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Polycystic ovary syndrome (PCOS) represents major endocrine and metabolic disorder among women largely characterized by hyperandrogenism and oligomenorrhea precipitates serious complications such as type 2 diabetes, early atherosclerosis, infertility, and endometrial cancer. Several etiological theories were proposed to define the exact cause of the PCOS, which is characterized, by the hypothalamic-pituitary axis, ovarian morphology, and release of adrenal steroid hormones, metabolic syndrome, and hereditary factors. The review explored the role of dysbiosis and the mechanisms through which microbial dysbiosis can affect PCOS development. In recent time, various research groups highlighted the role of microbial gut dysbiosis associated with obesity as potential etiological factor for the PCOS. In the present review, we reviewed the mechanisms attributed to the microbial dysbiosis and treatment approaches to deal with the situation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Retinal drug delivery has witnessed significant advancements in recent years, mainly driven by the prevalence of retinal diseases and the need for more efficient and patient-friendly treatment strategies., Areas Covered: Advancements in nanotechnology have introduced novel drug delivery platforms to improve bioavailability and provide controlled/targeted delivery to specific retinal layers. This review highlights various treatment options for retinal diseases. Additionally, diverse strategies aimed at enhancing delivery of small molecules and antibodies to the posterior segment such as implants, polymeric nanoparticles, liposomes, niosomes, microneedles, iontophoresis and mixed micelles were emphasized. A comprehensive overview of the special technologies currently under clinical trials or already in the clinic was provided., Expert Opinion: Ideally, drug delivery system for treating retinal diseases should be less invasive in nature and exhibit sustained release up to several months. Though topical administration in the form of eye drops offers better patient compliance, its clinical utility is limited by nature of the drug. There is a wide range of delivery platforms available, however, it is not easy to modify any single platform to accommodate all types of drugs. Coordinated efforts between ophthalmologists and drug delivery scientists are necessary while developing therapeutic compounds, right from their inception.

Purpose: Some systemic medications are reported to be associated with dry eye disease (DED), yet their associations with the severity of DED signs and symptoms are not well studied. To evaluate these associations, we performed a secondary analysis of data from the DRy Eye Assessment and Management (DREAM) Study., Methods: Participants (N = 535) were assessed for DED signs using tear break-up time (TBUT), Schirmer testing, corneal fluorescein staining, conjunctival lissamine green staining, meibomian gland dysfunction (MGD), and tear osmolarity and DED symptoms using the Ocular Surface Disease Index (OSDI). We derived a composite signs severity score from the 6 DED signs and categorized participant-reported systemic medications into antidepressants, antihistamines, aspirin, corticosteroids, diuretics, nonsteroidal anti-inflammatory drugs, proton pump inhibitors, statins, vitamin D3, and medications for diabetes mellitus, hypertension, hypothyroidism, migraine, and seizure. Generalized linear models were used to compare DED symptom and sign scores between medication users and non-users, with adjustment for factors associated with DED severity., Results: Compared to non-users, antihistamine users had lower TBUT (p = 0.01) and higher OSDI score (p = 0.02); aspirin users had lower TBUT (p = 0.02); corticosteroid users had lower TBUT (p = 0.02), lower Schirmer test scores (p = 0.03), higher cornea fluorescein staining (p = 0.01), higher composite severity score (p = 0.01), and higher OSDI score (p = 0.03); seizure medication users had higher composite severity score (p = 0.02); vitamin D3 users had lower TBUT (p = 0.001) and greater MGD (p = 0.03); and diuretic users had less MGD (p = 0.03)., Conclusions: Certain systemic medications may be associated with more severe DED. This may guide prescription practices in patients with DED., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Uridine 5'-diphospho-glucuronosyltransferases (UGTs) have been considered as a family of enzymes responsible for the glucuronidation process, a crucial phase II detoxification reaction. Among the various UGT isoforms, UGTs A10 and B7 have garnered significant attention due to their broad substrate specificity and involvement in the metabolism of numerous compounds. Recent studies have suggested that certain vitamins may exert inhibitory effects on UGT activity, thereby influencing the metabolism of drugs, environmental toxins, and endogenous substances, ultimately impacting their biological activities. In the present study, the inhibition potential of vitamins (A, B1, B2, B3, B5, B6, B7, B9, D3, E, and C) on UGT1A10 and UGT2B7 was determined using in silico and in vitro approaches. A 3-dimensional model of UGT1A10 and UGT2B7 enzymes was built using Swiss Model, ITASSER, and ROSETTA and verified using Ramachandran plot and SAVES tools. Molecular docking studies revealed that vitamins interact with UGT1A10 and UGT2B7 enzymes by binding within the active site pocket and interacting with residues. Among all vitamins, the highest binding affinity predicted by molecular docking was - 8.61 kcal/mol with vitamin B1. The in vitro studies results demonstrated the inhibition of the glucuronidation activity of UGTs by vitamins A, B1, B2, B6, B9, C, D, and E, with IC 50 values of 3.28 ± 1.07 µg/mL, 24.21 ± 1.11 µg/mL, 3.69 ± 1.02 µg/mL, 23.60 ± 1.08 µg/mL, 6.77 ± 1.08 µg/mL, 83.95 ± 1.09 µg/ml, 3.27 ± 1.13 µg/mL and 3.89 ± 1.12 µg/mL, respectively. These studies provided the valuable insights into the mechanisms underlying drug-vitamins interactions and have the potential to guide personalized medicine approaches, optimizing therapeutic outcomes, and ensuring patient safety. Indeed, further research in the area of UGT (UDP-glucuronosyltransferase) inhibition by vitamins is essential to fully understand the clinical relevance and implications of these interactions. UGTs play a crucial role in the metabolism and elimination of various drugs, toxins, and endogenous compounds in the body. Therefore, any factors that can modulate UGT activity, including vitamins, can have implications for drug metabolism, drug-drug interactions, and overall health., Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00182-0., Competing Interests: Conflict of interestThe authors have no conflicts of interest to declare that are relevant to the content of this article., (© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

In December 2019, an outbreak emerged of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which leads to coronavirus disease 2019 (COVID-19). The World Health Organisation announced the outbreak a global health emergency on 30 January 2020 and by 11 March 2020 it was declared a pandemic. The spread and severity of the outbreak took a heavy toll and overburdening of the global health system, particularly since there were no available drugs against SARS-CoV-2. With an immediate worldwide effort, communication, and sharing of data, large amounts of funding, researchers and pharmaceutical companies immediately fast-tracked vaccine development in order to prevent severe disease, hospitalizations and death. A number of vaccines were quickly approved for emergency use, and worldwide vaccination rollouts were immediately put in place. However, due to several individuals being hesitant to vaccinations and many poorer countries not having access to vaccines, multiple SARS-CoV-2 variants quickly emerged that were distinct from the original variant. Uncertainties related to the effectiveness of the various vaccines against the new variants as well as vaccine specific-side effects have remained a concern. Despite these uncertainties, fast-track vaccine approval, manufacturing at large scale, and the effective distribution of COVID-19 vaccines remain the topmost priorities around the world. Unprecedented efforts made by vaccine developers/researchers as well as healthcare staff, played a major role in distributing vaccine shots that provided protection and/or reduced disease severity, and deaths, even with the delta and omicron variants. Fortunately, even for those who become infected, vaccination appears to protect against major disease, hospitalisation, and fatality from COVID-19. Herein, we analyse ongoing vaccination studies and vaccine platforms that have saved many deaths from the pandemic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chavda, Yao, Vora, Apostolopoulos, Patel, Bezbaruah, Patel and Chen.)

The gas-phase unimolecular reactions of C 2 D 5 CHFCl molecules with 94 kcal mol -1 of vibrational energy have been studied by the chemical-activation experimental technique and by electronic-structure computations. Products from the reaction of C 2 D 5 CHFCl molecules, formed by the recombination of C 2 D 5 and CHFCl radicals in a room temperature bath gas, were measured by gas chromatography-mass spectrometry. The 2,1-DCl (81%) and 1,1-HCl (17%) elimination reactions are the principal processes, but 2,1-DF and 1,1-HF elimination reactions also are observed. Comparison of experimental rate constants to calculated statistical rate constants provides threshold energies. The potential surfaces associated with C 2 D 5 (F)C: + HCl and C 2 D 5 (Cl)C: + HF reactions are of special interest because hydrogen-bonded adducts with HCl and HF with dissociation energies of 6.4 and 9.3 kcal mol -1 , respectively, are predicted by calculations. The relationship between the geometries and threshold energies of transition states for 1,1-HCl elimination and carbene:HCl adducts is complex, and previous studies of related molecules, such as CD 3 CHFCl, CD 2 ClCHFCl, C 2 D 5 CHCl 2 , and halogenated methanes are included in the computational analysis. Extensive calculations for CH 3 CHFCl as a model for 1,1-HCl reactions illustrate properties of the exit-channel potential energy surface. Since the 1,1-HCl transition state is submerged relative to dissociation of the adduct, inner and outer transition states should be considered for analysis of rate constants describing 1,1-HCl elimination and addition reactions of carbenes to HCl.

Objective: This study aimed to describe real-world experiences following a non-medical switch among adults with type 2 diabetes mellitus (T2DM) in the United States., Methods: For this cross-sectional study, patients with T2DM (N = 451) provided data on demographics, and how a non-medical switch of their anti-hyperglycemic agent (AHA) affected their general health, HbA1c levels and medication management, via an Internet-based survey. Patients self-reported their level of satisfaction with the original medication and emotional reactions to the non-medical switch. Patients who recently experienced a non-medical switch of their AHA(s) (n = 379) were asked about the consequences of switching and their satisfaction with the switch (vs. the original) medication., Results: Patients most frequently reported feeling very/extremely frustrated, surprised, upset and angry in reaction to a non-medical switch. Patients were somewhat satisfied with their original medication. Between 20% and 30% of patients reported the non-medical switch had a moderate/major effect on their general health, diabetes, mental well-being and control over their health. The blood glucose levels of recent switchers were somewhat/much worse (20.7%) and medication management was somewhat/much worse (12.9%) on the switch (vs. the original) medication. Some recent switchers reported old symptoms returning (7.7%) and experiencing new side-effects (14.2%)., Conclusions: Approximately one in five patients reported a moderate/major negative impact on their blood glucose level, diabetes, mental well-being, general health and control over their health following a non-medical switch. Findings suggest that a non-medical switch may have unintended negative health consequences and results in considerable burden across multiple domains for a sizeable minority of patients with T2DM.

Background: The randomized, double-blind CANTATA-SU (CANagliflozin Treatment And Trial Analysis Sulfonyl Urea) clinical trial compared the use of canagliflozin (100 mg or 300 mg) and maximally tolerated glimepiride (6-8 mg) over 104 weeks as add-on therapy for patients with type 2 diabetes mellitus (T2DM) inadequately controlled with metformin. Compared with glimepiride, canagliflozin use was associated with durable reductions in glycated hemoglobin (A1C), blood pressure (BP), and body weight. The aim of this post-hoc analysis of the CANTATA-SU trial was to assess the comparative efficacy of canagliflozin and glimepiride in the attainment of recently updated diabetes-related quality measures (QMs) for up to 104 weeks of treatment., Methods: This post-hoc analysis evaluated the proportions of patients achieving individual diabetes-related QMs using data from the randomized, double-blind, Phase 3 CANTATA-SU trial. Change in A1C from baseline, and proportions of the study population achieving QMs: A1C <7.0 %, <8.0 %, and >9.0 % were assessed. Secondary endpoints included change in BP from baseline, and the proportions of the study population achieving QMs related to BP and body weight., Results: The proportions of patients in the canagliflozin 100 mg, canagliflozin 300 mg, and glimepiride groups meeting criteria for all QMs were similar at baseline. At 52 and 104 weeks of treatment, canagliflozin 100 mg and canagliflozin 300 mg provided better or similar reductions in A1C from baseline and achievement of glycemic control QMs compared with glimepiride. At 52 and 104 weeks of treatment, the attainment of QMs related to reductions in body weight and BP all favored canagliflozin compared with glimepiride. Canagliflozin was associated with lower incidence of documented hypoglycemia and severe hypoglycemia compared with glimepiride., Conclusions: Using the recently adjusted and currently accepted diabetes-related QMs, this analysis observed superior glycemic control with canagliflozin compared with maximally tolerated glimepiride in patients with T2DM who were previously poorly controlled on metformin monotherapy. Compared with maximally tolerated glimepiride, canagliflozin resulted in better achievement of diabetes-related QMs related to weight loss and BP, and was associated with lower incidences of hypoglycemic events., Trial Registration: Clinical trial registry name: CANagliflozin Treatment And Trial Analysis-Sulfonylurea (CANTATA-SU) SGLT2 Add-on to Metformin Versus Glimepiride., Clinical Trial Registration Number: NCT00968812 , registered August 28, 2009.

Objective: Probiotic strain Lactobacillus sporogenes and Bifidobacteria bifidum were used to assess the anti-inflammatory properties in Carrageenan induced acute inflammatory model., Methods: Non-encapsulated and encapsulated Probiotic strain of Bifidobacteria bifidum and Lactobacillus sporogenes was given orally. Diclofenac sodium was used as standard drug at a concentration of 150 mg/kg of body weight. Edema was induced with 1% carrageenan to all the groups except group A after half an hour of the oral treatments. Paw thickness was checked at t = 1, 2, 4 and 24 h. Stair climbing score and motility score were assessed at t = 24 h., Results: Non-encapsulated and encapsulated Probiotic Bifidobacteria and Lactobacillus showed a statistically significant decrease in paw thickness at P < 0.05. The percentage inhibition in paw thickness of non-encapsulated and encapsulated probiotic L. sporogenes and B. bifidum is 37 ± 3% and 43 ± 2% after 24 h of treatment. They both significantly increased stair climbing and motility score., Conclusion: Probiotic B. bifidum and L. sporogenes significantly decreased the inflammatory reactions induced by carrageenan.

Objectives: We compared the efficacy and safety of alcohol septal ablation (ASA) for obstructive hypertrophic cardiomyopathy (HCM) in young, middle-aged, and elderly patients., Background: Intersociety guidelines suggest based on limited evidence that young patients with medically refractory symptoms of obstructive HCM should undergo surgical myectomy while elderly patients may be more appropriate for ASA., Methods: Data for 360 patients undergoing 389 ASAs were prospectively collected and retrospectively analyzed according to age., Results: Young (<45 years), middle-aged (45-64 years), and elderly (≥65 years) patients comprised 28, 40, and 32% of the study population, respectively. Young patients had thicker left ventricular septal walls at baseline, and elderly patients had more comorbidity and dyspnea. Resting, mean left ventricular outflow tract gradients (LVOTGs) were similar across the age groups at baseline (62, 66, and 68 mm Hg, respectively; P = NS for all comparisons). LVOTGs and dyspnea were significantly and similarly improved in all age groups immediately after ASA and through 12 months of follow-up (P < 0.001 for before and after comparisons; P = NS for intergroup comparisons). Complication rates were similar for young and middle-aged patients but higher for elderly patients (9.1 and 6.3% vs. 20.8%, respectively; P ≤ 0.016 for elderly vs. others). Mortality rates for young and middle-aged patients were lower than for elderly patients, but the differences were not statistically significant., Conclusions: Patients undergoing ASA had significant and similar improvements in LVOTGs and symptoms regardless of age. Procedural complications were increased in elderly patients, who had numerically but not statistically significantly higher mortality rates., (Copyright © 2012 Wiley Periodicals, Inc.)

An 18 yr old female referred to us as hypertrophic obstructive cardiomyopathy with bicuspid aortic valve. On Initial history and examination patient also had primary amenorrhoea, differential cyanosis in lower limbs, differential clubbing, polydactyly, syndactyly, high arched foot, pectus carinatum and scoliosis. Oxygen saturation was 94% at room air and complete blood count was suggestive of polycythaemia (Hb 20 g/dl, Hct 60%, Tc-16500, RBC count--6.29 million/cumm, Platelet count--1,88000). Imaging studies were done, ultrasonography showed absent uterus, absent left kidney, right ectopic kidney.X-ray foot showed 6th metatarsal with phalanx. 2D ECHO was suggestive of Patent Ductus Arteriosus with reversal of shunt with severe aortic stenosis and bicuspid aortic valve. All these anomalies form part of a syndrome complex called MURCS ASSOCIATION (Mullerian agenesis/aplasia, renal anomalies, and Cervicothoracic Somite deformities).

Background: Fractional flow reserve (FFR), the hyperemic ratio of distal (Pd) to proximal (Pa) coronary pressure, is used to identify the need for coronary revascularization. Changes in left ventricular end-diastolic pressure (LVEDP) might affect measurements of FFR., Methods and Materials: LVEDP was recorded simultaneously with Pd and Pa during conventional FFR measurement as well as during additional infusion of nitroprusside. The relationship between LVEDP, Pa, and FFR was assessed using linear mixed models., Results: Prospectively collected data for 528 cardiac cycles from 20 coronary arteries in 17 patients were analyzed. Baseline median Pa, Pd, FFR, and LVEDP were 73 mmHg, 49 mmHg, 0.69, and 18 mmHg, respectively. FFR<0.80 was present in 14 arteries (70%). With nitroprusside median Pa, Pd, FFR, and LVEDP were 61 mmHg, 42 mmHg, 0.68, and 12 mmHg, respectively. In a multivariable model for the entire population LVEDP was positively associated with FFR such that FFR increased by 0.008 for every 1-mmHg increase in LVEDP (beta=0.008; P<0.001), an association that was greater in obstructed arteries with FFR<0.80 (beta=0.01; P<0.001). Pa did not directly affect FFR in the multivariable model, but an interaction between LVEDP and Pa determined that LVEDP's effect on FFR is greater at lower Pa., Conclusions: LVEDP was positively associated with FFR. The association was greater in obstructive disease (FFR<0.80) and at lower Pa. These findings have implications for the use of FFR to guide revascularization in patients with heart failure., Summary for Annotated Table of Contents: The impact of left ventricular diastolic pressure on measurement of fractional flow reserve (FFR) is not well described. We present a hemodynamic study of the issue, concluding that increasing left ventricular diastolic pressure can increase measurements of FFR, particularly in patients with FFR<0.80 and lower blood pressure., (© 2013.)

Background- The prevalence of ischemia and its prediction of events are unclear in outpatients with diabetes mellitus in the modern era of intensive medical management. We sought to identify the prevalence of ischemia, subsequent cardiac events, and impact of sex, stress type, and symptom status on these findings in a cohort of stable outpatients with diabetes mellitus referred for single-photon emission computed tomography myocardial perfusion imaging (MPI). Methods and Results- The study cohort included 575 consecutive outpatients with diabetes mellitus who underwent quantitative, gated single-photon emission computed tomography MPI. Clinical information, stress MPI variables, and cardiac events were prospectively collected and analyzed. The study population was at intermediate risk of coronary artery disease or had known coronary artery disease (40.3%); 29% of patients were asymptomatic at the time of stress testing. Scintigraphic ischemia and significant (≥10%) left ventricular ischemia were present in 126 patients (21.9%) and 29 patients (5.0%), respectively, and <1% of patients had early revascularization. The risk of ischemia was increased >2-fold by male sex (P<0.001), but was not impacted by pharmacological stress (P=0.15) or presence of symptoms (P=0.89). During a median 4.4 years follow-up, the rate of cardiac death/nonfatal myocardial infarction was moderate at 2.6%/y (cardiac death 0.8%/y) in the total cohort, but was 5.7%/y in those with ischemia (P<0.001). Pharmacological stress predicted a higher cardiac event rate (P<0.001) but symptoms did not (P=0.55). Conclusions- This cohort of stable outpatients with diabetes mellitus referred for single-photon emission computed tomography had low rates of significant ischemia and early revascularization; an initially low cardiac event rate increased after 2 years. Independent predictors of cardiac death/nonfatal myocardial infarction were known coronary artery disease, pharmacological stress, and MPI ischemia. Nearly one third of those with events had a normal MPI, indicating a need for improved risk stratification.

This study sought to examine the safety of percutaneous coronary intervention (PCI) before and during de novo establishment of a transradial (TR) program at a teaching hospital. TR access remains underused in the United States, where cardiology fellowship programs continue to produce cardiologists with little TR experience. Establishment of TR programs at teaching hospitals may affect PCI safety. Starting in July 2009 a TR program was established at a teaching hospital. PCI-related data for academic years 2008 to 2009 (Y1) and 2009 to 2010 (Y2) were prospectively collected and retrospectively analyzed. Of 1,366 PCIs performed over 2 years, 0.1% in Y1 and 28.7% in Y2 were performed by TR access. No major complications were identified in 194 consecutive patients undergoing TR PCI, and combined bleeding and vascular complication rates were lower in Y2 versus Y1 (0.7% vs 2.0%, p = 0.05). Patients treated in Y2 versus Y1 and by TR versus transfemoral approach required slightly more fluoroscopy but similar contrast volumes and had similar procedural durations, lengths of stay, and predischarge mortality rates. PCI success rates were 97% in Y1, 97% in Y2, and 98% in TR cases. TR PCIs were performed by 13 cardiology fellows and 9 attending physicians, none of whom routinely performed TR PCI previously. In conclusion, de novo establishment of a TR program improved PCI safety at a teaching hospital. TR programs are likely to improve PCI safety at other teaching hospitals and should be established in all cardiology fellowship training programs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: The clinical significance of isolated calf vein thrombosis (ICVT) remains controversial. Several studies have shown that the majority of ICVT do not propagate above the knee while other studies have suggested ICVT propagation and recommend full anticoagulation. The purpose of this study was to determine the progression of ICVT, identify risk factors for clot propagation, and to evaluate further thrombotic events associated with it., Methods: This study consisted of 156 patients and a total of 180 limbs. All patients included had ICVT involving either the tibial, peroneal, gastrocnemius, or the soleal vein. After initial diagnosis, all patients were started on prophylactic dose of low molecular weight heparin (LMWH) or unfractionated heparin, unless already anticoagulated. All limbs were monitored using duplex ultrasonography scans at intervals of 2 to 3 days, 1 to 3 months, and 6 to 8 months from the initial time of diagnosis. Outcomes examined included lysis of clot, propagation to a proximal vein, and pulmonary emboli., Results: ICVT was detected in 180 limbs of 156 patients. No significant difference was noted in the gender of the patients or limb preference. Twenty-four patents had both limbs involved. The mean age was 77 years old and the mean follow-up was 5.1 months. The soleal vein was most commonly involved. The second most common vein involved was peroneal, followed by posterior tibial and then gastrocnemius. The least commonly involved vein was the anterior tibial with only one positive result on each side. Fifteen of 180 limbs (9%) had complete resolution of the thrombus within 72 hours. Of these, six were anticoagulated to a therapeutic level. All patients had a follow-up duplex scan within 1 to 3 months' time, and none had recurrence. At the 1 to 3-month follow-up, 11 of 180 patients (7%) had propagation to a proximal vein; all of whom were in a high-risk group to develop a deep vein thrombosis (DVT), either after an orthopedic procedure, stroke, or malignancy. Nine of 156 patients developed a pulmonary emboli also diagnosed within the 1 to 3-months' time period. At the 6 to 8-month follow-up, there was no further propagation of any additional limbs and no further incidences of pulmonary emboli., Conclusion: ICVT can be safely observed in asymptomatic patients without therapeutic anticoagulation. In our study, patients who have had orthopedic procedures, those with malignancy, and those that were immobile seemed to have a higher incidence of clot propagation. In this group, we recommend full anticoagulation until the patient is ambulatory or the follow-up duplex scan is negative. Our data also suggest that a follow-up duplex scan is not beneficial when performed within 72 hours or after 3 months., (Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Sea buckthorn (Hippophae rhamnoides L.) constitutes thorny nitrogen fixing deciduous shrub. Sea buckthorn(SBT) is primarily valued for its very rich vitamins A, B(1), B(12), C, E, K, and P; flavonoids, lycopene, carotenoids, and phytosterols. and therapeutically important since it is rich with potent antioxidants. Scientifically evaluated pharmacological actions of SBT are like inflammation inhibited by reduced permeability, loss of follicular aggregation of lymphocytes from the inflamed synovium and suppress lymphocyte proliferation. SBT-reduced recurrence of angina, ischemic electrocardiogram which might be due to decreased myocardial oxygen consumption and inhibition of platelet aggregation induced by collagen. SBT can kill both cancer cells of S180, P388, SGC7901 and lymphatic leukemia (L1200). The antiulcer activity may be related to reduce gastric empty time, inhibiting proteolytic activity and promoting wound reparation processes of mucosa. SBT exerts antihypertensive effect in part by blocking angiotensin-2 receptor on cell surface. SBT decreased the level of stress hormones and enhanced hypoxic tolerance in animals indicating its anti-stress, adaptogenic activity. A lot of research work is still needed to find cellular and molecular mechanisms of these activities and also yet to be explored for its activity in osteoporosis, hemorrhage, cataract, urinary stone, acne, psoriasis, polyneuritis, cheilosis, glossities, baldness, anti-obesity, gout, and chronic prostitis.

We created a cross-species display system that allows the display of the same antibody libraries on both prokaryotic phage and eukaryotic yeast without the need for molecular cloning. Using this cross-display system, a large, diverse library can be constructed once and subsequently used for display and selection in both phage and yeast systems. In this article, we performed the parallel phage and yeast selection of an antibody maturation library using this cross-display platform. This parallel selection allowed us to isolate more unique hits than single-species selection, with 162 unique clones from phage and 107 unique clones from yeast. In addition, we were able to shuttle yeast hits back to Escherichia coli cells for affinity characterization at a higher throughput.

A novel adapter-directed yeast display system with modular features was developed. This display system consists of two modules, a display vector and a helper vector, and is capable of displaying proteins of interest on the surface of Saccharomyces cerevisiae through the interaction of two small adapters that are expressed from the display and helper vectors. In this report, an anti-VEGF scFv antibody gene was cloned into the display vector and introduced alone into yeast S. cerevisiae cells. This led to the expression and secretion of a scFv antibody that was fused in-frame with the coiled-coil adapter GR1. For display purposes, a helper vector was constructed to express the second coiled-coil adapter GR2 that was fused with the outer wall protein Cwp2, and this was genetically integrated into the yeast genome. Co-expression of the scFv-GR1 and GR2-Cwp2 fusions in the yeast cells resulted in the functional display of anti-VEGF scFv antibodies on the yeast cell surfaces through pairwise interaction between the GR1 and GR2 adapters. Visualization of the co-localization of GR1 and GR2 on the cell surfaces confirmed the adapter-directed display mechanism. When the adapter-directed phage and yeast display modules are combined, it is possible to expand the adapter-directed display to a novel cross-species display that can shuttle between phage and yeast systems., (2010 Elsevier B.V. All rights reserved.)

Beta-amyloid (Abeta) degrading endopeptidases are thought to protect against Alzheimer's disease (AD) and are potentially therapeutic. Of particular interest are endopeptidases that are blocked by thiorphan and phosphoramidon (T/P), as these inhibitors rapidly induce Abeta deposition in rodents. Neprilysin (NEP) is the best known target of T/P; however neprilysin knockout results in only modest Abeta increases insufficient to induce deposition. Therefore, other endopeptidases targeted by T/P must be critical for Abeta catabolism. Another candidate is the T/P sensitive membrane metallo-endopeptidase-like protein (MMEL), a close homolog of neprilysin. The endopeptidase properties of beta and gamma splice forms of human MMEL were determined in HEK293T cells transduced with the human cDNAs for the two splice forms; this showed degradation of both Abeta(42) and Abeta(40) by hMMEL-beta but not hMMEL-gamma. hMMEL-beta activity was found at the extracellular surface with no significant secreted activity. hMMEL-gamma was not expressed at the extracellular surface. Finally, it was found that hMMEL cleaves Abeta near the alpha-secretase site (producing Abeta(1-17)>>Abeta(1-16)). These data establish hMMEL as a mediator of Abeta catabolism and raise the possibility of its involvement in the etiology of AD and as a target for intervention.

The selectivity of different Rho kinase (ROCK) inhibitors in the spontaneously tonic smooth muscle has not been investigated. We examined this issue using Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarbox anecarboxamide, 2HCl], H-1152 [(S)-(+)-(2-methyl-5-isoquinolinyl) sulfonylhomopiperazine, 2HCl], HA-1077 [(5 isoquinolinesulfonyl) homopiperazine, 2HCl], and ROCK inhibitor II [N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea]. We compared these inhibitors in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). ROCK, protein kinase C (PKC), and myosin light chain kinase (MLCK) activities were determined in the IAS, before and after different ROCK inhibitors. Y-27632 and H-1152 were approximately 30-fold more potent in the IAS (IC(50): 4.4 x 10(-7) and 7.9 x 10(-8) M, respectively) vs. the phasic rectal smooth muscle (RSM) (IC(50): 1.3 x 10(-5) and 2.5 x 10(-6) M, respectively). HA-1077 and ROCK inhibitor II were equipotent in the IAS vs. RSM. In the IAS, H-1152 was the most potent whereas ROCK inhibitor II is the least. Y-27632 and H-1152 caused concentration-dependent decrease in the IAS tone that correlates directly with the decreases in ROCK activity, without significant effect in the PKC and MLCK activities. This specifically selective correlation between ROCK activity and decrease in the IAS tone was absent in the case of HA-1077 and ROCK inhibitor II, which also inhibited PKC and MLCK. We conclude that the IAS tone is critically dependent on ROCK activity, and H-1152 and Y-27632 are the most selective and potent ROCK inhibitors in the IAS.

Vasoactive intestinal peptide and pituitary adenylate cyclase activating peptide have high affinity for VPAC1, VPAC2 and PAC1 receptors overexpressed on human cancer cells. Four potent analogues of these peptides, TP3939, TP3982, TP4200 and TP3805 were labeled with (64)Cu and evaluated ex vivo and in vivo to asses their biological activity and receptor specificity. The ultimate goal is to utilize (64)Cu analogues for positron emission tomography (PET) imaging of breast cancers in humans. Radiochemical purity of each analogue was >92%. The muscle relaxivity assay revealed IC(50) to be 5.3x10(-8) M, 4.4x10(-8) M, 8.1x10(-8) M, 8.1x10(-9) M and Kd values determined by receptor specific cell binding assays were 3.3 nM, 0.33 nM, 0.2 nM and 0.72 nM for TP3805, TP3939, TP3982, and TP4200 respectively. The receptor affinity, using human breast cancer tissues, was 10.93 times greater than normal breast tissues. RT-PCR confirmed increased VPAC1 receptor expression on human breast tumor cells over normal cells and corroborated with autoradiography data. The blood clearance was rapid and in vivo translocation of (64)Cu to plasma protein was <15%. Data demonstrate that these analogues are potent, have uncompromised biological activity and are worthy of further evaluation for accurate PET imaging of human breast cancers and in determining malignant and benign lesions.

Sustained contractions of smooth muscle cells (SMC) maintain basal tone in the internal anal sphincter (IAS). To examine the molecular bases for the myogenic tone in the IAS, the present studies focused on the role of RhoA/ROCK in the SMC isolated from the IAS vs. the adjoining phasic tissues of the rectal smooth muscle (RSM) and anococcygeus smooth muscle (ASM) of rat. We also compared cellular distribution of RhoA/ROCK, levels of RhoA-GTP, RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex formation, levels of p(Thr696)-MYPT1, and SMC relaxation caused by RhoA inhibition. Levels of RhoA/ROCK were higher at the cell membrane in the IAS SMC compared with those from the RSM and ASM. C3 exoenzyme (RhoA inhibitor) and Y 27632 (ROCK inhibitor) caused a concentration-dependent relaxation of the IAS SMC. In addition, active ROCK-II (primary isoform of ROCK in SMC) caused further shortening in the IAS SMC. C3 exoenzyme increased RhoA-RhoGDI binding and reduced the levels of RhoA-GTP and p(Thr696)-MYPT1. ROCK inhibitor attenuated PKC-induced contractions in IAS SMC. Conversely, a PKC inhibitor (Gö 6850, which causes partial relaxation of the SMC) had no significant effect on ROCK-II-induced contractions. Further experiments showed the highest levels of RhoA, active form of RhoA (RhoA-GTP), ROCK-II, 20-kDa myosin regulatory light chain (MLC(20)), phospho-MYPT1, and phospho-MLC(20) in the IAS vs. RSM and ASM SMC. However, the trend was the reverse with the levels of inactive RhoA (GDP-RhoA-RhoGDI complex) and MYPT1. We conclude that RhoA/ROCK play a critical role in maintenance of spontaneous tone in the IAS SMC via inhibition of myosin light chain phosphatase.

RhoA prenylation is a critical step for the translocation of RhoA to the membrane and its activation in response to agonist-induced sustained contraction of the smooth muscle. However, the effect and role of RhoA prenylation in the spontaneously tonic smooth muscle, such as internal anal sphincter (IAS), is not known. Present studies determined RhoA prenylation and its association with the basal tone in the IAS before and after the RhoA prenylation inhibitor, geranylgeranyl transferase inhibitor GGTI-297 [N-4-[2(R)-amino-3-mercaptopropyl]amino-2-naphthylbenzoyl-(L)-leucine,TFA]. Western blot analyses of cytosolic and membrane fractions determined the effects of RhoA prenylation inhibition on the cellular distribution of the RhoA. Additional studies were performed to determine the relationship between RhoA prenylation and Rho kinase (ROCK) activity. GGTI-297 decreased prenylation of RhoA, decreased ROCK activity, and caused a corresponding fall in the IAS tone. These inhibitory effects following RhoA prenylation blockade were demonstrated to be directly on the spontaneously contracted IAS smooth muscle cells. Western blot analysis revealed high levels of RhoA in the IAS smooth muscle cellular membrane in the basal state, and GGTI-297 shifted the RhoA localization to the cytosol. RhoA prenylation may play an important role in the translocation of RhoA to the smooth muscle cell membrane leading to its activation and for the maintenance of basal tone in the IAS.

Background & Aims: The present studies evaluated the role of H-ras and its implications in the RhoA/Rho kinase (ROCK) pathway in regulating basal tone in the internal anal sphincter (IAS)., Methods: Studies were performed in the IAS from the wild-type (H-ras(+/+)) and knock-out (H-ras(-/-)) mice. The basal tone of smooth muscle strips was measured by isometric force transducers. Length of smooth muscle cells (SMC) isolated from the IAS in the basal state was determined by phase contrast microscopy. Experiments were repeated in the presence of Y 27632, a ROCK inhibitor. Involvement of the RhoA/ROCK machinery was analyzed by reverse-transcription polymerase chain reaction, Western blot, and immunocytochemistry. Reversal of H-ras knock-out effect was evaluated by transfection of SMCs with the constitutively activated (G12V) mutant., Results: Basal tone of the H-ras(-/-) IAS was significantly higher and resistant to relaxation by Y 27632, compared with the H-ras(+/+) IAS. Similarly, the length of SMCs from H-ras(-/-) IAS was significantly shorter. Y 27632 eliminated this difference. RhoA immunoreactivity shifted from cytoplasm to plasma membrane in H-ras(-/-) SMCs, a change typically associated with contraction. Further, SMCs from H-ras(-/-) mice exhibited higher levels of the contractile proteins ROCK II, phosphorylated-MYPT(1) and phosphorylated-MLC(20). Transfection with the G12V mutant increased the length of H-ras(-/-) cells. Conversely, the dominant negative H-ras (S17N) mutant decreased the length of H-ras(+/+) cells., Conclusions: H-ras negatively regulates basal tone in the IAS by inhibiting RhoA/Rho-kinase machinery. Studies may have significant relevance in the pathophysiology and therapy of certain anorectal motility disorders associated with the IAS dysfunction.

The internal anal sphincter (IAS) tone is important for the rectoanal continence. The RhoA/Rho kinase (ROK) pathway has been associated with the agonist-induced sustained contraction of the smooth muscle, but its role in the spontaneously tonic smooth muscle is not known. Present studies compared expression of different components of the RhoA/ROK pathway between the IAS (a truly tonic SM), the rectal smooth muscle (RSM) (a mixture of phasic and tonic), and anococcygeus smooth muscle (ASM) (a purely phasic SM) of rat. RT-PCR and Western blot analyses were performed to determine RhoA, ROCK-II, CPI-17, MYPT1, and myosin light-chain 20 (MLC20). Phosphorylated CPI-17 at threonine-38 residue (p(Thr38)-CPI-17), MYPT1 at threonine-696 residue (p(Thr696)-MYPT1), and MLC20 at threonine-18/serine-19 residues (p(Thr18/Ser19)-MLC20) were also determined in the basal state and after pretreatment with the ROK inhibitor Y 27632. In addition, we compared the effect of Y 27632 on the basal isometric tension and ROK activity in the IAS vs. the RSM. Our data show the highest levels of RhoA, ROCK-II, CPI-17, MLC20, and of phospho-MYPT1, -CPI-17, and -MLC20 in the IAS followed by in the RSM and ASM. Conversely, MYPT1 levels were lowest in the IAS and highest in the ASM. In the IAS, Y 27632 caused a concentration-dependent decrease in the basal tone, levels of phospho-MYPT1, -CPI-17, and -MLC20, and ROK activity. We conclude that RhoA/ROK plays a critical role in the basal tone in the IAS by the inhibition of MLC phosphatase via the phosphorylation of MYPT1 and CPI-17.

Background & Aims: An increase in Rho kinase (ROK) activity has been associated with agonist-induced sustained contraction of the smooth muscle, but its role in the pathophysiology of spontaneously tonic smooth muscle is not known., Methods: Present studies examined the effects of ROK inhibitor Y-27632 in the tonic smooth muscle of the rat internal anal sphincter (IAS) versus in the flanking phasic smooth muscle of the rectum. In addition, studies were performed to determine the relationship between the decreases in the basal IAS tone and the ROK activity. Confocal microscopic studies determined the cellular distribution of the smooth muscle-predominant isoform of ROK (ROCK-II) in the smooth muscle cells (SMCs)., Results: In in vitro studies using neurohumoral inhibitors and tetrodotoxin and the use of SMCs demonstrate direct relaxation of the IAS SMCs by Y-27632. The ROK inhibitor was more potent in the IAS than in the rectal smooth muscle. The IAS relaxation by Y-27632 correlated specifically with the decrease in ROK activity. Confocal microscopy revealed high levels of ROCK-II toward the periphery of the IAS SMCs. In in vivo studies, the lower doses of Y-27632 caused a potent and selective decrease in the IAS pressures without any adverse cardiovascular systemic effects. The ROK inhibitor also caused potent relaxation of the hypertensive IAS., Conclusions: RhoA/ROK play a crucial role in the maintenance of the basal tone in the IAS, and ROK inhibitors have a therapeutic potential in the IAS dysfunction characterized by the hypertensive IAS.

Background & Aims: Inhibitory reflexes in the internal anal sphincter (IAS) are controlled by inhibitory nonadrenergic, noncholinergic innervation (i-NANC). We investigated the roles of 3 different neurohumoral agonists as possible i-NANC neurotransmitters: carbon monoxide (CO), nitric oxide (NO), and vasoactive intestinal peptide (VIP)., Methods: IAS smooth muscle strips were isolated from wild-type (WT), heme oxygenase (HO)-2 knockout (HO-2-/-) and neuronal NO synthase (nNOS) knockout (nNOS-/-) mice. Relaxation of IAS was induced by CO, NO, VIP, and electrical field stimulation (EFS) in the presence and absence of neurohumoral inhibitors (tin protoporphyrin IX [SnPP IX] for CO synthesis, N(omega)-nitro-L-arginine [L-NNA] for NO synthesis, and VIP(10-28) for VIP receptor). Western blot and immunohistochemistry were used to test the presence and localization of HO (for CO synthesis) types 1 (HO-1) and 2 (HO-2), neuronal NO synthase (nNOS, for NO synthesis), and VIP., Results: All 3 neurohumoral agonists produced relaxation (with no difference between WT and HO-2-/- IAS), but CO was over 100 times less potent than NO and VIP. EFS produced relaxation in WT and HO-2-/- IAS with the same intensity. L-NNA and nNOS deletion (approximately 80%) and VIP(10-28) (approximately 15%) significantly inhibited the relaxations, whereas SnPP IX had no effect. Positive immunoreactivities for HO-2, nNOS, and VIP were found in the myenteric plexus of WT IAS. HO-2-/- IAS did not express immunoreactivity for HO-2., Conclusions: i-NANC relaxations of mouse IAS are primarily mediated via NO (by nNOS activity) and partly via VIP. CO directly relaxes the mouse IAS but does not play any significant role in the i-NANC relaxation.

The structural maintenance of chromosome 3 protein (SMC3) is a component of the multimeric cohesin complex that holds sister chromatids together and prevents their premature separation during mitosis. By screening a human cDNA library for interacting proteins we have established that the proto-oncogene RET finger protein (RFP) interacts with SMC3. The sites of interaction map to part of the central coiled coil region of RFP and to the C-terminal region of the SMC3 globular hinge domain. SMC3/RFP interaction was confirmed in vivo by co-immunoprecipitation studies and by performing mammalian two-hybrid interaction assays. Cytoimmunolocalization experiments showed that SMC3 and RFP co-localize in the same cell substructures. Overexpression of RFP in NIH3T3 cells significantly increased the fraction of SMC3 recovered in the nucleus supporting the idea that RFP regulates the intracellular distribution of SMC3. These studies identify a novel SMC3-interacting protein that may affect SMC3 availability to complex with its cohesin partners.

Background: The structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase. The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions., Results: In order to identify proteins that by binding to SMC3 may interfere with the protein dimerization process, a human cDNA library was screened by the yeast two-hybrid system by using the hinge region of SMC3 as bait. This has lead to the identification of Hinderin, a novel five domains protein including two coiled-coil motifs and sharing a strikingly structural similarity to the SMC family of proteins. Hinderin is ubiquitously expressed in human tissues. Orthologue forms of the protein are present in other vertebrates but not in lower organisms. A mapping of the interaction sites revealed that the N- and C-terminal globular domains mediate the binding of Hinderin to SMC3. Hinderin/SMC3 complexes could be recovered by immunoprecipitation from cell lysates using an anti-SMC3 antibody, thus demonstrating that the two proteins interact in vivo. On the contrary, Hinderin did not interact with SMC1. In vivo the rate of SMC1/SMC3 interaction was decreased by the ectopic expression of Hinderin., Conclusions: Hinderin is a novel binding partner of SMC3. Based on its ability to modulate SMC1/SMC3 interaction we postulate that Hinderin affects the availability of SMC3 to engage in the formation of multimeric protein complexes.

Background: Cholesterol 27-hydroxylase, an enzyme expressed at high levels by human monocytes/macrophages, provides a first line of defense against the development of atherosclerosis. Prior studies have suggested that the cytokine interferon-gamma (IFN-gamma) promotes atherosclerosis. We therefore examined the effect of IFN-g on macrophage foam cell formation and on expression of the anti-atherogenic 27-hydroxylase in THP-1 human monocytes/macrophages., Material/methods: THP-1 monocytes and acetylated LDL-treated THP-1 macrophages were incubated in the presence or absence of IFN-gamma (500 U/ml) with or without the addition of IFN- gamma receptor blocking or neutralizing antibody. Foam cell formation was quantified based on percentage of macrophages harboring oil red O-stained globules. Cellular mRNA and protein were isolated. 27-Hydroxylase message was measured by RT-PCR and 27-hydroxylase protein by immunoblot., Results: IFN-gamma -treated THP-1 macrophages exhibit increased foam cell transformation compared to untreated cells under cholesterol loading conditions. IFN-gamma-promoted foam cell formation is abolished by pre-treatment with either IFN-gamma neutralizing or IFN-gamma receptor blocking antibody. IFN-gamma diminishes cholesterol 27-hydroxylase expression in THP-1, and this IFN-gamma -induced downregulation is prevented by pre-treating the cultured cells with either IFN-gamma neutralizing or IFN-gamma receptor blocking antibody., Conclusions: Imbalances in cellular cholesterol flux within macrophages lead to formation of lipid-laden foam cells, a critical step in the pathogenesis of atherosclerosis. We have demonstrated that IFN-gamma, acting through the IFN-gamma receptor, decreases expression of 27-hydroxylase and increases propensity to foam cell formation in the cell line THP-1. These observations suggest that one mechanism by which IFN-g promotes atherosclerosis may involve affecting expression of cholesterol 27-hydroxylase, a cholesterol homeostatic protein.

APJ, a member of the human G protein-coupled seven-transmembrane receptor family, has been shown to serve as a coreceptor for the entry of human immunodeficiency virus type I (HIV-1) and simian immunodeficiency virus (SIV), and it is dramatically expressed in central nervous system (CNS)-based cells. In this study, expression of APJ tagged with the green fluorescent protein (GFP) and a fluorescent peptide, 5-carboxyfluorescein (5-CF) conjugated Apelin-13, were utilized for studying receptor internalization and recycling, in stably expressing indicator cells, human neurons, primary CNS microvascular endothelial cells (MVECs), and astrocytes. Fusion of the C-terminus of APJ to the N-terminus of GFP did not alter receptor ligand binding and functions, including signaling and internalization. Using 293 cells stably expressing APJ-GFP, we demonstrated that rapid internalization of the APJ receptor was induced by stimulation with Apelin-36 and Apelin-13, in a dose-dependent manner. Furthermore, investigations showed that the internalized APJ was colocalized with transferrin receptors, suggesting that the internalization of APJ induced by Apelin is likely to be via clathrin-coated pits. Interestingly, we found that the internalized APJ molecules were recycled to the cell surface within 60 min after removal of Apelin-13, but most of the internalized APJ still remained in the cytoplasm, even 2 h after washout of Apelin-36. The intact cytoplasmic C-terminal domain was found to be required for ligand-induced APJ internalization. Human neurons were dramatically stained by the APJ-binding fluorescent peptides. Primary human fetal astrocytes were less strongly labeled with 5-CF-Apelin-13, and in primary human CNS MVECs only weak distribution of green fluorescence specific for APJ in the cytoplasm was observed. Apelin-36 blocked cell membrane fusion mostly due to steric interference, with only a very modest effect on receptor internalization. The CNS represents a unique reservoir site for HIV-1. As such, molecular therapeutics and small molecular inhibitors of HIV-1 entry via this unique CNS receptor are now able to be rationally designed.

Virion infectivity factor (Vif) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) in vivo, but its function remains uncertain. Recently, we have shown that Vif proteins are able to form multimers, including dimers, trimers, or tetramers. Because the multimerization of Vif proteins is required for Vif function in the viral life cycle, we propose that it could be a novel target for anti-HIV-1 therapeutics. Through a phage peptide display method, we have identified a set of 12-mer peptides containing a PXP motif that binds to HIV-1 Vif protein. These proline-enriched peptides potently inhibited the Vif-Vif interaction in vitro. We have also screened a set of synthesized Vif peptides (15-mer), which covers all the amino acids of the HIV-1 Vif protein sequence, for their ability to inhibit the Vif-Vif interaction in vitro. We demonstrated that Vif-derived proline-enriched peptides that contain the (161)PPLP(164) domain are able to inhibit the Vif-Vif interaction. Conversely, the deletion of the (161)PPLP(164) domain of Vif protein will significantly impair the capability of Vif proteins to interact with each other, indicating that the (161)PPLP(164) domain plays a key role in Vif multimerization. All these results demonstrate that the proline-enriched peptides block the multimerization of Vif through interfering with the polyproline interfaces of Vif formed by (161)PPLP(164) domain. Moreover, these peptides which inhibit the Vif-Vif interaction in vitro potently inhibit HIV-1 replication in the "nonpermissive" T-cells. We propose that this study starts a novel strategy to develop structural diverse inhibitors of Vif such as peptidomimetics or small organic molecules.

Our recent studies have demonstrated that extracellular, recombinant human immunodeficiency virus type I (HIV-1) Vpr protein is highly neurotoxic in the microenvironment of differentiated mature human neurons and undifferentiated neuronal precursors. Although most of the direct neurotoxic effects of HIV-1 have been attributed previously to the envelope gene product, gp120, and the Tat regulatory protein, it was demonstrated that Vpr protein caused apoptosis comparable to that induced by gp120 protein in a dose-dependent manner in the neuronal system. Having observed the neurocytopathic effects of extracellular Vpr protein previously, the effects of virally expressed Vpr on nondividing, terminally differentiated human neurons were investigated. An HIV-1-based three-plasmid expression vector system was utilized to study the effects of intracellularly expressed Vpr. These virion preparations were then used to transduce neurons generated from the human neuronal precursor NT2 cell-line. Intracellularly expressed Vpr induced apoptosis within terminally differentiated neurons, as demonstrated by TUNEL assays. Additionally, virions lacking Vpr expression did not significantly induce apoptosis within these neurons. These results suggest that HIV-1 Vpr may also be leading directly to selective neurotoxicity through intracellular expression. Furthermore, human apoptosis gene microarray comparisons exhibited an up-regulation of Bcl-2-related mRNA, as well as other apoptosis genes involved in the mitochondrial apoptotic pathway, for the Vpr-transduced neuronal cells, when compared to Vpr-negative controls. Thus, Vpr delivered intracellularly, as well as extracellularly, is involved in the induction of significant neuronal apoptosis and may be one of the molecular mechanisms in HIV-1-induced encephalopathy.

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) causes AIDS dementia complex (ADC) in certain infected individuals. Recent studies have suggested that patients with ADC have an increased incidence of neuronal apoptosis leading to neuronal dropout. Of note, a higher level of the HIV-1 accessory protein Vpr has been detected in the cerebrospinal fluid of AIDS patients with neurological disorders. Moreover, extracellular Vpr has been shown to form ion channels, leading to cell death of cultured rat hippocampal neurons. Based on these previous findings, we first investigated the apoptotic effects of the HIV-1 Vpr protein on the human neuronal precursor NT2 cell line at a range of concentrations. These studies demonstrated that apoptosis induced by both Vpr and the envelope glycoprotein, gp120, occurred in a dose-dependent manner compared to protein treatment with HIV-1 integrase, maltose binding protein (MBP), and MBP-Vpr in the undifferentiated NT2 cells. For mature, differentiated neurons, apoptosis was also induced in a dose-dependent manner by both Vpr and gp120 at concentrations ranging from 1 to 100 ng/ml, as demonstrated by both the terminal deoxynucleotidyltransferase (Tdt)-mediated dUTP-biotin nick end labeling and Annexin V assays for apoptotic cell death. In order to clarify the intracellular pathways and molecular mechanisms involved in Vpr- and gp120-induced apoptosis in the NT2 cell line and differentiated mature human neurons, we then examined the cellular lysates for caspase-8 activity in these studies. Vpr and gp120 treatments exhibited a potent increase in activation of caspase-8 in both mature neurons and undifferentiated NT2 cells. This suggests that Vpr may be exerting selective cytotoxicity in a neuronal precursor cell line and in mature human neurons through the activation of caspase-8. These data represent a characterization of Vpr-induced apoptosis in human neuronal cells, and suggest that extracellular Vpr, along with other lentiviral proteins, may increase neuronal apoptosis in the CNS. Also, identification of the intracellular activation of caspase-8 in Vpr-induced apoptosis of human neuronal cells may lead to therapeutic approaches which can be used to combat HIV-1-induced neuronal apoptosis in AIDS patients with ADC.

Objective: To compare outcomes in premature infants with respiratory distress syndrome who received surfactant replacement therapy and were treated with either high-frequency or conventional mechanical ventilation., Design: Retrospective chart review of patient series., Setting: Tertiary academic medical center., Patients: One hundred fourteen extremely low-birth-weight infants (< 1000 g) with respiratory distress syndrome treated with surfactant replacement therapy, consecutively admitted to the neonatal intensive care unit between September 1989 and August 1992., Interventions: Treatment with either high-frequency ventilation (n = 46) or conventional mechanical ventilation (n = 68) after surfactant replacement therapy., Main Outcome Measures: Intraventricular hemorrhage and neurodevelopmental status., Results: Infants who received high-frequency ventilation had significantly lower birth weights and were more premature than infants receiving conventional mechanical ventilation. Despite this, patients ventilated with high frequency had similar incidences of intraventricular hemorrhage and impaired neurodevelopmental outcomes when compared with the conventionally ventilated patients. As expected, the smaller and more premature infants receiving high-frequency ventilation required a longer duration of respiratory support (mechanical ventilation and nasopharyngeal continuous positive airway pressure). Additionally, multiple logistic regression analysis to control for differences in birth weight and gestational age between the two groups revealed a significant association between the combined use of high-frequency ventilation and antenatal corticosteroids and the absence of either intraventricular hemorrhage or pneumothorax., Conclusion: We conclude that high-frequency ventilation combined with surfactant therapy is as safe as conventional mechanical ventilation combined with surfactant therapy for treating respiratory distress syndrome in extremely low-birth-weight infants (< 1000 g) and does not increase the risk of either intraventricular hemorrhage or abnormal neurodevelopmental outcome.