The TRIM-NHL RNA-binding protein Brain Tumor coordinately regulates expression of the glycolytic pathway and vacuolar ATPase complex.

The essential Drosophila RNA-binding protein Brain Tumor (Brat) represses specific genes to control embryogenesis and differentiation of stem cells. In the brain, Brat functions as a tumor suppressor that diminishes neural stem cell proliferation while promoting differentiation. Though important Brat-regulated target mRNAs have been identified in these contexts, the full impact of Brat on gene expression remains to be discovered. Here, we identify the network of Brat-regulated mRNAs by performing RNA sequencing (RNA-seq) following depletion of Brat from cultured cells. We identify 158 mRNAs, with high confidence, that are repressed by Brat. De novo motif analysis identified a functionally enriched RNA motif in the 3' untranslated regions (UTRs) of Brat-repressed mRNAs that matches the biochemically defined Brat binding site. Integrative data analysis revealed a high-confidence list of Brat-repressed and Brat-bound mRNAs containing 3'UTR Brat binding motifs. Our RNA-seq and reporter assays show that multiple 3'UTR motifs promote the strength of Brat repression, whereas motifs in the 5'UTR are not functional. Strikingly, we find that Brat regulates expression of glycolytic enzymes and the vacuolar ATPase complex, providing new insight into its role as a tumor suppressor and the coordination of metabolism and intracellular pH.

Functional Validation of SAM Riboswitch Element A from Listeria monocytogenes.

SreA is one of seven candidate S-adenosyl methionine (SAM) class I riboswitches identified in Listeria monocytogenes, a saprophyte and opportunistic foodborne pathogen. SreA precedes genes encoding a methionine ATP-binding cassette (ABC) transporter, which imports methionine and is presumed to regulate transcription of its downstream genes in a SAM-dependent manner. The proposed role of SreA in controlling the transcription of genes encoding an ABC transporter complex may have important implications for how the bacteria senses and responds to the availability of the metabolite SAM in the diverse environments in which L. monocytogenes persists. Here we validate SreA as a functional SAM-I riboswitch through ligand binding studies, structure characterization, and transcription termination assays. We determined that SreA has both a structure and SAM binding properties similar to those of other well-characterized SAM-I riboswitches. Despite the apparent structural similarities to previously described SAM-I riboswitches, SreA induces transcription termination in response to comparatively lower (nanomolar) ligand concentrations. Furthermore, SreA is a leaky riboswitch that permits some transcription of the downstream gene even in the presence of millimolar SAM, suggesting that L. monocytogenes may "dampen" the expression of genes for methionine import but likely does not turn them "OFF".

Intercropping Alters Phytochemical Defenses Against Insect Herbivory.

Given the multiple possible mechanisms for interspecific chemical interaction between adjacent heterospecific plants, phytochemical defenses of pest-susceptible crop species could potentially be enhanced or altered by intercropping with phytochemically diverse neighbors. We assessed the influence of intercropping between phytochemically diverse plants on aerial volatile organic compound (VOC) emission profiles by intercropping Melilotus alba and Triticum aestivum with Silphium integrifolium in AMF-inoculated soil. We also assessed the impact of intercropping on induced plant defenses by conducting an in-situ, no-choice bioassay with Spodoptera frugiperda. Of eight compound classes we identified across the three plant species, prenol lipids (terpenoids) were upregulated in silflower plants when monocropped with wheat and when herbivory was induced. Carboxylic acids and organooxygen compounds were reduced in sweetclover when intercropped with silflower, but increased under herbivory. Uninfested wheat plants emitted more organooxygen compounds and fatty acyls than infested plants when intercropped with silflower, but not when monocropped. This study showed that VOC emissions of plants from three diverse taxa are altered by both intercropping and herbivory in ways that may impact their resistance to insect herbivory. Further research into the role of intercropping on pest resistance in agroecological systems could help farmers to design intercropping systems that optimize natural plant herbivory defenses, thus improving agricultural sustainability.

Laminar organization of visual responses in core and parabelt auditory cortex.

Audiovisual (AV) interaction has been shown in many studies of auditory cortex. However, the underlying processes and circuits are unclear because few studies have used methods that delineate the timing and laminar distribution of net excitatory and inhibitory processes within areas, much less across cortical levels. This study examined laminar profiles of neuronal activity in auditory core (AC) and parabelt (PB) cortices recorded from macaques during active discrimination of conspecific faces and vocalizations. We found modulation of multi-unit activity (MUA) in response to isolated visual stimulation, characterized by a brief deep MUA spike, putatively in white matter, followed by mid-layer MUA suppression in core auditory cortex; the later suppressive event had clear current source density concomitants, while the earlier MUA spike did not. We observed a similar facilitation-suppression sequence in the PB, with later onset latency. In combined AV stimulation, there was moderate reduction of responses to sound during the visual-evoked MUA suppression interval in both AC and PB. These data suggest a common sequence of afferent spikes, followed by synaptic inhibition; however, differences in timing and laminar location may reflect distinct visual projections to AC and PB.

Hierarchical differences in the encoding of amplitude modulation in the subcortical auditory system of awake nonhuman primates.

Sinusoidal amplitude modulation (SAM) is a key feature of complex sounds. Although psychophysical studies have characterized SAM perception, and neurophysiological studies in anesthetized animals report a transformation from the cochlear nucleus' (CN; brainstem) temporal code to the inferior colliculus' (IC; midbrain's) rate code, none have used awake animals or nonhuman primates to compare CN and IC's coding strategies to modulation-frequency perception. To address this, we recorded single-unit responses and compared derived neurometric measures in the CN and IC to psychometric measures of modulation frequency (MF) discrimination in macaques. IC and CN neurons often exhibited tuned responses to SAM in rate and spike-timing measures of modulation coding. Neurometric thresholds spanned a large range (2-200 Hz ΔMF). The lowest 40% of IC thresholds were less than or equal to psychometric thresholds, regardless of which code was used, whereas CN thresholds were greater than psychometric thresholds. Discrimination at 10-20 Hz could be explained by indiscriminately pooling 30 units in either structure, whereas discrimination at higher MFs was best explained by more selective pooling. This suggests that pooled CN activity was sufficient for AM discrimination. Psychometric and neurometric thresholds decreased as stimulus duration increased, but IC and CN thresholds were higher and more variable than behavior at short durations. This slower subcortical temporal integration compared with behavior was consistent with a drift diffusion model that reproduced individual differences in performance and can constrain future neurophysiological studies of temporal integration. These measures provide an account of AM perception at the neurophysiological, computational, and behavioral levels.NEW & NOTEWORTHY In everyday environments, the brain is tasked with extracting information from sound envelopes, which involves both sensory encoding and perceptual decision-making. Different neural codes for envelope representation have been characterized in midbrain and cortex, but studies of brainstem nuclei such as the cochlear nucleus (CN) have usually been conducted under anesthesia in nonprimate species. Here, we found that subcortical activity in awake monkeys and a biologically plausible perceptual decision-making model accounted for sound envelope discrimination behavior.

Laminar pattern of sensory-evoked dynamic high-frequency oscillatory activity in the macaque auditory cortex.

High-frequency (>60 Hz) neuroelectric signals likely have functional roles distinct from low-frequency (<30 Hz) signals. While high-gamma activity (>60 Hz) does not simply equate to neuronal spiking, they are highly correlated, having similar information encoding. High-gamma activity is typically considered broadband and poorly phase-locked to sensory stimuli and thus is typically analyzed after transformations into absolute amplitude or spectral power. However, those analyses discard signal polarity, compromising the interpretation of neuroelectric events that are essentially dipolar. In the spectrotemporal profiles of field potentials in auditory cortex, we show high-frequency spectral peaks not phase-locked to sound onset, which follow the broadband peak of phase-locked onset responses. Isolating the signal components comprising the high-frequency peaks reveals narrow-band high-frequency oscillatory events, whose instantaneous frequency changes rapidly from >150 to 60 Hz, which may underlie broadband high-frequency spectral peaks in previous reports. The laminar amplitude distributions of the isolated activity had two peak positions, while the laminar phase patterns showed a counterphase relationship between those peaks, indicating the formation of dipoles. Our findings suggest that nonphase-locked HGA arises in part from oscillatory or recurring activity of supragranular-layer neuronal ensembles in auditory cortex.

Development and validation of a HPLC method for the determination of chemical and radiochemical purity of O-(2-[18F]fluoroethyl-l-tyrosine ([18F]FET)), a PET radiotracer for the imaging of brain tumor.

A novel HPLC method was developed and validated to determine radiochemical identity, radiochemical purity and chemical purity for the analysis of O-(2-[18F]fluoroethyl-l-tyrosine ([18F]FET). In this method, an analytical Phenomenex Gemini C18 column was used with an isocratic eluent of 7 % ethanol and 93 % 50 mM potassium phosphate buffer (pH = 6.9). The flow rate was 1.0 mL/min and the injection volume was 10 µL. A photo-diode array detector set at 220 nm was used for UV mass detection and a single channel, high sensitivity radiation detector was used. The method validation assays including specificity, linearity, precision, accuracy, and robustness were evaluated. Results show that the method was suitable for qualitative and quantitative determination of radiochemical and chemical purity of [18F]FET. This system has been routinely used for the analysis of more than 120 batches of [18F]FET with radiochemical yield 23.7 ± 6 % (no decay corrected) and molar activity 593 ± 284 GBq/µmole in our facility to support human use.

uafR: An R package that automates mass spectrometry data processing.

Chemical information has become increasingly ubiquitous and has outstripped the pace of analysis and interpretation. We have developed an R package, uafR, that automates a grueling retrieval process for gas -chromatography coupled mass spectrometry (GC -MS) data and allows anyone interested in chemical comparisons to quickly perform advanced structural similarity matches. Our streamlined cheminformatics workflows allow anyone with basic experience in R to pull out component areas for tentative compound identifications using the best published understanding of molecules across samples (pubchem.gov). Interpretations can now be done at a fraction of the time, cost, and effort it would typically take using a standard chemical ecology data analysis pipeline. The package was tested in two experimental contexts: (1) A dataset of purified internal standards, which showed our algorithms correctly identified the known compounds with R2 values ranging from 0.827-0.999 along concentrations ranging from 1 × 10-5 to 1 × 103 ng/µl, (2) A large, previously published dataset, where the number and types of compounds identified were comparable (or identical) to those identified with the traditional manual peak annotation process, and NMDS analysis of the compounds produced the same pattern of significance as in the original study. Both the speed and accuracy of GC -MS data processing are drastically improved with uafR because it allows users to fluidly interact with their experiment following tentative library identifications [i.e. after the m/z spectra have been matched against an installed chemical fragmentation database (e.g. NIST)]. Use of uafR will allow larger datasets to be collected and systematically interpreted quickly. Furthermore, the functions of uafR could allow backlogs of previously collected and annotated data to be processed by new personnel or students as they are being trained. This is critical as we enter the era of exposomics, metabolomics, volatilomes, and landscape level, high-throughput chemotyping. This package was developed to advance collective understanding of chemical data and is applicable to any research that benefits from GC -MS analysis. It can be downloaded for free along with sample datasets from Github at github.org/castratton/uafR or installed directly from R or RStudio using the developer tools: 'devtools::install_github("castratton/uafR")'.

Using an Innovative Method for Self-Collection of Capillary Blood for HIV and Syphilis Testing Among Men Who Have Sex With Men Who Use Pre-exposure Prophylaxis in the Netherlands; Limburg4zero.

BACKGROUND: Home-based sampling could create accessible testing opportunities for men who have sex with men (MSM) who use pre-exposure prophylaxis (PrEP). Blood collection is required for the most reliable laboratory results for HIV and syphilis testing. An innovative blood collection method (Tasso+) creates a vacuum and semi-automatically collects larger volumes of blood from the upper arm. This study aimed to assess acceptability and feasibility of this device among PrEP-using MSM and the performance of blood collection. METHODS: Between August 2022 and January 2023, 47 MSM were recruited during their routine PrEP consultations at a Dutch Centre for Sexual Health. Participants tested the method directly after consultation, and an online questionnaire determined acceptability and feasibility. Blood and residual serum volumes were measured after sampling and after HIV and syphilis testing. RESULTS: Of the participants, 87% had a positive attitude toward use of the device, and 77% would use it again for self-sampling at home. Participants rated the use of the blood collection device as easy (96%). On average, 536 µL whole blood (244 µL serum) was collected. All samples were tested for HIV and syphilis, and most samples had sufficient blood for routine HIV (91%) and syphilis testing (89%). Most samples (85%) had 220 µL residual blood, sufficient for further testing (e.g., confirmation). CONCLUSIONS: Blood self-sampling with a method that creates a vacuum from the upper arm is highly acceptable by users and performs well in blood collection for multiple tests. This method has promising potential for use in home-based sexual health care for PrEP-using MSM.

The Sexual Health Clinic Role in Vaccine and Treatment Access During the 2022 Mpox Outbreak in King County, Washington.

BACKGROUND: Sexual health clinics were frontline providers in the 2022 US mpox public health response, though data on clinic-based mpox vaccine scale-up, diagnoses, and treatment are limited. We describe the role of a public health sexual health clinic (SHC) in King County's mpox response, between 5/23/22-10/31/22. METHODS: In July 2022, the SHC implemented a dedicated vaccine clinic and presumptive tecovirimat treatment (prior to laboratory confirmation) with on-site dispensation. We describe SHC's vaccine scale-up and contribution to clinical care by calculating the weekly number of vaccines administered by SHC and the total number of patients diagnosed and treated for mpox within SHC, and comparing to countywide data. We calculated time from symptom onset to testing and time from testing to treatment, and assessed temporal changes in these metrics using linear regression. RESULTS: The SHC provided ≥1 vaccine doses to 7,442 individuals (10,295 doses), administering 42% of the 24,409 vaccine doses provided countywide, with the greatest contribution in the first week of August (n = 1,562, 58% of countywide vaccinations that week). Of 598 patients evaluated for mpox and tested, 178 (30%) tested positive (37% of countywide cases), and 152 (85% of SHC patients with mpox) received tecovirimat (46% of treatment countywide). Median time from symptom onset to testing decreased from 12 to 6 days (p = 0.045); time from testing to treatment decreased from 4.5 days to 0 days (p < 0.001). CONCLUSION: The SHC was central to mpox vaccination and treatment scale-up, particularly in the first months of the 2022 epidemic.

Male elephant management in AZA institutions: Current status and priorities for the future.

Asian elephant (Elephas maximus) and African savanna elephant (Loxodonta africana) populations collectively managed by ex-situ facilities accredited by the Association of Zoos and Aquariums (AZA) face sustainability challenges. Among the priorities to strengthen animal wellbeing and population sustainability is male elephant management. We conducted a survey of AZA facilities currently housing male elephants to assess the status, challenges, and priorities in three areas of male elephant management: musth, socialization, and semen collection. Surveys were administered to elephant care teams at AZA-accredited institutions between November 2022 and February 2023, and we received responses from 34 institutions (91.9% of AZA-accredited facilities holding adult male elephants), housing 32 adult male Asians and 26 adult male Africans. Most facilities prioritized breeding and male socialization over musth management and semen collection (although most facilities acknowledged that all these efforts are important), citing leadership support and staffing as most important to achieve male management goals. Behaviors most commonly accompanying musth included reduced appetite, difficulty training or shifting, human-directed aggression, and interest in females. Musth timing was variable between males and facilities. Most males were well-socialized with females and/or other males, though elephant compatibility and facility design were limiting factors in managing socialization. Although 60.6% of facilities collected semen or were training for semen collection, very few male elephants could reliably provide viable semen samples, challenging assisted reproductive efforts that could bolster population sustainability in both species. Together, our results provide a better understanding of the state of male elephant management, offering specific areas deserving of research and development to enhance wellbeing and sustainability.

Histiocytoid Sweet's Syndrome in the Setting of Myelodysplastic Syndrome.

Acute febrile neutrophilic dermatosis, or Sweet's syndrome, is characterized by tender, edematous papules and plaques, favoring the upper extremities and the head and neck regions. The classic variant of Sweet's syndrome involves a predominantly neutrophilic dermal infiltrate on histopathology. However, histiocytoid Sweet's syndrome has been noted to have a primary histiocytoid mononuclear infiltrate and is typically found in patients with malignancies such as myelodysplasia. This case report discusses the treatment of histiocytoid Sweet's syndrome in an immunocompromised patient with a recent history of Mycobacterium avium complex infection and latent tuberculosis in the setting of myelodysplastic syndrome.

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling.

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly occurs in diseases marked by progressive vision loss, such as age-related macular degeneration (AMD). Research on AMD often relies on human donor eyes or induced pluripotent stem cells (iPSCs) to represent the RPE. However, these RPE sources require extended differentiation periods and substantial expertise for culturing. Additionally, some research institutions, particularly those in rural areas, lack easy access to donor eyes. While a commercially available immortalized RPE cell line (ARPE-19) exists, it lacks essential in vivo RPE features and is not widely accepted in many ophthalmology research publications. There is a pressing need to obtain representative primary RPE cells from a more readily available and cost-effective source. This protocol elucidates the isolation and subculture of primary RPE cells obtained post-mortem from porcine eyes, which can be sourced locally from commercial or academic suppliers. This protocol necessitates common materials typically found in tissue culture labs. The result is a primary, differentiated, and cost-effective alternative to iPSCs, human donor eyes, and ARPE-19 cells.

In vitro activity of gepotidacin and comparator antimicrobials against isolates of nontuberculous mycobacteria (NTM).

Novel antimicrobials are needed to treat rising nontuberculous mycobacteria (NTM) infections. Using standard broth microdilution methods, 68 NTM isolates were tested against gepotidacin, a new, first-in-class, oral triazaacenaphthylene bacterial topoisomerase inhibitor. MICs varied (0.25 to >64 µg/mL) with the lowest being M. fortuitum complex (0.25-8 µg/mL), M. mucogenicum complex (1-2 µg/mL), M. kansasii (0.25-8 µg/mL), and M. marinum (4-16 µg/mL). Testing greater numbers of some species is suggested to better understand gepotidacin activity against NTM.

Vegfc-expressing cells form heterotopic bone after musculoskeletal injury.

Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.

A randomized trial to compare smooth monofilament suture vs. barbed suture using the three-layer continuous closure technique in canine ovariohysterectomy in a high-quality high-volume spay/neuter clinic.

The primary objective of this study was to compare time efficiency and complication rates between smooth monofilament suture (SMS) and barbed suture (BS) using the three-layer continuous incisional closure pattern after ovariohysterectomy in a high-quality high-volume spay/neuter clinic. The study was designed as a randomized controlled trial enrolling 71 adult female dogs. Dogs were randomly assigned to SMS or BS treatments. The effect of closure with BS or SMS on closure time was tested through multilevel, multivariable linear regression in a generalized linear mixed model. Body condition score, weight, and pre-closure incision length were tested as covariates. Surgeon was included in the model as a random effect. Pre-closure incision length (p = 0.01) and method (p ≤ 0.0001) were associated with closure time. Adjusting for pre-closure incision length, the average time for closure with SMS was 6.5 min (range 3.70-10.31 min), and the average time for closure with BS was 4.91 min (range 3.05-8.05 min). Accounting for the closure method, the closure time increased by 39 s for each additional centimeter of incision length. BS was more efficient than SMS when performing the three-layer continuous suture pattern. No short-term telemedicine-assessed complications were noted with either treatment method. BS can improve efficiency in surgical closures, especially considering large volumes of animals, and appears to have a similar short-term, telemedicine-assessed complication rate when compared to SMS.

The HIF-1α/PLOD2 axis integrates extracellular matrix organization and cell metabolism leading to aberrant musculoskeletal repair.

While hypoxic signaling has been shown to play a role in many cellular processes, its role in metabolism-linked extracellular matrix (ECM) organization and downstream processes of cell fate after musculoskeletal injury remains to be determined. Heterotopic ossification (HO) is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues. Hypoxia and hypoxia-inducible factor 1α (HIF-1α) activation have been shown to promote HO. However, the underlying molecular mechanisms by which the HIF-1α pathway in mesenchymal progenitor cells (MPCs) contributes to pathologic bone formation remain to be elucidated. Here, we used a proven mouse injury-induced HO model to investigate the role of HIF-1α on aberrant cell fate. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics analyses of the HO site, we found that collagen ECM organization is the most highly up-regulated biological process in MPCs. Zeugopod mesenchymal cell-specific deletion of Hif1α (Hoxa11-CreERT2; Hif1afl/fl) significantly mitigated HO in vivo. ScRNA-seq analysis of these Hoxa11-CreERT2; Hif1afl/fl mice identified the PLOD2/LOX pathway for collagen cross-linking as downstream of the HIF-1α regulation of HO. Importantly, our scRNA-seq data and mechanistic studies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1α deletion. From a translational aspect, a pan-LOX inhibitor significantly decreased HO. A newly screened compound revealed that the inhibition of PLOD2 activity in MPCs significantly decreased osteogenic differentiation and glycolytic metabolism. This suggests that the HIF-1α/PLOD2/LOX axis linked to metabolism regulates HO-forming MPC fate. These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promising strategy to mitigate HO formation.

Intersections of Fibrodysplasia Ossificans Progressiva and Traumatic Heterotopic Ossification.

Heterotopic ossification (HO) is a debilitating pathology where ectopic bone develops in areas of soft tissue. HO can develop as a consequence of traumatic insult or as a result of dysregulated osteogenic signaling, as in the case of the orphan disease fibrodysplasia ossificans progressiva (FOP). Traumatic HO (tHO) formation is mediated by the complex interplay of signaling between progenitor, inflammatory, and nerve cells, among others, making it a challenging process to understand. Research into the pathogenesis of genetically mediated HO (gHO) in FOP has established a pathway involving uninhibited activin-like kinase 2 receptor (ALK2) signaling that leads to downstream osteogenesis. Current methods of diagnosis and treatment lag behind pre-mature HO detection and progressive HO accumulation, resulting in irreversible decreases in range of motion and chronic pain for patients. As such, it is necessary to draw on advancements made in the study of tHO and gHO to better diagnose, comprehend, prevent, and treat both.