Aberrant PGC-1α signaling in a lamb model of persistent pulmonary hypertension of the newborn.

BACKGROUND: Persistent Pulmonary Hypertension of the Newborn (PPHN) is characterized by elevated pulmonary vascular resistance (PVR), resulting in hypoxemia. Impaired angiogenesis contributes to high PVR. Pulmonary artery endothelial cells (PAECs) in PPHN exhibit decreased mitochondrial respiration and angiogenesis. We hypothesize that Peroxisome Proliferator-Activated Receptor Gamma Co-Activator-1α (PGC-1α) downregulation leads to reduced mitochondrial function and angiogenesis in PPHN. METHODS: Studies were performed in PAECs isolated from fetal lambs with PPHN induced by ductus arteriosus constriction, with gestation-matched controls and in normal human umbilical vein endothelial cells (HUVECs). PGC-1α was knocked downed in control lamb PAECs and HUVECs and overexpressed in PPHN PAECs to investigate the effects on mitochondrial function and angiogenesis. RESULTS: PPHN PAECs had decreased PGC-1α expression compared to controls. PGC-1α knockdown in HUVECs led to reduced Nuclear Respiratory Factor-1 (NRF-1), Transcription Factor-A of Mitochondria (TFAM), and mitochondrial electron transport chain (ETC) complexes expression. PGC-1α knockdown in control PAECs led to decreased in vitro capillary tube formation, cell migration, and proliferation. PGC-1α upregulation in PPHN PAECs led to increased ETC complexes expression and improved tube formation, cell migration, and proliferation. CONCLUSION: PGC-1α downregulation contributes to reduced mitochondrial oxidative phosphorylation through control of the ETC complexes, thereby affecting angiogenesis in PPHN. IMPACT: Reveals a novel mechanism for angiogenesis dysfunction in persistent pulmonary hypertension of the newborn (PPHN). Identifies a key mitochondrial transcription factor, Peroxisome Proliferator-Activated Receptor Gamma Co-Activator-1α (PGC-1α), as contributing to the altered adaptation and impaired angiogenesis function that characterizes PPHN through its regulation of mitochondrial function and oxidative phosphorylation. May provide translational significance as this mechanism offers a new therapeutic target in PPHN, and efforts to restore PGC-1α expression may improve postnatal transition in PPHN.

Male Gender and Competitive Athlete Status Are Associated With Better Outcomes Following Hip Arthroscopy In Patients With Global Acetabular Retroversion.

Purpose: To evaluate outcomes of hip arthroscopy in patients with global acetabular retroversion and to identify correlations between sex, radiographic measurements, athlete status, and return to play with patient-reported outcomes (PROs). Methods: Retrospective study of patients with global acetabular retroversion who underwent arthroscopic femoroacetabular impingement (FAI) surgery was performed. Global acetabular retroversion was defined by 3 criteria: the crossover sign, ischial spine sign, and posterior wall sign on an anteroposterior (AP) pelvic radiograph. Radiographs were used to measure lateral center edge angle, alpha angle, and anterior and posterior wall indices. Femoral version was measured with 3-dimensional computed tomography. Demographics included age, gender, athlete status, return to play, and reoperation. PROs included modified Harris Hip Score, Hip Outcome Score (HOS), Hip Disability and Osteoarthritis Outcome Score, visual analog scale (VAS), and Veterans RAND-12. Spearman correlation determined correlation with perioperative PROs. Generalized estimating equation determined independent predictors. Significance was set at P = .05. Results: From 2013 to 2019, 149 patients (65.0% female) with 160 hips with FAI and global acetabular retroversion underwent hip arthroscopy. Follow-up averaged 29.6 months. All PROs demonstrated significant improvement with the exception of the Veterans RAND-12 Mental. Female patients scored significantly lower on most postoperative PROs and had greater VAS scores (P = .0002-0.0402). A greater proportion of male subjects met the minimum clinically important difference for the modified Harris Hip Score (88.00% vs 78.79%) Low femoral version correlated with greater HOS ADL, HOS Sport, and Hip Disability and Osteoarthritis Outcome Score Sport scores (P = .0077-0.0177). Athletes reported lower preoperative VAS scores, and higher perioperative scores in multiple PROs (P = .0004-0.0486). Nine hips (5.63%) underwent reoperation. Conclusions: Patients with global acetabular retroversion and FAI undergoing hip arthroscopy report good outcomes at short-term follow-up. Male subjects and athletes had superior outcomes compared to female subjects and nonathletes. Radiographic measurements did not correlate with outcomes with exception of low femoral version. Athletes reported lower preoperative pain scores and greater postoperative PROs than nonathletes. Level of Evidence: Level IV, therapeutic case series.

Safe Elective Surgery Using Selective SARS-CoV-2 (COVID-19) Molecular Testing.

Resuming elective surgery amid shortages of rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tests and personal protective equipment, while protecting patient and staff safety, posed a challenge at the study institution. Many hospital systems implemented testing of all presurgical patients, using results from tests performed 3 or more days prior to surgery. Among asymptomatic persons, the percent positive rates of coronavirus disease 2019 (COVID-19) tests in the region did not appear to justify this practice. Instead of universal preoperative COVID-19 testing, the authors elected to mitigate risk by implementing a preoperative program including 14 days of recommended patient self-quarantine and social distancing. For those unable to complete this program and for those undergoing high-risk, aerosol-generating procedures, targeted rapid polymerase chain reaction testing within 2 days of surgery was performed. Data from the initial 4 months suggests that this approach was noninferior to universal preoperative testing with regard to postoperative COVID-19 detection and patient exposure-related COVID-19 cases among hospital staff.

Mammary Paget's Disease of the Male Breast: A Rare Case With an Unusual Immunohistochemical Profile.

Mammary Paget's disease is rare and comprises about 0.62% of all breast cancer cases, only 1.65% of which occur in male patients. This case report involves a 76-year-old man who presented to his primary care physician with an itching, scaly, unilateral lesion involving the nipple skin. He underwent wide local excision of the lesion for a diagnosis of Bowen's disease (squamous cell carcinoma in situ). Histologic examination of the specimen revealed mammary Paget's disease with ductal carcinoma in situ in the underlying breast tissue. A panel of immunohistochemical stains revealed the Paget cells to be positive for cytokeratin 7, MUC1, GATA3, and androgen receptor and negative for cytokeratins 5/6, p63, SOX10, and MART-1/Melan-A. Paget cells were also negative for estrogen receptor and progesterone receptor, and positive for HER2/neu. However, the underlying ductal carcinoma in situ was positive for both estrogen receptor and progesterone receptor and negative for HER2/neu. This discordance, supported by the current literature, suggests an alternative etiology for Paget's disease in certain cases that cannot be explained by the well-established epidermotropic and transformative theories of Paget's disease evolution.

Migrating eastern North Pacific gray whale call and blow rates estimated from acoustic recordings, infrared camera video, and visual sightings.

During the eastern North Pacific gray whale 2014-2015 southbound migration, acoustic call recordings, infrared blow detections, and visual sightings were combined to estimate cue rates, needed to convert detections into abundance. The gray whale acoustic call rate ranged from 2.3-24 calls/whale/day during the peak of the southbound migration with an average of 7.5 calls/whale/day over both the southbound and northbound migrations. The average daily calling rate increased between 30 December-13 February. With a call rate model, we estimated that 4,340 gray whales migrated south before visual observations began on 30 December, which is 2,829 more gray whales than used in the visual estimate, and would add approximately 10% to the abundance estimate. We suggest that visual observers increase their survey effort to all of December to document gray whale presence. The infrared camera blow rate averaged 49 blows/whale/hour over 5-8 January. Probability of detection of a whale blow by the infrared camera was the same at night as during the day. However, probability of detection decreased beyond 2.1 km offshore, whereas visual sightings revealed consistent whale densities up to 3 km offshore. We suggest that future infrared camera surveys use multiple cameras optimised for different ranges offshore.

Minimally invasive surgery and its impact on 30-day postoperative complications, unplanned readmissions and mortality.

BACKGROUND: A critical appraisal of the benefits of minimally invasive surgery (MIS) is needed, but is lacking. This study examined the associations between MIS and 30-day postoperative outcomes including complications graded according to the Clavien-Dindo classification, unplanned readmissions, hospital stay and mortality for five common surgical procedures. METHODS: Patients undergoing appendicectomy, colectomy, inguinal hernia repair, hysterectomy and prostatectomy were identified in the American College of Surgeons National Surgical Quality Improvement Program database. Non-parsimonious propensity score methods were used to construct procedure-specific matched-pair cohorts that reduced baseline differences between patients who underwent MIS and those who did not. Bonferroni correction for multiple comparisons was applied and P < 0·006 was considered significant. RESULTS: Of the 532 287 patients identified, 53·8 per cent underwent MIS. Propensity score matching yielded an overall sample of 327 736 patients (appendicectomy 46 688, colectomy 152 114, inguinal hernia repair 59 066, hysterectomy 59 066, prostatectomy 10 802). Within the procedure-specific matched pairs, MIS was associated with significantly lower odds of Clavien-Dindo grade I-II, III and IV complications (P ≤ 0·004), unplanned readmissions (P < 0·001) and reduced hospital stay (P < 0·001) in four of the five procedures studied, with the exception of inguinal hernia repair. The odds of death were lower in patients undergoing MIS colectomy (P < 0·001), hysterectomy (P = 0·002) and appendicectomy (P = 0·002). CONCLUSION: MIS was associated with significantly fewer 30-day postoperative complications, unplanned readmissions and deaths, as well as shorter hospital stay, in patients undergoing colectomy, prostatectomy, hysterectomy or appendicectomy. No benefits were noted for inguinal hernia repair.

Robotic-assisted laparoscopic bladder augmentation in the pediatric patient.

INTRODUCTION: Bladder augmentation is a common surgical intervention for neuropathic bladder dysfunction, and has conventionally been an open procedure. We present a robotic ileocystoplasty to demonstrate the feasibility of an entirely intracorporeal approach in a pediatric patient. METHODS: The patient was a 6 year old (18.5 kg) boy with a neurogenic bladder secondary to lumbar myelomeningocele. Urodynamics revealed a small capacity and poorly compliant bladder and he was incontinent between frequent catheterizations. A robotic augmentation cystoplasty was performed. RESULTS: At one-month postoperatively, a cystogram revealed no urine leak, and the suprapubic tube was removed. The patient resumed CIC every 3 h during the day and once overnight until postoperative urodynamic studies confirmed safe dynamics, after which the CIC interval was lengthened. CONCLUSION: Robotic bladder augmentation is safe and feasible in a select pediatric population. The entire procedure including preparation of the bowel segment can be completed intracorporeally, even in smaller children.

Hip Arthroscopy for Removal of an Acetabular Rim-Based Osteoid Osteoma and Concomitant Femoroacetabular Impingement Correction.

Minimally invasive percutaneous radiofrequency ablation (RFA) techniques are the standard of care for treating simple osteoid osteomas (OOs). Historically, OOs were treated with open en bloc resection or curettage of the nidus. RFA procedures have been linked to soft-tissue complications of varying severity. In addition, RFA may be a poor choice for periarticular OOs because of the potential for procedure-related articular cartilage damage. Hip arthroscopy is a widely accepted approach for the treatment of femoroacetabular impingement. We describe arthroscopic resection of an acetabular rim-based OO as part of a rim resection and labral repair. Early clinical follow-up suggests that arthroscopic resection of an OO in this unusual location is precise and predictable, allows for an additional evaluation of associated symptomatic pathology (i.e., femoroacetabular impingement), and results in prompt symptom resolution.

Disruption of ß-catenin/CBP signaling inhibits human airway epithelial-mesenchymal transition and repair.

The epithelium of asthmatics is characterized by reduced expression of E-cadherin and increased expression of the basal cell markers ck-5 and p63 that is indicative of a relatively undifferentiated repairing epithelium. This phenotype correlates with increased proliferation, compromised wound healing and an enhanced capacity to undergo epithelial-mesenchymal transition (EMT). The transcription factor ß-catenin plays a vital role in epithelial cell differentiation and regeneration, depending on the co-factor recruited. Transcriptional programs driven by the ß-catenin/CBP axis are critical for maintaining an undifferentiated and proliferative state, whereas the ß-catenin/p300 axis is associated with cell differentiation. We hypothesized that disrupting the ß-catenin/CBP signaling axis would promote epithelial differentiation and inhibit EMT. We treated monolayer cultures of human airway epithelial cells with TGFß1 in the presence or absence of the selective small molecule ICG-001 to inhibit ß-catenin/CBP signaling. We used western blots to assess expression of an EMT signature, CBP, p300, ß-catenin, fibronectin and ITGß1 and scratch wound assays to assess epithelial cell migration. Snai-1 and -2 expressions were determined using q-PCR. Exposure to TGFß1 induced EMT, characterized by reduced E-cadherin expression with increased expression of α-smooth muscle actin and EDA-fibronectin. Either co-treatment or therapeutic administration of ICG-001 completely inhibited TGFß1-induced EMT. ICG-001 also reduced the expression of ck-5 and -19 independent of TGFß1. Exposure to ICG-001 significantly inhibited epithelial cell proliferation and migration, coincident with a down regulation of ITGß1 and fibronectin expression. These data support our hypothesis that modulating the ß-catenin/CBP signaling axis plays a key role in epithelial plasticity and function.

Critical evaluation of ninhydrin for monitoring surgical instrument decontamination.

BACKGROUND: New Department of Health (England) Choice Framework for Local Policies and Procedures guidance (CFPP 0101) still states that ninhydrin can be used to check for efficient protein removal from surgical instruments processed in sterile services departments (SSDs). AIM: With the potential transfer of variant Creutzfeldt-Jakob disease (vCJD) via surgical procedures it is necessary to re-evaluate recommended methods for protein detection. METHODS: This paper reports studies on the sensitivity and applicability of ninhydrin for detecting proteins in laboratories and SSDs. The efficiency of protein removal by swabbing was also evaluated. FINDINGS: Ninhydrin showed poor sensitivity toward proteins. Limits of detection for bovine serum albumin (BSA) in solution were 205 µg/mL compared with arginine 6 µg/mL. A commercial kit could detect neither rat brain homogenate nor BSA at <1000 µg protein pipetted directly into the vials. Swabbing with water-wetted rayon swabs was inefficient at removing protein (50 µg) from instruments (N = 6) with 32 ± 4% BSA and 61 ± 5% fibrinogen remaining bound. Swabs dipped in 0.5% detergent (Triton X-100) solution had slightly better removal efficiency with 20 ± 3% BSA and 24 ± 2.8% fibrinogen remaining. CONCLUSIONS: Ninhydrin kits, currently used in SSDs, are ineffective at detecting residual proteins due not only to the insensitivity of ninhydrin towards proteins but also to the poor desorption of adhered proteins by swabbing. Overall ninhydrin, either as a laboratory reagent or as supplied in protein detection kits, does not provide sensitive detection of proteins and generates high numbers of false negatives when used in decontamination practices.

The airway epithelium in asthma: developmental issues that scar the airways for life?

While allergies are very common, affecting ∼40% of the population in most Western countries, only a proportion of allergic people develop asthma. This highlights the importance of tissue and cell specific mechanisms that contribute to the disease. As the interface between the inhaled environment and the internal environment of the lung, the epithelium normally possesses numerous mechanisms to maintain an effective protective barrier. However, the inability of the airway epithelium of asthmatics to effectively defend the lung against normally innocuous inhaled agents strongly suggests that asthma must involve defects in the epithelial barrier rather than being primarily an allergic disease. Evidence is accumulating that in asthma, the epithelium does not go through normal stages of development and differentiation and as a consequence, remain somewhat "immature". This in turn leads to a chronic cycle of dysregulated damage and repair which ultimately impacts on the airways function by increasing inflammation, but also by initiating processes that ultimately lead to changes to the structure and function of the airway.

Training induces nonuniform increases in eNOS content along the coronary arterial tree.

Exercise training produces enhanced nitric oxide (NO)-dependent, endothelium-mediated vasodilator responses of porcine coronary arterioles but not conduit coronary arteries. The purpose of this study was to test the hypothesis that exercise training increases the amount of endothelial NO synthase (eNOS) in the coronary arterial microcirculation but not in the conduit coronary arteries. Miniature swine were either exercise trained or remained sedentary for 16--20 wk. Exercise-trained pigs exhibited increased skeletal muscle oxidative capacity, exercise tolerance, and heart weight-to-body weight ratios. Content of eNOS protein was determined with immunoblot analysis in conduit coronary arteries (2- to 3-mm ID), small arteries (301- to 1,000-microm ID), resistance arteries (151- to 300-microm ID), and three sizes of coronary arterioles [large (101- to 150-microm ID), intermediate (51- to 100-microm ID), and small (<50-microm ID)]. Immunoblots revealed increased eNOS protein in some sizes of coronary arteries and arterioles but not in others. Content of eNOS was increased by 60--80% in small and large arterioles, resistance arteries, and small arteries; was increased by 10--20% in intermediate-sized arterioles; and was not changed or decreased in conduit arteries. Immunohistochemistry revealed that eNOS was located in the endothelial cells in all sizes of coronary artery. We conclude that exercise training increases eNOS protein expression in a nonuniform manner throughout the coronary arterial tree. Regional differences in shear stress and intraluminal pressures during exercise training bouts may be responsible for the distribution of increased eNOS protein content in the coronary arterial tree.

Monocyte chemoattractant protein-1: receptor interactions and calcium signaling mechanisms.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the Cys-Cys chemokine family. Two related MCP-1 receptors have been identified (CC-CKR2A and CC-CKR2B), although the precise kinetics of ligand binding and calcium signaling of these receptors has yet to be investigated. To examine this more closely, the human MCP-1 receptors were cloned and expressed in Chinese hamster ovary (CHO) cells. Membranes prepared from cells expressing CC-CKR2B bind MCP-1 selectively and with high affinity (Kd = 120 pM). MCP-1 stimulation of recombinant CHO cells expressing CC-CKR2B induces a rapid increase in intracellular Ca2+ through both receptor-operated Ca2+ channels and mobilization of Ca2+ from intracellular stores, and leads to a rapid temperature-dependent internalization of the ligand/receptor complexes. In contrast, recombinant CHO cells expressing CC-CKR2A, and membranes prepared from these cells, fail to bind detectable levels of MCP-1. However, MCP-1 stimulation of cells expressing CC-CKR2A induces a small but significant increase in intracellular Ca2+. Repeated stimulation of these cells with MCP-1 leads to a potentiation of the response to a level comparable to that seen in cells expressing CC-CKR2B. These observations suggest that the levels of cell surface CC-CKR2A are controlled by novel mechanisms.

Myelin gene expression in immortalized Schwann cells: relationship to cell density and proliferation.

Myelin gene expression was investigated in the immortalized S16 Schwann cell line grown in the presence and absence of serum and at different densities. Protein expression was monitored by western blotting, and message levels were determined by RNase protection assays. To study cell proliferation rates at different cell densities and serum conditions, [3H]thymidine uptake assays and cell counts were performed. Although serum deprivation decreased cell proliferation as expected, the proliferation of S16 cells was unchanged or slightly increased at high density under the conditions of our experiments in either serum-containing or serum-free medium. This increased cell division at high density appeared to be due to greater release of an autocrine growth factor to the medium by dense cell populations. For both sparse and dense cells, substantially more P0 glycoprotein (P0) and myelin-associated glycoprotein (MAG) per milligram of total cellular protein were expressed when the cells were proliferating slowly in defined medium in comparison with more rapidly proliferating cells in serum-containing medium. Furthermore, in both serum-containing and defined media, dense cell populations expressed more MAG and PO than sparse ones. PO mRNa and MAG mRNA levels generally paralleled protein levels. The level of mRNA for peripheral myelin protein-22 (PMP-22) was also increased at high cell density but did not change much when proliferation was decreased by serum deprivation. PMP-22 protein was not detected under any of the growth conditions. The changes in expression of these genes with growth conditions may be specific for myelin proteins, because the expression of a nonmyelin glycoprotein, L1, remained constant. The level of cyclic AMP in the cells did not change with the different growth conditions tested. The results indicate that the S16 Schwann cell line mimics primary or secondary Schwann cells by down-regulating myelin gene expression when it proliferates more rapidly in the presence of serum. Furthermore, in both the presence and absence of serum, there was greater expression of myelin genes at high cell density that was not associated with a decreased proliferative rate. Because evidence for a role of secretory factors in affecting myelin gene expression was not obtained by treating sparse S16 cells with medium conditioned by dense S16 cells, the results suggest that the higher expression of myelin genes at high density may be mediated by cell-to-cell contact.

High level expression of human MCP-1 using the LCR/MEL expression system.

We have expressed human monocyte chemoattractant protein-1 (hMCP-1) in preerythroid mouse erythroleukemia (MEL) C88 cells using the locus control region/MEL expression system and studied the biological activity of the purified protein in a range of in vitro experimental systems. The recombinant hMCP-1 is expressed at high levels (approximately 10 mg/liter) in this system and is modified in a manner which is very similar to native hMCP-1. We have developed a simple high-yielding two-step purification route employing dye ligand and ion exchange chromatographies which enables us to separate glycosylated and unglycosylated hMCP-1. The purified glycosylated and unglycosylated forms of hMCP-1 have equivalent biological activities in all of the assay systems tested.

Identification of multiple human calcitonin receptor isoforms: heterologous expression and pharmacological characterization.

The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, h alpha CTR and h beta CTR. The two cDNAs are identical except that the h alpha CTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (h alpha CTR Kd 0.09 nM, h beta CTR Kd 0.12 nM). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the h beta CTR isoform was expressed at tenfold higher levels than the h alpha CTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in beta-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.

Computer-generated presentations: current status and future directions.

The personal computer allows the user to create professional presentations as good as any created by commercially available services. With the new generation of inexpensive software, myriad fonts, layouts, graphics, and imported images can all be used by the novice. The technology is currently available that can project images directly from the computer, obviating the need for slides. This presentation will discuss the relative merits and costs of such systems and the problems that remain concerning implementation at a national level.