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The centre of the Milky Way Galaxy hosts a black hole with a solar mass of about 4 million (Sagittarius A * (Sgr A)) that is very quiescent at present with a luminosity many orders of magnitude below those of active galactic nuclei 1 . Reflection of X-rays from Sgr A * by dense gas in the Galactic Centre region offers a means to study its past flaring activity on timescales of hundreds and thousands of years 2 . The shape of the X-ray continuum and the strong fluorescent iron line observed from giant molecular clouds in the vicinity of Sgr A * are consistent with the reflection scenario 3-5 . If this interpretation is correct, the reflected continuum emission should be polarized 6 . Here we report observations of polarized X-ray emission in the direction of the molecular clouds in the Galactic Centre using the Imaging X-ray Polarimetry Explorer. We measure a polarization degree of 31% ± 11%, and a polarization angle of -48° ± 11°. The polarization angle is consistent with Sgr A * being the primary source of the emission, and the polarization degree implies that some 200 years ago, the X-ray luminosity of Sgr A * was briefly comparable to that of a Seyfert galaxy., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background : Outpatient primary care clerkships are an important part of medical students' education.Traditional clerkships usually partner a student with a single preceptor in that physician's clinic. However, it can be quite difficult for the preceptor to balance the educational needs of the students, the expectations of the patients and the organizational demands of the clinic practice. Objective : An innovative scheduling model (named "Patients as Teachers" [PAT] clinic) was developed as part of our third-year Family Medicine clerkship. The goal was to increase the students' opportunities for independence and improve their satisfaction without negatively impacting the flow of the clinic or patient satisfaction. Design : The third-year medical students spent part of their clerkship working in the PAT clinic and part of the time working with an individual preceptor in that preceptor's clinic in the traditional, usual fashion (PAU clinic-precepting as usual). The students completed patient-logs regarding the patients they saw and their level of participation. They also completed a voluntary survey regarding their experiences. Results : Students performed more independent interviews (90.3 vs 59.0%) and independent exams (96.2 vs 63.3%) in the PAT clinic than while working with their traditional preceptor (both p<0.01). Students were highly satisfied with the experience with 89.5% stating they would recommend it and 87.7% finding the PAT clinic to be an equal or superior experience to the PAU experience. Conclusions : Using a combination of time in the PAT clinic and time with a one on one preceptor in the usual fashion was successful in increasing opportunities for student autonomy and achieving a high level of student satisfaction in our third-year Family Medicine clerkship. Additional opportunities for innovative scheduling could be considered for meeting a variety of clerkship and clinic needs.

Background: Use of liposomal bupivacaine (LB) in surgery is reported with decreased postoperative opioid requirements. The efficacy of LB versus standard bupivacaine injections at laparoscopic port sites during bariatric surgery is unknown., Objectives: To determine whether there was a difference in postoperative hospital opioid requirements after port site injections of LB versus standard bupivacaine during laparoscopic bariatric surgeries. Primary endpoint was total in hospital opioid use expressed as morphine-equivalent use. Secondary endpoints included home opioid use, pain scores, hospital length of stay, and adverse events., Setting: Academic-affiliated private practice., Methods: A 2-group randomized, double-blinded trial from November 2017 to August 2018 with patients randomly assigned to receive either LB or bupivacaine alone at trocar site injections during laparoscopic Roux-en-Y gastric bypass (LRYGB) or vertical sleeve gastrectomy (VSG). All patients underwent enhanced recovery after bariatric surgery protocols., Results: All patients undergoing LRYGB or VSG assessed for eligibility. Of 682 patients undergoing LRYGB or VSG, 231 met inclusion criteria, 52 patients excluded intraoperatively. Among 231 patients (mean age, 39.2 years; 79% women; mean body mass index 45.0), 179 patients (77%) completed the trial. Patients randomly assigned to receive either LB (n = 89) or bupivacaine alone (n = 90) at trocar site injection during LRYGB or VSG. Postoperative morphine-equivalent use were similar (LB 8.3 [standard deviation 4.0-13.9] versus bupivacaine group 7.5 [standard deviation 3.6-13.1] P = .94) with highest requirement in first 4 hours after surgery. There was no significant difference in length of stay, pain scores, or complications. There were more patients in the bupivacaine group that did not take pain medications on postoperative days 2 to 4 (P = .032, P = .23, P = .005, respectively). There were more patients in the bupivacaine group 48.1% (n = 39) compared with the LB group 34.2% (n = 27) that did not consume any narcotic tablets at home but this not found to be statistically significant (P value = .07)., Conclusions: Among patients undergoing primary bariatric surgery under enhanced recovery after bariatric surgery protocol, there was no significant difference in postoperative hospital opioid use in those receiving LB compared with standard bupivacaine. A greater percentage of patients in the standard bupivacaine group did not require any narcotics at home, which was significant on postoperative days 2 to 4. To become completely opioid free after bariatric surgery, resources should be focused on multimodal approaches instead of reliance on type of anesthetic medication used., (Copyright © 2019 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.)

Background: Prior randomized studies have shown a survival benefit using combined androgen deprivation therapy (ADT) and radiation therapy for intermediate-risk prostate cancer. However, these studies either used low doses of radiation (66.6 Gy to isocenter) or imaged guidance was not available. This study reports the initial differences for high dose image guided radiation with or without ADT., Methods: From 2012 to 2014, 56 patients were treated with and 60 patients without 6 months of ADT (N = 116) in our phase III randomized trial for intermediate-risk prostate cancer. The primary endpoints of the current analysis are Expanded Prostate Cancer Index Composite (EPIC) scores, International Prostate Symptom Score (IPSS) scores, and bowel or urinary adverse events (AEs, graded using CTCAE v4) with and without ADT. Treatment consisted of 81 Gy in 45 treatments (tx) or 100 Gy Pd-103 implant followed by 45 Gy in 25 tx with or without ADT. Cone-beam fiducial-based guidance was done. Statistical analysis included Fisher's exact test, chi-square test, and ANCOVA., Results: Median follow-up for both groups was 2.6 years. Acute or chronic urinary and acute or chronic bowel toxicities were similar with or without ADT (acute urinary: 16 vs 25 G0-1, 39 vs 35 G2 and 1 vs 0 G3, p = 0.17; chronic urinary: 40 vs 45 G1 and 16 vs 15 G2 toxicities, p = 0.68; acute bowel: 56 vs 59 G1 and 0 vs 1 G2 toxicities, p = 0.99; chronic bowel: 56 vs 59 G1 and 0 vs 1 G2 toxicities, p = 0.99). One patient had grade 3 urinary AE (1/116 or 0.8%). No patient had grade 3 bowel AE. With the use of ADT, a temporary decline in the EPIC sexual (p = 0.004) and hormonal scores (p = 0.02) were seen for the first 3 to 6 months after the completion of radiation, but the scores recovered by 12 months. Brachytherapy plus external beam radiation was compared to external beam radiation alone; brachytherapy EPIC urinary irritative scores were temporarily lower at 3 months, 76 vs. 84 (p = 0.006), had higher IPSS scores at 3 months, 15 vs 12 (p = 0.01), and had increased acute urinary AEs (p<0.001). No difference in failures were seen with or without ADT or associated with the use of brachytherapy., Significance: Low toxicity and minimal temporary bother as measured by EPIC and IPSS were seen in both arms. ADT was well-tolerated and associated with temporary changes., (Copyright © 2019. Published by Elsevier Ltd.)

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Background: The oxygenation index (OI = mean airway pressure, MAP × FiO2 × 100 : PaO2) is used to assess the severity of hypoxic respiratory failure (HRF) and persistent pulmonary hypertension of the newborn (PPHN). An indwelling arterial line or arterial punctures are necessary to obtain PaO2 for the calculation of OI. Oxygenation can be continuously and noninvasively assessed using pulse oximetry. The use of the oxygen saturation index (OSI = MAP × FiO2 × 100 : SpO2) can be an alternate method of assessing the severity of HRF., Objective: To evaluate the correlation between OSI and OI in the following: (1) neonates with HRF and (2) a lamb model of meconium aspiration syndrome., Methods: Human neonates: a retrospective chart review of 74 ventilated late preterm/term neonates with indwelling arterial access and SpO2 values in the first 24 h of life was conducted. OSI and OI were calculated and correlated. Lamb model: arterial blood gases were drawn and preductal SpO2 was documented in 40 term newborn lambs with asphyxia and meconium aspiration. OI and OSI were calculated and correlated with pulmonary vascular resistance (PVR)., Results: Mean values of OSI and OI showed a correlation coefficient of 0.952 in neonates (mean value of 308 observations in 74 neonates) and 0.948 in lambs (mean value of 743 observations in 40 lambs). In lambs, with increasing PVR, there was a decrease in OI and OSI., Conclusion: OSI correlates significantly with OI in infants with HRF. This noninvasive measure may be used to assess the severity of HRF and PPHN in neonates without arterial access., (© 2015 S. Karger AG, Basel)

Purpose: Evaluate outcomes and prognostic factors in men with localized prostate cancer., Methods and Materials: A total of 3760 patients have undergone prostate seed implantation at our institution. This review is of our initial 304 consecutive patients treated before January 30, 2001. A total of 124 patients were treated with (125)I implant monotherapy and 180 with (103)Pd implant combined with 45-Gy external beam radiation therapy., Results: The median followup was 10.3 years. A 10-year biochemical control for low risk (LR) was 98% , intermediate risk (IR) 94%, high risk (HR) 78%, and HR with one HR factor 88% (p < 0.001); cause-specific survival was 99%, 98%, and 84% for LR, IR, and HR, respectively (p < 0.001); No significant difference in outcome was seen for LR and IR patients (p > 0.3). On multivariate analysis, only pretreatment PSA, Gleason score, and T-stage were significant for biochemical control. Most biochemical failures occurred within 5 years (93%)., Conclusions: With a minimum followup of 10 years, results are excellent and do not differ for LR or IR prostate cancer patients. HR patients are a very heterogeneous group, and excellent results can still be achieved for HR patients with only one HR feature., (Copyright © 2013 American Brachytherapy Society. Published by Elsevier Inc. All rights reserved.)

Confocal laser endomicroscopy (CLE) is an advanced imaging technique which combines conventional white light endoscopy (WLE) with an integrated or probe based confocal microscope. This allows microscopic examination of the surface epithelium and in vivo diagnosis during endoscopy. Established CLE applications include the diagnosis of Barrett's oesophagus, gastric intestinal metaplasia, coeliac disease and microscopic colitis. CLE can differentiate hyperplastic from adenomatous polyps in the colon and may obviate the need to biopsy all polyps at endoscopy. CLE is particularly helpful in surveillance endoscopy in inflammatory bowel disease where it has been shown to reduce the number of biopsies required and improve the detection of dysplasia. The future of CLE may be with new contrast agents to allow for molecular tagging and improved endoscopic diagnoses. The aim of this review is to describe the technology and techniques involved in CLE, and discuss the evolving applications in obtaining "virtual biopsy" throughout the GI tract.

Purpose: The lungs in congenital diaphragmatic hernia (CDH) are hypoplastic and immature making respiratory support one of the most challenging aspects of caring for these neonates. Vitamin A is essential for normal lung growth and development. It also promotes alveolarization. The aim of this study is to investigate the effects of antenatal vitamin A on lung growth and alveolarization and ventilation in the lamb model of CDH., Methods: This study was approved by the Animal Care Committee of the State University of New York at Buffalo, and conforms to the National Institute of Health guidelines. Diaphragmatic defects were created at 79-81 days gestation. Group 1 lambs (CDH, n = 5) were untreated. In group 2 (CDH + vitamin A, n = 6) and group 3 lambs (control + vitamin A, n = 3) right jugular venous catheters were inserted at 118-120 days and retinyl palmitate (vitamin A) was administered until 135 days. The control group (n = 5) consisted of twin littermates. Lambs were delivered at 136-139 days and ventilated for 2 h according to a set protocol. The left lungs were harvested and fixed for histology., Results: Lung compliance was significantly higher in CDH + vitamin A (median 0.27, range 0.1-0.48 ml/cmH(2)O/kg) versus CDH lambs (median 0.07, range 0.07-0.18 ml/cmH(2)O/kg), P < 0.05. At 1 h CDH + vitamin A lambs experienced significantly lower PaCO(2) (median 115, range 35-194 mmHg vs. median 192, range 168-234 mmHg) and higher arterial pH (median 7.0, range 6.74-7.35 vs. median 6.73, range 6.5-6.81) than CDH lambs, P < 0.05. The lung weight to body weight ratio of CDH + vitamin A lambs was significantly less than that of CDH lambs (P < 0.05). Histology showed small thick walled air-spaces and no true alveoli in CDH lambs. In contrast, true alveoli and thinning of the inter-alveolar septums were seen in CDH + vitamin A lambs., Conclusion: This is the first study to demonstrate an improvement in lung function and structural maturation when antenatal vitamin A is given in a surgical model of CDH.

INTRATHORACIC GOITERS ARE DIVIDED INTO TWO CATEGORIES: primary and secondary. Intrathoracic goiters (IG) can cause upper airway obstruction. The presence of obstructive symptoms secondary to increased thyroid growth and tracheal compression is major indication for surgery; however, goiters do not always require immediate surgical attention. In addition, although some diagnostic tests indicate upper airway obstruction, many patients remain asymptomatic. Surgeries to remove IG are performed routinely however, they are not without risk. In some cases, intrathoracic goiters present as thyroid cancers. Very rare cancers such as Hürthle cell carcinoma (HCC) can create a challenge for the surgeon when surgical intervention is vital.

Background: Perforated marginal ulcer (PMU) after laparoscopic Roux-en-Y gastric bypass (LRYGB) is a serious complication, but its incidence and etiology have rarely been investigated. Therefore, a retrospective review of all patients undergoing LRYGB at the authors' center was conducted to determine the incidence of PMU and whether any causative factors were present., Methods: A prospectively kept database of all patients at the authors' bariatric center was retrospectively reviewed. The complete records of patients with a PMU were examined individually for accuracy and analyzed for treatment, outcome, and possible underlying causes of the marginal perforation., Results: Between April 1999 and August 2007, 1% of the patients (35/3,430) undergoing laparoscopic gastric bypass experienced one or more perforated marginal ulcers 3 to 70 months (median, 18 months) after LRYGB. The patients with and without perforation were not significantly different in terms of mean age (37 vs 41 years), weight (286 vs 287 lb), body mass index (BMI) (46 vs 47), or female gender (89% vs 83%). Of the patients with perforations, 2 (6%) were taking steroids, 10 (29%) were receiving nonsteroidal antiinflammatory drugs (NSAIDs) at the time of the perforation, 18 (51%) were actively smoking, and 6 of the smokers also were taking NSAIDs. Eleven of the patients (31%) who perforated did not have at least one of these possible risk factors, but 4 (36%) of the 11 patients in this group had been treated after bypass for a marginal ulcer. Only 7 (20%) of the 35 patients who had laparoscopic bypass, or 7 (0.2%) in the entire group of 3,430 patients, perforated without any warning. There were no deaths, but three patients reperforated., Conclusions: The incidence of a marginal ulcer perforating after LRYGB was significant (>1%) and appeared to be related to smoking or the use of NSAIDs or steroids. Because only 0.2% of all patients acutely perforated without some risk factor or warning, long-term ulcer prophylaxis or treatment may be necessary for only a select group of high-risk patients.

The epididymal lumen represents a unique extracellular environment because of the active sperm maturation process that takes place within its confines. Although much focus has been placed on the interaction of epididymal secretory proteins with spermatozoa in the lumen, very little is known regarding how the complex epididymal milieu as a whole is maintained, including mechanisms to prevent or control proteins that may not stay in their native folded state following secretion. Because some misfolded proteins can form cytotoxic aggregate structures known as amyloid, it is likely that control/surveillance mechanisms exist within the epididymis to protect against this process and allow sperm maturation to occur. To study protein aggregation and to identify extracellular quality control mechanisms in the epididymis, we used the cystatin family of cysteine protease inhibitors, including cystatin-related epididymal spermatogenic and cystatin C as molecular models because both proteins have inherent properties to aggregate and form amyloid. In this chapter, we present a brief summary of protein aggregation by the amyloid pathway based on what is known from other organ systems and describe quality control mechanisms that exist intracellularly to control protein misfolding and aggregation. We then present a summary of our studies of cystatin-related epididymal spermatogenic (CRES) oligomerization within the epididymal lumen, including studies suggesting that transglutaminase cross-linking may be one mechanism of extracellular quality control within the epididymis.

Objective: Infants with congenital diaphragmatic hernia (CDH) are susceptible to ventilation-induced lung injury. Vitamin A may protect the lung from injury during ventilation. The authors investigated the effects of antenatal vitamin A on ventilation-induced lung injury in CDH lambs using lung myeloperoxidase (MPO) activity as an indicator of lung injury., Methods: Left-sided diaphragmatic defects were created in 10 lambs at 79-81 days' gestation. Six CDH lambs had right jugular venous catheters inserted at 120 days' gestation and were given vitamin A until 135 days' gestation. Four CDH lambs were not treated. Twin littermates (n = 3) served as controls. All lambs were delivered at 136-139 days of gestation and ventilated for 2 hours. Lambs were sacrificed following ventilation and samples of left lung were snap frozen. MPO was extracted from lung tissue and MPO activity was assayed., Results: CDH lambs treated with antenatal vitamin A demonstrated significantly lower MPO activity than untreated CDH lambs (0.0477 +/- 0.0150 vs. 0.1106 +/- 0.0230 units/mg protein, p < 0.05)., Conclusion: This is the first study to look at the effect of vitamin A on lung injury in CDH. In the lamb model of CDH, antenatal vitamin A decreases ventilation-induced lung injury.

Nitric oxide causes dilation of the pulmonary circulation and reduction in net lung liquid production in the fetal lamb, two critical perinatal events. Phosphodiesterase inhibition alone causes similar changes and also enhances the effects of nitric oxide. To better define the cyclic guanosine 5'-monophosphate (GMP) pathway in these events, we studied the effects of a specific phosphodiesterase inhibitor, E4021, on pulmonary arteries and veins isolated from near-term fetal lambs, as well as in intact, chronically instrumented late-gestation fetal lambs. In the in vitro experiments, both pulmonary arteries and veins relaxed to E4021 in a dose-dependent manner, although pulmonary veins were significantly more sensitive to E4021. Pretreatment with N(G)-nitro-l-arginine (L-NNA) abolished this response in arteries but not in veins. In both arteries and veins, pretreatment with beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate blunted relaxations to E4021. In the in vivo experiments, E4021 infusion into either the pulmonary artery or central venous circulation increased pulmonary blood flow and decreased pulmonary vascular resistance, and these responses were blunted by pretreatment with L-NNA. Net lung liquid production, measured by a dye-dilution technique using blue dextran, decreased when E4021 was infused directly into the pulmonary artery and this effect was not altered by L-NNA. There was no effect on lung liquid production when E4021 was infused into the central venous circulation. Taken together, these results suggest that the pulmonary hemodynamic effects of E4021 involve the cyclic GMP pathway and are primarily nitric oxide synthase dependent. In contrast, the effects on E4021 on net lung liquid production appear to be independent of nitric oxide synthase, suggesting that these two critical perinatal events might be modulated independently.

The objectives of this study were to determine the influence of pH and MyHC isoforms on myofibrillar and actin-activated myosin subfragment 1 (S1) ATPase activity and the protective effect of actin. Red (RST) semitendinosus and white (WST) semitendinosus myofibrils were incubated at pH 7, 6, or 5.5 with 0 or 2mM ATP. RST and WST S1 isolates were incubated at pH 7, 6, or 5.5 in the presence or absence of actin. Maximum calcium-activated myofibrillar and actin-activated S1-ATPase activity were then assayed at pH 7. Incubation of myofibrils with ATP caused ATPase activity of myofibrils to decrease (p<0.05) with the pH of the incubation. RST myofibrils maintained a higher (p<0.0001) relative activity than WST myofibrils after incubation at pH 6 with ATP. Myofibrils incubated without ATP exhibited higher (p<0.001) activities than those incubated with ATP following pH 5.5 treatments. WST myofibrils had a lower (p<0.05) relative activity than RST following incubation at pH 5.5 without ATP. S1 ATPase activities decreased (p<0.05) with incubation pH in WST samples, but not in RST samples. WST S1 activity was higher (p<0.01) in samples exposed to pH 6 and 5.5 with actin bound compared to those incubated without actin. RST S1 exhibited a higher (p<0.01) relative activity than WST samples following pH 5.5 treatment with bound actin. These data show that low pH inactivates myofibrils by altering actin-activated S1 ATPase. Furthermore, these results suggest that muscles with high proportions of fast fibers are more susceptible to pH inactivation of ATPase activity and that the protective effect of actin binding to myosin is less in fast fibers.

The objective of this study was to determine the effects of postmortem muscle pH and temperature declines on the actomyosin ATPase activity of muscle fibers expressing different MyHC isoforms. Using a quantitative histochemical procedure to determine ATPase activity, the maximum actomyosin ATPase activity was determined on individual fibers classified by MyHC expression. Samples were collected from the red (RST) and white (WST) semitendinosus muscles at 3 min and 24 h postmortem from electrically stimulated (ES) and control (NS) pork carcasses. In samples taken at 3 min postmortem, type I fibers had the lowest ATPase activity staining and type 2X and 2B had the highest activity staining, with type 2A fibers intermediate. Postmortem time and carcass treatment did not influence the ATPase activity staining of type I muscle fibers. ATPase activity staining of 2A fibers was lower (p<0.001) in 24 h samples than in 3 min samples from ES carcasses. In 3 min and NS-24 h samples, RST type 2A fibers had lower (p<0.05) activities than type 2A fibers from the WST. In type 2X fibers, ATPase activity staining decreased (p<0.01) from 3 min to 24 h postmortem in ES carcasses. This decrease was more severe in WST 2X fibers compared to RST 2X fibers. ATPase activity staining in type 2B fibers did not decrease from 3 min to 24 h postmortem in NS carcasses. In ES carcasses, activity staining of 2B fibers decreased (p<0.0001) with time postmortem. The results of the experiment indicate that fibers expressing fast MyHC isoforms have a higher ATPase activity early postmortem than slow muscle fibers but are more prone to inactivation by a rapid pH decline.

The pH and Ca(2+) sensitivity of myofibrillar ATPase activity plays an integral role in regulating postmortem muscle ATP utilization and likely paces postmortem glycolysis. The objective of this study was to determine the influence of pH and Ca(2+) concentration on the ATPase activity of myofibrils from red semitendinosus (RST) and white semitendinosus (WST) porcine muscles. Myofibrillar ATPase was measured at 39 °C over a pH range 5-7.5 and a [Ca(2+)] range pCa 4-9 (10(-4)-10(-9)M). At maximum Ca(2+)-dependent activation (pCa 4), RST myofibrils had lower (p<0.0001) ATPase activity than WST myofibrils. This maximum activity of myofibrils from both muscle regions was not influenced from pH 7.5 to 6.5, declined between pH 6.5 and 5.75 (Hill coefficient, n(H)=2.7-3.4; pH at half maximum activity, pH(50)=5.97) and was near zero at pH 5.5. At pH 7, pCa-activity relationships showed that RST required less Ca(2+) for half-maximum activation (higher pCa(50); 6.50) than WST myofibrils (pCa(50)=6.35) but had no difference in n(H). At pH 7, both RST and WST myofibrils had maximum Ca(2+)-dependent, actin-activated ATPase activity at pCa ⩽6 and Ca(2+)-independent myosin ATPase activity at pCa ⩾6.75. pCa-activity relationships at different pH levels indicated that pCa(50) decreased with pH from pH 6.5 to 6.125 in both RST and WST myofibrils. At pH <5.75, [Ca(2+)] did not influence ATPase activity in RST or WST myofibrils. These data show that myofibrils with predominantly fast MyHC (WST) have a higher actin-activated myosin ATPase activity than myofibrils with primarily slow MyHC isoforms (RST) at Ca(2+) concentrations and pH values characteristic of postmortem muscle.

The objective of this study was to determine the influence of myofibril isolation procedures and myosin heavy chain (MyHC) isoform composition on myofibrillar ATPase activity as related to postmortem muscle metabolism. Myofibrils from the red (RST) and white (WST) portions of semitendinosus muscles were isolated using two different methods (A and B) at 3 min and 24 h postmortem in control (NS) and electrically stimulated (ES) pork carcasses. Comparison of the relative MyHC isoform profiles between the two different myofibril isolation methods and myosin extracts from the RST and WST at 3 min showed that method B myofibrils were more similar to the myosin extract than method A. Myofibrillar ATPase activity remained constant or increased (P<0.01) from 3 min to 24 h postmortem in NS carcasses and decreased (P<0.0001) in ES carcasses. From the RST, method A myofibrils had higher (P<0.0001) ATPase activity compared to method B across sampling time and carcass treatment. In the WST, method A myofibrils had lower (P<0.01) activity at 3 min, were not different at 24 h in NS carcasses, but had higher (P<0.05) activity at 24 h in ES carcasses versus method B myofibrils. Compared to method B, isolation method A biased the isoform profile of myofibril samples more towards faster MyHC (2A and 2X) in the RST and towards MyHC 2X in the WST. Results suggest that the ATPase activity and MyHC isoform profile of isolated myofibril samples are influenced by method of myofibril isolation, postmortem sampling time, and the rate of postmortem metabolism. Thus, differences in MyHC isoform profile and method of myofibril isolation must be taken into account to determine accurately the relationship between myofibrillar ATPase activity and rate of postmortem metabolism.

Spectroscopic methods were used to monitor the unfolding of the leucine specific (LS) and the leucine-isoleucine-valine (LIV) binding proteins. Our studies indicate that ligand-free protein undergoes a simple two-state unfolding, whereas the protein-ligand complex undergoes a three-state unfolding model. Ligand binding causes significant stabilization of both proteins. There is correlation between ligand hydrophobicity and protein stabilization: the most hydrophobic ligand, isoleucine, causes the most significant stabilization of LIV protein. A disulfide bond present in N-domain of both proteins makes a large contribution to the protein stability of these periplasmic binding receptors., (Copyright 2003 Wiley-Liss, Inc.)

Rho-kinase was recently found to phosphorylate the myosin-binding subunit (MBS) of myosin phosphatase (MP) and to regulate MP activity. Although myosin light chain (MLC) phosphorylation in pulmonary arterial smooth muscle cells (PASMCs) is thought to be the cellular/molecular basis for hypoxic pulmonary vasoconstriction (HPV), very little is known about the role that Rho-kinase/MP plays in HPV. Rat PASMCs were cultured and made hypoxic (PO2 = 23 +/- 2 mm Hg). Cells exposed to normoxia (PO2 approximately 148 mm Hg) served as controls. PASMCs exposed to hypoxia showed a significant increase in MLC and MBS phosphorylation, and a significant decrease in MP activity. Rho-kinase inhibitors (HA1077 or Y-27632) blocked hypoxia-induced MP inactivation and inhibited the hypoxia-induced MLC phosphorylation. Hypoxia was also found to induce stress fiber formation and actin polymerization in cultured PASMCs. In summary, these data show that MP inhibition in PASMCs is linked to activation of Rho-kinase, and that hypoxia inhibits the MP signaling pathway via Rho-kinase.

Two periplasmic binding proteins of E. coli, the leucine specific-binding protein (LS) and leucine-isoleucine-valine binding protein (LIV), have high similarity in their structure and function, but show different substrate specificity. A key difference between these proteins is residue 18 in the binding pocket, a tryptophan residue in the LS and a tyrosine residue in the LIV. To examine the role of this residue in binding specificity, we used fluorescence and (19)F NMR to monitor ligand binding to three mutants: LSW18Y, LSW18F and LIVY18W. We observed leucine binding to all proteins. LS binds L-phenylalanine but the mutation from Trp to Tyr or Phe disallows this ligand and expands the binding repertoire to L-isoleucine and L-valine. The LIVY18W mutant still retains the ability to bind L-isoleucine and also binds L-phenylalanine.

Background: Laparoscopic donor nephrectomy (LDN) preferentially involves the left kidney to optimize vessel length, but occasionally, right nephrectomy is preferred. Right LDN differs markedly in anatomic relations and the need for a fourth port. This retrospective study compares donor outcomes and graft function of right and left LDN and describes the technique., Methods: Consecutive patients undergoing right LDN from March 26, 1996 to December 31, 2000 were compared with those undergoing left LDN. Age, height, weight, body mass index, creatinine, creatinine clearance, operative time, warm ischemia time, analgesic requirements, serial postoperative creatinine, time to diet resumption, and hospital stay were compared. A second cohort matched for age, gender, race, and temporal left LDN also were compared with the group undergoing right LDN., Results: No significant differences were found for any of the parameters measured., Conclusion: This study demonstrates that despite substantial differences in the procedures, donor outcome and graft survival are similar for right and left LDN.

Background: In vitro studies suggest that polymorphonuclear neutrophils (PMN) can damage endothelial cells (EC) by releasing hydrogen peroxide. In vivo this can lead to anasarca secondary to capillary leakage of fluid, protein, and electrolytes. The result is multiple organ dysfunction syndrome, which is associated with high mortality. In vivo, circulating PMN-EC interactions take place in the presence of plasma, and we have shown previously that plasma affords protection to EC from PMN-mediated damage., Methods: Human umbilical vein endothelial cells were primed with cytokines, cultured to a confluent monolayer, and coincubated with normal human PMNs. Cytotoxicity was assayed by gamma scintigraphy, plasma C5 was determined by sepharose column elution, and H(2)O(2) was assayed by R-Phycoerythrin fluorescence., Results: Addition of C5, but not C3, to RPMI resulted in EC cytoprotection equivalent to adding whole serum. Removal of C5 from serum using F(ab')(2) rabbit IgG anti-human C5 coupled to CNBr-activated 4 sepharose beads resulted in significant loss of EC cytoprotection against H(2)O(2)-mediated damage, whereas adding back C5 restored the cytoprotection. C5 also reduced H(2)O(2)-mediated destruction of R-Phycoerythrin., Conclusions: The data suggest that the protection of EC against hydrogen peroxide-mediated damage is partly mediated through complement component C5.

Rho-associated serine/threonine kinase (Rho-kinase) is a downstream effector of small GTPase RhoA that has recently been shown to play an important role in regulating smooth muscle contraction. The present study investigated the role of Rho/ Rho-kinase in hypoxia-induced pulmonary vasoconstriction (HPV). Small pulmonary resistance vessels and cultured pulmonary arterial smooth muscle cells (PASMCs) from the rat were used. PASMCs exposed to hypoxia (PO(2) = 26 +/- 2 mm Hg) showed a significant increase in Rho-kinase activity. Exposure to hypoxia for 20, 40, 60, 90, and 120 min also resulted in a significant increase in myosin light chain (MLC) phosphorylation at all time points in PASMCs. Hypoxia-induced MLC phosphorylation was inhibited by Y-27632 (a Rho-kinase inhibitor), exoenzyme C3 (a specific Rho inhibitor), or toxin B (an inhibitor for Rho proteins). In addition, hypoxia-induced Rho-kinase activation was blocked by C3 and toxin B. Small rat intrapulmonary arterial rings, which were made hypoxic (PO(2) = 30 +/- 3 mm Hg), showed a slow sustained contraction, and Y-27632 caused a significant relaxation during the sustained phase of HPV in a concentration-dependent manner. In summary, the data show that Rho-kinase is activated by hypoxia in PASMCs, and Rho/Rho-kinase is functionally linked to hypoxia-induced MLC phosphorylation and plays a role in the sustained phase of HPV.

We investigated the pulmonary vascular effects of superoxide dismutase (SOD) alone and in combination with inhaled nitric oxide (iNO) in newborn lambs with persistent pulmonary hypertension (PPHN) following prenatal ligation of the ductus arteriosus. In in vitro experiments, pretreatment with SOD significantly enhanced vascular relaxation in response to the NO donor S-nitrosyl-acetylpenicillamine (SNAP) in fifth-generation pulmonary arteries isolated from lambs with PPHN. In vivo treatment of fully instrumented newborn lambs with a single intratracheal dose of recombinant human CuZn SOD (rhSOD; 5 mg/kg) produced selective dilation of the pulmonary circulation. Further studies, of the combination of rhSOD and iNO, showed enhancement of the pulmonary vascular effects of iNO after brief periods of inhalation of 5 ppm and 80 ppm NO. We conclude that rhSOD reduces pulmonary vascular resistance and facilitates the action of iNO in a lamb model of PPHN. This suggests that rhSOD may prove to be an effective adjunctive treatment for newborns with PPHN.

The present study examined the effects of Ca(2+) and strongly bound cross-bridges on tension development induced by changes in the concentration of MgADP. Addition of MgADP to the bath increased isometric tension over a wide range of [Ca(2+)] in skinned fibers from rabbit psoas muscle. Tension-pCa (pCa is -log [Ca(2+)]) relationships and stiffness measurements indicated that MgADP increased mean force per cross-bridge at maximal Ca(2+) and increased recruitment of cross-bridges at submaximal Ca(2+). Photolysis of caged ADP to cause a 0.5 mM MgADP jump initiated an increase in isometric tension under all conditions examined, even at pCa 6.4 where there was no active tension before ADP release. Tension increased monophasically with an observed rate constant, k(ADP), which was similar in rate and Ca(2+) sensitivity to the rate constant of tension re-development, k(tr), measured in the same fibers by a release-re-stretch protocol. The amplitude of the caged ADP tension transient had a bell-shaped dependence on Ca(2+), reaching a maximum at intermediate Ca(2+) (pCa 6). The role of strong binding cross-bridges in the ADP response was tested by treatment of fibers with a strong binding derivative of myosin subfragment 1 (NEM-S1). In the presence of NEM-S1, the rate and amplitude of the caged ADP response were no longer sensitive to variations in the level of activator Ca(2+). The results are consistent with a model in which ADP-bound cross-bridges cooperatively activate the thin filament regulatory system at submaximal Ca(2+). This cooperative interaction influences both the magnitude and kinetics of force generation in skeletal muscle.

1. At low levels of activation, unloaded shortening of skinned skeletal muscle fibres takes place in two phases: an initial phase of high-velocity shortening followed by a phase of low-velocity shortening. The basis for Ca(2+) dependence of unloaded shortening velocity (V(o)) in the low-velocity phase was investigated by varying the level of thin filament activation with Ca(2+) and N-ethyl-maleimide myosin subfragment-1 (NEM-S1), a non-tension-generating, strong binding derivative of subfragment-1. V(o) was measured with the slack-test method. 2. Treatment of skinned fibres with 5 microM NEM-S1 eliminated the low-velocity phase of shortening but had no effect on the high-velocity phase of shortening during submaximal activation with Ca(2+), or on V(o) during maximal activation with Ca(2+). 3. Extensive washout of NEM-S1 from the treated fibres restored the low-velocity phase of shortening and returned low-velocity V(o) to pre-treatment values. 4. The effect of NEM-S1 to increase low-velocity V(o) can be explained in terms of a model in which strong binding myosin cross-bridges activate the thin filament to a state in which the rate of ADP release from the actin-myosin-ADP complex and the rate of cross-bridge detachment from actin are accelerated during unloaded shortening.

Microtubules constitute one of the main cytoskeletal components in eukaryotic cells. Recent studies have shown that microtubule disruption induced significant vasoconstriction or enhanced agonist-induced contraction in vascular smooth muscle. However, the underlying mechanisms are not clear. We hypothesize that microtubule disruption may affect contractile signaling in vascular smooth muscle and lead to the enhanced contraction. The present study demonstrates that both colchicine and nocodazole induced a small but sustained contraction (4-6% P0) in rat aortic rings. This microtubule disruption-induced contraction was abolished by co-treatment with either HA 1077 or Y-27632, both of which are relatively specific Rho-kinase inhibitors. However, co-treatment with ML-9, an inhibitor of myosin light chain kinase, (MLCK) did not have a significant effect on the colchicine-induced contraction. The enhanced KCl-induced contraction due to treatment with colchicine was also blocked by inhibition of Rho-kinase, but not by inhibition of MLCK. These results indicate that microtubule disruption modulates contractile signaling in vascular smooth muscle, mainly through the Rho-kinase pathway, but not MLCK. Interestingly, the colchicine-enhanced, phenylephrine-induced contraction was not completely blocked by inhibition of Rho-kinase suggesting that other signaling pathways might also be involved.

Objective: Recent in vivo studies of both septic humans and animals demonstrate that leukocyte delivery is attenuated to sites remote from the primary infection. The mechanisms for this are not entirely clear. L-selectin is integral to rolling, the first step in leukocyte recruitment to an inflammatory site. L-selectin is shed from leukocytes in sepsis, resulting in increased levels of soluble L-selectin in plasma (2.33 microg/mL). This study investigates the effects of soluble L-selectin at levels found in sepsis on leukocyte trafficking in vivo., Design: Prospective, controlled trial., Setting: Surgical research laboratory in a university hospital., Subjects: Swiss white male mice of 25-35 g., Interventions: Mice were randomized to one of three study groups: soluble L-selectin 2.33, soluble L-selectin 8.0, or albumin. Intravital microscopy was performed on postcapillary venules of 20-40 microm in diameter in the cremaster muscle of mice. Leukocyte-endothelial cell interactions (rolling, adherence, and rolling velocity) were measured pre- and post- (1, 15, 30, and 45 mins) intravenous infusion of human recombinant soluble L-selectin (2.33 and 8.0 microg/mL) or human albumin (8.0 microg/mL)., Measurements and Main Results: The intravenous administration of soluble L-selectin to a systemic concentration of 2.33 microg/mL diminished rolling significantly. Soluble L-selectin at 8.0 microg/mL decreased rolling and increased rolling velocity to a greater degree. Injection of albumin did not alter leukocyte-endothelial cell interactions at any time point. No difference between groups in blood pressure, shear rate, or leukocyte counts was detected., Conclusions: Soluble L-selectin diminishes leukocyte rolling at levels present in sepsis (2.33 microg/mL). This effect is dose dependent, and could not be explained by differences in blood pressure, shear rate, or leukocyte counts. These findings identify increased soluble L-selectin levels as one of the mechanisms for decreased leukocyte delivery and exudation to remote sites in septic patients.

Chronic alveolar hypoxia is the major cause of pulmonary hypertension. The cellular mechanisms involved in hypoxia- induced pulmonary arterial remodeling are still poorly understood. Mitogen-activated protein kinase (MAPK) is a key enzyme in the signaling pathway leading to cellular growth and proliferation. The purpose of this investigation was to determine the roles that MAPKs, specifically Jun-N-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 kinase, play in the hypoxia-induced pulmonary arterial remodeling. Rats were exposed to normobaric hypoxia (10% O(2)) for 1, 3, 7, or 14 d. Hypoxia caused significant remodeling in the pulmonary artery characterized by thickening of pulmonary arterial wall and increases in tissue mass and total RNA. JNK, ERK, and p38 kinase tyrosine phosphorylations and their activities were significantly increased by hypoxia. JNK activation peaked at Day 1 and ERK/p38 kinase activation peaked after 7 d of hypoxia. The results from immunohistochemistry show that hypoxia increased phospho-MAPK staining in both large and small intrapulmonary arteries. Hypoxia also upregulated vascular endothelial growth factor messenger RNA (mRNA) and platelet-derived growth factor receptor mRNA levels in pulmonary artery with a time course correlated to the activation of ERK and p38 kinase. The gene expressions of c-jun, c-fos, and egr-1, known as downstream effectors of MAPK, were also investigated. Hypoxia upregulated egr-1 mRNA but downregulated c-jun and c-fos mRNAs. These data suggest that hypoxia-induced activation of JNK is an early response to hypoxic stress and that activation of ERK and p38 kinase appears to be associated with hypoxia-induced pulmonary arterial remodeling.

Background: Previous in vitro studies have demonstrated that the host response to intra-abdominal infection produces increased generalized polymorphonuclear neutrophil (PMN) adherence to vascular endothelial cells (ECs), which may lead to subsequent endothelial damage, leaky capillaries, and organ dysfunction. There are scant data to demonstrate this enhanced systemic PMN adherence in vivo or the influence of PMN rolling on PMN endothelial adherence., Hypothesis: Systemic PMN adherence in the animal with sepsis is increased., Design: In vivo murine model of a 2-front infection using intravital microscopy of the cremasteric muscle to quantify PMN-EC adherence in a septic response., Setting: Basic science laboratory and animal surgical facility., Patients or Other Participants: One hundred CD1 male mice., Interventions: Animals underwent cecal ligation and puncture peritonitis, cremasteric muscle Escherichia coli infection, both infections, or neither (controls). Eighteen hours later, the mice underwent exteriorization of the cremasteric muscle under an intravital microscope for measurement of PMN-EC interactions. Blood was then drawn for calculation of circulating PMN counts., Main Outcome Measures: Adherence of PMNs, PMN rolling flux, PMN rolling velocity, and circulating PMN counts., Results: Circulatory mechanics did not differ between the groups. Unlike static in vitro systems, we could not detect an increase in PMN adherence after peritonitis with this dynamic in vivo model. A local (cremasteric) infection was associated with marked PMN adherence. Peritonitis was associated with reduced PMN adherence at a local infection site as well as reduced rolling adhesion and PMN rolling velocity., Conclusions: The data suggest that intra-abdominal infection does not increase remote PMN adherence, and may actually result in reduction of systemic adherence via modulation of PMN rolling.

Contraction of skeletal muscle is regulated by calcium at the level of the thin filament via troponin and tropomyosin. Studies have indicated that strong cross-bridge binding is also involved in activation of the thin filament. To further test this, myofibrils were incubated with a wide range of fluorescent myosin subfragment 1(fS1) at pCa 9 or pCa 4 with or without ADP. Sarcomere fluorescence intensity and the fluorescence intensity ratio (non-overlap region/overlap region) were measured to determine the amount and location of bound fS1 in the myofibril. There was lower sarcomere fluorescence intensity with ADP compared to without ADP for both calcium levels. Similar data were obtained from biochemical measures of bound fS1, validating the fluorescence microscopy measurements. The intensity ratio, which is related to activation of the thin filament, increased with increasing [fS1] with or without ADP. At pCa 9, the fluorescence intensity ratio was constant until 80-160 nM fS1 without ADP conditions, then it went up dramatically and finally attained saturation. The dramatic shift of the ratio demonstrated the cooperative character of strong cross-bridge binding, and this was not observed at high calcium. A similar pattern was observed with ADP in that the ratio was right-shifted with respect to total [fS1]. Saturation was obtained with both the fluorescence intensity and ratio data. Plots of intensity ratio as a function of normalized sarcomere intensity (bound fS1) showed little difference between with and without ADP. This suggests that the amount of strongly bound fS1, not fS1 state (with or without ADP) is related to activation of the thin filament.

The aim of the present study was to assess with ultrasound the ductus venosus flow velocity in newborn lambs with increasing pulmonary artery pressures and to evaluate whether this is a useful method to detect elevated pulmonary artery pressure. The ductus venosus flow velocity was studied with pulsed-wave Doppler echocardiography in nine newborn lambs < or = 30 h old. The lambs were anesthetized, mechanically ventilated, and instrumented to measure mean airway pressure and pulmonary artery and arterial blood pressures. A vascular occluder was placed around the main pulmonary artery. With mean pressures ranging from 20 to 50 mm Hg in the pulmonary artery, the ductus venosus flow velocity was examined. In seven lambs, the mean portal pressure and central venous pressure were also measured. With a stepwise increase in the pulmonary artery pressure, the minimum ductus venosus flow velocity during atrial systole decreased to a reversed flow, and the duration of this reversed flow component increased. The systolic forward peak flow velocity signal also gradually decreased. No changes were detected in the mean central venous or in the portal pressure with increasing pulmonary artery pressure or changes in ductus venosus flow. The flow velocity in the ductus venosus, which is higher than in other precordial veins, shows a reduction and even reversal of the nadir and an increase of the duration of reversed flow during atrial systole as a response to increased pulmonary artery pressure. Thus, Doppler ultrasound of the ductus venosus flow velocity may be a useful noninvasive diagnostic supplement to detect pulmonary hypertension of the newborn.