Transcription factor binding specificities of the oomycete Phytophthora infestans reflect conserved and divergent evolutionary patterns and predict function.

BACKGROUND: Identifying the DNA-binding specificities of transcription factors (TF) is central to understanding gene networks that regulate growth and development. Such knowledge is lacking in oomycetes, a microbial eukaryotic lineage within the stramenopile group. Oomycetes include many important plant and animal pathogens such as the potato and tomato blight agent Phytophthora infestans, which is a tractable model for studying life-stage differentiation within the group. RESULTS: Mining of the P. infestans genome identified 197 genes encoding proteins belonging to 22 TF families. Their chromosomal distribution was consistent with family expansions through unequal crossing-over, which were likely ancient since each family had similar sizes in most oomycetes. Most TFs exhibited dynamic changes in RNA levels through the P. infestans life cycle. The DNA-binding preferences of 123 proteins were assayed using protein-binding oligonucleotide microarrays, which succeeded with 73 proteins from 14 families. Binding sites predicted for representatives of the families were validated by electrophoretic mobility shift or chromatin immunoprecipitation assays. Consistent with the substantial evolutionary distance of oomycetes from traditional model organisms, only a subset of the DNA-binding preferences resembled those of human or plant orthologs. Phylogenetic analyses of the TF families within P. infestans often discriminated clades with canonical and novel DNA targets. Paralogs with similar binding preferences frequently had distinct patterns of expression suggestive of functional divergence. TFs were predicted to either drive life stage-specific expression or serve as general activators based on the representation of their binding sites within total or developmentally-regulated promoters. This projection was confirmed for one TF using synthetic and mutated promoters fused to reporter genes in vivo. CONCLUSIONS: We established a large dataset of binding specificities for P. infestans TFs, representing the first in the stramenopile group. This resource provides a basis for understanding transcriptional regulation by linking TFs with their targets, which should help delineate the molecular components of processes such as sporulation and host infection. Our work also yielded insight into TF evolution during the eukaryotic radiation, revealing both functional conservation as well as diversification across kingdoms.

Genome resequencing reveals population divergence and local adaptation of blacklegged ticks in the United States.

Tick vectors and tick-borne disease are increasingly impacting human populations globally. An important challenge is to understand tick movement patterns, as this information can be used to improve management and predictive modelling of tick population dynamics. Evolutionary analysis of genetic divergence, gene flow and local adaptation provides insight on movement patterns at large spatiotemporal scales. We develop low coverage, whole genome resequencing data for 92 blacklegged ticks, Ixodes scapularis, representing range-wide variation across the United States. Through analysis of population genomic data, we find that tick populations are structured geographically, with gradual isolation by distance separating three population clusters in the northern United States, southeastern United States and a unique cluster represented by a sample from Tennessee. Populations in the northern United States underwent population contractions during the last glacial period and diverged from southern populations at least 50 thousand years ago. Genome scans of selection provide strong evidence of local adaptation at genes responding to host defences, blood-feeding and environmental variation. In addition, we explore the potential of low coverage genome sequencing of whole-tick samples for documenting the diversity of microbial pathogens and recover important tick-borne pathogens such as Borrelia burgdorferi. The combination of isolation by distance and local adaptation in blacklegged ticks demonstrates that gene flow, including recent expansion, is limited to geographical scales of a few hundred kilometres.

MyD88-mediated signaling is critical for the generation of seizure responses and cognitive impairment in a model of anti-N-methyl-D-aspartate receptor encephalitis.

OBJECTIVE: We previously demonstrated that interleukin-1 receptor-mediated immune activation contributes to seizure severity and memory loss in anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. In the present study, we assessed the role of the myeloid differentiation primary response gene 88 (MyD88), an adaptor protein in Toll-like receptor signaling, in the key phenotypic characteristics of anti-NMDAR encephalitis. METHODS: Monoclonal anti-NMDAR antibodies or control antibodies were infused into the lateral ventricle of MyD88 knockout mice (MyD88-/-) and control C56BL/6J mice (wild type [WT]) via osmotic minipumps for 2 weeks. Seizure responses were measured by electroencephalography. Upon completion of the infusion, the motor, anxiety, and memory functions of the mice were assessed. Astrocytic (glial fibrillary acidic protein [GFAP]) and microglial (ionized calcium-binding adaptor molecule 1 [Iba-1]) activation and transcriptional activation for the principal inflammatory mediators involved in seizures were determined using immunohistochemistry and quantitative real-time polymerase chain reaction, respectively. RESULTS: As shown before, 80% of WT mice infused with anti-NMDAR antibodies (n = 10) developed seizures (median = 11, interquartile range [IQR] = 3-25 in 2 weeks). In contrast, only three of 14 MyD88-/- mice (21.4%) had seizures (0, IQR = 0-.25, p = .01). The WT mice treated with antibodies also developed memory loss in the novel object recognition test, whereas such memory deficits were not apparent in MyD88-/- mice treated with anti-NMDAR antibodies (p = .03) or control antibodies (p = .04). Furthermore, in contrast to the WT mice exposed to anti-NMDAR antibodies, the MyD88-/- mice had a significantly lower induction of chemokine (C-C motif) ligand 2 (CCL2) in the hippocampus (p = .0001, Sidak tests). There were no significant changes in the expression of GFAP and Iba-1 in the MyD88-/- mice treated with anti-NMDAR or control antibodies. SIGNIFICANCE: These findings suggest that MyD88-mediated signaling contributes to the seizure and memory phenotype in anti-NMDAR encephalitis and that CCL2 activation may participate in the expression of these features. The removal of MyD88 inflammation may be protective and therapeutically relevant.

Evaluating the definition and distribution of spring ephemeral wildflowers in eastern North America.

PREMISE: The herbaceous layer accounts for the majority of plant biodiversity in eastern North American forests, encompassing substantial variation in life history strategy and function. One group of early-season herbaceous understory species, colloquially referred to as spring ephemeral wildflowers, are ecologically and culturally important, but little is known about the prevalence and biogeographic patterns of the spring ephemeral strategy. METHODS: We used observations collected by the Global Biodiversity Information Facility (GBIF) to quantify the ephemerality of 559 understory forb species across eastern North America and classify them according to a continuous ephemerality index (ranging from 0 = never ephemeral to 1 = always ephemeral). We then used this information to model where ephemeral forbs were most common across the landscape with the goal of identifying geographic and environmental drivers important to their distributions and ranges. RESULTS: Only 3.4% of all understory wildflower species were spring ephemerals in all parts of their range, and 18.4% (103 species) were ephemeral in at least part of their range. Spring ephemerals peaked in absolute species richness and relative proportion at mid latitudes. CONCLUSIONS: Spring ephemeral phenology is an important shade-avoidance strategy for a large segment of the total understory species in temperate deciduous forests. In North America, the strategy is relatively most important for forest understories at mid latitudes. The definitions of spring ephemerality we provide here serve as an important ecological context for conservation priorities and to evaluate responses of this biodiverse group to future environmental change.

Discovery of circulating blood biomarkers in patients with and without Modic changes of the lumbar spine: a preliminary analysis.

PURPOSE: The following study aimed to determine the existence of blood biomarkers in symptomatic patients with or without lumbar Modic changes (MC). METHODS: A cross-sectional sub-analyses of a prospective cohort was performed. Fasting blood samples were collected from patients with and without lumbar MC who had undergone spinal fusion or microdiscectomy. An 80-plex panel and CCL5/RANTES were used to assess preoperative plasma cytokine concentrations. Patient demographics and imaging phenotypes were also assessed. RESULTS: Thirty-one subjects were analysed (n = 18 no MC; n = 13 MC). No significant differences were found in age, sex, body mass index, smoking and alcohol history, and surgical procedure (i.e. fusion, decompression) between the two groups (p > 0.05). Several statistically significant blood biomarkers in MC patients were identified, including elevated levels of C-C Motif Chemokine Ligand 5 (CCL5, p = 0.0006), while Macrophage Migration Inhibitory Factor (MIF) was significantly lower (p = 0.009). Additionally, C-X-C Motif Chemokine Ligand 5 (CXCL5, p = 0.052), Pentraxin 3 (PTX3, p = 0.06) and Galectin-3 (Gal-3, p = 0.07) showed potential relevance. Moreover, MC patients exhibited significantly higher levels of disc degeneration (p = 0.0001) and displacement severity (p = 0.020). Based on multivariate analyses and controlling for disc degeneration/displacement, CCL5 (OR 1.02; 95% CI 1.002-1.033; p = 0.028) and MIF (OR 0.60; 95% CI 0.382-0.951; p = 0.030) were independently associated with MC patients. CONCLUSION: This "proof-of-concept" study is the first to identify specific and significantly circulating blood biomarkers associated with symptomatic patients with lumbar MC, independent of disc alterations of degeneration and/or bulges/herniations. Specifically, differences in CCL5 and MIF protein levels were significantly noted in MC patients compared to those without MC.

HIV Infection Drives Foam Cell Formation via NLRP3 Inflammasome Activation.

Persistent immune activation is linked to an increased risk of cardiovascular disease (CVD) in people with HIV (PWH) on antiretroviral therapy (ART). The NLRP3 inflammasome may contribute to elevated CVD risk in PWH. This study utilized peripheral blood mononuclear cells (PBMCs) from 25 PWH and 25 HIV-negative controls, as well as HIV in vitro infections. Transcriptional changes were analyzed using RNAseq and pathway analysis. Our results showed that in vitro HIV infection of macrophages and PBMCs from PWH had increased foam cell formation and expression of the NLRP3 inflammasome components and downstream cytokines (caspase-1, IL-1ß, and IL-18), which was reduced with inhibition of NLRP3 activity using MCC950. Transcriptomic analysis revealed an increased expression of multiple genes involved in lipid metabolism, cholesterol storage, coronary microcirculation disorders, ischemic events, and monocyte/macrophage differentiation and function with HIV infection and oxLDL treatment. HIV infection and NLRP3 activation increased foam cell formation and expression of proinflammatory cytokines, providing insights into the mechanisms underlying HIV-associated atherogenesis. This study suggests that HIV itself may contribute to increased CVD risk in PWH. Understanding the involvement of the inflammasome pathway in HIV atherosclerosis can help identify potential therapeutic targets to mitigate cardiovascular risks in PWH.

Mitochondrial DNA Instability Supersedes Parkin Mutations in Driving Mitochondrial Proteomic Alterations and Functional Deficits in Polg Mutator Mice.

Mitochondrial quality control is essential in mitochondrial function. To examine the importance of Parkin-dependent mechanisms in mitochondrial quality control, we assessed the impact of modulating Parkin on proteome flux and mitochondrial function in a context of reduced mtDNA fidelity. To accomplish this, we crossed either the Parkin knockout mouse or ParkinW402A knock-in mouse lines to the Polg mitochondrial mutator line to generate homozygous double mutants. In vivo longitudinal isotopic metabolic labeling was followed by isolation of liver mitochondria and synaptic terminals from the brain, which are rich in mitochondria. Mass spectrometry and bioenergetics analysis were assessed. We demonstrate that slower mitochondrial protein turnover is associated with loss of mtDNA fidelity in liver mitochondria but not synaptic terminals, and bioenergetic function in both tissues is impaired. Pathway analysis revealed loss of mtDNA fidelity is associated with disturbances of key metabolic pathways, consistent with its association with metabolic disorders and neurodegeneration. Furthermore, we find that loss of Parkin leads to exacerbation of Polg-driven proteomic consequences, though it may be bioenergetically protective in tissues exhibiting rapid mitochondrial turnover. Finally, we provide evidence that, surprisingly, dis-autoinhibition of Parkin (ParkinW402A) functionally resembles Parkin knockout and fails to rescue deleterious Polg-driven effects. Our study accomplishes three main outcomes: (1) it supports recent studies suggesting that Parkin dependence is low in response to an increased mtDNA mutational load, (2) it provides evidence of a potential protective role of Parkin insufficiency, and (3) it draws into question the therapeutic attractiveness of enhancing Parkin function.

Robert Rosenthal (1933-2024).

Robert Rosenthal died on January 5, 2024, in Riverside, California, at the age of 90. Born March 2, 1933, in Giessen, Germany, just as the Nazis came to power, the young Bob-he always insisted that everyone call him "Bob"-and his family fled in 1939 to Rhodesia (a British African protectorate) before making it to New York and then Los Angeles. Bob's dissertation derived from the Thematic Apperception Test (TAT). It compared projection in paranoid schizophrenic patients versus a normal control group after an experimental manipulation. Bob analyzed his dissertation pretest data (before the intervention) and found that his groups already differed in the direction that would support his expected result. Thus was launched the career and field of experimenter expectancy effects-"the Rosenthal effect." Saying that God also loved p < .06, Bob helped lead the charge against the ridiculous but long-standing practice whereby psychology journals would reject articles where significance testing did not reach the magical .05 level, regardless of the quality and importance of the research. With over 500 publications and hundreds of thousands of citations of his work, he forever transformed the fields of psychology and education. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

ArcTiCA: Arctic tidal constituents atlas.

Tides in the Arctic Ocean affect ocean circulation and mixing, and sea ice dynamics and thermodynamics. However, there is a limited network of available in situ tidal coefficient data for understanding tidal variability in the Arctic Ocean; e.g., the global TICON-3 database contains only 111 sites above 60°N and 21 above 70°N. At the same time, the presence of sea ice and latitude limits of satellite altimetry complicate altimetry-based retrievals of Arctic tidal coefficients. This leads to a reliance on ocean tide models whose accuracy depend on having sufficient in situ data for validation and assimilation. Here, we present a comprehensive new dataset of tidal constituents in the Arctic region, combining analyses of in situ measurements from tide gauges, ocean bottom pressure sensors and GNSS interferometric reflectometry. The new dataset contains 914 measurement sites above 60°N and 399 above 70°N, with each site being quality-assessed and expert guidance provided to help maximise the usage of the dataset. We also compare the dataset to recent tide models.

Replacement of Lys27 by asparagine in the SERCA regulator myoregulin: A Ca2+ affinity modulator or a catalytic activity switch?

Myoregulin (MLN) is a protein that regulates the activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) without affecting its affinity for Ca2+. MLN's residue Lys27 is located at a site where other SERCA regulators control Ca2+ affinity. Therefore, we conducted atomistic simulations and ATPase activity experiments to determine whether replacing Lys27 with asparagine, a conserved residue found in various muscle SERCA regulators, would enable MLN to modulate both the Ca2+ affinity and catalytic activity of SERCA. Our findings indicate that replacing Lys27 with Asn significantly enhances the inhibitory potency of MLN, but it does not affect SERCA's affinity for Ca2+. We suggest that the SERCA site modulating Ca2+ affinity also acts as a catalytic activity switch. Therefore, this site is a key element contributing to the functional divergence among homologous SERCA regulators. This study paves the way for future investigations to explore how biological function diverges during the evolution of the SERCA regulator family.

Probing the formation of a hetero-dimeric membrane transport complex with dual in vitro and in silico mutagenesis.

The reversible association of transmembrane helices is a fundamental mechanism in how living cells convey information and respond to physiological events. The cardiac calcium transport regulator phospholamban (PLN) is an example of a single-span transmembrane protein that populates a variety of reversible and competing oligomeric states. PLN primarily forms monomers and pentamers in the membrane, where the PLN pentamer is a storage form and the PLN monomer forms a hetero-dimeric inhibitory complex with SERCA. The binding affinity and free-energy of formation of the SERCA-PLN complex in a membrane have not been determined. As is the case for most transmembrane protein interactions, measuring these quantities experimentally is extremely challenging. In this study, we estimated binding affinities by employing in silico alchemical free-energy calculations for all PLN transmembrane alanine substitutions in a membrane bilayer. The binding affinities were calculated separately for the SERCA-PLN complex, a PLN monomer, and a PLN pentamer and compared to in vitro functional measurements of SERCA regulation by the PLN alanine substitutions. Initially, the changes in SERCA inhibition by PLN alanine substitutions were compared to the changes in free energy for the SERCA-PLN complex formed from the PLN monomer. However, the functional data for the PLN alanine substitutions were better explained by the formation of the SERCA-PLN complex directly from the PLN pentamer. This finding points to an inhibitory mechanism favoring conformational selection of SERCA and the interaction of a PLN pentamer with SERCA for 'delivery' of a PLN monomer to the inhibitory site. The implications of these findings suggest that the energetics of helix exchange between homo- and hetero-oligomeric signaling complexes is favored over an intermediate involving a free monomeric helix in the membrane bilayer.

Missense variants in phospholamban and cardiac myosin binding protein identified in patients with a family history and clinical diagnosis of dilated cardiomyopathy.

As the genetic landscape of cardiomyopathies continues to expand, the identification of missense variants in disease-associated genes frequently leads to a classification of variant of uncertain significance (VUS). For the proper reclassification of such variants, functional characterization is an important contributor to the proper assessment of pathogenic potential. Several missense variants in the calcium transport regulatory protein phospholamban have been associated with dilated cardiomyopathy. However, >40 missense variants in this transmembrane peptide are currently known and most remain classified as VUS with little clinical information. Similarly, missense variants in cardiac myosin binding protein have been associated with hypertrophic cardiomyopathy. However, hundreds of variants are known and many have low penetrance and are often found in control populations. Herein, we focused on novel missense variants in phospholamban, an Ala15-Thr variant found in a 4-year-old female and a Pro21-Thr variant found in a 60-year-old female, both with a family history and clinical diagnosis of dilated cardiomyopathy. The patients also harbored a Val896-Met variant in cardiac myosin binding protein. The phospholamban variants caused defects in the function, phosphorylation, and dephosphorylation of this calcium transport regulatory peptide, and we classified these variants as potentially pathogenic. The variant in cardiac myosin binding protein alters the structure of the protein. While this variant has been classified as benign, it has the potential to be a low-risk susceptibility variant because of the structural change in cardiac myosin binding protein. Our studies provide new biochemical evidence for missense variants previously classified as benign or VUS.

Validation of a novel algorithm with a high specificity in ruling out MDS.

INTRODUCTION: A previously published web-based App using Gradient-boosted models (GBMs) of eight laboratory parameters was established by Oster et al. to facilitate diagnosis or exclusion of myelodysplastic syndromes (MDS) in patients. METHODS: To validate their algorithm, we compared 175 anemic patients with MDS diagnosis from our German MDS Registry with 1378 non-MDS anemic patients who consulted various specialties in the Düsseldorf university hospital. RESULTS: Based on hemoglobin level, leukocyte and platelet count, mean corpuscular volume, absolute neutrophil count, absolute monocyte count, glucose and creatinine, plus the patients' gender and age, we could not reproduce a high negative predictive value (NPV), but confirmed a useful specificity of 90.9% and a positive predictive value (PPV) of 77.1%. 1192 of 1378 controls were correctly categorized as "probably not MDS (pnMDS)" patients. A total of 65 patients were wrongly classified as "probable MDS (pMDS)," of whom 48 had alternative explanations for their altered laboratory results. In a second analysis, we included 29 patients with chronic myelomonocytic leukemia (CMML) resulting in only one label as possible MDS, suggesting that highly proliferative bone marrow disorders are correctly excluded. CONCLUSION: The possibility of reliably excluding MDS from differential diagnosis based on peripheral blood lab work appears to be attractive for patients and physicians alike while the confirmation of MDS diagnosis still requires a bone marrow biopsy.

Practice Patterns in Asymmetric Sensorineural Hearing Loss: Survey Data.

OBJECTIVE: Although screening protocols for patients who present with asymmetric sensorineural hearing loss (ASNHL) exist, there are no clear guidelines to direct practitioners. In particular, various thresholds have been proposed for the degree of hearing loss that should prompt MRI studies, but the topic remains understudied. This project aims to compare protocols followed by practitioners to guide their imaging practices. STUDY DESIGN: Web-based survey. SETTING: Otolaryngology faculty at academic medical centers. METHODS: A list of 530 otolaryngologists (276 otology/neurotology specialists, 254 general otolaryngologists) was compiled. A survey consisting of three parts: demographics, general practice patterns, and simulated patient cases was distributed. RESULTS: A total of 468 surveys were successfully distributed, resulting in 88 (18.8%) responses. The majority of respondents (63.8%) self-reported their definition of ASNHL as ">30 dB hearing asymmetry at one frequency OR >20 dB hearing asymmetry at two continuous frequencies OR >10 dB hearing asymmetry at three contiguous frequencies." Overall, general otolaryngologists were more likely to observe asymmetric findings with serial audiogram alone, whereas otology/neurotology specialists were more likely to obtain imaging. CONCLUSION: There is significant variability between providers with regard to managing patients with ASNHL and evidence-based guidelines would be useful in guiding imaging practices. LEVEL OF EVIDENCE: N/A Laryngoscope, 2024.

"Emergency Room Incision and Drainage of Perirectal Abscess is Equivalent to Incision and Drainage in the Operating Room".

BACKGROUND: Current guidelines for management of anorectal abscesses make no recommendations for operative vs bedside incision and drainage (I&D). The purpose of this study was to determine if management in the operating room is necessary to adequately drain anorectal abscesses and prevent short-term complications for patients presenting to the emergency department (ED). METHODS: Patients with perirectal abscesses were identified and divided into two groups based on intervention type: "bedside" or "operative." Demographic, laboratory, and encounter data were obtained from the medical record. Study outcomes included 30-day complications (return to the ED, reintervention, and readmission). Data were analyzed with univariate and multivariate analyses using SPSS (version 28). RESULTS: A total of 113 patients with anorectal abscesses were identified. Sixty-six (58%) underwent bedside I&D and 47 (42%) operative I&D. The overall complication rate was 10%. A total of 9 patients (6 bedside and 3 operative) returned to the ED. Six of these patients required reintervention (5 bedside and 1 operative), and 1 was readmitted. Two patients from the bedside group required a second I&D during their index admission. Pre-procedure SIRS (P = .02) was found to be associated with 30-day complications. Provider specialty and training level were not associated with 30-day complications. DISCUSSION: In this study, for patients presenting to the ED, bedside drainage was found to be an adequate management strategy to achieve complete drainage without a significant increase in the rate of complications when compared to operative drainage.

Attosecond and nano-Coulomb electron bunches via the Zero Vector Potential mechanism.

The commissioning of multi-petawatt class laser facilities around the world is gathering pace. One of the primary motivations for these investments is the acceleration of high-quality, low-emittance electron bunches. Here we explore the interaction of a high-intensity femtosecond laser pulse with a mass-limited dense target to produce MeV attosecond electron bunches in transmission and confirm with three-dimensional simulation that such bunches have low emittance and nano-Coulomb charge. We then perform a large parameter scan from non-relativistic laser intensities to the laser-QED regime and from the critical plasma density to beyond solid density to demonstrate that the electron bunch energies and the laser pulse energy absorption into the plasma can be quantitatively described via the Zero Vector Potential mechanism. These results have wide-ranging implications for future particle accelerator science and associated technologies.

Microglia and macrophages alterations in the CNS during acute SIV infection: a single-cell analysis in rhesus macaques.

Human Immunodeficiency Virus (HIV) is widely acknowledged for its profound impact on the immune system. Although HIV primarily affects peripheral CD4 T cells, its influence on the central nervous system (CNS) cannot be overlooked. Within the brain, microglia and CNS-associated macrophages (CAMs) serve as the primary targets for HIV, as well as for the simian immunodeficiency virus (SIV) in nonhuman primates. This infection can lead to neurological effects and the establishment of a viral reservoir. Given the gaps in our understanding of how these cells respond in vivo to acute CNS infection, we conducted single-cell RNA sequencing (scRNA-seq) on myeloid cells from the brains of three rhesus macaques 12-days after SIV infection, along with three uninfected controls. Our analysis revealed six distinct microglial clusters including homeostatic microglia, preactivated microglia, and activated microglia expressing high levels of inflammatory and disease-related molecules. In response to acute SIV infection, the population of homeostatic and preactivated microglia decreased, while the activated and disease-related microglia increased. All microglial clusters exhibited upregulation of MHC class I molecules and interferon-related genes, indicating their crucial roles in defending against SIV during the acute phase. All microglia clusters also upregulated genes linked to cellular senescence. Additionally, we identified two distinct CAM populations: CD14lowCD16hi and CD14hiCD16low CAMs. Interestingly, during acute SIV infection, the dominant CAM population changed to one with an inflammatory phenotype. Notably, specific upregulated genes within one microglia and one macrophage cluster were associated with neurodegenerative pathways, suggesting potential links to neurocognitive disorders. This research sheds light on the intricate interactions between viral infection, innate immune responses, and the CNS, providing valuable insights for future investigations.