Investigation of the effects of ceramide-C16 and n-decane on the polymorphism of phosphatidylethanolamine : a 2H and 31P solid-state NMR and differential scanning calorimetry study

Les céramides, dont la structure comporte une chaîne hexadecanoyle (CerC16), une chaîne considérée comme de longueur moyenne, sont des sphingolipides impliqués dans de nombreuses pathologies telles que l’obésité, le diabète, la maladie de Parkinson ainsi que certains types de cancer. La première partie de mon projet de M.Sc. a consisté à utiliser la calorimétrie différentielle à balayage (DSC de l'anglais differential scanning calorimetry) et la spectroscopie RMN séquentielle à l'état solide du deutérium (2H) et du 31P afin d’étudier l'impact du CerC16 (<20 mol%) sur le polymorphisme de la 1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphoéthanolamine (POPE). Les résultats montrent que le CerC16 a un impact majeur. Il élargit la transition de phase lamellaire gel-fluide (Lβ‒Lα) et la décale vers les hautes températures, ce qui entraîne la création de larges régions de coexistence Lβ/Lα dans le diagramme de phase du système. La présence du céramide entraîne également un élargissement de la transition de phase Lα‒hexagonale inverse (HII) ainsi qu’une diminution de la température qui lui est associée. Lorsque la proportion de CerC16 dans la membrane de POPE est supérieure à 12-13 mol%, les deux transitions se chevauchent, ce qui entraîne la coexistence des trois phases. Ce chevauchement conduit à une transition de phase directe Lβ‒HII. De manière générale, le céramide entraîne donc une multitude d’effets sur les propriétés de la membrane lipidique, de la variation de sa fluidité à la modulation de son rayon spontané de courbure. Certains alcanes de longueurs variables ont le potentiel de favoriser la transition Lα-HII de différentes phosphatidyléthanolamines (PE). Dans la seconde partie de mon M.Sc., j'ai caractérisé les impacts du n-décane-d22 (10 mol%), un alcane à courte chaîne, sur le polymorphisme de la POPE, en exploitant les mêmes techniques de spectroscopie RMN que précédemment. J’ai montré que, malgré une diminution significative de la température associée au début de transition Lα‒HII, la température de la fin de transition restait quant à elle sensiblement inchangée. Globalement, la transition est donc élargie. Nous proposons que les molécules de n-décane se localisent au niveau des bouts de chaînes acyles de la POPE en phase Lα, ce qui engendre des compressions et des contraintes. Il en résulte un décalage du début de transition Lα‒HII vers les basses températures. Lorsque la température augmente, de plus en plus de molécules de n-décane sont transférées vers la phase HII, ce qui réduit les contraintes sur les chaînes acyles de la phase Lα. Le décane comble les espaces entre les cylindres HII et favorise aussi de cette manière la phase non-lamellaire.Ceramide C16 (CerC16), a mid-chain sphingolipid bearing a hexadecanoyl chain, is involved in several diseases such as diabetes, obesity, Parkinson, and certain type of cancers. In the first part of my M.Sc. project, I employed differential scanning calorimetry (DSC) and sequential 2H and 31P static solid-state NMR spectroscopy to study the role of CerC16 ( ≤ 20 mol%) in modifying the polymorphism of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). The results demonstrate that CerC16 has major impacts. It broadens the gel‒fluid lamellar (Lβ‒Lα) phase transition and shifts it toward higher temperatures. This effect leads to the creation of a wide Lβ/Lα coexistence region in the phase diagram of the system. CerC16 also broadens the Lα‒inverted hexagonal (HII) phase transition and shifts it towards lower temperatures. When CerC16 proportion in POPE bilayers is ≥ 12-13 mol%, the two transitions overlap, leading to a three-phase coexistence line in the phase diagram. Such a phenomenon leads to a direct Lβ‒HII phase transition. Globally, CerC16 can have a wide range of effects on membrane properties, from varying its fluidity to modulating its curvature. Alkanes of various chain lengths are able to promote the Lα‒HII transition of different phosphatidylethanolamines (PE). In the second part of my M.Sc. thesis, I investigated the impacts of 10 mol% n-decane-d22, a short alkane, on the polymorphism of POPE, using sequential 2H and 31P solid-state NMR spectroscopy. The results show that although, n-decane causes a significant downshift in the onset temperature of the Lα‒HII transition, it does not shift considerably the end of the transition. As a consequence, a very broad Lα‒HII transition is obtained. It is inferred that n-decane molecules are found within the lower part of the POPE acyl chains in the Lα phase. This location increases the chain packing stress and consequently, initiates the Lα‒HII transition at lower temperatures. Upon increasing temperature, more and more n-decane molecules are transferred to the HII phase; the relocation not only decreases the chain packing stress in the Lα phase, but also allows the decane molecules to fill the interstitial void spaces between the HII cylinders

Relationship of Conjunctival and Corneal Calcification with Secondary Hyperparathyroidism in Hemodialysis Patients

Background/Objective: Hyperphosphatemia is the consequence of end stage renal failure. Inadequate control of serum phosphorus results in elevated Ca×P product with subsequent soft tissue deposition in the form of conjunctival and corneal calcification. In this study, we evaluated the relationship of conjunctival and corneal calcification with secondary hyperparathyroidism in hemodialysis patients. Patients and methods: This is a descriptive-analytic study performed on 24 hemodialysis patients. We measured serum calcium, phosphorus, alkaline phosphatase, iPTH and conjunctival and corneal calcification using slit-lamp microscope according to a modification of Porter's criteria. The duration of hemodialysis was 30.7± 21.7 months. Results: 24 patients participated in this study. The biochemical values were: Ca: 9.1 ± 0.8 mg/dl, P: 6.5 ± 2.2 mg/dl, ipTH: 488 ± 326 pg/ml, Ca×P: 51.5 ± 16.6 The mean of conjunctival and corneal calcification score was 7.1±4. There was a positive correlation between conjunctival and corneal calcification with a duration of hemodialysis (p=0.033, r-0.436), Ca×P product (p=0.007, r=0.538). P (p=0.006, r=548) and iPTH (p= 0.028, r=0.449). There was no correlation between conjunctival and corneal calcification with the age of the patients, serum calcium and alkaline phosphatase. Conclusion: There is a positive correlation of serum phosphorus, Ca×P product and iPTH with conjunctival and corneal calcification and no significant correlation with serum calcium implying that there is a central role for phosphorus in calcium-phosphorus deposition in soft tissues like cornea and conjunctiva, underscoring further attention to phosphorus control in hemodialysis patients

Relationship of Conjunctival and Corneal Calcification with Secondary Hyperpara-thyroidism in Hemodialysis Patients

Abstract Background/Objective: Hyperphosphatemia is the consequence of end stage renal failure. Inadequate control of serum phosphorus results in elevated Ca×P product with subsequent soft tissue deposition in the form of conjunctival and corneal calcification. In this study, we evaluated the relationship of conjunctival and corneal calcification with secondary hyperparathyroidism in hemodialysis patients. Patients and methods: This is a descriptive–analytic study performed on 24 hemodialysis patients. We measured serum calcium, phosphorus, alkaline phosphatase, iPTH and conjunctival and corneal calcification using slit–lamp microscope according to a modification of Porter's criteria. The duration of hemodialysis was 30.7 ± 21.7 months. Results: 24 patients participated in this study. The biochemical values were: Ca: 9.1 ± 0.8 mg/dl, P: 6.5 ± 2.2 mg/dl, ipTH: 488 ± 326 pg/ml, Ca×P : 51.5 ± 16.6 The mean of conjunctival and corneal calcification score was 7.1±4. There was a positive correlation between conjunctival and corneal calcification with a duration of hemodialysis (p=0.033, r=0.436), Ca×P product (p=0.007, r=0.538). P (p=0.006, r=548) and iPTH (p= 0.028 , r=0.449). There was no correlation between conjunctival and corneal calcification with the age of the patients, serum calcium and alkaline phosphatase. Conclusion: There is a positive correlation of serum phosphorus, Ca×P product and iPTH with conjunctival and corneal calcification and no significant correlation with serum calcium implying that there is a central role for phosphorus in calcium-phosphorus deposition in soft tissues like cornea and conjunctiva, underscoring further attention to phosphorus control in hemodialysis patients

Motivations and Predictors of Cheating in Pharmacy School

Objective. To assess the prevalence, methods, and motivations for didactic cheating among pharmacy students and to determine predictive factors for cheating in pharmacy colleges and schools. Methods. A 45-item cross-sectional survey was conducted at all four doctor of pharmacy programs in Northern California. For data analysis, t test, Fisher exact test, and logistic regression were used. Results. Overall, 11.8% of students admitted to cheating in pharmacy school. Primary motivations for cheating included fear of failure, procrastination, and stress. In multivariate analysis, the only predictor for cheating in pharmacy school was a history of cheating in undergraduate studies. Conclusion. Cheating occurs in pharmacy schools and is motivated by fear of failure, procrastination, and stress. A history of past cheating predicts pharmacy school cheating. The information presented may help programs better understand their student population and lead to a reassessment of ethical culture, testing procedures, and prevention programs

Gender-Based Differences Among Pharmacy Students Involved in Academically Dishonest Behavior

Objective. To determine whether differences based on gender exist among pharmacy students involved in cases of admitted cheating or other academic dishonesty and to assess perceptions of academic dishonesty. Methods. Two cohorts of second-year male and female pharmacy students from four Northern California pharmacy programs were invited to complete a 45-item cross-sectional survey. Descriptive statistics and Pearson’s chi-squared test were used for statistical analysis. Results. There were 330 surveys completed with a 59% response rate. No significant gender-based differences were found regarding admitted cheating in pharmacy school and in regards to participating in various forms of academically dishonest behavior. Female students were more likely than male students to report witnessing a classmate copying another student’s assignment. Male students were less likely than female students to perceive a student who distributed a stolen exam as a cheater. Conclusion. No gender-based differences were noted in cases of admitted cheating or with regards to taking part in various forms of academically dishonest behavior. However, female students report witnessing cheating more than male students, and male students may have a more lenient perception toward academically dishonest behavior than female students. The information gathered from this study may provide further insight to pharmacy programs and educators regarding academic dishonesty at their institution

The Effect of Zinc Sulfate on Malnutrition in Hemodialysis Patients

Background: Malnutrition is common in hemodialysis patients and it must be controlled quickly. This study aimed to investigate the effect of zinc sulfate on malnutrition in dialysis patients. Methods: This study was a randomized controlled trial on 84 hemodialysis patients referred to Imam Hossain Hospital in Shahroud (northeastern of Iran). Patients were randomly divided into two case and control groups. For the intervention group, one tablet of zinc sulfate 220 mg was administered daily for 8 weeks. Both groups were subjected to standard dialysis three times in the week and all patients were assessed for malnutrition using a standard questionnaire, lab tests, and necessary examinations in the first stage, one month and two months after the treatment. &nbsp; Results: Of 84 patients, 39 cases (46.4%) were female and rest was male. The mean age of the patients was 59.1±27.2 years. The mean total duration of dialysis was 2.9±2.3 years. The severity and extent of malnutrition at the beginning and one month after the study did not differ between the two groups, but after the second month, there was a significant decrease of malnutrition in the intervention group (Pvalue=0.015). Also, malnutrition variables were significantly associated with BMI less than 18 kg/m2 (Pvalue&lt;0.039), and serum creatinine less than 3 mg/dl (Pvalue&lt;0.011) and hemoglobin less than 11 g/dl (Pvalue&lt;0.001), Conclusions: The results of this study showed that zinc sulfate consumption for at least 2 months could significantly reduce the severity of malnutrition in hemodialysis patients. Keywords: Malnutrition, Zinc sulfate, Hemodialysis

The Effect of Zinc Sulfate on Malnutrition in Hemodialysis Patients

Background: Malnutrition is common in hemodialysis patients and it must be controlled quickly. This study aimed to investigate the effect of zinc sulfate on malnutrition in dialysis patients. Methods: This study was a randomized controlled trial on 84 hemodialysis patients referred to Imam Hossain Hospital in Shahroud (northeastern of Iran). Patients were randomly divided into two case and control groups. For the intervention group, one tablet of zinc sulfate 220 mg was administered daily for 8 weeks. Both groups were subjected to standard dialysis three times in the week and all patients were assessed for malnutrition using a standard questionnaire, lab tests, and necessary examinations in the first stage, one month and two months after the treatment. &nbsp; Results: Of 84 patients, 39 cases (46.4%) were female and rest was male. The mean age of the patients was 59.1±27.2 years. The mean total duration of dialysis was 2.9±2.3 years. The severity and extent of malnutrition at the beginning and one month after the study did not differ between the two groups, but after the second month, there was a significant decrease of malnutrition in the intervention group (Pvalue=0.015). Also, malnutrition variables were significantly associated with BMI less than 18 kg/m2 (Pvalue&lt;0.039), and serum creatinine less than 3 mg/dl (Pvalue&lt;0.011) and hemoglobin less than 11 g/dl (Pvalue&lt;0.001), Conclusions: The results of this study showed that zinc sulfate consumption for at least 2 months could significantly reduce the severity of malnutrition in hemodialysis patients. Keywords: Malnutrition, Zinc sulfate, Hemodialysis

Novel insights into the cardio-protective effects of FGF21 in lean and obese rat hearts

Aims: Fibroblast growth factor 21 (FGF21) is a hepatic metabolic regulator with pleotropic actions. Its plasma concentrations are increased in obesity and diabetes; states associated with an increased incidence of cardiovascular disease. We therefore investigated the direct effect of FGF21 on cardio-protection in obese and lean hearts in response to ischemia. Methods and Results: FGF21, FGF21-receptor 1 (FGFR1) and beta-Klotho (βKlotho) were expressed in rodent, human hearts and primary rat cardiomyocytes. Cardiac FGF21 was expressed and secreted (real time RT-PCR/western blot and ELISA) in an autocrine-paracrine manner, in response to obesity and hypoxia, involving FGFR1-βKlotho components. Cardiac-FGF21 expression and secretion were increased in response to global ischemia. In contrast βKlotho was reduced in obese hearts. In isolated adult rat cardiomyocytes, FGF21 activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt), ERK1/2(extracellular signal-regulated kinase) and AMPK (AMP-activated protein kinase) pathways. In Langendorff perfused rat [adult male wild-type wistar] hearts, FGF21 administration induced significant cardio-protection and restoration of function following global ischemia. Inhibition of PI3K/Akt, AMPK, ERK1/2 and ROR-α (retinoic-acid receptor alpha) pathway led to significant decrease of FGF21 induced cardio-protection and restoration of cardiac function in response to global ischemia. More importantly, this cardio-protective response induced by FGF21 was reduced in obesity, although the cardiac expression profiles and circulating FGF21 levels were increased. Conclusion: In an ex vivo Langendorff system, we show that FGF21 induced cardiac protection and restoration of cardiac function involving autocrine-paracrine pathways, with reduced effect in obesity. Collectively, our findings provide novel insights into FGF21-induced cardiac effects in obesity and ischemia

S100A4 as a Target of the E3-Ligase Asb2β and Its Effect on Engineered Heart Tissue

Background: S100A4 has recently emerged as an important player in cardiac disease, affecting phenotype development in animal models of myocardial infarction and pathological cardiac hypertrophy, albeit it is unclear whether S100A4 exerts a detrimental or beneficial function. The goal of the current study was to analyze S100A4 expression in models of cardiac pathology, investigate its degradation by the ubiquitin-proteasome system (UPS), and furthermore examine the functional effects of S100A4 levels in a 3D model of engineered heart tissue (EHT).Methods and Results: S100A4 mRNA and protein levels were analyzed in different models of cardiac pathology via quantitative RT-PCR and Western blot, showing a higher S100A4 steady-state protein concentration in hearts of Mybpc3-knock-in (KI) hypertrophic cardiomyopathy (HCM) mice. COS-7 cells co-transfected with plasmids encoding mutant (MUT) Asb2β lacking the E3 ligase activity in combination with V5-tagged S100A4 plasmid presented higher S100A4-V5 protein steady-state concentrations than cells co-transfected with the Asb2β wild type (WT) plasmid. This effect was blunted by treatment with the specific proteasome inhibitor epoxomicin. Adeno-associated virus serotype 6 (AAV6)-mediated S100A4 overexpression in a 3D model of EHT did not affect contractile parameters. Immunofluorescence analysis showed a cytosolic and partly nuclear expression pattern of S100A4. Gene expression analysis in EHTs overexpressing S100A4-V5 showed markedly lower steady-state concentrations of genes involved in cardiac fibrosis and pathological cardiac hypertrophy.Conclusion: We showed that S100A4 protein level is higher in cardiac tissue of Mybpc3-KI HCM mice probably as a result of a lower degradation by the E3 ligase Asb2β. While an overexpression of S100A4 did not alter contractile parameters in EHTs, downstream gene expression analysis points toward modulation of signaling cascades involved in fibrosis and hypertrophy

Acute hypoxia induces apoptosis of pancreatic β-cell by activation of the unfolded protein response and upregulation of CHOP

The success of pancreatic β-cells transplantation to treat type 1 diabetes has been hindered by massive β-cell dysfunction and loss of β-cells that follows the procedure. Hypoxia-mediated cell death has been considered one of the main difficulties that must be overcome for transplantation to be regarded as a reliable therapy. Here we have investigated the mechanisms underlying β-cell death in response to hypoxia (1% O2). Our studies show that mouse insulinoma cell line 6 (Min6) cells undergo apoptosis with caspase-3 activation occurring as early as 2 h following exposure to hypoxia. Hypoxia induces endoplasmic reticulum stress in Min6 cells leading to activation of the three branches of the unfolded protein response pathway. In response to hypoxia the pro-apoptotic transcription factor C/EBP homologous protein (CHOP) is upregulated. The important role of CHOP in the apoptotic process was highlighted by the rescue of Min6 cells from hypoxia-mediated apoptosis observed in CHOP-knockdown cells. Culturing isolated pancreatic mouse islets at normoxia showed intracellular hypoxia with accumulation of hypoxia-inducible factor-1α and upregulation of CHOP, the latter one occurring as early as 4 h after isolation. Finally, we observed that pancreatic islets of type 2 db/db diabetic mice were more hypoxic than their counterpart in normoglycemic animals. This finding indicates that hypoxia-mediated apoptosis may occur in type 2 diabetes