T staging esophageal tumors with x rays.

With histopathology results typically taking several days, the ability to stage tumors during interventions could provide a step change in various cancer interventions. X-ray technology has advanced significantly in recent years with the introduction of phase-based imaging methods. These have been adapted for use in standard labs rather than specialized facilities such as synchrotrons, and approaches that enable fast 3D scans with conventional x-ray sources have been developed. This opens the possibility to produce 3D images with enhanced soft tissue contrast at a level of detail comparable to histopathology, in times sufficiently short to be compatible with use during surgical interventions. In this paper we discuss the application of one such approach to human esophagi obtained from esophagectomy interventions. We demonstrate that the image quality is sufficiently high to enable tumor T staging based on the x-ray datasets alone. Alongside detection of involved margins with potentially life-saving implications, staging tumors intra-operatively has the potential to change patient pathways, facilitating optimization of therapeutic interventions during the procedure itself. Besides a prospective intra-operative use, the availability of high-quality 3D images of entire esophageal tumors can support histopathological characterization, from enabling "right slice first time" approaches to understanding the histopathology in the full 3D context of the surrounding tumor environment.

Epicardial Pacing Outcomes in Infants with Heart Block: Lead and Device Complications from a Multicenter Experience.

BACKGROUND: Infants with complete heart block (CHB) require epicardial pacemaker (PM) insertion. Prior studies described epicardial pacing outcomes in infants and children though were limited by small and/or heterogeneous populations. OBJECTIVE: To explore patient and procedural-level associations with device complications in infants with CHB who received a permanent PM. METHODS: Multicenter, retrospective cohort study including infants receiving an epicardial PM between 2000-2021 for CHB. The primary outcome was time to device-related adverse event (DRAE): (1) lead failure requiring revision; (2) pocket infection; (3) exit block requiring increased pacing output; or (4) lead-related coronary artery compression. Time to event analysis was performed using the Kaplan-Meier method with a multivariable Cox proportional hazards model. RESULTS: 174 infants received an epicardial PM (282 bipolar, 39 unipolar leads) for CHB. Median age and weight at PM were 93.5 days and 4.5 kg, respectively. Pacing indication was postoperative CHB in 63% and congenital CHB in 37%. The median follow-up was 2.1 years. The primary outcome occurred in 26 infants at a median time to event of 0.6 years. Age ≤90 days at PM was the most significant risk factor for DRAE (HR 7.02, p<0.001), primarily driven by pocket infections. Lead failure occurred in 3% of leads with a 5- and 10-year freedom from failure of 93% and 83%, respectively. CONCLUSIONS: Device complications affect 15% of infants receiving a permanent PM for heart block. Age ≤90 days at PM implant is especially associated with infectious complications. Epicardial lead durability appears similar to previously reported pediatric experiences.

Serum AZD7442 (tixagevimab-cilgavimab) concentrations and in vitroIC50 values predict SARS-CoV-2 neutralising antibody titres.

Objectives: The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates rapid methods for assessing monoclonal antibody (mAb) potency against emerging variants. Authentic virus neutralisation assays are considered the gold standard for measuring virus-neutralising antibody (nAb) titres in serum. However, authentic virus-based assays pose inherent practical challenges for measuring nAb titres against emerging SARS-CoV-2 variants (e.g. storing infectious viruses and testing at biosafety level-3 facilities). Here, we demonstrate the utility of pseudovirus neutralisation assay data in conjunction with serum mAb concentrations to robustly predict nAb titres in serum. Methods: SARS-CoV-2 nAb titres were determined via authentic- and lentiviral pseudovirus-based neutralisation assays using serological data from three AZD7442 (tixagevimab-cilgavimab) studies: PROVENT (NCT04625725), TACKLE (NCT04723394) and a phase 1 dose-ranging study (NCT04507256). AZD7442 serum concentrations were assessed using immunocapture. Serum-based half-maximal inhibitory concentration (IC50) values were derived from pseudovirus nAb titres and serum mAb concentrations, and compared with in vitro IC50 measurements. Results: nAb titres measured via authentic- and lentiviral pseudovirus-based neutralisation assays were strongly correlated for the ancestral SARS-CoV-2 virus and SARS-CoV-2 Alpha. Serum AZD7442 concentrations and pseudovirus nAb titres were strongly correlated for multiple SARS-CoV-2 variants with all Spearman correlation coefficients ≥ 0.78. Serum-based IC50 values were similar to in vitro IC50 values for AZD7442, for ancestral SARS-CoV-2 and Alpha, Delta, Omicron BA.2 and Omicron BA.4/5 variants. Conclusions: These data highlight that serum mAb concentrations and pseudovirus in vitro IC50 values can be used to rapidly predict nAb titres in serum for emerging and historical SARS-CoV-2 variants.

Increase in methicillin-resistant Staphylococcus spp. colonization among pregnant individuals during COVID-19 pandemic.

Methicillin-resistant Staphylococcus (MRS) has been associated with neonatal infections, with colonization of the anovaginal tract being the main source of vertical transmission. The COVID-19 pandemic has altered the frequency of antibiotic usage, potentially contributing to changes in the dynamics of bacterial agents colonizing humans. Here we determined MRS colonization rates among pregnant individuals attending a single maternity in Rio de Janeiro, Brazil before (January 2019-March 2020) and during (May 2020-March 2021) the COVID-19 pandemic. Anovaginal samples (n = 806 [521 samples before and 285 during the pandemic]) were streaked onto chromogenic media. Colonies were identified by MALDI-TOF MS. Detection of mecA gene and SCCmec typing were assessed by PCR and antimicrobial susceptibility testing was done according to CLSI guidelines. After the onset of the pandemic, MRS colonization rates increased significantly (p < 0.05) from 8.6% (45) to 54.7% (156). Overall, 215 (26.6%) MRS isolates were detected, of which S. haemolyticus was the most prevalent species (MRSH, 84.2%; 181 isolates). SCCmec type V was the most frequent among MRS (63.3%; 136), and 31.6% (68) of MRS strains had a non-typeable SCCmec, due to new combinations of ccr and mecA complexes. Among MRS strains, 41.9% (90) were resistant to at least 3 different classes of antimicrobial agents, and 60% (54) of them were S. haemolyticus harboring SCCmec V. MRS colonization rates and the emergence of multidrug-resistant variants detected in this study indicate the need for continuing surveillance of this important pathogen within maternal and child populations.

Potassium-Alkali-Enriched Diet, Hypertension, and Proteinuria following Uninephrectomy.

BACKGROUND: Losing or donating a kidney is associated with risks of developing hypertension and albuminuria. Few studies address mechanisms or interventions. We investigate potential benefits of a K+- alkali-enriched diet and the mechanisms underlying proteinuria. METHODS: Male Sprague Dawley rats were fed either a 2% NaCl + 0.95% KCl diet (HNa-LK) or a 0.74% NaCl + 3% K+-alkali diet (HK-alk) for 3 wk prior to uninephrectomy then maintained on respective diets for 12 wk. Blood pressure (by tail-cuff), urine, blood and kidney proteins were analyzed Pre- and Post-uninephrectomy. RESULTS: Pre-uninephrectomy, HK-alk vs. HNa-LK fed rats exhibited similar blood pressures and plasma [K+], [Na+], but lower proximal (NHE3, NBCe1, NaPi2) and higher distal (NCC, ENaC, pendrin) transporter abundance, a pattern facilitating K+ and HCO3- secretion. Post-uninephrectomy, single nephron GFR rose 50% and Li+ clearance doubled with both diets; in HK-alk vs HNa-LK: the rise in blood pressure was less and ammoniagenesis was lower, abundance of proximal tubule transporters remained lower, ENaC-α fell and NCCp rose consistent with K+ conservation. Post-uninephrectomy, independent of diet, albuminuria increased 8-fold and abundance of endocytic receptors was reduced (megalin by 44%, dab2 by 25-35%) and KIM-1 was increased. CONCLUSIONS: The K-alkali-enriched diet blunted post-uninephrectomy hypertension and facilitated acid clearance by suppressing proximal Na+ transporters and increasing K+ -alkali secretion. Further, uninephrectomy associated proteinuria could be attributed, at least in part, to elevated SNGFR coupled to downregulation of megalin which reduced fractional protein endocytosis and Vmax.

AZD3152 neutralizes SARS-CoV-2 historical and contemporary variants and is protective in hamsters and well tolerated in adults.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in variants that can escape neutralization by therapeutic antibodies. Here, we describe AZD3152, a SARS-CoV-2-neutralizing monoclonal antibody designed to provide improved potency and coverage against emerging variants. AZD3152 binds to the back left shoulder of the SARS-CoV-2 spike protein receptor binding domain and prevents interaction with the human angiotensin-converting enzyme 2 receptor. AZD3152 potently neutralized a broad panel of pseudovirus variants, including the currently dominant Omicron variant JN.1 but has reduced potency against XBB subvariants containing F456L. In vitro studies confirmed F456L resistance and additionally identified T415I and K458E as escape mutations. In a Syrian hamster challenge model, prophylactic administration of AZD3152 protected hamsters from weight loss and inflammation-related lung pathologies and reduced lung viral load. In the phase 1 sentinel safety cohort of the ongoing SUPERNOVA study (ClinicalTrials.gov: NCT05648110), a single 600-mg intramuscular injection of AZD5156 (containing 300 mg each of AZD3152 and cilgavimab) was well tolerated in adults through day 91. Observed serum concentrations of AZD3152 through day 91 were similar to those observed with cilgavimab and consistent with predictions for AZD7442, a SARS-CoV-2-neutralizing antibody combination of cilgavimab and tixagevimab, in a population pharmacokinetic model. On the basis of its pharmacokinetic characteristics, AZD3152 is predicted to provide durable protection against symptomatic coronavirus disease 2019 caused by susceptible SARS-CoV-2 variants, such as JN.1, in humans.

Extracellular Microenvironment Alterations in Ductal Carcinoma In Situ and Invasive Breast Cancer Pathologies by Multiplexed Spatial Proteomics.

Ductal carcinoma in situ (DCIS) is a heterogeneous breast disease that remains challenging to treat due to its unpredictable progression to invasive breast cancer (IBC). Contemporary literature has become increasingly focused on extracellular matrix (ECM) alterations with breast cancer progression. However, the spatial regulation of the ECM proteome in DCIS has yet to be investigated in relation to IBC. We hypothesized that DCIS and IBC present distinct ECM proteomes that could discriminate between these pathologies. Tissue sections of pure DCIS, mixed DCIS-IBC, or pure IBC (n = 22) with detailed pathological annotations were investigated by multiplexed spatial proteomics. Across tissues, 1,005 ECM peptides were detected in pathologically annotated regions and their surrounding extracellular microenvironments. A comparison of DCIS to IBC pathologies demonstrated 43 significantly altered ECM peptides. Notably, eight fibrillar collagen peptides could distinguish with high specificity and sensitivity between DCIS and IBC. Lesion-targeted proteomic imaging revealed heterogeneity of the ECM proteome surrounding individual DCIS lesions. Multiplexed spatial proteomics reported an invasive cancer field effect, in which DCIS lesions in closer proximity to IBC shared a more similar ECM profile to IBC than distal counterparts. Defining the ECM proteomic microenvironment provides novel molecular insights relating to DCIS and IBC.

Single Cell Analysis Reveals Novel Immune Perturbations in Fibrotic Hypersensitivity Pneumonitis.

Rationale: Fibrotic hypersensitivity pneumonitis is a debilitating interstitial lung disease driven by incompletely understood immune mechanisms. Objectives: To elucidate immune aberrations in fibrotic hypersensitivity pneumonitis in single-cell resolution. Methods: Single-cell 5' RNA sequencing was conducted on peripheral blood mononuclear cells and bronchoalveolar lavage cells obtained from 45 patients with fibrotic hypersensitivity pneumonitis, 63 idiopathic pulmonary fibrosis, 4 non-fibrotic hypersensitivity pneumonitis, and 36 healthy controls in the United States and Mexico. Analyses included differential gene expression (Seurat), transcription factor activity imputation (DoRothEA-VIPER), and trajectory analyses (Monocle3/Velocyto-scVelo-CellRank). Measurements and Main Results: Overall, 501,534 peripheral blood mononuclear cells from 110 patients and controls and 88,336 bronchoalveolar lavage cells from 19 patients were profiled. Compared to controls, fibrotic hypersensitivity pneumonitis has elevated classical monocytes (adjusted-p=2.5e-3) and are enriched in CCL3hi/CCL4hi and S100Ahi classical monocytes (adjusted-p<2.2e-16). Trajectory analyses demonstrate that S100Ahi classical monocytes differentiate into SPP1hi lung macrophages associated with fibrosis. Compared to both controls and idiopathic pulmonary fibrosis, fibrotic hypersensitivity pneumonitis patient cells are significantly enriched in GZMhi cytotoxic T cells. These cells exhibit transcription factor activities indicative of TGFß and TNFα/NFκB pathways. These results are publicly available at https://ildimmunecellatlas.org. Conclusions: Single-cell transcriptomics of fibrotic hypersensitivity pneumonitis patients uncovered novel immune perturbations, including previously undescribed increases in GZMhi cytotoxic CD4+ and CD8+ T cells - reflecting this disease's unique inflammatory T-cell driven nature - as well as increased S100Ahi and CCL3hi/CCL4hi classical monocytes also observed in idiopathic pulmonary fibrosis. Both cell populations may guide the development of new biomarkers and therapeutic interventions.

Noninvasive assessment of the lung inflammation-fibrosis axis by targeted imaging of CMKLR1.

Precision management of fibrotic lung diseases is challenging due to their diverse clinical trajectories and lack of reliable biomarkers for risk stratification and therapeutic monitoring. Here, we validated the accuracy of CMKLR1 as an imaging biomarker of the lung inflammation-fibrosis axis. By analyzing single-cell RNA sequencing datasets, we demonstrated CMKLR1 expression as a transient signature of monocyte-derived macrophages (MDMφ) enriched in patients with idiopathic pulmonary fibrosis (IPF). Consistently, we identified MDMφ as the major driver of the uptake of CMKLR1-targeting peptides in a murine model of bleomycin-induced lung fibrosis. Furthermore, CMKLR1-targeted positron emission tomography in the murine model enabled quantification and spatial mapping of inflamed lung regions infiltrated by CMKLR1-expressing macrophages and emerged as a robust predictor of subsequent lung fibrosis. Last, high CMKLR1 expression by bronchoalveolar lavage cells identified an inflammatory endotype of IPF with poor survival. Our investigation supports the potential of CMKLR1 as an imaging biomarker for endotyping and risk stratification of fibrotic lung diseases.

Understanding the processes underpinning IMPlementing IMProved Asthma self-management as RouTine (IMP2ART) in primary care: study protocol for a process evaluation within a cluster randomised controlled implementation trial.

BACKGROUND: Providing supported self-management for people with asthma can reduce the burden on patients, health services and wider society. Implementation, however, remains poor in routine clinical practice. IMPlementing IMProved Asthma self-management as RouTine (IMP2ART) is a UK-wide cluster randomised implementation trial that aims to test the impact of a whole-systems implementation strategy, embedding supported asthma self-management in primary care compared with usual care. To maximise opportunities for sustainable implementation beyond the trial, it is necessary to understand how and why the IMP2ART trial achieved its clinical and implementation outcomes. METHODS: A mixed-methods process evaluation nested within the IMP2ART trial will be undertaken to understand how supported self-management was implemented (or not) by primary care practices, to aid interpretation of trial findings and to inform scaling up and sustainability. Data and analysis strategies have been informed by mid-range and programme-level theory. Quantitative data will be collected across all practices to describe practice context, IMP2ART delivery (including fidelity and adaption) and practice response. Case studies undertaken in three to six sites, supplemented by additional interviews with practice staff and stakeholders, will be undertaken to gain an in-depth understanding of the interaction of practice context, delivery, and response. Synthesis, informed by theory, will combine analyses of both qualitative and quantitative data. Finally, implications for the scale up of asthma self-management implementation strategies to other practices in the UK will be explored through workshops with stakeholders. DISCUSSION: This mixed-methods, theoretically informed, process evaluation seeks to provide insights into the delivery and response to a whole-systems approach to the implementation of supported self-management in asthma care in primary care. It is underway at a time of significant change in primary care in the UK. The methods have, therefore, been developed to be adaptable to this changing context and to capture the impact of these changes on the delivery and response to research and implementation processes.

Talking with clinicians about online cancer information: a survey of cancer patients and surrogate information seekers.

OBJECTIVES: To describe patients' and surrogate information seekers' experiences talking to clinicians about online cancer information. To assess the impact of clinicians telling patients or surrogate seekers not to search for information online. DESIGN: Cross-sectional survey. SAMPLE: A total of 282 participants, including 185 individuals with cancer and 97 surrogate seekers. METHODS: Individuals were recruited through a broad consent registry and completed a 20-min survey. FINDINGS: Cancer patients and surrogate seekers did not differ significantly in their experiences talking with clinicians about online cancer information. Nearly all patients and surrogate seekers who were told by a clinician not to go online for cancer information did so anyway. IMPLICATIONS: Interventions for improving cancer information seeking and communication with clinicians should target both patients and surrogate seekers. Clinicians should be educated about effective ways to communicate with patients and surrogate seekers about online cancer information.

SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

Pilot Study to Optimize Goal-directed Hemodynamic Management During Pancreatectomy.

INTRODUCTION: Intraoperative goal-directed hemodynamic therapy (GDHT) is a cornerstone of enhanced recovery protocols. We hypothesized that use of an advanced noninvasive intraoperative hemodynamic monitoring system to guide GDHT may decrease intraoperative hypotension (IOH) and improve perfusion during pancreatic resection. METHODS: The monitor uses machine learning to produce the Hypotension Prediction Index to predict hypotensive episodes. A clinical decision-making algorithm uses the Hypotension Prediction Index and hemodynamic data to guide intraoperative fluid versus pressor management. Pre-implementation (PRE), patients were placed on the monitor and managed per usual. Post-implementation (POST), anesthesia teams were educated on the algorithm and asked to use the GDHT guidelines. Hemodynamic data points were collected every 20 s (8942 PRE and 26,638 POST measurements). We compared IOH (mean arterial pressure <65 mmHg), cardiac index >2, and stroke volume variation <12 between the two groups. RESULTS: 10 patients were in the PRE and 24 in the POST groups. In the POST group, there were fewer minimally invasive resections (4.2% versus 30.0%, P = 0.07), more pancreaticoduodenectomies (75.0% versus 20.0%, P < 0.01), and longer operative times (329.0 + 108.2 min versus 225.1 + 92.8 min, P = 0.01). After implementation, hemodynamic parameters improved. There was a 33.3% reduction in IOH (5.2% ± 0.1% versus 7.8% ± 0.3%, P < 0.01, a 31.6% increase in cardiac index >2.0 (83.7% + 0.2% versus 63.6% + 0.5%, P < 0.01), and a 37.6% increase in stroke volume variation <12 (73.2% + 0.3% versus 53.2% + 0.5%, P < 0.01). CONCLUSIONS: Advanced intraoperative hemodynamic monitoring to predict IOH combined with a clinical decision-making tree for GDHT may improve intraoperative hemodynamic parameters during pancreatectomy. This warrants further investigation in larger studies.

The Antiretroviral Pregnancy Registry: Three decades of prospective monitoring for birth defects.

PURPOSE: Antiretrovirals (ARVs) are life-saving drugs used for the treatment and prevention of HIV infection and antiviral drugs (AVs) for the treatment of chronic HBV infection. ARVs have proven highly effective in reducing perinatal HIV transmission, however the risk of birth defects from prenatal exposure to ARVs/AVs is an ongoing concern. The Antiretroviral Pregnancy Registry (APR), an international, prospective exposure-registration cohort study, monitors ARV and AV use in pregnancy for early signals of teratogenicity. This communication reports results of 30-years' experience of ARV/AV exposure during pregnancy and lessons learned through continuous quality improvement. METHODS AND RESULTS: Birth defect prevalence is estimated and compared to internal and external groups. Statistical inference is based on exact methods for binomial proportions. Between 2006 and 2023, cumulative enrollment more than tripled from 6893 to 25 960 pregnancies and ARVs/AVs monitored increased from 29 to 222. Through January 2023, there were 21 636 live births and 631 outcomes with birth defects, for overall prevalence of 2.9/100 live births (95% CI 2.7, 3.2). The birth defect prevalence was 3.0% (95% CI 2.7%, 3.3%) among first trimester exposures and 2.8% (95% CI 2.5%, 3.2%) among second/third trimester exposures (prevalence ratio 1.04 [95% CI 0.89, 1.21]). CONCLUSIONS: Birth defect prevalence is not statistically significantly different between first trimester ARV/AV pregnancy exposures compared to second/third trimester exposures and is also not different from two population-based surveillance systems: 2.72/100 live births reported in the Metropolitan Atlanta Congenital Defects Program (MACDP); and 4.17/100 live births from the Texas Birth Defects Registry (TBDR).

Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase.

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

Asymptomatic neonatal herpes simplex virus infection in mice leads to long-term cognitive impairment.

Neonatal herpes simplex virus (nHSV) is a devastating infection impacting approximately 14,000 newborns globally each year. Infection is associated with high neurologic morbidity and mortality, making early intervention and treatment critical. Clinical outcomes of symptomatic nHSV infections are well-studied, but little is known about the frequency of, or outcomes following, sub-clinical or asymptomatic nHSV. Given the ubiquitous nature of HSV infection and frequency of asymptomatic shedding in adults, subclinical infections are underreported, yet could contribute to long-term neurological damage. To assess potential neurological morbidity associated with subclinical nHSV infection, we developed a low-dose (100 PFU) HSV infection protocol in neonatal C57BL/6 mice. At this dose, HSV DNA was detected in the brain by PCR but was not associated with acute clinical symptoms. However, months after initial inoculation with 100 PFU of HSV, we observed impaired mouse performance on a range of cognitive and memory performance tasks. Memory impairment was induced by infection with either HSV-1 or HSV-2 wild-type viruses, but not by a viral mutant lacking the autophagy-modulating Beclin-binding domain of the neurovirulence gene γ34.5. Retroviral expression of wild type γ34.5 gene led to behavioral pathology in mice, suggesting that γ34.5 expression may be sufficient to cause cognitive impairment. Maternal immunization and HSV-specific antibody treatment prevented offspring from developing neurological sequelae following nHSV-1 infection. Altogether, these results support the idea that subclinical neonatal infections may lead to cognitive decline in adulthood, with possible profound implications for research on human neurodegenerative disorders such as Alzheimer's Disease.

Unlocking the epigenome: Stress and exercise induced Bdnf regulation in the prefrontal cortex.

Aversive caregiving in early life is a risk factor for aberrant brain and behavioral development. This outcome is related to epigenetic dysregulation of the brain-derived neurotrophic factor (Bdnf) gene. The Bdnf gene encodes for BDNF, a neurotrophin involved in early brain development, neural plasticity, learning, and memory. Recent work suggests that exercise may be neuroprotective in part by supporting BDNF protein and gene expression, making it an exciting target for therapeutic interventions. To our knowledge, exercise has never been studied as a therapeutic intervention in preclinical rodent models of caregiver maltreatment. To that end, the current study investigated the effect of an adult voluntary wheel running intervention on Bdnf methylation and expression in the prefrontal cortex of rats who experienced aversive caregiving in infancy. We employed a rodent model (Long Evans rats) wherein rat pups experienced intermittent caregiver-induced stress from postnatal days 1-7 and were given voluntary access to a running wheel (except in the control condition) from postnatal days 70-90 as a young adulthood treatment intervention. Our results indicate that maltreatment and exercise affect Bdnf gene methylation in an exon, CG site, and sex-specific manner. Here we add to a growing body of evidence of the ability for our experiences, including exercise, to permeate the brain. Keywords: Early life stress, Bdnf, exercise, prefrontal cortex.

Multifunctional host-defense peptides isolated from skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae).

Five host-defense peptides (figainin 2PL, hylin PL, raniseptin PL, plasticin PL, and peptide YL) were isolated from norepinephrine-stimulated skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae) collected in Trinidad. Raniseptin PL (GVFDTVKKIGKAVGKFALGVAKNYLNS.NH2) and figainin 2PL (FLGTVLKLGKAIAKTVVPMLTNAMQPKQ. NH2) showed potent and rapid bactericidal activity against a range of clinically relevant Gram-positive and Gram-negative ESKAPE + pathogens and Clostridioides difficile. The peptides also showed potent cytotoxic activity (LC50 values < 30 µM) against A549, MDA-MB-231 and HT29 human tumor-derived cell lines but appreciably lower hemolytic activity against mouse erythrocytes (LC50 = 262 ± 14 µM for raniseptin PL and 157 ± 16 µM for figainin 2PL). Hylin PL (FLGLIPALAGAIGNLIK.NH2) showed relatively weak activity against microorganisms but was more hemolytic. The glycine-leucine-rich peptide with structural similarity to the plasticins (GLLSTVGGLVGGLLNNLGL.NH2) and the non-cytotoxic peptide YL (YVPGVIESLL.NH2) lacked antimicrobial and cytotoxic activities. Hylin PL, raniseptinPL and peptide YL stimulated the rate of release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥100 nM. Peptide YL was the most effective (2.3-fold increase compared with basal rate at 1 µM concentration) and may represent a template for the design of a new class of incretin-based anti-diabetic drugs.

Dendritic Cell - Fibroblast Crosstalk via TLR9 and AHR Signaling Drives Lung Fibrogenesis.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive scarring and loss of lung function. With limited treatment options, patients succumb to the disease within 2-5 years. The molecular pathogenesis of IPF regarding the immunologic changes that occur is poorly understood. We characterize a role for non-canonical aryl-hydrocarbon receptor signaling (ncAHR) in dendritic cells (DCs) that leads to production of IL-6 and IL-17, promoting fibrosis. TLR9 signaling in myofibroblasts is shown to regulate production of TDO2 which converts tryptophan into the endogenous AHR ligand kynurenine. Mice with augmented ncAHR signaling were created by crossing floxed AHR exon-2 deletion mice (AHR Δex2 ) with mice harboring a CD11c-Cre. Bleomycin was used to study fibrotic pathogenesis. Isolated CD11c+ cells and primary fibroblasts were treated ex-vivo with relevant TLR agonists and AHR modulating compounds to study how AHR signaling influenced inflammatory cytokine production. Human datasets were also interrogated. Inhibition of all AHR signaling rescued fibrosis, however, AHR Δex2 mice treated with bleomycin developed more fibrosis and DCs from these mice were hyperinflammatory and profibrotic upon adoptive transfer. Treatment of fibrotic fibroblasts with TLR9 agonist increased expression of TDO2. Study of human samples corroborate the relevance of these findings in IPF patients. We also, for the first time, identify that AHR exon-2 floxed mice retain capacity for ncAHR signaling.

Dedifferentiated early postnatal lung myofibroblasts redifferentiate in adult disease.

Alveolarization ensures sufficient lung surface area for gas exchange, and during bulk alveolarization in mice (postnatal day [P] 4.5-14.5), alpha-smooth muscle actin (SMA)+ myofibroblasts accumulate, secrete elastin, and lay down alveolar septum. Herein, we delineate the dynamics of the lineage of early postnatal SMA+ myofibroblasts during and after bulk alveolarization and in response to lung injury. SMA+ lung myofibroblasts first appear at ∼ P2.5 and proliferate robustly. Lineage tracing shows that, at P14.5 and over the next few days, the vast majority of SMA+ myofibroblasts downregulate smooth muscle cell markers and undergo apoptosis. Of note, ∼8% of these dedifferentiated cells and another ∼1% of SMA+ myofibroblasts persist to adulthood. Single cell RNA sequencing analysis of the persistent SMA- cells and SMA+ myofibroblasts in the adult lung reveals distinct gene expression profiles. For instance, dedifferentiated SMA- cells exhibit higher levels of tissue remodeling genes. Most interestingly, these dedifferentiated early postnatal myofibroblasts re-express SMA upon exposure of the adult lung to hypoxia or the pro-fibrotic drug bleomycin. However, unlike during alveolarization, these cells that re-express SMA do not proliferate with hypoxia. In sum, dedifferentiated early postnatal myofibroblasts are a previously undescribed cell type in the adult lung and redifferentiate in response to injury.