An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region

The mitotic spindle is a microtubular assembly required for chromosome segregation during mitosis. Additionally, a spindle matrix has long been proposed to assist this process, but its nature has remained elusive. By combining live-cell imaging with laser microsurgery, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy in Drosophila melanogaster S2 cells, we uncovered a microtubule-independent mechanism that underlies the accumulation of molecules in the spindle region. This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells. Supported by mathematical modeling, we demonstrate that organelle exclusion by a membrane system causes spatio-temporal differences in molecular crowding states that are sufficient to drive accumulation of mitotic regulators, such as Mad2 and Megator/Tpr, as well as soluble tubulin, in the spindle region. This membranous “spindle envelope” confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation. Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function.     

Optimization Studies on Gastroretentive Floating System Using Response Surface Methodology

The aim of the present investigation was to develop and optimize gastroretentive floating system of amoxicillin for the efficient treatment of peptic ulcer induced by Helicobacter pylori infection. Floating microballoons were developed using central composite design (CCD), and optimization was done by employing response surface methodology. The selected independent variables were cellulose acetate phthalate, drug–Eudragit S100 ratio, and the ratio of dichloromethane/ethanol/isopropyl alcohol. The selected dependent variables were yield, mean particle size, buoyancy, encapsulation efficiency, and drug release within 8 h. A quadratic polynomial model was generated which had linear, interaction, and quadratic terms to predict and evaluate the independent variables with respect to the dependent variables. Results showed that selected independent variables significantly affect the yield (30.53–82.71%), particle size (31.62–47.03 μm), buoyancy (42.68–95.75%), encapsulation efficiency (56.96–93.13%), and cumulative drug release from the microballoons (34.01–74.65%). The interaction and quadratic terms were also found to affect the process variables. An excellent agreement was found between the actual value and predicted value. In conclusion, it can be said that CCD is a valuable second-degree design to develop and optimize GFS of amoxicillin which in turn provides a basis to localize the drug release in the gastric region for effective treatment of H. pylori-mediated infection.     

Rice germ oil as multifunctional excipient in preparation of self-microemulsifying drug delivery system (SMEDDS) of tacrolimus

Surmounting the constraints of limited solubilization efficiency and prime requisite of antioxidant for conventional lipid formulations, the research work explores an edge over formulation utilizing potential applicability of rice germ oil (RGO) as a multifunctional excipient. Self-microemulsifying drug delivery system (SMEDDS) of tacrolimus (TAC) was formulated with RGO, an indigenous source of gamma-oryzanol. Being the same biological source, RGO and rice bran oil (RBO) were compared and it was found that RGO have more solubilization potential for TAC (2.2-fold) as well as higher antioxidant activity (8.06-fold) than the RBO. TAC-SMEDDS was prepared using RGO/Capmul PG8 (2:3) as an oil phase, Cremophore EL as a surfactant, and Transcutol P as a cosurfactant. The approximate particle size of TAC-SMEDDS was found to be 38 nm by dynamic light scattering and 12 nm by small angle neutron scattering. The in vitro dissolution studies showed complete and rapid drug release in 30 min compared to a plain drug (<5%) and marketed capsule (<50%). AUC and C _max were found to be 45.05 ± 15.64 ng h/ml and 3.91 ± 1.2 ng/ml for TAC-SMEDDS, 12.59 ± 5.54 ng h/ml and 0.48 ± 0.12 ng/ml for plain TAC, and 30.23 ± 10.34 ng h/ml and 2.31 ± 0.68 ng/ml for marketed formulation, respectively. The improved pharmacokinetic profile of TAC-SMEDDS is correlating to the dissolution results. Thus, gamma-oryzanol-enriched RGO acts as a potential multifunctional excipient for lipid formulations.     

Detection, isolation and confirmation of Crimean-Congo hemorrhagic fever virus in human, ticks and animals in Ahmadabad, India, 2010-2011

Background

In January 2011, human cases with hemorrhagic manifestations in the hospital staff were reported from a tertiary care hospital in Ahmadabad, India. This paper reports a detailed epidemiological investigation of nosocomial outbreak from the affected area of Ahmadabad, Gujarat, India.

Principal Findings

Samples from 3 suspected cases, 83 contacts, Hyalomma ticks and livestock were screened for Crimean-Congo hemorrhagic fever (CCHF) virus by qRT-PCR of which samples of two medical professionals (case C and E) and the husband of the index case (case D) were positive for CCHFV. The sensitivity and specificity of indigenous developed IgM ELISA to screen CCHFV specific antibodies in human serum was 75.0% and 97.5% respectively as compared to commercial kit. About 17.0% domestic animals from Kolat, Ahmadabad were positive for IgG antibodies while only two cattle and a goat showed positivity by qRT-PCR. Surprisingly, 43.0% domestic animals (Buffalo, cattle, sheep and goat) showed IgG antibodies in the adjoining village Jivanpara but only one of the buffalo was positive for CCHFV. The Hyalomma anatolicum anatolicum ticks were positive in PCR and virus isolation. CCHFV was isolated from the blood sample of case C, E in Vero E-6 cells and Swiss albino mice. In partial nucleocapsid gene phylogeny from CCHFV positive human samples of the years 2010 and 2011, livestock and ticks showed this virus was similar to Tajikistan (strain TAJ/{"type":"entrez-nucleotide","attrs":{"text":"H08966","term_id":"873788","term_text":"H08966"}}H08966), which belongs in the Asian/middle east genetic lineage IV.

Conclusions

The likely source of CCHFV was identified as virus infected Hyalomma ticks and livestock at the rural village residence of the primary case (case A). In addition, retrospective sample analysis revealed the existence of CCHFV in Gujarat and Rajasthan states before this outbreak. An indigenous developed IgM ELISA kit will be of great use for screening this virus in India.     

Site specific chemical delivery of NSAIDs to inflamed joints: synthesis, biological activity and gamma-imaging studies of quaternary ammonium salts of tropinol esters of some NSAIDs or their active metabolites

Quaternized tropinol ester derivatives of some commonly used non-steroidal anti-inflammatory drugs (NSAIDs) or their active metabolites, were prepared and studied for their anti-inflammatory activity in a chronic inflammation model and for inflamed tissue tropism. The quaternized esters were radiolabeled with 99mTechnetium (99mTc) and their selective localization in the inflamed tissue was traced using scintigraphy. In the chronic arthritis rodent model, most of the quaternized esters exhibited anti-inflammatory effect comparable to their respective parent drugs. In the gamma-imaging studies only the quaternary derivatives exhibited selective accumulation into the inflamed tissue unlike the parent NSAIDs or the unquaternized tropinol esters. This work is a step ahead in the direction of use of quaternary ammonium ester derivatives for site specific chemical delivery of commonly used NSAIDs to the inflamed tissues to minimize their GIT side effect or other systemic toxicities.     

Comparative analysis of neonatal and adult rat carotid body responses to chronic intermittent hypoxia.

Previous studies suggest that carotid body responses to long-term changes in environmental oxygen differ between neonates and adults. In the present study we tested the hypothesis that the effects of chronic intermittent hypoxia (CIH) on the carotid body differ between neonates and adult rats. Experiments were performed on neonatal (1-10 days) and adult (6-8 wk) males exposed either to CIH (9 episodes/h; 8 h/day) or to normoxia. Sensory activity was recorded from ex vivo carotid bodies. CIH augmented the hypoxic sensory response (HSR) in both groups. The magnitude of CIH-evoked hypoxic sensitization was significantly greater in neonates than in adults. Seventy-two episodes of CIH were sufficient to evoke hypoxic sensitization in neonates, whereas as many as 720 CIH episodes were required in adults, suggesting that neonatal carotid bodies are more sensitive to CIH than adult carotid bodies. CIH-induced hypoxic sensitization was reversed in adult rats after reexposure to 10 days of normoxia, whereas the effects of neonatal CIH persisted into adult life (2 mo). Acute intermittent hypoxia (IH) evoked sensory long-term facilitation of the carotid body activity (sensory LTF, i.e., increased baseline neural activity following acute IH) in CIH-exposed adults but not in neonates. The effects of CIH were associated with hyperplasia of glomus cells in neonatal but not in adult carotid bodies. These observations demonstrate that responses to CIH differ between neonates and adults with regard to the magnitude of sensitization of HSR, susceptibility to CIH, induction of sensory LTF, reversibility of the responses, and morphological remodeling of the chemoreceptor tissue.     

Synthesis,antioxidant, antifungal,molecular docking and ADMET studies of some thiazolyl hydrazones

Some thiazolyl hydrazones were synthesized by one pot reaction of thiophene-2-carbaldehyde or 2, 4-dichlorobenzaldehyde, thiosemicarbazide and various phenacyl bromides which were preliminarily screened for in vitro antioxidant and antifungal activities. Excellent DPPH and H₂O₂ radical scavenged antioxidant activities were observed with almost all the tested compounds. Compounds 4a, 4b, 4c, 4e, 4f and 4i showed comparable DPPH scavenged antioxidant potential (90.26–96.56%) whereas H₂O₂ scavenged antioxidant activity (90.98–92.08%) was noticeable in case of 4a and 4f; showing significant antioxidant potential comparable with the standard ascorbic acid (95.3%). In vitro antifungal activity of synthesized compounds against fungal species Candida albicance, Aspergillus niger and Aspergillus flavus was found to be moderate to good as compared with the standard fluconazole and MIC values were found in the range of 3.12–25 μg/mL. Molecular docking studies revealed that the compounds 4a, 4b and 4c have a potential to become lead molecules in drug discovery process. In silico ADMET study was also performed for predicting pharmacokinetic and toxicity profile of the synthesized antioxidants which expressed good oral drug like behaviour and non-toxic nature.     

Fluorescence‐based sensor for selective and sensitive detection of amoxicillin (Amox) in aqueous medium: Application to pharmaceutical and biomedical analysis

We here for the first time demonstrate an analytical approach for the highly selective and sensitive detection of amoxicillin (Amox) in aqueous medium based on the fluorescence quenching of quantum dots (QDs). The change in fluorescence intensity of mercaptopropionic acid‐capped cadmium sulphide (MPA‐CdS) QDs is attributed to the increasing concentration of Amox. The results show that the fluorescence quenching of QDs by Amox takes place through both static and dynamic types of quenching mechanism. The fluorescence quenching of QDs with increase in concentration of Amox shows the linear range between 5 μg ml^?1 and 30 μg ml^?1 and the limit of detection (LOD) is 5.19 μg ml^?1. There is no interference of excipients, which are commonly present in pharmaceutical formulation and urine samples. For the practical application approach, the developed method has been successfully applied for the determination of Amox in pharmaceutical formulations and urine samples with acceptable results.     

Probing the mechanism of interaction of metoprolol succinate with human serum albumin by spectroscopic and molecular docking analysis

In the present work, the mechanism of the interaction between a β1 receptor blocker, metoprolol succinate (MS) and human serum albumin (HSA) under physiological conditions was investigated by spectroscopic techniques, namely fluorescence, Fourier transform infra‐red spectroscopy (FT‐IR), fluorescence lifetime decay and circular dichroism (CD) as well as molecular docking and cyclic voltammetric methods. The fluorescence and lifetime decay results indicated that MS quenched the intrinsic intensity of HSA through a static quenching mechanism. The Stern–Volmer quenching constants and binding constants for the MS–HSA system at 293, 298 and 303 K were obtained from the Stern–Volmer plot. Thermodynamic parameters for the interaction of MS with HSA were evaluated; negative values of entropy change (ΔG°) indicated the spontaneity of the MS and HSA interaction. Thermodynamic parameters such as negative ΔH° and positive ΔS° values revealed that hydrogen bonding and hydrophobic forces played a major role in MS–HSA interaction and stabilized the complex. The binding site for MS in HSA was identified by competitive site probe experiments and molecular docking studies. These results indicated that MS was bound to HSA at Sudlow's site I. The efficiency of energy transfer and the distance between the donor (HSA) and acceptor (MS) was calculated based on the theory of Fosters' resonance energy transfer (FRET). Three‐dimensional fluorescence spectra and CD results revealed that the binding of MS to HSA resulted in an obvious change in the conformation of HSA. Cyclic voltammograms of the MS–HSA system also confirmed the interaction between MS and HSA. Furthermore, the effects of metal ions on the binding of MS to HSA were also studied.