic formation of melanotic masses in the absence of an external challenge. Furthermore, knockdown of downstream targets of these signaling pathways reduced melanotic mass formation caused by SMN loss. Importantly, we identify SMN as a negative regulator of an ubiquitylation complex that includes Traf6, Bendless and Diap2, and plays a pivotal role in several signaling networks., Conclusions: In alignment with recent research on other neurodegenerative diseases, these findings suggest that hyperactivation of innate immunity contributes to SMA pathology. This work not only provides compelling evidence that hyperactive innate immune signaling is a primary effect of SMN depletion, but it also suggests that the SMN complex plays a regulatory role in this process in vivo . In summary, immune dysfunction in SMA is a consequence of reduced SMN levels and is driven by cellular and molecular mechanisms that are conserved between insects and mammals.

Introduction: Intradialytic cycling is often performed during the first half of hemodialysis because of concerns regarding increased frequency of intradialytic hypotension (IDH) late in hemodialysis. This increases exercise program resource needs and limits utility of intradialytic cycling to treat dialysis-related symptoms., Methods: This multicenter, randomized, crossover trial compared IDH rate when cycling during the first half versus the second half of hemodialysis in 98 adults on maintenance hemodialysis. Group A cycled during the first half of hemodialysis for 2 weeks and subsequently during the second half for 2 weeks. In group B, the cycling schedule was reversed. Blood pressure (BP) was measured every 15 minutes throughout hemodialysis. Primary outcome was IDH rate (systolic BP [SBP] decrease of >20 mm Hg or SBP <90 mm Hg). Secondary outcomes included symptomatic IDH rate and time to recover post hemodialysis. Data were analyzed using negative binomial and gamma distribution mixed regression., Results: Mean age 64.7 (SD 12.0) and 64.7 (SD 14.2) years in group A ( n  = 52) and group B ( n  = 46), respectively. Proportions of females were 33% in group A and 43% in group B. Median time on hemodialysis was 4.1 (interquartile range [IQR] 2.5, 6.1]) years in group A and 3.9 years (IQR 2.5, 6.7) in group B. IDH rate per 100 hemodialysis hours (95% confidence interval [CI]) was 34.2 (26.4, 42.0) and 36.0 (28.9, 43.1) during early and late intradialytic cycling, respectively ( P  = 0.53). Timing of intradialytic cycling was not associated with symptomatic IDH (relative risk [RR]: 1.07 [0.75-1.53]) or time to recover post hemodialysis (odds ratio: 0.99 [0.79-1.23])., Conclusion: We found no association between the rate of overall or symptomatic IDH and the timing of intradialytic cycling in patients enrolled in an intradialytic cycling program. Increased use of cycling late in hemodialysis may optimize intradialytic cycling program resource use and should be studied as a possible treatment for symptoms common in late hemodialysis., (© 2023 Published by Elsevier Inc. on behalf of the International Society of Nephrology.)

Genetic disruptions to the biogenesis of spliceosomal small-nuclear ribonucleoproteins in Drosophila cause wide-spread alternative splicing changes, including changes to the splicing of pre-mRNA for Ribosomal protein S21 (RpS21). Using a transposon mutant for the Phosphorylated adaptor for RNA export (Phax) gene, we demonstrate that changes in the splicing of RpS21 transcripts have a strong influence on the developmental progression of PhaxSH/SH mutants. Different alleles of the Drosophila RpS21 gene are circulating in common laboratory strains and cell lines. These alleles exhibit differences in RpS21 intron retention and splicing efficiency. Differences in the splicing of RpS21 transcripts account for prior conflicting observations of the phenotypic severity of PhaxSH/SH mutant stocks. The alleles uncover a strong splicing enhancer in RpS21 transcripts that can fully suppress the larval lethality and partially suppress the pupal lethality exhibited by PhaxSH/SH mutant lines. In the absence of the splicing enhancer, the splicing of RpS21 transcripts can be modulated in trans by the SR-rich B52 splicing factor. As PhaxSH/SH mutants exhibit wide-spread splicing changes in transcripts for other genes, findings here establish the importance of a single alternative splicing event, RpS21 splicing or intron retention, to the developmental progression of Drosophila., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Chromatin dynamics are central to the regulation of gene expression and genome stability. In order to improve understanding of the factors regulating chromatin dynamics, the genes encoding these factors are deleted and the differential gene expression profiles are determined using approaches such as RNA sequencing. Here, we analyzed a gene expression dataset aimed at uncovering the function of the relatively uncharacterized chromatin regulator, Set4, in the model system Saccharomyces cerevisiae (budding yeast). The main theme of this paper focuses on identifying the highly differentially expressed genes in cells deleted for Set4 (referred to as Set4 Δ mutant dataset) compared to the wild-type yeast cells. The Set4 Δ mutant data produce a spiky distribution on the log-fold changes of their expressions, and it is reasonably assumed that genes which are not highly differentially expressed come from a mixture of two normal distributions. We propose an adaptive local false discovery rate (FDR) procedure, which estimates the null distribution of the log-fold changes empirically. We numerically show that, unlike existing approaches, our proposed method controls FDR at the aimed level (0.05) and also has competitive power in finding differentially expressed genes. Finally, we apply our procedure to analyzing the Set4 Δ mutant dataset., (© 2021 Wiley-VCH GmbH.)

The yeast chromatin protein Set4 is a member of the Set3-subfamily of SET domain proteins which play critical roles in the regulation of gene expression in diverse developmental and environmental contexts. We previously reported that Set4 promotes survival during oxidative stress and regulates expression of stress response genes via stress-dependent chromatin localization. In this study, global gene expression analysis and investigation of histone modification status identified a role for Set4 in maintaining gene repressive mechanisms within yeast subtelomeres under both normal and stress conditions. We show that Set4 works in a partially overlapping pathway to the SIR complex and the histone deacetylase Rpd3 to maintain proper levels of histone acetylation and expression of stress response genes encoded in subtelomeres. This role for Set4 is particularly critical for cells under hypoxic conditions, where the loss of Set4 decreases cell fitness and cell wall integrity. These findings uncover a new regulator of subtelomeric chromatin that is key to stress defense pathways and demonstrate a function for Set4 in regulating repressive, heterochromatin-like environments., (© 2021 Jethmalani et al.)

A common way of illustrating phylogeographic results is through the use of haplotype networks. While these networks help to visualize relationships between individuals, populations, and species, evolutionary studies often only quantitatively analyze genetic diversity among haplotypes and ignore other network properties. Here, we present a new metric, haplotype network branch diversity (HBd), as an easy way to quantifiably compare haplotype network complexity. Our metric builds off the logic of combining genetic and topological diversity to estimate complexity previously used by the published metric haplotype network diversity (HNd). However, unlike HNd which uses a combination of network features to produce complexity values that cannot be defined in probabilistic terms, thereby obscuring the values' implication for a sampled population, HBd uses frequencies of haplotype classes to incorporate topological information of networks, keeping the focus on the population and providing easy-to-interpret probabilistic values for randomly sampled individuals. The goal of this study is to introduce this more intuitive metric and provide an R script that allows researchers to calculate diversity and complexity indices from haplotype networks. A group of datasets, generated manually (model dataset) and based on published data (empirical dataset), were used to illustrate the behavior of HBd and both of its terms, haplotype diversity, and a new index called branch diversity. Results followed a predicted trend in both model and empirical datasets, from low metric values in simple networks to high values in complex networks. In short, the new combined metric joins genetic and topological diversity of haplotype networks, into a single complexity value. Based on our analysis, we recommend the use of HBd, as it makes direct comparisons of network complexity straightforward and provides probabilistic values that can readily discriminate situations that are difficult to resolve with available metrics., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: None

Heptachlor has been widely used as an insecticide. It is a GABA-A antagonist and causes seizures. It also increases peripheral benzodiazepine receptors (PBRs) in brain. PBRs are found on the outer mitochondrial membrane in glia, rather than in neurons, and are necessary for steroidogenesis in brain. We compared the effects of acute oral administration of heptachlor (60 mg/ml oil/kg body wt) at 10 ages from postnatal day (PND) 0 to 60 on brain PBR expression and seizure severity in both male and female rats at 1 and 2 hr after administration. From PND 10 through 60, brain PBR expression was increased about 175-225% of controls at both 1 and 2 hr after heptachlor in females. In males however, PBRs were only increased at 30-60 days at 1 hr but not at any age at 2 hr. At 2 hr after heptachlor at 30-60 days in males, PBRs were significantly lower than at 1 hr and even tended to be lower than control levels. By contrast, seizure intensity was greater in males than in females from 10 through 20 days of age at 1 hr and was even greater at 2 hr from 16 through 30 days of age, reflecting the lower PBR levels at 2 hr than at 1 hr in males. Thus, the gender difference in PBR expression was the opposite of the gender difference in seizure intensity. PBRs in brain synthesize several neurosteroids, including allopregnanolone, which is a potent anticonvulsant agent. We hypothesize that the gender differences in seizure intensity after heptachlor were due to the action of heptachlor in greatly increasing PBR expression in females but not in males. Thus the greater expression of PBRs in females would result in more synthesis of allopregnanolone than in males. Therefore, because of allopregnanolone's anticonvulsant effects, seizure intensity was less in females than in males. By comparison, maximal electroshock (MES) caused seizures and increased PBRs in brain in both male and female developing rats with no gender differences at 10-20 days of age.