SARS-CoV-2 infection perturbs the gastrointestinal tract and induces modest microbial translocation across the intestinal barrier.

SARS-CoV-2 infects via the respiratory tract, but COVID-19 includes an array of non-respiratory symptoms, among them gastrointestinal (GI) manifestations such as vomiting and diarrhea. Here we investigated the GI pathology of SARS-CoV-2 infections in rhesus macaques and humans. Macaques experienced mild infection with USA-WA1/2020 and shed viral RNA in the respiratory tract and stool, including subgenomic RNA indicative of replication in the GI tract. Intestinal immune cell populations were disturbed, with significantly fewer proliferating (Ki67+) jejunal B cells in SARS-CoV-2-infected macaques than uninfected ones. Modest translocation of bacteria/bacterial antigen was observed across the colonic epithelium, with a corresponding significant increase in plasma soluble CD14 (sCD14) that may be induced by LPS. Human plasma demonstrated significant decreases in interleukin (IL)-6 and sCD14 upon recovery from COVID-19, suggesting resolution of inflammation and response to translocated bacteria. sCD14 significantly positively correlated with zonulin, an indicator of gut barrier integrity, and IL-6. These results demonstrate that GI perturbations such as microbial translocation can occur in even mild SARS-CoV-2 infections and may contribute to the COVID-19 inflammatory state.IMPORTANCEThis study investigates gastrointestinal (GI) barrier disruption in SARS-CoV-2 infections and how it may contribute to disease. We observed bacteria or bacterial products crossing from the colon interior (the lumen) to the lamina propria during SARS-CoV-2 infection in macaques. Bacteria/bacterial products are tolerated in the lumen but may induce immune responses if they translocate to the lamina propria. We also observed a significant increase in soluble CD14, which is associated with an immune response to bacterial products. In addition, we observed that humans recovering from COVID-19 experienced a significant decrease in soluble CD14, as well as the inflammatory marker interleukin (IL)-6. IL-6 and sCD14 correlated significantly across macaque and human samples. These findings suggest that SARS-CoV-2 infection results in GI barrier disruption that permits microbial translocation and a corresponding immune response. These findings could aid in developing interventions to improve COVID-19 patient outcomes.

Cooperative synaptic and intrinsic plasticity in a disynaptic limbic circuit drive stress-induced anhedonia and passive coping in mice.

Stress promotes negative affective states, which include anhedonia and passive coping. While these features are in part mediated by neuroadaptations in brain reward circuitry, a comprehensive framework of how stress-induced negative affect may be encoded within key nodes of this circuit is lacking. Here, we show in a mouse model for stress-induced anhedonia and passive coping that these phenomena are associated with increased synaptic strength of ventral hippocampus (VH) excitatory synapses onto D1 medium spiny neurons (D1-MSNs) in the nucleus accumbens medial shell (NAcmSh), and with lateral hypothalamus (LH)-projecting D1-MSN hyperexcitability mediated by decreased inwardly rectifying potassium channel (IRK) function. Stress-induced negative affective states are prevented by depotentiation of VH to NAcmSh synapses, restoring Kir2.1 function in D1R-MSNs, or disrupting co-participation of these synaptic and intrinsic adaptations in D1-MSNs. In conclusion, our data provide strong evidence for a disynaptic pathway controlling maladaptive emotional behavior.

Development of a patient specific cartilage graft using magnetic resonance imaging and 3D printing.

OBJECTIVES: The goal of this project was to develop and validate a patient-specific, anatomically correct graft for cartilage restoration using magnetic resonance imaging (MRI) data and 3-dimensional (3D) printing technology. The specific aim was to test the accuracy of a novel method for 3D printing and implanting individualized, anatomically shaped bio-scaffolds to treat cartilage defects in a human cadaveric model. We hypothesized that an individualized, anatomic 3D-printed scaffold designed from MRI data would provide a more optimal fill for a large cartilage defect compared to a generic flat scaffold. METHODS: Four focal cartilage defects (FCDs) were created in paired human cadaver knees, age <40 years, in the weight-bearing surfaces of the medial femoral condyle (MFC), lateral femoral condyle (LFC), patella, and trochlea of each knee. MRIs were obtained, anatomic grafts were designed and 3D printed for the left knee as an experimental group, and generic flat grafts for the right knee as a control group. Grafts were implanted into corresponding defects and fixed using tissue adhesive. Repeat post-implant MRIs were obtained. Graft step-off was measured as the distance in mm between the surface of the graft and the native cartilage surface in a direction perpendicular to the subchondral bone. Graft contour was measured as the gap between the undersurface of the graft and the subchondral bone in a direction perpendicular to the joint surface. RESULTS: Graft step-off was statistically significantly better for the anatomic grafts compared to the generic grafts in the MFC (0.0 â€‹± â€‹0.2 â€‹mm vs. 0.7 â€‹± â€‹0.5 â€‹mm, p â€‹< â€‹0.001), LFC (0.1 â€‹± â€‹0.3 â€‹mm vs. 1.0 â€‹± â€‹0.2 â€‹mm, p â€‹< â€‹0.001), patella (-0.2 â€‹± â€‹0.3 â€‹mm vs. -1.2 â€‹± â€‹0.4 â€‹mm, p â€‹< â€‹0.001), and trochlea (-0.4 â€‹± â€‹0.3 vs. 0.4 â€‹± â€‹0.7, p â€‹= â€‹0.003). Graft contour was statistically significantly better for the anatomic grafts in the LFC (0.0 â€‹± â€‹0.0 â€‹mm vs. 0.2 â€‹± â€‹0.4 â€‹mm, p â€‹= â€‹0.022) and trochlea (0.0 â€‹± â€‹0.0 â€‹mm vs. 1.4 â€‹± â€‹0.7 â€‹mm, p â€‹< â€‹0.001). The anatomic grafts had an observed maximum step-off of -0.9 â€‹mm and a maximum contour mismatch of 0.8 â€‹mm. CONCLUSION: This study validates a process designed to fabricate anatomically accurate cartilage grafts using MRI and 3D printing technology. Anatomic grafts demonstrated superior fit compared to generic flat grafts. LEVEL OF EVIDENCE: Level IV.

Pdgfrß+ lineage cells transiently increase at the site of Achilles tendon healing.

The purpose of this study was to track platelet-derived growth factor receptor-ß (Pdgfr-ß) lineage cells at the site of Achilles tendon injury over time. Pdgfr-ß-CreERT2 :Ai9 mice were generated to track Pdgfr-ß lineage cells in adult mice. A surgical Achilles transection injury model was employed to examine the presence of Pdgfr-ß lineage cells in the healing tendon over time, with five mice per time point at 3, 7, 14, 28, and 56 days postoperatively. Histology and immunohistochemistry for tdTomato (Pdgfr-ß lineage cells), PCNA (proliferating cell nuclear antigen, cell proliferation), and α-SMA (α-smooth muscle actin, myofibroblasts) were performed. The percentage of cells at the healing tendon site staining positive for tdTomato and PCNA were quantified. Over 75% of cells at the injury site were Pdgfr-ß lineage cells at Days 3, 7, and 14, and this percentage decreased significantly by Days 28 and 56 postinjury. Cell proliferation at the injury site peaked on Day 7 and decreased thereafter. Immunohistochemistry for α-SMA demonstrated minimal colocalization of myofibroblasts with Pdgfr-ß lineage cells. This study demonstrates that in a mouse model of Achilles tendon injury, Pdgfr-ß lineage cells' presence at the injury site is transient. Thus, we conclude that they are unlikely to be the cells that differentiate into myofibroblasts and directly contribute to tendon fibrous scar formation. Clinical Significance: This study provides some insight into the presence of Pdgfr-ß lineage cells (including pericytes) following Achilles injury, furthering our understanding of tendon healing.

Pulmonary Co-Infections Detected Premortem Underestimate Postmortem Findings in a COVID-19 Autopsy Case Series.

Bacterial and fungal co-infections are reported complications of coronavirus disease 2019 (COVID-19) in critically ill patients but may go unrecognized premortem due to diagnostic limitations. We compared the premortem with the postmortem detection of pulmonary co-infections in 55 fatal COVID-19 cases from March 2020 to March 2021. The concordance in the premortem versus the postmortem diagnoses and the pathogen identification were evaluated. Premortem pulmonary co-infections were extracted from medical charts while applying standard diagnostic definitions. Postmortem co-infection was defined by compatible lung histopathology with or without the detection of an organism in tissue by bacterial or fungal staining, or polymerase chain reaction (PCR) with broad-range bacterial and fungal primers. Pulmonary co-infection was detected premortem in significantly fewer cases (15/55, 27%) than were detected postmortem (36/55, 65%; p < 0.0001). Among cases in which co-infection was detected postmortem by histopathology, an organism was identified in 27/36 (75%) of cases. Pseudomonas, Enterobacterales, and Staphylococcus aureus were the most frequently identified bacteria both premortem and postmortem. Invasive pulmonary fungal infection was detected in five cases postmortem, but in no cases premortem. According to the univariate analyses, the patients with undiagnosed pulmonary co-infection had significantly shorter hospital (p = 0.0012) and intensive care unit (p = 0.0006) stays and significantly fewer extra-pulmonary infections (p = 0.0021). Bacterial and fungal pulmonary co-infection are under-recognized complications in critically ill patients with COVID-19.

Rehabilitation Principles to Consider for Anterior Cruciate Ligament Repair.

CONTEXT: Injury to the anterior cruciate ligament (ACL) is among the most common orthopaedic injuries, and reconstruction of a ruptured ACL is a common orthopaedic procedure. In general, surgical intervention is necessary to restore stability to the injured knee, and to prevent meniscal damage. Along with surgery, intense postoperative physical therapy is needed to restore function to the injured extremity. ACL reconstruction (ACLR) has been the standard of care in recent decades, and advances in surgical technology have reintroduced the prospect of augmented primary repair of the native ACL via a variety of methods. EVIDENCE ACQUISITION: A search of PubMed database of articles and reviews available in English was performed through 2020. The search terms ACLR, anterior cruciate ligament repair, bridge enhanced acl repair, suture anchor repair, dynamic intraligamentary stabilization, internal bracing, suture ligament augmentation, and internal brace ligament augmentation were used. STUDY DESIGN: Clinical review. LEVEL OF EVIDENCE: Level 5. RESULTS: No exact consensus exists on effective rehabilitation protocols after ACL repair techniques, as the variation in published protocols seem even greater than the variation in those for ACLR. For some techniques such as internal bracing and dynamic interligamentary stabilization, it is likely permissible for the patients to progress to full weightbearing and discontinue bracing sooner. However, caution should be applied with regard to earlier return to sport than after ACLR as to minimize risk for retear. CONCLUSION: More research is needed to address how physical therapies must adapt to these innovative repair techniques. Until that is accomplished, we recommend that physical therapists understand the differences among the various ACL surgery techniques discussed here and work with the surgeons to develop a rehabilitation protocol for their mutual patients. STRENGTH OF RECOMMENDATION TAXONOMY (SORT): C.

Alignment Between Resident Medical Condition Regard Scale Scores and Desire to Treat Patients With Opioid Use Disorder.

OBJECTIVES: To (1) evaluate attitudes of resident physicians towards patients with opioid use disorder (OUD) and (2) identify characteristics associated with residents' desire to treat patients with OUD. METHODS: We administered the validated medical condition regard scale (MCRS), a question regarding desire to treat patients with OUD, and a demographic questionnaire to residents in multiple specialties at the University of New Mexico (family medicine, psychiatry, emergency medicine, internal medicine, anesthesiology, general surgery, obstetrics/gynecology). RESULTS: One hundred sixty-three of 307 residents (53%) responded to the survey; 146 provided complete responses to the "desire" and MCRS questions. Response rates, MCRS, and desire to care for patients with OUD varied between specialties ( P < 0.001); family medicine had highest MCRS and desire to care scores; surgery, anesthesiology had low scores. MCRS and resident "desire" scores were highly correlated on univariate analysis ( r = 0.73, P < 0.001); resident demographics were not. On logistic regression, resident desire to care for OUD increased with MCRS scores ( P < 0.001). The predicated probability of desire to care for OUD was ≥80% with MCRS >57; MCRS classification skill on receiver operator curve analysis was excellent (area under curve = 0.81 [95% confidence interval 0.74, 0.88], and specialty-adjusted MCRS area under curve = 0.85 [95% confidence interval 0.79, 0.91]). CONCLUSIONS: High resident regard for patients with OUD on MCRS was directly related to resident's desire to provide OUD care. MCRS may offer a tool to alter or individualize OUD education, potentially influencing the OUD workforce of the future.

Hip Fracture With Elevated Troponin: Harbinger of Mortality or Need for Accelerated Surgery?

OBJECTIVE: To determine the association of elevated troponin levels with time to surgery and the risk of mortality and other key clinical outcomes among elderly patients with hip fracture who had measured troponin levels at hospital admission. DESIGN: Retrospective cohort study. SETTING: Single academic trauma center. PATIENTS: We included 299 consecutive patients 60 years of age or older with a hip fracture and cardiac troponin levels measured at the time of hospital admission. INTERVENTION: Patients with elevated cardiac troponin levels at hospital admission (n = 43) compared with patients with normal troponin levels at admission (n = 256). MAIN OUTCOME MEASURES: Time to surgery, 90-day mortality, and major complications within 90 days of injury. RESULTS: The median age of the cohort was 80 years (interquartile range, 70-87 years), 59% were female, and 86% were living independently before their injury. Elevated troponin levels were associated with a 21-hour [95% confidence interval (CI), 12 to 32, P < 0.001] increase in the median time from admission to surgery (43 vs. 22 hours). Elevated troponin levels were also associated with a 14% (95% CI, 0% to 29%, P = 0.01) absolute increase in 90-day mortality (28% vs. 14%). Patients with elevated troponins were 15% (95% CI, -1% to 30%, P = 0.06) more likely to have a major complication (37% vs. 23%); however, the difference did not reach statistical significance. CONCLUSIONS: Among patients with a hip fracture and measured troponin levels, elevated troponin levels were associated with significant delays in surgery and increased 90-day mortality. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

SARS-CoV-2 mRNA vaccine induced higher antibody affinity and IgG titers against variants of concern in post-partum vs non-post-partum women.

BACKGROUND: Limited knowledge exists in post-partum women regarding durability of SARS-CoV-2 vaccine-induced antibody responses and their neutralising ability against SARS-CoV-2 variants of concern (VOC). METHODS: We elucidated longitudinal mRNA vaccination-induced antibody profiles of 13 post-partum and 13 non-post-partum women (control). FINDINGS: The antibody neutralisation titres against SARS-CoV-2 WA-1 strain were comparable between post-partum and non-post-partum women and these levels were sustained up to four months post-second vaccination in both groups. However, neutralisation titers declined against several VOCs, including Beta and Delta. Higher antibody binding was observed against SARS-CoV-2 receptor-binding domain (RBD) mutants with key VOC amino acids when tested with post-second vaccination plasma from post-partum women compared with controls. Importantly, post-vaccination plasma antibody affinity against VOCs RBDs was significantly higher in post-partum women compared with controls. INTERPRETATION: This study demonstrates that there is a differential vaccination-induced immune responses in post-partum women compared with non-post-partum women, which could help inform future vaccination strategies for these groups. FUNDING: The antibody characterisation work described in this manuscript was supported by FDA's Medical Countermeasures Initiative (MCMi) grant #OCET 2021-1565 to S.K and intramural FDA-CBER COVID-19 supplemental funds.

Perspectives on pharmacy access to hormonal contraception among rural New Mexico women.

OBJECTIVE: In 2017, New Mexico approved an amendment allowing pharmacists to prescribe and dispense hormonal contraception. We interviewed rural New Mexico women to determine their perceptions of pharmacy access to hormonal contraception. STUDY DESIGN: We conducted semi-structured telephone interviews with women recruited from rural New Mexico communities. The interview guide explained the amendment followed by questions about the advantages and disadvantages of pharmacy access to hormonal contraception within rural communities. RESULTS: Between November 2017 and May 2018, we recruited 32 women to participate. Participants were young (26/32 18-29 years old), gravid (27/31), employed (30/32), white (22/32) and Hispanic (26/31). The majority used Medicaid as their primary insurance (16/28). Most participants were supportive of pharmacy access to hormonal contraception. Participants saw their rural communities as facing health care barriers, some of which could be alleviated by pharmacy access. Perceived benefits of pharmacy access included convenience of pharmacy hours, shorter wait times, and no need for an appointment. Participants expressed concerns about lack of privacy in their pharmacies. Many expressed trust in their pharmacist to review side effects and explain usage of contraception- a role that was considered separate from that of a primary care provider who offers regular medical visits for routine screening and nuanced or complex discussions about contraception. Some participants expressed that pharmacy access could be especially beneficial for teens. CONCLUSIONS: Rural New Mexico women were supportive of pharmacy access to contraception and accept pharmacists as trusted members of the health care team. IMPLICATIONS: Rural New Mexico women find benefit in pharmacy access to hormonal contraception, citing improved access to contraceptives in their communities.

A Novel Presentation of Bladder Mass: Self-palpation by a Patient With Cystocele.

INTRODUCTION: This case describes diagnosis of a T2 transitional cell carcinoma in an 89-year-old woman with known cystocele and urinary retention managed with clean intermittent self-catheterization. CASE: While self-catheterizing, the patient noted a palpable mass in her cystocele. She eventually pursued urologic evaluation of this mass, which ultimately led to her diagnosis. This is the first reported case of transitional cell carcinoma being found on self-examination by palpating a cystocele.

Pathway- and Cell-Specific Kappa-Opioid Receptor Modulation of Excitation-Inhibition Balance Differentially Gates D1 and D2 Accumbens Neuron Activity.

Endogenous dynorphin signaling via the kappa-opioid receptor (KOR) in the nucleus accumbens (NAcc) powerfully mediates negative affective states and stress reactivity. Excitatory inputs from the hippocampus and amygdala play a fundamental role in shaping the activity of both NAcc D1 and D2 MSNs, which encode positive and negative motivational valences, respectively. However, a circuit-based mechanism by which KOR modulation of excitation-inhibition balance modifies D1 and D2 MSN activity is lacking. Here, we provide a comprehensive synaptic framework wherein presynaptic KOR inhibition decreases the excitatory drive of D1 MSN activity by the amygdala, but not the hippocampus. Conversely, presynaptic inhibition by KORs of inhibitory synapses on D2 MSNs enhances integration of excitatory drive by the amygdala and hippocampus. In conclusion, we describe a circuit-based mechanism showing differential gating of afferent control of D1 and D2 MSN activity by KORs in a pathway-specific manner.

Circuit specificity in the inhibitory architecture of the VTA regulates cocaine-induced behavior.

Afferent inputs to the ventral tegmental area (VTA) control reward-related behaviors through regulation of dopamine neuron activity. The nucleus accumbens (NAc) provides one of the most prominent projections to the VTA; however, recent studies have provided conflicting evidence regarding the function of these inhibitory inputs. Using optogenetics, cell-specific ablation, whole cell patch-clamp and immuno-electron microscopy, we found that NAc inputs synapsed directly onto dopamine neurons, preferentially activating GABAB receptors. GABAergic inputs from the NAc and local VTA GABA neurons were differentially modulated and activated separate receptor populations in dopamine neurons. Genetic deletion of GABAB receptors from dopamine neurons in adult mice did not affect general or morphine-induced locomotor activity, but markedly increased cocaine-induced locomotion. Collectively, our findings demonstrate notable selectivity in the inhibitory architecture of the VTA and suggest that long-range GABAergic inputs to dopamine neurons fundamentally regulate behavioral responses to cocaine.

C1D family proteins in coordinating RNA processing, chromosome condensation and DNA damage response.

Research on the involvement of C1D and its yeast homologues Rrp47 (S. cerevisiae) and Cti1 (S. pombe) in DNA damage repair and RNA processing has remained mutually exclusive, with most studies predominantly concentrating on Rrp47. This review will look to reconcile the functions of these proteins in their involvement with the RNA exosome, in the regulation of chromatin architecture, and in the repair of DNA double-strand breaks, focusing on non-homologous end joining and homologous recombination. We propose that C1D is situated in a central position to maintain genomic stability at highly transcribed gene loci by coordinating these processes through the timely recruitment of relevant regulatory factors. In the event that the damage is beyond repair, C1D induces apoptosis in a p53-dependent manner.

Labeling of biotin antibodies with horseradish peroxidase using cyanuric chloride.

In this report, we describe a two-step protocol for labeling of an affinity-purified antibody to biotin with horseradish peroxidase (HRP) using cyanuric chloride (CC) as a bridge. The enzyme was first modified with CC, and following chromatography on a PD-10 column, the activated HRP was incubated with the antibody to effect coupling of the two proteins. Assessment of the conjugate product was carried out using ELISA and SDS-PAGE electrophoresis where evidence for high antibody activity, high specific activity of the conjugate preparation, coupling of nearly all the antibody and over 90% of the enzyme was shown. The titer of the conjugate exceeded 1/100,000. High molecular weight complexes were observed in the SDS-PAGE results, indicating an efficient conjugation procedure. The presence of high molecular weight complexes indicated an efficient conjugation procedure. The protocol is simple, and the conjugation steps can be completed in 27 h once the preparatory phase has been carried out; the method is entirely generic and may be applied to labeling of any antibody.