The preparation of silica gel thin layers with a new apparatus.

An apparatus for the preparation of uniform thin layer chromatography gel layers is described. The apparatus employs rubber cushion runners to compensate for differences in plate thickness and a gel applicator which functions independently of plate edge variability. Consistently uniform layers are prepared by careful establishment of the plate-to-applicator distance. Silica gel layers averaged 87% of the applied thickness with a variability of +/-2% within a single run and +/-8% between independent runs.     

Parallel plate equipment for preparation of uniform gel sheets

A method for the preparation of uniform gel-disks for enzyme and cell immobilisation, as well as for characterisation of gel mechanical stability, is described. The apparatus comprises a stainless steel base unit and glass parallel plates, designed to permit easy and fast production of multiple homogeneous gel sheets of variable thickness.     

Quantitative analysis of 6-hydroxymelatonin in human urine by gas chromatography-negative chemical ionization mass spectrometry

A method for assay of urinary 6-hydroxymelatonin, the major metabolite of the pineal hormone melatonin is described. After addition of an internal standard of deuterated 6-hydroxymelatonin sulfate, human urine was hydrolyzed enzymatically and free 6-hydroxymelatonin extracted, reacted to form a stable t-butyldimethylsilylpentafluoropropionyl derivative which was separated on silica gel column chromatography, and quantified using electron capture negative-ion chemical ionization mass spectrometry. Intrassay variability over an 18-h period was 5.4% [53.8 ng/3 ml urine ± 2.94 (SD)] and interassay variability over a 2-week period was 2.1% [51.8 ng/3 ml urine ± 1.08 (SD)].     

Terbinafine hydrochloride loaded liposome film formulation for treatment of onychomycosis: in vitro and in vivo evaluation

Onychomycosis is a fungal infection of nail unit that is caused by dermatophytes. Oral Terbinafine hydrochloride (TBF-HCl) is being used for the treatment of onychomycosis since 24 years. The side effects caused by the systemic application and limitations of topical administration of this drug regarding the diffusion through nail lead to the development of a new formulation based on, TBF-HCl-loaded liposome. The newly obtained film formulations were prepared and characterized via several parameters, such as physical appearance, drug content, thickness, bioadhesive properties and tensile strength. In vitro and ex vivo permeation studies were performed to select an optimum film formulation for antifungal activity to show the efficiency of formulations regarding the treatment of onychomycosis. The in vitro release percentages of drug were found 71.6?±?3.28, 54.4?±?4.26, 56.1?±?7.48 and 46.0?±?2.43 for liposome loaded pullulan films (LI-P, LII-P) and liposome loaded Eudragit films (LI-E, LII-E), respectively. The accumulated drug in the nail plates were found 31.16?±?4.22, 24.81?±?5.35, 8.17?±?1.81 and 8.92?±?3.37 for LI-P, LII-P, LI-E and LII-E, respectively, which within therapeutic range for all film formulations. The accumulated drug in the nail plate was found within therapeutic range for all film formulations. The efficacy of the selected TBF-HCl-loaded liposome film formulation was compared with TBF-HCl-loaded liposome, ethosome, liposome poloxamer gel and ethosome chitosan gel formulations. It was found that TBF-HCl-loaded liposome film formulation had better antifungal activity on fungal nails which make this liposome film formulation promising for ungual therapy of fungal nail infection.     

Progesterone-loaded nanosized transethosomes for vaginal permeation enhancement: formulation,statistical optimization,and clinical evaluation in anovulatory polycystic ovary syndrome

Bio-identical progesterone (PRG) is an exogenous female steroidal hormone which is used for treatment of polycystic ovary syndrome (PCOS). However, it suffers from poor bioavailability due to hepatic metabolism and poor solubility. The target of this work was to evaluate and statistically optimize PRG-loaded nanovesicle transethosomes (NVTEs) based in mucoadhesive gel for transvaginal delivery of PRG as potential luteal-phase support. A 2⁴ full factorial design was used to explore the effect of phosphatidylcholine (PC), Tween 80, cetyltrimethyl ammonium bromide and ethanol concentration on particle size, entrapment efficiency (EE%), % in vitro PRG release after 24?h and transvaginal flux. PRG-loaded NVTEs were prepared by injection sonication method. The results revealed that the mean particle sizes ranged from 133.3?±?3.42 to 349.5?±?1.24?nm, zeta potential ranged from –23.5?±?3.84 to +74.6?±?4.97?mV, EE% ranged from 87.93?±?3.58 to 97.05?±?2.61%, % PRG release ranged from 50.9?±?2.75 to 90.69?±?2.07 and transvaginal flux ranged from 0.274?±?0.03 to 0.531?±?0.04?mg/cm²/h. The optimized formulation was subjected to transmission electron microscope for morphological examination and then incorporated in the mucoadhesive vaginal gel using Carbopol 974, hydroxyl propyl methylcellulose and sodium alginate. The optimized formulation was clinically studied in anovulatory PCOS and showed a significant increase in the serum PRG, endometrial thickness, echogenicity degree and the pregnancy rate. Briefly, PRG-loaded NVTEs vaginal gel might be a promising formulation for luteal phase support and increase pregnancy rate in anovulatory PCOS.     

An automated apparatus for the real-time monitoring of bioluminescence in plants

We developed an automated, high-throughput, bioluminescence-monitoring apparatus that can monitor 1920 individual plant seedlings under uniform light conditions. The apparatus is composed of five units: (i) a plate platform that can hold 20 96-well microplates under uniform light conditions, (ii) a scintillation counter, (iii) a robot that conveys plates between the plate platform and a scintillation counter, (iv) a sequence controller, and (v) an external computer that collects and analyzes bioluminescence data automatically. The apparatus gave reproducible and reliable results for both bioluminescence photon counts and period length of bioluminescence rhythms; neither was affected by the well position in a plate or the plate position on the platform. The apparatus is a powerful tool for both large-scale detailed analysis of gene expression and large-scale screening of mutants.     

Improvements of DNA sequencing gels

We describe three simple modifications of DNA sequencing gels which all result in improved oligonucleotide resolution as visualized by autoradiography. First, it was possible to reduce the thickness of the gel to 0.2 mm by using new gel molding techniques. Second, the gel could be dried without any distortions of its dimensions by prior binding of the gel to the surface of the glass plate. Third, a uniform high temperature was obtained in all parts of the gel during electrophoresis by replacing one of the glass plates with an inexpensive thermostating plate with circulating water. The use of this heating plate resulted in a straight band pattern all over the gel and also in the resolution of such bands which were not resolved in other electrophoresis systems.     

Processing Stripping Film Autoradiographs of Citrus Buds; Avoidance of Entrapped Air between Tissues and Developed Emulsion

Autoradiography was used on paraffin sections for investigating DNA synthesis in vegetative and flower buds of Citrus sinensis (L.) Osbeck. We preferred stripping film to liquid emulsion to obtain a more uniform thickness of the silver grain layer, as this would not vary more than ±10%. Furthermore, Baserga and Nemeroff (1962) have observed a lower sensitivity of fluid emulsion in comparison to stripping film together with occasionally erratic grain count and a slightly higher incidence of mechanical fogging. However, on using the customary procedure it was found that close contact between the surface of the section and the developed emulsion was not maintained when dehydration and clearing was done in the usual manner. The following modifications were therefore introduced to prevent the formation of air pockets between these two layers.     

Effect of Husk morphology on grain development and topography in rice

Kernels grown within loosened glumes in three varieties of paddy were darker in color and had a smoother surface than those grown under normal conditions. The thickness of the pericarp plus seed coat layers was 33.6 ±2.8 µm, and the thickness of the aleurone layers was 21.7 ± 2.5 µm in grains of the first type, while in the normal grains, these dimensions were 13.0 ± 1.4 and 26.9 ± 2.9 µm respectively. The kernels which developed within loosened glumes tended to taper towards the distal end. They were lighter in weight than normal grains by 32 to 67 percent, the weight loss being less in the bolder variety. The lemma-palea interlocking depth was positively correlated with the groove depth on the kernel and with the clearance between husk and kernel. All three parameters showed a positive correlation with grain breadth. A low lemma-palea interlocking depth and a smaller clearance between husk and kernel are technologically desirable characteristics in rice. The reclasping of the two glume components after pollination was essential for the normal development of the rice grain.     

Investigating the penetrating potential of nanocomposite β-cycloethosomes: development using central composite design,in vitro and ex vivo characterization

Context: Atopic dermatitis (AD) is a chronic skin disease characterized by inflammation of the skin and has exhibited remarkable repercussions on human life across the globe. Fluocinolone acetonide (FA), a topical corticosteroid is employed in the treatment of atopic dermatitis, but suffers from limited penetration into deeper epidermis of atopic skin.

Objective: The present investigation was focused to explore the utility of β-cylcoethosomes in improvising the penetration deep into the skin.

Materials and methods: β-Cylcoethosomes developed using β-cycloamylose by injection method were evaluated for vesicle size, entrapment efficiency and in vitro release. Central Composite design employed for the preparation depicted FA8 as an optimized formulation which was then formulated as dermatological gel using carbomer 934P as a gel base. The gels were characterized for pH, viscosity, drug content and in vitro permeability.

Results and discussion: Optimized formulation (FA8) showed maximum desirability (0.795) with vesicle size of 228.33?±?1.23?nm), EE (82.49?±?1.21%) and CDR (90.90?±?0.29%). FA8-loaded gels showed maximum in vitro permeability as found in BG and BGP (83.22?±?0.72% and 84.02?±?0.87). BG was selected as an optimized gel and compared with optimized reference ethosomal gel and control gel. CLSM studies depicted deeper uniform penetration of fluorescent dye deep into the epidermis via BG. Improved penetration was observed due to the synergistic effect exerted by ethanol and β-cycloamylose.

Conclusion: β-cylcoethosomes proved to be a promising carrier for improvised penetration of fluocinolone acetonide via topical gel.     

A simple electronic apparatus for the analysis of radioactively labeled gel electrophoretograms

Radioactively labeled gel electrophoretograms are generally analyzed either by slicing the gel and dissolving the individual pieces in a fluor for scintillation counting or by exposure to photographic emulsions. In this report, we describe a technique for fast quantitative analysis of such electrophoretograms, based on counting individually β-or γ-particles emerging from the gel surface and locating their position to ±0.6 mm. The apparatus which employs a position-sensitive single-wire gas proportional counter is simple to construct and operate with readily available electronics.     

盾叶薯蓣四倍体与二倍体叶表皮及气孔器显微结构的比较

对盾叶薯蓣四倍体和二倍体阴叶和阳叶表皮及气孔器的显微形态结构进行了比较。结果显示:叶片表皮细胞及气孔器的形态结构因染色体倍性及光照的不同而存在明显差异。表皮细胞有规则多边形与不规则细胞壁内褶2种类型;气孔器为典型的双子叶植物无规则型气孔器,仅分布在下表皮。四倍体表皮细胞密度、气孔器密度及气孔指数平均值分别为476.28±6.87个.mm-2、78.22±3.1个.mm-2、14.11,较二倍体的分别小4.60%、17.95%和11.98%。前者气孔器长、宽及保卫细胞宽度平均值分别为32.78±2.09μm、26.07±1.55μm、9.63±1.14μm,较后者的分别大10.73%、3.90%和18.01%,差异极显著。前者阴叶的较其阳叶的分别大5.77%、6.00%、8.72%;后者的则分别大1.72%、1.74%、2.41%。前者叶片保卫细胞中的叶绿体数目为23.93±3.19个,较后者的多74.00%。表皮细胞及气孔器密度、气孔指数,气孔器大小,保卫细胞叶绿体数目、尤其是保卫细胞宽度,可作为倍性鉴定的参考依据。四倍体高产有其良好的叶片结构基础。     

A Novel Apremilast Nail Lacquer Formulation for the Treatment of Nail Psoriasis

The objective was to prepare a novel nail lacquer formulation to improve the ungual and trans-ungual delivery of apremilast for the potential treatment of nail psoriasis. Nail lacquer formulation was prepared using Eudragit® S 100 as a film-forming polymer and the mixture of ethanol, ethyl acetate, and water as a solvent system. As a result of high-throughput screening studies, dexpanthenol and salicylic acid were found to be the potential penetration enhancers. After 7 days of in vitro studies, the cumulative amount of apremilast delivered by the nail lacquer formulation across the nail plate was found to be ~3-fold (0.52 ± 0.07 μg/cm²) more compared to control (nail lacquer formulation without enhancers) (0.19 ± 0.02 μg/cm²). The cumulative amount of apremilast retained in the nail plate in the case of nail lacquer formulation was 1.26 ± 0.18 μg/mg which was found to be ~2-fold more compared to control (0.57 ± 0.07 μg/mg). Human subject studies were performed on the nails of thumb and index finger of six volunteers for 15 days. As a result, the cumulative amount of apremilast retained in the free distal edge of the nail plate in the case of nail lacquer was found to be ~2-fold (0.93 ± 0.14 μg/mg) more related to control (0.41 ± 0.04 μg/mg). As a conclusion, nail lacquer formulation was found to be capable of delivering a substantial amount of apremilast into the nail apparatus; thus, it can be a potential option for the treatment of nail psoriasis.     

Digital twin of a continuous chromatography process for mAb purification: Design and model-based control

Model-based design of integrated continuous train coupled with online process analytical technology (PAT) tool can be a potent facilitator for monitoring and control of Critical Quality Attributes (CQAs) in real time. Charge variants are product related variants and are often regarded as CQAs as they may impact potency and efficacy of drug. Robust pooling decision is required for achieving uniform charge variant composition for mAbs as baseline separation between closely related variants is rarely achieved in process scale chromatography. In this study, we propose a digital twin of a continuous chromatography process, integrated with an online HPLC-PAT tool for delivering real time pooling decisions to achieve uniform charge variant composition. The integrated downstream process comprised continuous multicolumn capture protein A chromatography, viral inactivation in coiled flow inverter reactor (CFIR), and multicolumn CEX polishing step. An online HPLC was connected to the harvest tank before protein A chromatography. Both empirical and mechanistic modeling have been considered. The model states were updated in real time using online HPLC charge variant data for prediction of the initial and final cut point for CEX eluate, according to which the process chromatography was directed to switch from collection to waste to achieve the desired charge variant composition in the CEX pool. Two case studies were carried out to demonstrate this control strategy. In the first case study, the continuous train was run for initially 14 h for harvest of fixed charge variant composition as feed. In the second case study, charge variant composition was dynamically changed by introducing forced perturbation to mimic the deviations that may be encountered during perfusion cell culture. The control strategy was successfully implemented for more than ±5% variability in the acidic variants of the feed with its composition in the range of acidic (13%–17%), main (18%–23%), and basic (59%–68%) variants. Both the case studies yielded CEX pool of uniform distribution of acidic, main and basic profiles in the range of 15 ± 0.8, 31 ± 0.3, and 53 ± 0.5%, respectively, in the case of empirical modeling and 15 ± 0.5, 31 ± 0.3, and 53 ± 0.3%, respectively, in the case of mechanistic modeling. In both cases, process yield for main species was >85% and the use of online HPLC early in the purification train helped in making quicker decision for pooling of CEX eluate. The results thus successfully demonstrate the technical feasibility of creating digital twins of bioprocess operations and their utility for process control.     

Regional Intra‐Subject Variability in Abdominal Adiposity Limits Usefulness of Computed Tomography

Objective: Computed tomography (CT) and magnetic resonance imaging, the most accurate methods of abdominal fat measurement, have been applied using a number of protocols, ranging from single‐slice area determination to multiple‐slice volume calculation. The aim of this study was to assess the validity of single‐slice CT for abdominal fat area measurement by estimating the intra‐subject variability in abdominal fat areas and comparing the ranking of subjects across four contiguous abdominal levels. Research Methods and Procedures: Nineteen premenopausal women (age, 35.3 ± 1.4 years; mean ± SE) were studied. CT was used to measure intra‐abdominal fat (IAF) area, percentage of total intra‐abdominal area (%IAF), subcutaneous abdominal fat (SAF) area, and IAF/SAF at four adjacent cross‐sectional lumbar levels (L2–L4). Intra‐subject variability (percentage) was defined as SD/mean × 100. Total body fat was measured by DXA, which was further analyzed for central abdominal fat. Results: Mean body mass index was 24.9 ± 1.0 kg/m². The average (range) intra‐subject variability was 28% (8% to 61%) for IAF, 46% (19% to 124%) for %IAF, 26% (14% to 38%) for SAF area, and 19% (7% to 71%) for IAF/SAF. The pattern of this variability was not uniform between subjects, because their ranking by IAF area was markedly different at each CT level. Discussion: We demonstrated significant intra‐subject variability in CT‐measured adipose tissue areas across four predetermined sites. This resulted in a difference in the ordering of subjects by IAF at each of the four imaging sites, suggesting that the usefulness of single‐slice CT in the assessment of abdominal adiposity in premenopausal women may be limited, particularly when performed for the purpose of making comparisons between subjects based on abdominal fat area.     

Potential application of immobilized streptokinase extracted from Streptococcus equinus VIT_VB2

Streptokinase purified from Streptococcus equinus VIT_VB2 isolated from bovine milk sample was immobilized in various solid supports namely entrapment in agarose gel, calcium alginate beads and gelatin gel by cross-linking with formaldehyde. Immobilization of streptokinase in calcium alginate beads showed maximum efficiency (81.8?±?1.06%) when compared with entrapment with agarose gel (55.6?±?2.17%) and cross-linked gelatin formaldehyde gel (71.0?±?1.54%). The purified SK activity was expressed maximum in calcium alginate (1%) and gelatin gel (0.25%) with 1292.68?±?1.33 and 1121.9?±?1.2?U?mL^?1, respectively. Similarly, SK entrapped in gelatin gel and calcium alginate showed maximum in vitro blood clot lysis activity with 77.67?±?2.64% and 76.16?±?2.72%, respectively. The immobilized SK in gelatin gel showed complete clot lysis within 15?min; hence, this application of the study could be used in the treatment of superficial thrombophlebitis, phlebitis, and venous thrombosis. These beads were used for three repeated cycles to check the conversion of substrates into their products, and we concluded that SK can be immobilized in the suitable matrices. Therefore, this helps in the drug-delivery strategies in highly efficient way, moreover, economically competent process in the pharmaceutics.     

Topical Delivery of Fenoprofen Calcium <Emphasis Type="Italic">via</Emphasis> Elastic Nano-vesicular Spanlastics: Optimization Using Experimental Design and <Emphasis Type="Italic">In Vivo</Emphasis> Evaluation

The aim of this study was to investigate the potential of surfactant-based nanovesicular system (spanlastics) for topical delivery of fenoprofen calcium (FPCa) to eliminate its oral gastrointestinal adverse effects. FPCa-loaded spanlastics were prepared by thin film hydration (TFH) technique according to a full factorial design to investigate the influence of formulation variables on the drug entrapment efficiency (%EE), particle size (PS), deformability index (DI), and the % drug released after 24 h through the cellulose membrane (Q24h) using Design-Expert® software. The optimized formula (composed of Span 60 and Tween 60 as an edge activator at weight ratio of 8: 2 in presence of Transcutol P as a cosolvent in the hydration media) exhibited the highest %EE (49.91 ± 2.60%), PS of 536.1 ± 17.14 nm, DI of 5.07 ± 0.06 g, and Q24h of 61.11 ± 2.70%; it was also characterized for morphology and physical stability. In vitro release study of FPCa-loaded spanlastic gel and conventional FPCa gel through a synthetic membrane and hairless rat skin were evaluated. The skin permeation study revealed that spanlastic gel exhibited both consistent and prolonged action. Finally, the % inhibition of carrageenan-induced rat paw edema of spanlastic gel was three times higher than the conventional FPCa gel after 24 h. In conclusion, spanlastic-based gel could be a great approach for improving topical delivery of fenoprofen calcium, providing both prolonged and enhanced anti-inflammatory activity in the treatment of arthritis.