Preparation of budesonide and budesonide-PLA microparticles using supercritical fluid precipitation technology

The objective of this study was to prepare and characterize microparticles of budesonide alone and budesonide and polylactic acid (PLA) using supercritical fluid (SCF) technology. A precipitation with a compressed antisolvent (PCA) technique employing supercritical CO₂ and a nozzle with 100-μm internal diameter was used to prepare microparticles of budesonide and budesonide-PLA. The effect of various operating variables (temperature and pressure of CO₂ and flow rates of drug-polymer solution and/or CO₂) and formulation variables (0.25%, 0.5%, and 1% budesonide in methylene chloride) on the morphology and size distribution of the microparticles was determined using scanning electron microscopy. In addition, budesonide-PLA particles were characterized for their surface charge and drug-polymer interactions using a zeta meter and differential scanning calorimetry (DSC), respectively. Furthermore, in vitro budesonide release from budesonide-PLA microparticles was determined at 37°C. Using the PCA process, budesonide and budesonide-PLA microparticles with mean diameters of 1 to 2 μm were prepared. An increase in budesonide concentration (0.25%–1% wt/vol) resulted in budesonide microparticles that were fairly spherical and less aggiomerated. In addition, the size of the microparticles increased with an increase in the drug-polymer solution flow rate (1.4–4.7 mL/min) or with a decrease in the CO₂ flow rate (50–10 mL/min). Budesonide-PLA microparticles had a drug loading of 7.94%, equivalent to ∼80% encapsulation efficiency. Budesonide-PLA microparticles had a zeta potential of— 37±4 mV, and DSC studies indicated that SCF processing of budesonide-PLA microparticles resulted in the loss of budesonide crystallinity. Finally, in vitro drug release studies at 37°C indicated 50% budesonide release from the budesonide-PLA microparticles at the end of 28 days. Thus, the PCA process was successful in producing budesonide and budesonide-PLA microparticles. In addition, budesonide-PLA microparticles sustained budesonide release for 4 weeks.     

Development of a simple fed-batch process for the high-yield production of recombinant Japanese encephalitis virus protein

Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. Optimization of media was carried out for enhanced production of recombinant JE virus envelope domain III (EDIII) protein in Escherichia coli. Furthermore, batch and fed-batch cultivation process in E. coli was also developed in optimized medium. Expression of this protein in E. coli was induced with 1 mM isopropyl-β-thiogalactoside and yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 8 M urea, and the protein was purified under denaturing conditions using Ni-NTA affinity chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell dry weight and purified protein about 36.45 g l⁻¹ and 720 mg l⁻¹ of culture, respectively. The purity of the recombinant JE virus EDIII protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, and reactivity of this protein was determined by Western blotting and ELISA with JE virus-infected human serum samples. These results establish the application of this protein to be used for the diagnosis of JE virus infection or for further studies in vaccine development. This process may also be suitable for the high-yield production of other recombinant viral proteins.     

Structural prediction and comparative docking studies of psychrophilic ��- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes

Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.     

Comprehensive small animal imaging strategies on a clinical 3 T dedicated head MR-scanner; adapted methods and sequence protocols in CNS pathologies

Background

Small animal models of human diseases are an indispensable aspect of pre-clinical research. Being dynamic, most pathologies demand extensive longitudinal monitoring to understand disease mechanisms, drug efficacy and side effects. These considerations often demand the concomitant development of monitoring systems with sufficient temporal and spatial resolution.

Methodology and Results

This study attempts to configure and optimize a clinical 3 Tesla magnetic resonance scanner to facilitate imaging of small animal central nervous system pathologies. The hardware of the scanner was complemented by a custom-built, 4-channel phased array coil system. Extensive modification of standard sequence protocols was carried out based on tissue relaxometric calculations. Proton density differences between the gray and white matter of the rodent spinal cord along with transverse relaxation due to magnetic susceptibility differences at the cortex and striatum of both rats and mice demonstrated statistically significant differences. The employed parallel imaging reconstruction algorithms had distinct properties dependent on the sequence type and in the presence of the contrast agent. The attempt to morphologically phenotype a normal healthy rat brain in multiple planes delineated a number of anatomical regions, and all the clinically relevant sequels following acute cerebral ischemia could be adequately characterized. Changes in blood-brain-barrier permeability following ischemia-reperfusion were also apparent at a later time. Typical characteristics of intra-cerebral haemorrhage at acute and chronic stages were also visualized up to one month. Two models of rodent spinal cord injury were adequately characterized and closely mimicked the results of histological studies. In the employed rodent animal handling system a mouse model of glioblastoma was also studied with unequivocal results.

Conclusions

The implemented customizations including extensive sequence protocol modifications resulted in images of high diagnostic quality. These results prove that lack of dedicated animal scanners shouldn''t discourage conventional small animal imaging studies.     

Recombinant dengue virus type 3 envelope domain III protein from Escherichia coli

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. The envelope protein of dengue virus is the major antigen to elicit neutralizing antibody response and protective immunity in hosts. Optimization of culture media was carried out for enhanced production of recombinant dengue virus type 3 envelope domain III (rDen 3 EDIII) protein in E. coli. Further, batch and fed-batch cultivation process were also developed in optimized medium. After fed-batch cultivation, the dry cell weight was about 22.80 g/L of culture. The rDen 3 EDIII protein was purified using immobilized metal affinity chromatography. This process produced ～649 mg of purified rDen 3 EDIII protein per liter of culture. The purity of the protein was determined by SDS-PAGE analysis and the reactivity was checked by Western blotting as well as ELISA. These results show that the purified protein may be used for the dengue diagnosis or further prophylactic studies for dengue infection.     

Cerebrospinal Fluid Catecholamine Levels in Japanese Encephalitis Patients with Movement Disorders

Norepinephrine and dopamine have important role in movement disorders but their role in movement disorders associated with Japanese encephalitis (JE) has not been evaluated. Therefore, in the present study, cerebrospinal fluid (CSF) catecholamine levels and its metabolites in JE patients with movement disorders were compared with those without JE. CSF was collected by lumbar puncture and analyzed by HPLC-ED. Norepinephrine, dopamine and homovanillic acid concentrations were significantly (P<0.005) lower in JE patients compared to control groups. Low levels of catecholamines in JE associated movement disorders compared to idiopathic Parkinson’s disease and other extrapyramidal symptoms may be due to severe structural damage to thalamus, basal ganglia and brainstem in JE patients as revealed by MRI findings.     

Chronic kidney disease and automatic reporting of estimated glomerular filtration rate: a position statement

The systematic staging of chronic kidney disease (CKD) by glomerular filtration measurement and proteinuria has allowed the development of rational and appropriate management plans. One of the barriers to early detection of CKD is the lack of a precise, reliable and consistent measure of kidney function.The most common measure of kidney function is currently serum creatinine concentration. It varies with age, sex, muscle mass and diet, and interlaboratory variation between measurements is as high as 20%.The reference interval for serum creatinine concentration includes up to 25% of people (particularly thin, elderly women) who have an estimated glomerular filtration rate (eGFR) that is significantly reduced (< 60 mL/min/1.73 m). The recent publication of a validated formula (MDRD) to estimate GFR from age, sex, race and serum creatinine concentration, without any requirement for measures of body mass, allows pathology laboratories to "automatically" generate eGFR from data already acquired. Automatic laboratory reporting of eGFR calculated from serum creatinine measurements would help to identify asymptomatic kidney dysfunction at an earlier stage. eGFR correlates well with complications of CKD and an increased risk of adverse outcomes such as cardiovascular morbidity and mortality. We recommend that pathology laboratories automatically report eGFR each time a serum creatinine test is ordered in adults. As the accuracy of eGFR is suboptimal in patients with normal or near-normal renal function, we recommend that calculated eGFRs above 60 mL/min/1.73 m be reported by laboratories as "> 60 mL/min/1.73 m", rather than as a precise figure.     

Expression, purification and evaluation of diagnostic potential and immunogenicity of dengue virus type 3 domain III protein

Dengue hemorrhagic fever and dengue shock syndrome are the severe manifestations of dengue infection. The quest for reliable dengue diagnostics and a dengue vaccine remained elusive for decades. Domain III of dengue virus envelope contains multiple conformation dependant neutralizing epitopes, thus making it an attractive diagnostic and vaccine candidate. In this report we show the expression of dengue virus type 3 envelope domain III protein (D3EDIII) and demonstrate its potential as a diagnostic and vaccine candidate. Accordingly, D3EDIII was expressed to high levels in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified protein was used to develop an in-house plate ELISA and was further tested with a panel of 40 dengue infected serum samples previously characterized by commercially available serological tests. The in-house results were in excellent agreement with the commercial kits. D3EDIII was refolded by rapid dilution method and the refolded monomer protein was purified by Ion exchange chromatography. Further, the recombinant protein was biologically functional and found to inhibit dengue virus type 3 plaque formation on LLC-MK2 cells demonstrating its function of receptor interaction. Furthermore, D3EDIII in combination with Freund's complete adjuvant induced high antibody titers in BALB/c mice and these antibodies efficiently neutralized dengue 3 virus. Additionally, D3EDIII induced expression of Th1 cytokines that can inhibit the intracellular viral infections. Thus, our results demonstrate that D3EDIII protein has tremendous potential both in diagnosis of dengue infections and in vaccine development.     

Intra- and inter-subunit disulfide bond formation is nonessential in adeno-associated viral capsids

The capsid proteins of adeno-associated viruses (AAV) have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s) of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.