Thioredoxin fusion construct enables high-yield production of soluble, active matrix metalloproteinase-8 (MMP-8) in Escherichia coli.

Matrix metalloproteinases (MMPs) are crucial proteases in maintaining the health and integrity of many tissues, however their dysregulation often facilitates disease progression. In disease states these remodeling and repair functions support, for example, metastasis of cancer by both loosening the matrix around tumors to enable cellular invasion and by affecting proliferation and apoptosis, and they promote degradation of biological restorations by weakening the substrate to which the restoration is attached. As such, MMPs are important therapeutic targets. MMP-8 participates in cancer, arthritis, asthma and failure of dental fillings. MMP-8 differs from other MMPs in that it has an insertion that enlarges its active site. To elucidate the unique features of MMP-8 and develop selective inhibitors to this therapeutic target, a stable and active form of the enzyme is needed. MMP-8 has been difficult to express at high yield in a soluble, active form. Typically recombinant MMPs accumulate in inclusion bodies and complex methods are applied to refold and purify protein in acceptable yield. Presented here is a streamlined approach to produce in Escherichia coli a soluble, active, stable MMP-8 fusion protein in high yield. This fusion shows much greater retention of activity when stored refrigerated without glycerol. A variant of this construct that contains the metal binding claMP Tag was also examined to demonstrate the ability to use this tag with a metalloprotein. SDS-PAGE, densitometry, mass spectrometry, circular dichroism spectroscopy and an activity assay were used to analyze the chemical integrity and function of the enzyme.

Future specialization interests among medical students in southern India.

BACKGROUND: A consideration of the future specialization interests of undergraduate medical students might help to understand the needs of higher medical education and future manpower availability for healthcare. METHODS: A cross-sectional study was conducted among 373 undergraduate students of a medical college in southern India using a self-administered questionnaire. RESULTS: Of the 373 students, 188 (50.4%) were men. Almost all of them (370 [99.2%]) wanted to pursue postgraduation. Of these, 267 (72.4%) wanted to pursue postgraduation in India. Overall, the first choice subject was surgery (120 [32.2%]) followed by internal medicine (85 [22.8%]) and paediatrics (43 [11.5%]). The third preference for men and women differed, with men choosing orthopaedics and women choosing obstetrics and gynaecology. The factors that influenced the choice of specialization were interest in the speciality (Likert scale score 4.7), job satisfaction (4.6), employment opportunities (4.0), job security (4) and high income potential (3.9). CONCLUSION: It was evident from the proportion of students desiring to do postgraduation and their choice of specialties that most of them will end up working at hospitals instead of at primary healthcare centres. The deficiencies of certain specialists such as ophthalmologists are likely to persist. This is a cause for concern as the majority of our population lives in rural areas and there is already a maldistribution of doctors.

Treatment data and technical process challenges for practical big data efforts in radiation oncology.

The term Big Data has come to encompass a number of concepts and uses within medicine. This paper lays out the relevance and application of large collections of data in the radiation oncology community. We describe the potential importance and uses in clinical practice. The important concepts are then described and how they have been or could be implemented are discussed. Impediments to progress in the collection and use of sufficient quantities of data are also described. Finally, recommendations for how the community can move forward to achieve the potential of big data in radiation oncology are provided.

Development and evaluation of a microemulsion formulation for transdermal delivery of terbinafine.

The aim of the present study is to develop and evaluate microemulsion formulations for Terbinafine (TB) with a view to enhance its permeability through the skin and provide release for 24 h. Various o/w microemulsions were prepared by the spontaneous emulsification method. Oleic acid was chosen as the oil phase, Caprylo caproyl macrogol-8- glyceride (Labrasol S) and purified diethylene glycol monoethyl ether (Transcutol P) were used as surfactant and cosurfactant, respectively, on the basis of solubility studies. Pseudoternary phase diagrams were constructed to obtain the concentration range of oil, surfactant, cosurfactant, and water for microemulsion formulation. The optimized microemulsion consisted of 2% w/w TB, 8% w/w oleic acid, 31% w/w labrasol S, 31% w/w transcutol P, and 30% w/w distilled water. Permeability parameters like Jss and Kp were found to be significantly higher for formulation F4 as compared to other formulations (P < 0.05). Microbiological studies of TB in microemulsion showed better anti-fungal activity against Candida albicans and Aspergillus flavus as compared to marketed product (P < 0.05).

Modeling Suggests a Mechanism of Synergy Between Hepatitis C Virus Entry Inhibitors and Drugs of Other Classes.

Hepatitis C virus (HCV) entry inhibitors (EIs) act synergistically with drugs targeting other stages of the HCV lifecycle. The origin of this synergy remains unknown. Here, we argue that the synergy may arise from the complementary activities of the drugs across cell subpopulations expressing different levels of HCV entry receptors. We employ mathematical modeling of viral kinetics in vitro, where cells with a distribution of entry receptor expression levels are exposed to HCV with or without drugs. The drugs act independently in each cell, as expected in the absence of underlying interactions. Yet, at the cell population level our model predicts that the drugs exhibit synergy. EIs effectively block infection of cells with low receptor levels. With high receptor levels, where EIs are compromised, other drugs are potent. This novel mechanism of synergy, arising at the cell population level may facilitate interpretation of drug activity and treatment optimization.

Genotyping of CYP21, linked chromosome 6p markers, and a sex-specific gene in neonatal screening for congenital adrenal hyperplasia.

We investigated the feasibility and diagnostic utility of genotyping 9 CYP21 mutations, linked chromosome 6p markers, and a dimorphic X-Y marker from neonatal screening samples. Blood-impregnated filter papers (Guthrie cards) from 603 randomly chosen New Zealand neonates were genotyped blind to 17-hydroxyprogesterone (17-OHP) levels. Another 50 samples from Swiss and North American infants with correlative hormonal data were also genotyped. DNA was extracted, and gene-specific PCR was performed. CYP21 PCR products were subjected to ligase detection reaction, simultaneously analyzing 9 CYP21 mutations; PCR products of other genes were subjected to direct gel analysis. CYP21 genotyping indicated a heterozygote rate of 2.8% for classic mutations (excluding CYP21 deletions), and 2.0% for nonclassic mutations in New Zealanders. Ten full-term affected neonates showed a wide range of 17-OHP levels (15-1400 nmol/L). Sick or preterm infants or infants screened on the first day of life with high 17-OHP proved genetically unaffected. Genetic linkage disequilibrium was found between two CYP21 mutations and chromosome 6p markers. Guthrie cards can be used to accurately genotype CYP21 and other relevant markers, potentially enhancing the specificity and sensitivity of congenital adrenal hyperplasia screening. CYP21 heterozygote frequency for classic mutations is higher than expected based on genotype compared with that predicted by hormonal newborn screening.

Study of a kindred with classic congenital adrenal hyperplasia: diagnostic challenge due to phenotypic variance.

We sought to determine the concordance of the phenotype and genotype in a kindred with classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency. The variation in phenotypic expression within this family underscores the difficulty of establishing the diagnosis in the absence of newborn screening, even with a heightened index of suspicion. Steroidogenic profiles were obtained for the three affected siblings. The available clinical history of the two affected aunts was retrieved. Genotyping was performed on several members of the kindred. Detailed sequencing of the entire CYP21 gene of two clinically dissimilar subjects in this family was undertaken to explore the possibility of other mutations or polymorphisms. PCR with ligase detection reaction analysis of CYP21 revealed that the affected family members III-2, III-3, III-4, II-3, and II-4, all were compound heterozygotes carrying the intron 2 point mutation known to interfere with splicing (nucleotide 656 A to G) and the exon 4 point mutation causing a nonconservative substitution of asparagine for isoleucine at codon 172 (I172N). Detailed sequencing of the gene was performed for the two most phenotypically dissimilar subjects. A single silent polymorphism was found in the third nucleotide for codon 248 in patient II-4, but not in patient III-4, and no additional mutations were found. Classic congenital adrenal hyperplasia remains a difficult diagnosis to make in the absence of newborn screening because of the variability of phenotypic expression. Likewise, the variable degree of genital ambiguity in affected females in this family serves to question universal advocacy of prenatal steroid treatment in pregnancies at risk for congenital adrenal hyperplasia. Extensive molecular exploration did not provide an explanation of the phenotypic heterogeneity and supports the possibility of influences other than the CYP21 gene for the observed divergence.

Steroid 21-hydroxylase expression and activity in human lymphocytes.

Steroid 21-hydroxylase encoded by CYP21 is expressed in adrenal cortex. Mutations in CYP21 cause potentially lethal congenital adrenal hyperplasia (CAH). Earlier observations suggested alternative sources of 21-hydroxylase activity, although its genetic source remains unclear. We found a novel source of CYP21 expression in normal human cultured B lymphocytes. The quantity of 21-hydroxylase transcript was reduced in B cell lines of CAH subjects compared with that in normal B-lymphoblastoid cells. No CYP21 transcript was detected in lymphocytes from a CAH patient with homozygous CYP21 deletion. Cultured lymphoid cells, including those carrying homozygous CYP21 deletion, and peripheral blood leukocytes converted both 17-hydroxyprogesterone to 11-deoxycortisol and progesterone to deoxycorticosterone. We conclude that lymphocytes express CYP21, but also possess a 21-hydroxylase distinct from CYP21. Activity of this isozyme may partially compensate for severe adrenal 21-hydroxylase deficiency.

Kinetics of crystallization in hard-sphere colloidal suspensions.

We propose a kinetic model for describing crystal nucleation kinetics in hard-sphere colloidal suspensions. The model captures the interplay between the enhanced thermodynamic driving force and the reduced particle diffusivity in determining crystal nucleation rates as the particle density is increased in hard-sphere suspensions. Model calculations of nucleation rates and crystal growth velocities agree quantitatively with experimental observations. The dependence of the critical cluster size on volume fraction that emerges differs qualitatively from predictions of classical theories allowing for an experimental validation of the mechanism of crystal nucleation in colloidal suspensions.

Multi-vessel coronary stenting--procedural results and late clinical outcomes: a comparison with single-vessel stenting.

The purpose of this study was to assess the 1-year clinical outcome of patients with multi-vessel coronary artery disease (CAD) who underwent coronary stenting, and to compare the results with single-vessel coronary stenting carried out during the same period. We evaluated the in-hospital and 12-month clinical outcomes [death, Q-wave myocardial infarction (MI) and repeat revascularization rates at one year] in 384 consecutive patients treated with coronary stents in 2 (92% of patients) or 3 of the native coronary arteries and compared the outcome to 624 consecutive patients undergoing stenting in a single coronary artery between January 1, 1997 and January 31, 1999. The overall procedural success was obtained in 99% of patients with 2- or 3-vessel stenting and 98% of patients with single-vessel stenting. Procedural complications were similar (2.9% vs 2.6%; p = 0.12). During follow-up, target lesion revascularization was 16% in multi-vessel and 14% in single-vessel stenting (p = 0.38) and repeat revascularization was also similar for both groups (19% vs. 20%; p = 0.73). There was no difference in death (0.8% vs. 1.3%; p = 0.31) and Q-wave MI (0.7% vs. 1.4%; p = 0. 16) in the 2 groups. Overall cardiac event-free survival was similar for both groups (76% vs. 78%; p = 0.54). Multi-vessel stenting in carefully selected patients in our experience had a high procedural success with very low complication rates. The one-year clinical outcomes were acceptable and were similar to the results of single-vessel stenting.

Seasonal variation in serum levels of steroid hormones and their relation with seminal quality and libido in buffalo bulls.

Blood samples from 15 breeding male Murrah buffaloes were collected during the winter, summer and monsoon seasons. Seminal characteristics and sexual behaviour were also studied. Serum samples were analysed for testosterone, progesterone and estradiol-17beta levels by radioimmunoassay. The studies showed significantly lower values for testosterone during winter (0.53 +/- 0.06 ng/ml) than during summer (1.22 +/- 0.19 ng/ml) and monsoon (1.06 +/- 0.12 ng/ml). The progesterone level was lowest during monsoon (84 +/- 9 pg/ml), intermediate during winter (115 +/- 14 pg/ml) and highest during summer (224 +/- 24 pg/ml). The mean level of estradiol-17beta was almost double (9 +/- 0.7 pg/ml) during monsoon as compared to winter (5 +/- 0.1 pg/ml). The correlations between hormone levels, seminal characteristics and sexual behaviour were of low magnitude.

Seasonal variations in serum levels of thyroid hormones and their relation with seminal quality and libido in buffalo bulls.

Blood samples from 15 Murrah buffalo bulls, (10- to 15- years-old) were collected during the summer, monsoon and winter seasons. The serum samples were analysed for thyroxine (T4) and triiodothyronine (T3) by RIA. Semen samples from these bulls were evaluated for various attributes. Sexual behaviour of these bulls was also recorded during the different seasons and was expressed as reaction time and refusal response. T4, T3, (T4 + T3) level and T4:T3 ratio did not reveal significant difference between seasons. Similarly, seminal characteristics did not exhibit any seasonal variation except for the percentage of live spermatozoa. However, the refusal response (no interest in mounting and ejaculating) was highest during summer months. T4 was significantly correlated to T(3) (r=0.53). Overall, T4 showed a positive correlation with seminal volume and initial motility while T3 exhibited a positive correlation with total sperm concentration and percentage of live spermatozoa. T3 was negatively correlated with refusal response only during the monsoon season. Correlation with other seminal and behavioural characteristics were not significant. The results indicated that although the sexual interest of buffalo bulls is reduced during the summer, the bulls can produce semen throughout the year under appropriate feeding and management conditions.

A twenty-four hour temporal variation in peripheral levels of testosterone and thyroid hormones in male buffaloes.

Hourly serum samples from four adult Murrah buffalo bulls of 5 to 6 years of age were analysed for testosterone, thyroxine and triiodothyronine by radioimmunoassay during a period of 24 hours. All four bulls exhibited three episodic peaks for testosterone with some variation in the time, duration and peak concentration of the hormone. The average testosterone concentration varied from 0.30 to 3.50 ng/ml of serum. Thyroxine levels varied from 20 to 40 ng/ml of serum among the four bulls. One clear-cut peak was observed between 2 and 5 a.m. in three of the four bulls. One animal showed a characteristic peak at 10 p.m. Triiodothyronine levels ranged from 1 to 2 ng/ml of serum and followed a similar trend as that of thyroxine except for an additional small peak between 6 and 9 p.m.

Outcomes of primary stenting for acute myocardial infarction.

Primary coronary stenting is being increasingly used in patients undergoing primary coronary angioplasty for acute myocardial infarction. In this prospective study we analysed our experience of direct angioplasty in 76 patients with acute myocardial infarction of whom 65 received intracoronary stents using high pressure deployment (> or = 12 atm) with adjunctive aspirin and ticlopidine therapy but without coumadin. All patients underwent pre-discharge angiography. Stent implantation was successful in all patients. Stent thrombosis was not seen in any patient. However, TIMI grade 3 flow was obtained in only 58 (89.2%) patients with evidence of slow-flow present in the remaining seven patients. Pre-discharge angiograms showed no-stent thrombosis but five out of the seven (71%) patients with slow-flow phenomenon persisted to have slow-flow. These patients had lower left ventricular ejection fraction as compared to patients with TIMI 3 flow at pre-discharge angiography (27.5 +/- 10.2% vs 42.1 +/- 15.2%; p < 0.001) and a high mortality (2 out of 7) within 30 days. Primary stenting is safe and feasible in the majority of patients with good short-term outcome. But persistent slow-flow phenomenon with adverse clinical outcome is seen in a small but significant number of patients.

Elective stenting of extracranial carotid arteries.

OBJECTIVES: In symptomatic and asymptomatic patients with significant carotid artery stenosis, surgical endarterectomy has been shown to be beneficial when compared with medical management. Carotid stenting is evolving as an alternative technique for treating such patients. This prospective study was designed to assess the feasibility and safety of carotid angioplasty and stenting. METHODS: Fourteen patients (15 carotid arteries) with significant carotid artery stenosis were enrolled. These patients were in the age range 46 to 84 years (mean 60.9 +/- 7 years) and there were 12 males (86%). All of these patients were symptomatic with either TIA (n = 8) or stroke (n = 6). Wallstents were used in all the cases to stent the carotid arteries. One patient underwent bilateral carotid artery stenting. RESULTS: Carotid angioplasty and stenting was successful in 13 out of 14 (92.8%) patients and 14 out of 15 (93.3%) carotid arteries, with reduction in mean (+/- SD) stenosis from 86 +/- 6% to 3 +/- 3%. There was one episode of minor stroke, no major stroke or death during the initial hospitalization. Another patient had a minor stroke with patent ipsilateral carotid artery (on repeat angiography) during the first 30 days after the procedure. This patient was also found to have asymptomatic thrombus formation in the contralateral carotid stent which resolved with intravenous anticoagulation. During a mean follow up of 6 +/- 2 months there has been no recurrence of symptoms. CONCLUSIONS: Based upon our limited experience we believe that percutaneous carotid angioplasty with stenting is feasible with low periprocedural complication rate.