Comparative Pathogenesis of Two Lineages of Powassan Virus Reveals Distinct Clinical Outcome, Neuropathology, and Inflammation.

Tick-borne flaviviruses (TBFV) can cause severe neuroinvasive disease which may result in death or long-term neurological deficit in over 50% of survivors. Multiple mechanisms for invasion of the central nervous system (CNS) by flaviviruses have been proposed including axonal transport, transcytosis, endothelial infection, and Trojan horse routes. Flaviviruses may utilize different or multiple mechanisms of neuroinvasion depending on the specific virus, infection site, and host variability. In this work we have shown that the infection of BALB/cJ mice with either Powassan virus lineage I (Powassan virus) or lineage II (deer tick virus) results in distinct spatial tropism of infection in the CNS which correlates with unique clinical presentations for each lineage. Comparative transcriptomics of infected brains demonstrates the activation of different immune pathways and downstream host responses. Ultimately, the comparative pathology and transcriptomics are congruent with different clinical signs in a murine model. These results suggest that the different disease presentations occur in clinical cases due to the inherent differences in the two lineages of Powassan virus.

Discovery of an Azabicyclo[2.1.1]hexane Piperazinium Salt and Its Application in Medicinal Chemistry via a Rearrangement.

Herein we report the discovery of an azabicyclo[2.1.1]hexane piperazinium methanesulfonate salt from an unexpected rearrangement reaction in the preparation of ligand-directed degraders (LDDs). This bench-stable compound was found to be a versatile electrophile in a ring-opening reaction with various types of nucleophiles. Its utility as a versatile medicinal chemistry building block is further demonstrated in the synthesis of an LDD compound targeting degradation of the androgen receptor.

Early intestinal ultrasound in severe ulcerative colitis identifies patients at increased risk of 1-year treatment failure and colectomy.

BACKGROUND AND AIMS: Reliable and easily accessible objective markers of disease activity to predict long-term treatment outcomes in severe ulcerative colitis (UC) are missing. We aimed to investigate if intestinal ultrasound (IUS) might predict long-term outcomes in hospitalized patients with severe UC treated with intravenous corticosteroids. METHODS: Hospitalized patients with severe UC and IUS inflammation (bowel wall thickness (BWT)>3.0mm) starting IV corticosteroids were recruited at three university hospitals in Denmark. IUS was performed before treatment, 48±24 hours (h), 6±1 days, and 3 months after treatment initiation. Time until colectomy or need for new interventions was registered together with Mayo score at 3 months and partial Mayo score (pMayo) at 12-months. Follow-up time was 12 months. RESULTS: Fifty-six patients were included in the final analysis. Forty-five (80%) patients needed intervention, including 9 colectomies, during the 12-month follow-up. After 48±24h: No patient with a BWT<3mm needed a colectomy, p=0.04. BWT≥4mm showed an increased risk of colectomy (odds ratio 9.5 (95%CI 1.5-186), p=0.03), while a BWT≥3mm showed an increased risk of intervention (3.6 (1.1-12.5), p=0.03). A BWT≥4mm resulted in a significantly shorter time until both colectomy, p=0.03, and treatment intensification (mean days 75 (95%CI24-127) vs. 176 (119-233), p=0.005. However, neither IUS parameters nor pMayo score, CRP, hemoglobin, or p-albumin could predict remission at 3- and 12-months. CONCLUSION: BWT assessed at 48h post intravenous corticosteroid initiation in patients hospitalized with severe UC may identify patients with an increased risk of short- and long-term colectomy and predict a more aggressive short-term disease course.

Sequenced treatment effectiveness for posttraumatic stress (STEPS) trial: A protocol for a pragmatic comparative effectiveness trial with baseline results.

BACKGROUND: There have only been two efficacy trials reporting a head-to-head comparison of medications and psychotherapy for posttraumatic stress disorder (PTSD), and neither was conducted in primary care. Therefore, in this pragmatic trial we compare outcomes of primary care patients randomized to initially receive a brief trauma-focused psychotherapy or a choice of three antidepressants. In addition, because there are few trials examining the effectiveness of subsequent treatments for patients not responding to the initial treatment, we also compare the outcomes of those switching or augmenting treatments. METHOD: Patients screening positive for PTSD (n = 700) were recruited from the primary care clinics of 7 Federally Qualified Health Centers (FQHC) and 8 Department of Veterans Affairs (VA) Medical Centers and randomized in the ratio 1:1:2 to one of three treatment sequences: 1) selective serotonin reuptake inhibitor (SSRI) followed by augmentation with Written Exposure Therapy (WET), 2) SSRI followed by a switch to serotonin-norepinephrine reuptake inhibitor (SNRI), or 3) WET followed by a switch to SSRI. Participants complete surveys at baseline, 6 months, and 12 months. The primary outcome is PTSD symptom severity as measured by the PTSD Checklist (PCL-5). RESULTS: The average PCL-5 score was 52.8 (SD = 11.1), indicating considerable severity. The most common bothersome traumatic event for VA enrollees was combat (47.8%), and for FQHC enrollees was other (28.2%), followed by sexual assault (23.4%), and child abuse (19.8%). Only 22.4% were taking an antidepressant at baseline. CONCLUSION: Results will help healthcare systems and clinicians make decisions about which treatments to offer to patients. CLINICALTRIALS: govID - NCT04597190.

The Effect of Concurrent Use of Opioids and Gabapentin on Fall Risk in Older Adults.

Falls pose a significant threat to older adults, resulting in injuries and mortality. Concurrently prescribed opioids and gabapentin for pain management may increase fall risks in older patients. This study aimed to estimate fall risks associated with the concurrent use of gabapentin and opioids, comparing them to opioid monotherapy in older adults. A retrospective case-control study of 1,813 patients aged 65-89 on chronic opioid therapy (2017-2020), excluding those with a fall history, analysis focused on the first fall occurrence. Logistic regression assessed the association between concurrent gabapentin and opioid use and fall events. Out of eligible patients, 122 (6.73%) experienced falls during opioid therapy, with 232 (12.80%) having concurrent gabapentin use. Concurrent use significantly increased fall risk (AOR = 1.73; 95% CI: 1.08-2.78). Being female, aged ≥81, and having more chronic conditions also increased risk. Mitigating fall risk in older adults requires education on prevention, exploring alternative pain management, and careful consideration of prescribing. Further research is crucial to understand adverse events linked to combined opioid and gabapentin use in the geriatric population.

mRNA Display in Cell Lysates Enables Identification of Cyclic Peptides Targeting the BRD3 Extraterminal Domain.

mRNA display is a powerful technology to screen libraries of >1012 cyclic peptides against a protein target, enabling the rapid discovery of high affinity ligands. These cyclic peptides are particularly well suited to challenging protein targets that have been difficult to drug with small molecules. However, target choice can still be limited as screens are typically performed against purified proteins which often demands the use of isolated domains and precludes the use of aggregation-prone targets. Here, we report a method to perform mRNA display selections in mammalian cell lysates without the need for prior target purification, vastly expanding the potential target scope of mRNA display. We have applied the methodology to identify low to sub-nanomolar peptide binders for two targets: a NanoLuc subunit (LgBiT) and full-length bromodomain-containing protein 3 (BRD3). Our cyclic peptides for BRD3 were found to bind to the extraterminal (ET) domain of BRD3 and the closely related BRD proteins, BRD2 and BRD4. While many chemical probes exist for the bromodomains of BRD proteins, the ET domain is relatively underexplored, making these peptides valuable additions to the BRD toolbox.

Intrinsic Gata4 expression sensitizes the aortic root to dilation in a Loeys-Dietz syndrome mouse model.

Loeys-Dietz syndrome (LDS) is an aneurysm disorder caused by mutations that decrease transforming growth factor-ß (TGF-ß) signaling. Although aneurysms develop throughout the arterial tree, the aortic root is a site of heightened risk. To identify molecular determinants of this vulnerability, we investigated the heterogeneity of vascular smooth muscle cells (VSMCs) in the aorta of Tgfbr1 M318R/+ LDS mice by single cell and spatial transcriptomics. Reduced expression of components of the extracellular matrix-receptor apparatus and upregulation of stress and inflammatory pathways were observed in all LDS VSMCs. However, regardless of genotype, a subset of Gata4-expressing VSMCs predominantly located in the aortic root intrinsically displayed a less differentiated, proinflammatory profile. A similar population was also identified among aortic VSMCs in a human scRNAseq dataset. Postnatal VSMC-specific Gata4 deletion reduced aortic root dilation in LDS mice, suggesting that this factor sensitizes the aortic root to the effects of impaired TGF-ß signaling.

Characterizing Modulators of Protease-activated Receptors with a Calcium Mobilization Assay Using a Plate Reader.

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of Gαq signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

All-Soft-Tissue Meniscus Allograft Transplantation With Circumferential Suture Tape Augmentation to Mitigate Hoop Stress and Promote Centralization.

Meniscus allograft transplantation (MAT) is a technically challenging procedure. Bone plugs, slot techniques, and all-soft-tissue fixation techniques have been described in the past. Each technique comes with advantages and disadvantages. Native menisci have circumferential collagen fibers to help resist hoop stress during loading cycles. Although hoop stress resistance is a known function of the menisci, its recreation in MAT has only been targeted indirectly through anatomic root placement. The authors describe the use of a high-tensile suture tape (i.e. InternalBrace) to promote centralization by directly mitigating hoop stresses through recreation of peripheral meniscus tensioning in MAT.

In Vitro Cell Surface Marker Expression on Mesenchymal Stem Cell Cultures does not Reflect Their Ex Vivo Phenotype.

Cell surface marker expression is one of the criteria for defining human mesenchymal stem or stromal cells (MSC) in vitro. However, it is unclear if expression of markers including CD73 and CD90 reflects the in vivo origin of cultured cells. We evaluated expression of 15 putative MSC markers in primary cultured cells from periosteum and cartilage to determine whether expression of these markers reflects either the differentiation state of cultured cells or the self-renewal of in vivo populations. Cultured cells had universal and consistent expression of various putative stem cell markers including > 95% expression CD73, CD90 and PDPN in both periosteal and cartilage cultures. Altering the culture surface with extracellular matrix coatings had minimal effect on cell surface marker expression. Osteogenic differentiation led to loss of CD106 and CD146 expression, however CD73 and CD90 were retained in > 90% of cells. We sorted freshly isolated periosteal populations capable of CFU-F formation on the basis of CD90 expression in combination with CD34, CD73 and CD26. All primary cultures universally expressed CD73 and CD90 and lacked CD34, irrespective of the expression of these markers ex vivo indicating phenotypic convergence in vitro. We conclude that markers including CD73 and CD90 are acquired in vitro in most 'mesenchymal' cells capable of expansion. Overall, we demonstrate that in vitro expression of many cell surface markers in plastic-adherent cultures is unrelated to their expression prior to culture.

Chemical Environment and Structural Variations in High Entropy Oxide Thin Film Probed with Electron Microscopy.

We employ analytical transmission electron microscopy (TEM) to correlate the structural and chemical environment variations within a stacked epitaxial thin film of the high entropy oxide (HEO) Mg0.2Co0.2Ni0.2Cu0.2Zn0.2O (J14), with two layers grown at different substrate temperatures (500 and 200 °C) using pulsed laser deposition (PLD). Electron diffraction and atomically resolved STEM imaging reveal the difference in out-of-plane lattice parameters in the stacked thin film, which is further quantified on a larger scale using four-dimensional STEM (4D-STEM). In the layer deposited at a lower temperature, electron energy loss spectroscopy (EELS) mapping indicates drastic changes in the oxidation states and bonding environment for Co ions, and energy-dispersive X-ray spectroscopy (EDX) mapping detects more significant cation deficiency. Ab initio density functional theory (DFT) calculations validate that vacancies on the cation sublattice of J14 result in significant electronic and structural changes. The experimental and computational analyses indicate that low temperatures during film growth result in cation deficiency, an altered chemical environment, and reduced lattice parameters while maintaining a single phase. Our results demonstrate that the complex correlation of configurational entropy, kinetics, and thermodynamics can be utilized for accessing a range of metastable configurations in HEO materials without altering cation proportions, enabling further engineering of functional properties of HEO materials.

The Arctic marine soundscape of the Amundsen Gulf, Western Canadian Arctic.

The underwater soundscape, a habitat component for Arctic marine mammals, is shifting. We examined the drivers of the underwater soundscape at three sites in the Amundsen Gulf, Northwest Territories, Canada from 2018 to 2019 and estimated the contribution of abiotic and biotic sources between 20 Hz and 24 kHz. Higher wind speeds and the presence of bearded seal (Erignathus barbatus) vocalizations led to increased SPL (0.41 dB/km/h and 3.87 dB, respectively), while higher ice concentration and air temperature led to decreased SPL (-0.39 dB/% and - 0.096 dB/°C, respectively). Other marine mammals did not significantly impact the ambient soundscape. The presence of vessel traffic led to increased SPLs (12.37 dB) but was quieter at distances farther from the recorder (-2.57 dB/log m). The presence of high frequency and broadband signals produced by ice led to increased SPLs (7.60 dB and 10.16 dB, respectively).

Non-TGFß profibrotic signaling in ulcerative colitis after in vivo experimental intestinal injury in humans.

Although impaired regeneration is important in many gastrointestinal diseases including ulcerative colitis (UC), the dynamics of mucosal regeneration in humans are poorly investigated. We have developed a model to study these processes in vivo in humans. Epithelial restitution (ER) and extracellular matrix (ECM) regulation after an experimental injury of the sigmoid colonic mucosa was assessed by repeated high-resolution endoscopic imaging, histological assessment, RNA sequencing, deconvolution analysis, and 16S rDNA sequencing of the injury niche microbiome of 19 patients with UC in remission and 20 control subjects. Human ER had a 48-h lag before induction of regenerative epithelial cells [wound-associated epithelial (WAE) and transit amplifying (TA) cells] along with the increase of fibroblast-derived stem cell growth factor gremlin 1 mRNA (GREM1). However, UC deconvolution data showed rapid induction of inflammatory fibroblasts and upregulation of major structural ECM collagen mRNAs along with tissue inhibitor of metalloproteinase 1 (TIMP1), suggesting increased profibrotic ECM deposition. No change was seen in transforming growth factor ß (TGFß) mRNA, whereas the profibrotic cytokines interleukin 13 (IL13) and IL11 were upregulated in UC, suggesting that human postinjury responses could be TGFß-independent. In conclusion, we found distinct regulatory layers of regeneration in the normal human colon and a potential targetable profibrotic dysregulation in UC that could lead to long-term end-organ failure, i.e., intestinal damage.NEW & NOTEWORTHY The study reveals the regulatory dynamics of epithelial regeneration and extracellular matrix remodeling after experimental injury of the human colon in vivo and shows that human intestinal regeneration is different from data obtained from animals. A lag phase in epithelial restitution is associated with induction of stromal cell-derived epithelial growth factors. Postinjury regeneration is transforming growth factor ß-independent, and we find a profibrotic response in patients with ulcerative colitis despite being in remission.

Advancing the Blueprint to Mental Health Parity Reform.

This Viewpoint discusses the current state of mental health parity reform, proposed amendments, and the challenges and considerations in play.

Manipulation of Gaseous Ions with Acoustic Fields at Atmospheric Pressure.

The ability to controllably move gaseous ions is an essential aspect of ion-based spectrometry (e.g., mass spectrometry and ion mobility spectrometry) as well as materials processing. At higher pressures, ion motion is largely governed by diffusion and multiple collisions with neutral gas molecules. Thus, high-pressure ion optics based on electrostatics require large fields, radio frequency drives, complicated geometries, and/or partially transmissive grids that become contaminated. Here, we demonstrate that low-power standing acoustic waves can be used to guide, block, focus, and separate beams of ions akin to electrostatic ion optics. Ions preferentially travel through the static-pressure regions ("nodes") while neutral gas does not appear to be impacted by the acoustic field structure and continues along a straight trajectory. This acoustic ion manipulation (AIM) approach has broad implications for ion manipulation techniques at high pressure, while expanding our fundamental understanding of the behavior of ions in gases.

ST-2560, a selective inhibitor of the NaV1.7 sodium channel, affects nocifensive and cardiovascular reflexes in non-human primates.

BACKGROUND AND PURPOSE: The voltage-gated sodium channel isoform NaV1.7 is a high-interest target for the development of non-opioid analgesics due to its preferential expression in pain-sensing neurons. NaV1.7 is also expressed in autonomic neurons, yet its contribution to involuntary visceral reflexes has received limited attention. The small molecule inhibitor ST-2560 was advanced into pain behaviour and cardiovascular models to understand the pharmacodynamic effects of selective inhibition of NaV1.7. EXPERIMENTAL APPROACH: Potency of ST-2560 at NaV1.7 and off-target ion channels was evaluated by whole-cell patch-clamp electrophysiology. Effects on nocifensive reflexes were assessed in non-human primate (NHP) behavioural models, employing the chemical capsaicin and mechanical stimuli. Cardiovascular parameters were monitored continuously in freely-moving, telemetered NHPs following administration of vehicle and ST-2560. KEY RESULTS: ST-2560 is a potent inhibitor (IC50 = 39 nM) of NaV1.7 in primates with ≥1000-fold selectivity over other isoforms of the human NaV1.x family. Following systemic administration, ST-2560 (0.1-0.3 mg·kg-1, s.c.) suppressed noxious mechanical- and chemical-evoked reflexes at free plasma concentrations threefold to fivefold above NaV1.7 IC50. ST-2560 (0.1-1.0 mg·kg-1, s.c.) also produced changes in haemodynamic parameters, most notably a 10- to 20-mmHg reduction in systolic and diastolic arterial blood pressure, at similar exposures. CONCLUSIONS AND IMPLICATIONS: Acute pharmacological inhibition of NaV1.7 is antinociceptive, but also has the potential to impact the cardiovascular system. Further work is merited to understand the role of NaV1.7 in autonomic ganglia involved in the control of heart rate and blood pressure, and the effect of selective NaV1.7 inhibition on cardiovascular function.

Diagnostic Yield of Decubitus CT Myelography for Detection of CSF-Venous Fistulas.

BACKGROUND AND PURPOSE: Various imaging techniques have been described to detect CSF-Venous Fistulas (CVFs) in the setting of Spontaneous Intracranial Hypotension (SIH), including decubitus CT myelography (dCTM). The expected diagnostic yield of dCTM for CVF detection is not fully established. The purpose of this study was to assess the yield of dCTM among consecutive patients presenting for evaluation of possible SIH, and to examine what impact brain MRI findings of SIH had on diagnostic yield. MATERIALS AND METHODS: Single-center, retrospective cohort of consecutive patients presenting over a one-year period who underwent CTM and had no CSF identified in the epidural space. Patients with epidural CSF leaks were included in a secondary cohort. Subjects were grouped according to positioning for the myelogram, either decubitus or prone, and the presence of imaging findings of SIH on pre-procedure brain MRI. Diagnostic yields for each subgroup were calculated, and the yield of dCTM was compared to prone CTM. RESULTS: The study cohort included 302 subjects, including 247 patients with no epidural fluid. Diagnostic yield of dCTM for CVF detection among subjects with positive brain MRI and no epidural fluid was 73%. No CVFs were identified among subjects with negative brain imaging. Among subjects with epidural leak, brain MRI was negative for signs of SIH in 22%. Prone CTM identified a CVF less commonly than dCTM (43% vs. 73%, p=0.19), although the difference was not statistically significant in this small subgroup. CONCLUSIONS: We found a diagnostic yield of dCTM to be similar to the yield previously reported for digital subtraction myelography among patients with positive brain imaging. No CVFs were identified in patients with negative brain imaging; epidural CSF leaks accounted for all cases of patients who had SIH with negative brain imaging. This study provides useful data for counseling patients and helps establish a general benchmark for dCTM yield for CVF detection.ABBREVIATIONS: SIH = spontaneous intracranial hypotension; CVF = CSF-Venous Fistula; CTM = CT Myelography; dCTM = decubitus CT myelography; EBP = epidural blood patch.

A Computational and Spectroscopic Analysis of Solvate Ionic Liquids Containing Anions with Long and Short Perfluorinated Alkyl Chains.

Anion-driven, nanoscale polar-apolar structural organization is investigated in a solvate ionic liquid (SIL) setting by comparing sulfonate-based anions with long and short perfluorinated alkyl chains. Representative SILs are created from 1,2-bis(2-methoxyethoxy)ethane ("triglyme" or "G3"), lithium nonafluoro-1-butanesulfonate, and lithium trifluoromethanesulfonate. Molecular dynamics simulations, density functional theory computations, and vibrational spectroscopy provide insight into the overall liquid structure, cation-solvent interactions, and cation-anion association. Significant competition between G3 and anions for cation-binding sites characterizes the G3-LiC4F9SO3 mixtures. Only 50% of coordinating G3 molecules form tetradentate complexes with Li+ in [(G3)1Li][C4F9SO3]. Moreover, the SIL is characterized by extensive amounts of ion pairing. Based on these observations, [(G3)1Li][C4F9SO3] is classified as a "poor" SIL, similar to the analogous [(G3)1Li][CF3SO3] system. Even though the comparable basicity of the CF3SO3- and C4F9SO3- anions leads to similar SIL classifications, the hydrophobic fluorobutyl groups support extensive apolar domain formation. These apolar moieties permeate throughout [(G3)1Li][C4F9SO3] and persist even at relatively low dilution ratios of [(G3)10Li][C4F9SO3]. By way of comparison, the CF3 group is far too short to sustain polar-apolar segregation. This demonstrates how chemically modifying the anions to include hydrophobic groups can impart unique nanoscale organization to a SIL. Moreover, tuning these nano-segregated fluorinated domains could, in principle, control the presence of dimensionally ordered states in these mixtures without changing the coordination of the lithium ions.