AAV9-based PMM2 gene replacement augments PMM2 expression and improves glycosylation in primary fibroblasts of patients with phosphomannomutase 2 deficiency (PMM2-CDG).

Inherited deficiency of phosphomannomutase 2 (PMM2) (aka PMM2-CDG) is the most common congenital disorders of glycosylation (CDG) and has no cure. With debilitating morbidity and significant mortality, it is imperative to explore novel, safe, and effective therapies for the disease. Our Proof-of-Concept study showed that AAV9-PMM2 infection of patient fibroblasts augmented PMM2 expression and improved glycosylation. Thus, AAV9-PMM2 gene replacement is a promising therapeutic strategy for PMM2-CDG patients.

Salubrinal enhances eIF2α phosphorylation and improves fertility in a mouse model of Classic Galactosemia.

Loss of galactose-1 phosphate uridylyltransferase (GALT) activity in humans results in Classic Galactosemia, and the GalT-deficient (GalT-/-) mouse mimics the patient condition. GalT-/- ovaries display elevated endoplasmic reticulum (ER) stress marker, BiP, and downregulated canonical phosphatidylinositol 3-kinase (Pi3k)/protein kinase B (Akt) growth/pro-survival signaling. Numbers of primordial follicles are reduced in the mutants, recapitulating the accelerated ovarian aging seen in human patients. We previously found that oral administration of the compound Salubrinal (an eIF2α phosphatase inhibitor), resulted in reduction of ovarian BiP expression, rescued Pi3k/Akt signaling, and a doubling of primordial follicles in GalT-/- adults. Here, we further characterized galactosemic stress in GalT-/- mice versus wild-type (WT) controls, and examined whether Salubrinal treatment improved broader reproductive parameters. We assessed the expression levels of factors of the unfolded protein response (UPR), and found that BiP, phospho-Perk, and phospho-eIF2α were all elevated in GalT-/- ovaries. However, neither IKK activation (NFκB pathway) nor alternative Xbp1 splicing downstream of ER membrane protein Ire1α activation was induced, suggesting an Xbp1-independent UPR in galactosemic stress. Moreover, Salubrinal treatment significantly increased the number of ovulated eggs in mutant animals after gonadotrophic superovulation. Salubrinal treatment also normalized estrus cycle stage lengths and resulted in significantly larger litter sizes than vehicle-treated mutants. Overall, we show that Salubrinal protects against galactosemia-induced primordial follicle loss in a fashion that includes suppressing the de-phosphorylation of eIF2α, and that intervention in this way significantly improves and extends ovarian function, fertility, and fecundity.

Immunomodulatory activity of glycodelin: implications in allograft rejection.

Glycodelin is an immunomodulator, indispensable for the maintenance of pregnancy in humans. The glycoprotein induces apoptosis in activated CD4+ T cells, monocytes and natural killer (NK) cells, and suppresses the activity of cytotoxic T cells, macrophages and dendritic cells. This study explores the immunosuppressive property of glycodelin for its possible use in preventing graft rejection. Because glycodelin is found only in certain primates, the hypothesis was investigated in an allograft nude mouse model. It is demonstrated that treatment of alloactivated mononuclear cells with glycodelin thwarts graft rejection. Glycodelin decreases the number of activated CD4+ and CD8+ cells and down-regulates the expression of key proteins known to be involved in graft demise such as granzyme-B, eomesodermin (EOMES), interleukin (IL)-2 and proinflammatory cytokines [tumour necrosis factor (TNF)-α and IL-6], resulting in a weakened cell-mediated immune response. Immunosuppressive drugs for treating allograft rejection are associated with severe side effects. Glycodelin, a natural immunomodulator in humans, would be an ideal alternative candidate.

Reversal of aberrant PI3K/Akt signaling by Salubrinal in a GalT-deficient mouse model.

Classic Galactosemia is an autosomal recessive disorder caused by deleterious mutations in the GALT gene, which encodes galactose-1 phosphate uridylyltransferase enzyme (GALT: EC 2.7.7.12). Recent studies of primary skin fibroblasts isolated from the GalT-deficient mice demonstrated a slower growth rate, a higher level of endoplasmic reticulum (ER) stress, and down-regulation of the Phosphoinositide 3 kinase/Protein kinase B (PI3K/Akt) signaling pathway. In this study, we compared the expression levels of the PI3K/Akt signaling pathway in normal and GalT-deficient mouse tissues. In mutant mouse ovaries, phospho-Akt [pAkt (Ser473)] and pGsk3ß were reduced by 62.5% and 93.5%, respectively (p<0.05 versus normal controls). In mutant cerebella, pAkt (Ser473) and pGsk3ß were reduced by 62%, 50%, respectively (p<0.05). To assess the role of ER stress in the down-regulation of PI3K/Akt signaling, we examined if administration of Salubrinal, a chemical compound that alleviates ER stress, to GalT-deficient fibroblasts and animals could normalize the pathway. Our results demonstrated that Salubrinal effectively reversed the down-regulated PI3K/Akt signaling pathway in the mutant cells and animals to levels close to those of their normal counterparts. Moreover, we revealed that Salubrinal can significantly slow down the loss of Purkinje cells in the cerebella, as well as the premature loss of primordial ovarian follicles in young mutant mice. These results open the door for a new therapeutic approach for the patients with Classic Galactosemia.

Bubbles in the Heart: A Case of Venous Air Thromboembolism.

INTRODUCTION: Venous air embolism (VAE) due to central venous catheter (CVC) placement is a rare but preventable complication which is potentially fatal. We describe a case highlighting unique patient characteristics which increase the risk of developing VAE. CASE DESCRIPTION: A sixty-year-old gentleman was admitted to the hospital with dyspnea and altered mental status. His comorbidities include cancer of the neck and tongue, currently in remission, and schizophrenia. On presentation, he was found to be in acute respiratory failure, due to pneumonia, and required mechanical ventilation. Following extubation, his CVC was removed from the right internal jugular vein. While ambulating around the unit, he experienced a coughing fit and dizziness. He rapidly developed cardiopulmonary collapse requiring re-intubation and vasopressor support. Chest x-ray demonstrated a radiolucent column along the lateral aspect of the right neck. Due to concern for VAE, an echocardiogram was performed, revealing multiple air-bubbles in the right and left chambers of the heart. DISCUSSION: Our patient was predisposed to developing VAE due to the extensive radiation induced skin changes, from his cancer treatment, on the neck and upper thorax. This resulted in loss of underlying subcutaneous tissue and decreased skin pliability. He had a large, open puncture wound at the catheter site on his neck, probably resulting in air entry. Anxiety and agitation, due to schizophrenia, made it difficult to maintain our patient in a supine or Trendelenburg position following CVC removal. This case highlights the importance of recognizing patient factors that may increase the risk of VAE.

Dimethyl 1,8-bis-(4-methyl-phen-yl)-11-oxatri-cyclo-[6.2.1.0(2,7)]undeca-2,4,6,9-tetra-ene-9,10-di-carboxyl-ate.

The title compound, C28H24O5, consists of a fused tricyclic system containing two five-membered rings and one six-membered ring. The five-membered rings both exhibit an envelope conformation with the O atom at the flap, whereas the six-membered ring adopts a boat conformation. The dihedral angle between the 4-methyl-phenyl rings at the 1,8-positions is 76.4 (1)°. In the crystal, mol-ecules are stacked in columns along the a axis through C-H⋯O inter-actions.

Dimethyl 1-(4-methyl-phen-yl)-8-(thio-phen-2-yl)-11-oxatricyclo-[6.2.1.0(2,7)]undeca-2,4,6,9-tetra-ene-9,10-dicarboxy-late.

The title compound, C25H20O5S, is the product of a Diels-Alder reaction. The mol-ecule consists of a fused tricyclic system containing two five-membered rings and one six-membered ring. The five-membered rings both show an envelope conformation with the O atom at the flap, whereas the six-membered ring adopts a boat conformation. The thio-phene ring is disordered over two sets of sites with an occupancy ratio of 0.53 (1):0.47 (1). The dihedral angles between the 4-methyl-phenyl ring and the major and minor components of the thio-phene ring are 66.3 (1) and 67.9 (1)°, respectively, while the dihedral angle between the disordered thio-phenyl components is 3.1 (1)°. The mean plane of the tricyclic ring system makes dihedral angles of 35.8 (1), 30.8 (1) and 32.8 (1)°, respectively, with the 4-methyl-phenyl ring and the major and minor components of the thio-phenyl ring. In the crystal, inversion dimers are formed through pairs of C-H⋯π inter-actions. In addition, C-H⋯O inter-actions are observed.

Diethyl 1,8-bis-(4-methyl-phen-yl)-11-oxatricyclo-[6.2.1.0(2,7)]undeca-2,4,6-triene-9,10-dicarboxyl-ate.

The title compound, C30H30O5, is the Diels-Alder adduct from 1,3-diphenyl-benzo[c]furan and diethyl maleate. The mol-ecule comprises a fused tricyclic system containing two five-membered rings, which are in envelope conformations with the O atom at the flap, and a six-membered ring adopting a boat conformation. The dihedral angle between the 4-methyl-phenyl substituents in the 1- and 8-positions is 62.1 (1)°. The ethyl group of one ester group and the eth-oxy group of the other ester group are disordered over two sets of sites, with occupancy ratios of 0.43 (2):0.57 (2) and 0.804 (7):0.196 (7), respectively. In the crystal, inversion dimers are formed through pairs of C-H⋯O inter-actions.

5-(3-Meth-oxy-phen-yl)-3-phenyl-1,2-oxazole.

In the title compound, C16H13NO2, the isoxazole ring makes dihedral angles of 17.1 (1)° with the 3-meth-oxy-phenyl ring and 15.2 (1)° with the phenyl group. Centrosymmetric dimers that are realised by pairs of C-H⋯π inter-actions are observed in the crystal structure.

Diethyl 1,8-diphenyl-11-oxatricyclo-[6.2.1.0(2,7)]undeca-2,4,6-triene-9,10-dicarboxyl-ate.

The title compound, C28H26O5, is the Diels-Alder adduct from 1,3-diphenyl-benzo[c]furan and diethyl maleate. The mol-ecule comprises of a fused tricyclic system containing two five-membered rings, which are in envelope conformations with the O atom at the flap, and a six-membered ring adopting a boat conformation. The dihedral angle between phenyl substituents in the 1,8-positions is 55.1 (1)°. The ethyl groups are disordered over two sets of sites, with occupancy ratios of 0.648 (9):0.352 (9) and 0.816 (1):0.184 (1). In the crystal, pairs of C-H⋯π inter-actions link the mol-ecules into inversion dimers.

Analgesic and anti-inflammatory activity of root bark of Grewia asiatica Linn. in rodents.

BACKGROUND: Grewia asiatica Linn. (Family: Tiliaceae), called Phalsa in Hindi is an Indian medicinal plant used for a variety of therapeutic and nutritional uses. The root bark of the plant is traditionally used in rheumatism (painful chronic inflammatory condition). AIMS: The present study demonstrates the analgesic and anti-inflammatory activity of root bark of G. asiatica in rodents. SETTINGS AND DESIGN: The methanolic extract of Grewia asiatica (MEGA) and aqueous extract of Grewia asiatica (AEGA) of the bark were prepared and subjected to phytochemical tests and pharmacological screening for analgesic and anti-inflammatory effect in rodents. MATERIALS AND METHODS: Analgesic effect was studied using acetic acid-induced writhing in mice and hot plate analgesia in rats while anti-inflammatory activity was investigated using carrageenan-induced paw oedema in rats. The MEGA or AEGA was administered orally in doses of 200 and 400 mg/kg/day of body weight. STATISTICAL ANALYSIS: Data were analysed by one-way analysis of variance followed by Dunnett's test. RESULTS: The extracts showed a significant inhibition of writhing response and increase in hot plate reaction time and also caused a decrease in paw oedema. The effects were comparable with the standard drugs used. CONCLUSIONS: The present study indicates that root bark of G. asiatica exhibits peripheral and central analgesic effect and anti-inflammatory activity, which may be attributed to the various phytochemicals present in root bark of G. asiatica.

Secretion and transfer of the thyroid hormone binding protein transthyretin by human placenta.

CONTEXT: The thyroid hormone and retinol binding protein transthyretin (TTR) is synthesised by human trophoblasts. Polarised JEG-3 choriocarcinoma cells grown in bicameral chambers secrete TTR predominantly apically but also basally and these cells and human trophoblasts also take up TTR suggesting that there may be a placental TTR shuttle that participates in materno-fetal transfer of thyroid hormones and retinol. OBJECTIVES AND METHODS: Our objective was to investigate TTR secretion into the maternal and fetal circuits of the ex vivo dually perfused placental lobule to confirm that placenta secretes TTR into the fetal circulation. We also investigated translocation of Alexa Fluor-594 labelled TTR from incubation medium into the fetal placental capillaries in early (14-15 weeks) and term placental villus explants. RESULTS: The perfused placental lobule secretes TTR into the maternal and fetal circuits. Secretion in both circuits is linear with time and is predominantly into the maternal circuit (mean maternal/fetal ratio 99.4 ± 25.6). The mean data fitted well to a three compartment mathematical model (maternal circuit, placenta and fetal circuit, constant secretion of TTR and return of maternal circuit TTR to the placental compartment). Explants from early (14-15 weeks) and late (38-40 weeks) placentas translocated fluorescently labelled TTR from medium to villus (fetal) capillaries. CONCLUSIONS: Our results confirm that human placenta secretes TTR into maternal and fetal circulations and supports the hypothesis that placental TTR secreted into the maternal placental circulation can be taken up by trophoblasts and translocated to the fetal circulation, forming a TTR shuttle system. This may have important implications for materno-fetal transfer of thyroid hormones, retinol/retinol binding protein and xenobiotics (such as polychlorinated biphenyls) all of which bind to TTR.

Transfer of gangliosides across the human placenta.

OBJECTIVES: Gangliosides are structural and functional glycosphingolipids, considered to have important roles in neuronal development in fetal and neonatal development and in memory formation. In this report, we have investigated the ability of bovine milk-derived gangliosides GM3 and GD3 to cross the human placenta. STUDY DESIGN: We have employed the ex-vivo model of dually-perfused isolated human placental lobules. RESULTS: There was significant uptake of both GD3 and GM3 from the maternal perfusate. There was significant increase of GM3 in the fetal side and a non-statistically significant trend for GD3 to increase on the fetal side. CONCLUSIONS: Hence an apparent preference for GM3 release into fetal circulation. We suggest that gangliosides consumed by the mother enter her circulation, can be transferred across the placenta and may be available to the developing fetus for building neural connections.

Intracardiac expression of markers of endothelial damage/dysfunction, inflammation, thrombosis, and tissue remodeling, and the development of postoperative atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is a common complication of coronary artery bypass grafting (CABG), and may have an inflammatory and/or thrombotic etiology. We sought to determine the expression of inflammatory (interleukin [IL]-6), thrombotic (tissue factor and von Willebrand factor [VWF]) and remodeling (matrix metalloproteinase [MMP]-9 and tissue inhibitor of metalloproteinase [TIMP]-1) markers by left atrial appendage (LAA) and right atrial appendage (RAA) tissue in the prediction of postoperative AF. We determined whether the tissue expression of markers of certain different pathophysiologic mechanisms predicted the development of AF after CABG. METHODS: LAA and RAA tissue was excised during CABG in 100 patients free of AF and inflammation. Tissue marker expression was quantified by immunohistochemistry and was related to 30-day postoperative AF. RESULTS: Overall, there were no significant differences in staining intensity of any marker between LAA tissue and RAA tissue. However, more intense expression of VWF by LAA tissue predicted the 30 patients with postoperative AF as compared with those free of AF (P = 0.006). IL-6, MMP-9 and TIMP-1 expression by RAA and LAA epicardial tissue was stronger than expression by endocardium or cardiomyocytes (all P < 0.025) but failed to predict AF. CONCLUSION: In this study, one of the largest to investigate tissue expression of pathophysiologic markers in relation to postoperative AF, we show that more intense expression of VWF by LAA tissue is a significant predictor of postoperative AF. This points towards a possible role of endothelial damage/dysfunction (as reflected by VWF changes) in the pathogenesis of postoperative AF.

5-(3-Methyl-phen-yl)-3-phenyl-1,2-oxazole.

In the title compound, C(16)H(13)NO, the isoxazole ring makes dihedral angles of 16.64 (7)° with 3-methyl-phenzyl ring and 17.60 (7)° with the unsubstituted phenyl ring.

Passage of 4-nonylphenol across the human placenta.

4-nonylphenol (4-NP) is an estrogenic endocrine active chemical that is present in detergents and is known to contaminate food and drinking water. The aim of the present study was to evaluate whether 4-NP crosses the human placenta and if so, to what extent. Human placentae obtained from healthy term singleton pregnancies were utilized in a dual ex-vivo re-circulating model of placental perfusion. Six placentae were perfused for 180 min following addition of 4-NP (30 ng/mL) to the maternal perfusate. Antipyrine and FITC dextran were used as positive and negative controls respectively to validate the integrity of the circuits. 4-NP was measured by High Performance Liquid Chromatography coupled with Fluorescence Detection (HPLC-FD). After 180 min of perfusion the transfer percentages for antipyrine and 4-NP were 25.6 ± 1.4% (mean ± s.e.m, n = 6) and 22.75 ± 3.76% respectively and the transfer index for 4-NP was 0.8. We conclude that the intact form of 4-NP at environmentally relevant concentrations can transfer across the human placenta albeit at a slow rate.

4,9-Dioxa-1,3(1,2)-dibenzena-2(4,5)-1,3-oxazolidinacyclononaphane.

The oxazole ring in the title compound, C(20)H(23)NO(3), adopts an envelope conformation while the 12-membered ring is in a chair conformation. The dihedral angle between the benzene rings is 37.8 (1)°. The crystal structure displays inter-molecular C-H⋯O hydrogen bonding.

Increased levels of plasma haemoxygenase-1 in prostate cancer.

Angiogenesis, a key component of cancer, may be driven by angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and angiopoietin-2. Haemoxygenase-1 (HO-1), a haem-degrading enzyme, may have alternative roles in angiogenesis. Levels of plasma HO-1 have not been reported in prostate cancer. We tested the hypothesis of abnormal HO-1 in 30 men with early prostate cancer, compared with 22 men with benign prostate disease (BPD) and 26 men free of prostate disease, and that HO-1 levels would correlate with VEGF, angiopoietin-2, von Willebrand factor (vWf, marking endothelial perturbation) and PSA. Plasma HO-1 was twofold higher in prostate cancer than in the two control groups, while vWf, VEGF and PSA were also raised (all P<0.02). In the subjects free of prostate disease and in the BPD groups, HO-1 correlated significantly with VEGF (r>0.5, P<0.02) but the correlation in prostate cancer was not significant (r=0.117, P=0.537). There were no correlations with PSA or the Gleason stage. We conclude that HO-1 is associated with VEGF in health and BPD, but in the presence of prostate cancer, raised levels of both HO-1 and VEGF fail to correlate. This observation may have implications for the pathogenesis of prostate cancer.

Comparative evaluation of two polymerase chain reactions targeting different genomic regions to detect Mycobacterium tuberculosis in sputum.

PURPOSE: Tuberculosis remains an important health problem all over the world, especially in resource poor settings like India. The Ziehl-Neelsen (ZN) staining of sputum smear is still the method of choice in the diagnosis of tuberculosis in spite of its low sensitivity and specificity. This paper evaluates comparison of two different polymerase chain reaction (PCR) assays with sputum smear findings to detect Mycobacterium tuberculosis. MATERIALS AND METHODS: A total of 191 sputum samples were collected from 84 patients attending a tertiary care hospital, who were suspected of having pulmonary tuberculosis, were examined by PCR targeting two different genomic regions, namely, TRC 4 by non-nested format and IS6110 insertion element by nested format in comparison to ZN staining of sputum smears. RESULTS: Among the patients tested, 20.24% (Mid-p 95%CI: 31.5-52.4) were smear positive, 7.14% (Mid-p 95%CI: 2.94-14.26) were positive by TRC 4 PCR and 41.67% (Mid-p 95%CI: 12.7-29.8) were positive by IS6110 nested PCR (nPCR). The median age of overall positive cases was 42 years. Among the nPCR positives, the median for age of rural and peri-urban community was 46 and 32 years, respectively. The kappa coefficient between smear findings and TRC4 PCR findings was 0.27 and an agreement of 0.83 was observed (Z = 2.99; one-tailed P = 0.001). TRC 4 PCR picked two unique positives that were negative by smear and IS6110 nPCR. CONCLUSION: The non-nested TRC 4 PCR showed inability for accurate detection of M. tuberculosis in sputum samples. The study concluded that the nPCR targeting IS6110 is superior and more sensitive than TRC 4 PCR.

Transplacental transfer and biotransformation of genistein in human placenta.

OBJECTIVE: To study transplacental transfer and biotransformation of genistein in the human placenta. STUDY DESIGN AND OUTCOMES: Human placentae obtained from healthy term singleton pregnancies were utilised in a dual re-circulating model of ex-vivo placental perfusion. Four placentae were perfused for 180min following addition of genistein (10ng/mL) to the maternal perfusate. Antipyrine and FITC dextran were used as positive and negative controls respectively to validate integrity of the circuits. Concentrations of genistein and its conjugates were determined by liquid chromatography-mass spectrometry (LC-MS). RESULTS: The transfer percentage for antipyrine and genistein was 25.6+/-1.40% and 22.1+/-1.61% respectively and the transfer index for genistein was 0.90+/-0.04 after 180min of perfusion. 12.0+/-2.40% of genistein in the fetal compartment and 7.36+/-4.73% of genistein in the maternal compartment were in the conjugated form. CONCLUSIONS: Genistein can transfer across the human placenta at environmentally relevant levels. Placental metabolizing enzymes conjugate a small fraction of genistein into the glucuronide/sulphate form, which is devoid of estrogenic action.