Understanding the correlation between structure and dynamics of clocortolone pivalate by solid state NMR measurement

Structural characteristics of clocortolone pivalate are unique in the topical corticosteroid field having high penetration power through the stratum corneum of skin as well as low corticosteroid-related adverse effects. The molecule was thoroughly studied by ¹³C 2DPASS CP MAS NMR and spin–lattice relaxation time measurements. Molecular correlation time at different carbon nuclei positions was calculated by assuming that the chemical shift anisotropy interaction and heteronuclear dipole–dipole interaction play vital roles in the ¹³C spin–lattice relaxation mechanism. The CSA parameters are substantially varied at different carbon nuclei sites. This suggests that the electronic distribution surrounding the carbon nuclei varies widely when the same carbon atom is placed in different chemical surroundings of the molecule. The spinning CSA sideband pattern for C11, C17, C26 nuclei is axially symmetric. The asymmetry parameter is very small (≤0.3) for C2, C5, C10, C22, C23, C24 nuclei, and it is reasonably high (≥0.9) for C3, C4, C6, C18, C19, C21 nuclei. The anisotropy parameter is very high for double bonded C14, C15, C18, C19, C21, C22, and C23 nuclei. Spin–lattice relaxation time and molecular correlation time are also varied substantially for carbon nuclei situated at various positions of the molecule. The spin–lattice relaxation time is slow for carbon nuclei residing at the carbon ring, and it is very fast for C12, C17, C16, C26 carbon nuclei situated at the side portion of the molecule. Molecular correlation time is of the order of 10⁻⁴ s for those carbon nuclei attached with neighbouring carbon or oxygen atoms by double bonds like C14, C15, C18, C19, C21, C22, and C23. It implies that the molecular correlation time is very high for those carbon nuclei associated with high values of the chemical shift anisotropy parameter. In contrast, the molecular correlation time is of the order of 10⁻⁸ s for C12, C16, and C17 carbon nuclei. From these studies, it is clear that the various portions of the molecule exhibit different degrees of motion and the dynamics is related with the structural characteristics of the molecule. These investigations on important drug clocortolone pivalate by solid state NMR will help researchers to understand the structure and dynamics of the molecule, which will give a direction to develop advance corticosteroids.

Structural characteristics of clocortolone pivalate are unique in the topical corticosteroid field having high penetration power through the stratum corneum of skin as well as low corticosteroid-related adverse effects.     

Transmembrane allosteric coupling of the gates in a potassium channel

It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98–103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.Potassium channel activation and inactivation is fundamental to many physiological functions including muscle contraction and the generation of synaptic action potentials (1). KcsA is a 160-residue pH-activated homotetrameric K⁺ channel isolated from the soil bacterium Streptomyces lividans (2, 3) with high sequence homology and functional similarity to mammalian potassium channels (4). It has provided an excellent model for studies of ion-conduction by X-ray crystallography (3, 5, 6), electrophysiology (7, 8), and NMR (9–21). Like many potassium channels, it exhibits (4, 6, 22, 23) slow, spontaneous inactivation involving the residues near the extracellular selectivity filter subsequent to channel activation. Recent results from X-ray crystallography and molecular dynamics suggest that the gates are coupled and that inactivation is prompted by channel opening, mediated via a series of intrasubunit steric contacts involving F103 with T74, T75, and M96 and an intersubunit contact with the neighboring I100 side chain (4–6, 24, 25). In separate experiments, the extracellular gate has been observed to respond directly to ambient [K⁺]: at high [K⁺] it exists in a conductive form, and at low K⁺ it collapses into a nonconductive state (3). Our NMR studies suggest that the low [K⁺] state and the low pH inactivated state may be similar; this conclusion is supported by the effect of the mutation E71A and the pattern of chemical shift perturbations in the selectivity filter when the ion is depleted (9, 19). Meanwhile, X-ray crystallography studies suggest that mutants (E71A) unable to undergo inactivation are also unable to expel ions (26).An established similarity of the low pH and the low [K⁺] states would clarify the importance of allosteric coupling and have the practical consequence that the well-behaved low K⁺ state could serve as a useful structural proxy for the otherwise fleeting inactivated state. For these reasons we tested this correspondence using NMR experiments. If the low K⁺ state is similar to the inactivated state of KcsA achieved by lowering the pH, it is expected that structural changes indicative of channel opening observed at low [K⁺] would occur not only in the selectivity filter but also in the pH gate and the hinge region. However, some studies imply that these two gates might be uncoupled or weakly coupled. For example, X-ray crystallographic studies of KcsA, where K⁺ sensitivity was largely isolated to the selectivity filter (3). In this work, we asked whether, by contrast, full-length wild-type KcsA (160 aa) reconstituted into hydrated lipid bilayers exhibits global structural changes upon ion expulsion suggestive of channel opening. To accomplish this, nearly complete ¹³C and ¹⁵N chemical shift assignments were obtained for the transmembrane and loop regions from four-dimensional (4D) solid-state nuclear magnetic resonance (SSNMR) (27), providing numerous reporters for conformational change during ion binding. In low [K⁺] ion conditions at neutral pH, not only does KcsA expel the K⁺ ions from the inner selectivity filter sites, but the channel also exhibits chemical shift perturbations at the pH gate and the hinge of the inner transmembrane helix, suggesting features akin to the inhibited state that is present at low pH and high [K⁺]. That these two distinct conditions result in a nearly identical state of the channel offers strong evidence for transmembrane allostery in the inactivation process.     

Hepatoportal sclerosis (obliterative portal venopathy) and nodular regenerative hyperplasia in a patient with myasthenia gravis: A case report and review of the published work

Nodular regenerative hyperplasia (NRH) and hepatoportal sclerosis, also known as obliterative portal venopathy (OPV), are two causes of non‐cirrhotic portal hypertension (NCPH). NCPH is an increasingly recognized entity that can be seen in association with collagen vascular diseases and with the use of medications such as azathioprine and didanosine, but oftentimes the etiology remains unidentified. We herein report a case of NCPH occurring due to OPV and NRH in a 64‐year‐old woman with myasthenia gravis (MG), status post‐thymectomy. Portal hypertension was diagnosed incidentally on computed tomography in the absence of predisposing factors. Extensive work‐up to determine the etiology of any underlying liver disease was unrevealing. NRH and OPV were identified on liver biopsy. Subsequently, the patient had variceal bleeding that necessitated transjugular intrahepatic portosystemic shunt placement. A few similar cases of NCPH occurring in the setting of MG have been previously reported, suggesting that the immunological mechanisms involved in the pathogenesis of myasthenia may also have contributed to the development of NCPH.     

The Application of Proteomics to Traumatic Brain and Spinal Cord Injuries

Traumatic brain injury (TBI) and traumatic spinal cord injury (SCI), collectively termed neurotrauma, are two parallel neurological conditions that can cause long-lasting neurological impairment and other comorbidities in patients, while at the same time, can create a high burden to society. To date, there are still no FDA-approved therapeutic interventions for either TBI or SCI. Recent advances in proteomic technologies, including tandem mass spectrometry, as well as imaging mass spectrometry, have enabled new approaches to study the differential proteome in TBI and SCI with the use of either animal disease models and/or biosamples from clinical observational studies. Thus, the applications of state-of-the-art proteomic method hold promises in shedding light on identifying clinically useful neurotrauma “biomarkers” and/or in identifying distinct and, otherwise, unobvious systems pathways or “key drivers” that can be further exploited as new therapeutic intervention targets.     

Dimeric organization of the yeast oligosaccharyl transferase complex

The enzyme complex oligosaccharyl transferase (OT) catalyzes N-glycosylation in the lumen of the endoplasmic reticulum. The yeast OT complex is composed of nine subunits, all of which are transmembrane proteins. Several lines of evidence, including our previous split-ubiquitin studies, have suggested an oligomeric organization of the OT complex, but the exact oligomeric nature has been unclear. By FLAG epitope tagging the Ost4p subunit of the OT complex, we purified the OT enzyme complex by using the nondenaturing detergent digitonin and a one-step immunoaffinity technique. The digitonin-solubilized OT complex was catalytically active, and all nine subunits were present in the enzyme complex. The purified OT complex had an apparent mass of approximately 500 kDa, suggesting a dimeric configuration, which was confirmed by biochemical studies. EM showed homogenous individual particles and revealed a dimeric structure of the OT complexes that was consistent with our biochemical studies. A 3D structure of the dimeric OT complex at 25-A resolution was reconstructed from EM images. We suggest that the dimeric structure of OT might be required for effective association with the translocon dimer and for its allosteric regulation during cotranslational glycosylation.     

Explanatory Models of Health and Disease Among South Asian Immigrants in Chicago

To identify concepts of health and disease as part of a study on designing culturally-targeted heart disease prevention messages for South Asians. We conducted qualitative, semi-structured interviews in English, Hindi and Urdu with 75 respondents from a federally qualified health center and at a community center for South Asian immigrants in Chicago, Illinois. Age ranged from 20 to 70 years; 60% were women; 60% held advanced degrees; 70% migrated to the US in the last 10 years; and 60% of the interviews were in Hindi or Urdu. Concepts of health and disease fell into four domains: behavioral, physical, psycho-social and spiritual. Muslim participants consistently evoked spiritual factors such as faith and prayer. Women more frequently included performing home duties and positive affect in their concept of health. Men more frequently cited behavioral factors such as smoking and drinking as the cause of disease. Many South Asians have a holistic conceptualization of health and disease, incorporating spiritual, physical and psycho-social factors. Health promotion strategies aimed at South Asians in the US should take into account this holistic model of health and disease, while also recognizing that variations exist within South Asians, by gender and religion.     

Student pharmacists' perceptions of community pharmacy residency programs

ObjectivesTo compare penultimate-year (next-to-last) and final-year student pharmacists' perceptions of the educational value of community pharmacy residency programs (CPRPs) and to compare student pharmacists' perceptions of the educational value of CPRPs and health-system residency programs (HSRPs).MethodsA self-administered online survey was sent to administrators at 119 Accreditation Council for Pharmacy Education–accredited schools of pharmacy for ultimate distribution to penultimate- and final-year student pharmacists. The survey included demographic measures and a 20-item residency program “perceived value of skill development” scale developed for this study.Results1,722 completed surveys were received and analyzed. Penultimate-year students attributed greater value to CPRPs more frequently than final-year students. Students more often attributed higher value to CPRPs for skills related to business management, practice management, and medication therapy management, while they attributed higher value to HSRPs for skills related to teaching, research, and clinical knowledge.ConclusionThe results of this study suggest students' perceived value of CPRPs may be related to their year of pharmacy school and the pharmacy practice skill in question.