Expert Recommendations on the Usage of Non-vitamin K Antagonist Oral Anticoagulants (NOACs) from India: Current Perspective and Future Direction.

Non-vitamin K antagonist oral anticoagulants (NOACs) are a new class of anticoagulant drugs used in the prevention and treatment of venous thromboembolism (VTE) and atrial fibrillation (AF). Anticoagulation requires the integration of the correct type and dose of oral anticoagulants based on patient characteristic, and therefore therapy needs to be individualized for each patient. Growing scientific evidence from studies on NOACs has led to a better understanding of their benefits and safety. A large amount of available data creates a necessity for an adaptable practical document for the usage of NOACs in India. The current consensus, developed by experts from India, aims to give recommendations on various frequently raised clinical questions with regards to NOACs and its usage. This practical document provides a platform upon which future guidelines, policies, training, and education for the use of NOACs can be tailored.

Incubation and its effect on Leishman stain.

BACKGROUND: Leishman stain is the universal stain used in staining of peripheral blood smears all over the world. AIM: To study the effect of incubation of buffer solution, slide alone and slide & buffer both on standard Leishman staining. The staining at times causes difficulty particularly in rainy season when there is increased moisture. METHOD: The study comprised of twenty peripheral blood smears selected consecutively in batches of ten each for two successive days . Minor modification in standard Leishman stain was done by either incubating slide or buffer solution or both. The staining characteristics were scored using system by NG et al and the statistical analysis was done. RESULTS: The highest mean score for background (1.5), nuclear (1.55), cytoplasmic features (1.5) and granules visualization (1.8) were seen in technique involving incubation of both slide and buffer. The results were statistically significant. CONCLUSION: We found that incubating glass slides or buffer solution or both yield better stained slide. Our study showed that there was better staining features seen in incubated slide verses routine method.

Epigallocatechin-3-Gallate Upregulates miR-221 to Inhibit Osteopontin-Dependent Hepatic Fibrosis.

Osteopontin (OPN) promotes hepatic fibrosis, and developing therapies targeting OPN expression in settings of hepatic injury holds promise. The polyphenol epigallocatechin-3-gallate (EGCG), found in high concentrations in green tea, downregulates OPN expression through OPN mRNA degradation, but the mechanism is unknown. Previous work has shown that microRNAs can decrease OPN mRNA levels, and other studies have shown that EGCG modulates the expression of multiple microRNAs. In our study, we first demonstrated that OPN induces hepatic stellate cells to transform into an activated state. We then identified three microRNAs which target OPN mRNA: miR-181a, miR-10b, and miR-221. In vitro results show that EGCG upregulates all three microRNAs, and all three microRNAs are capable of down regulating OPN mRNA when administered alone. Interestingly, only miR-221 is necessary for EGCG-mediated OPN mRNA degradation and miR-221 inhibition reduces the effects of EGCG on cell function. In vivo experiments show that thioacetamide (TAA)-induced cell cytotoxicity upregulates OPN expression; treatment with EGCG blocks the effects of TAA. Furthermore, chronic treatment of EGCG in vivo upregulates all three microRNAs equally, suggesting that in more chronic treatment all three microRNAs are involved in modulating OPN expression. We conclude that in in vitro and in vivo models of TAA-induced hepatic fibrosis, EGCG inhibits OPN-dependent injury and fibrosis. EGCG works primarily by upregulating miR-221 to accelerate OPN degradation. EGCG may therefore have utility as a protective agent in settings of liver injury.

Mutational analysis of telomere complex genes in Indian population with acquired aplastic anemia.

BACKGROUND: Acquired aplastic anemia (AAA) is rare disorders caused due to the profound or almost complete bone marrow failure. It is a life threatening hematopoietic stem cells disorder, which is characterized by pancytopenia or complete loss of blood-forming cells. The aim of the present study is to screen for the mutations in telomerase complex genes, and to establish a molecular and hematological profile of Indian sub population. METHODOLOGY: We have conducted a case control study of total 70 participants; 50 patients, who fulfilled the blood count and bone marrow criteria of the International agranulocytosis & AAA, and 20 healthy controls. These samples were selected from hematology clinics at Jaipur, India, during the period of two years (January 2012-December 2013). We screened four telomere complex genes; TERT, DKC1, NOP10 and NHP2 of mutations at single base pair in sampled blood and bone marrows. We have predicated the consequences of mutations on protein structure using 3D multilevel modeling protein structure software Phyre2, PolyPhen2 and YASARA. RESULTS: The hematological and molecular basis of acquired aplastic anemia was investigated in 50 anemia patients and 20 healthy controls. AAA patients showed hematologic abnormalities (macrocytic anemia, thrombocytopenia, & granulocytopenia) in peripheral blood and severe hypoplastic bone marrows. Screening of telomere complex genes TERT, DKC1, NOP10 and NHP2 in AAA patients and controls revealed; novel and reported mutations in TERT and DKC1, whereas, no pathogenic mutations were observed in NOP10 and NHP2 genes. In TERT gene, one non-synonymous mutation (Chr5: 1287,825 C→T; Arg979Trp) was identified in exon 12 and two heterozygous non-synonymous mutations (Chr X: 153,994,542 T→K; Val105Gly & Chr X: 153,994,591 T→K; Ser121Arg) were found in exon 5 of DKC1 gene. To determine and visualize the possible effect of TERT and DKC1 mutations on protein structure YASARA with FoldX functionality has been used and many structural consequences were found that might destabilize the protein. Predicated structural consequences may destabilize the TERT and DKC1 proteins ultimately causing blood disorders.. CONCLUSION: The present study indicates the mutation spectrum in the genes implicated in AAA, i.e. TERT, DKC1, NOP10 and NHP2 on small case-control group in an Indian sub population.

Urothelial cells undergo epithelial-to-mesenchymal transition after exposure to muscle invasive bladder cancer exosomes.

Bladder cancer, the fourth most common noncutaneous malignancy in the United States, is characterized by high recurrence rate, with a subset of these cancers progressing to a deadly muscle invasive form of disease. Exosomes are small secreted vesicles that contain proteins, mRNA and miRNA, thus potentially modulating signaling pathways in recipient cells. Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells. EMT has been implicated in the initiation of metastasis for cancer progression. We investigated the ability of bladder cancer-shed exosomes to induce EMT in urothelial cells. Exosomes were isolated by ultracentrifugation from T24 or UMUC3 invasive bladder cancer cell conditioned media or from patient urine or bladder barbotage samples. Exosomes were then added to the urothelial cells and EMT was assessed. Urothelial cells treated with bladder cancer exosomes showed an increased expression in several mesenchymal markers, including α-smooth muscle actin, S100A4 and snail, as compared with phosphate-buffered saline (PBS)-treated cells. Moreover, treatment of urothelial cells with bladder cancer exosomes resulted in decreased expression of epithelial markers E-cadherin and ß-catenin, as compared with the control, PBS-treated cells. Bladder cancer exosomes also increased the migration and invasion of urothelial cells, and this was blocked by heparin pretreatment. We further showed that exosomes isolated from patient urine and bladder barbotage samples were able to induce the expression of several mesenchymal markers in recipient urothelial cells. In conclusion, the research presented here represents both a new insight into the role of exosomes in transition of bladder cancer into invasive disease, as well as an introduction to a new platform for exosome research in urothelial cells.

Osteopontin mediates an MZF1-TGF-ß1-dependent transformation of mesenchymal stem cells into cancer-associated fibroblasts in breast cancer.

Interactions between tumor cells and cancer-associated fibroblasts (CAFs) in the tumor microenvironment significantly influence cancer growth and metastasis. Transforming growth factor-ß (TGF-ß) is known to be a critical mediator of the CAF phenotype, and osteopontin (OPN) expression in tumors is associated with more aggressive phenotypes and poor patient outcomes. The potential link between these two pathways has not been previously addressed. Utilizing in vitro studies using human mesenchymal stem cells (MSCs) and MDA-MB231 (OPN+) and MCF7 (OPN-) human breast cancer cell lines, we demonstrate that OPN induces integrin-dependent MSC expression of TGF-ß1 to mediate adoption of the CAF phenotype. This OPN-TGF-ß1 pathway requires the transcription factor, myeloid zinc finger 1 (MZF1). In vivo studies with xenotransplant models in NOD-scid mice showed that OPN expression increases cancer growth and metastasis by mediating MSC-to-CAF transformation in a process that is MZF1 and TGF-ß1 dependent. We conclude that tumor-derived OPN engenders MSC-to-CAF transformation in the microenvironment to promote tumor growth and metastasis via the OPN-MZF1-TGF-ß1 pathway.

Protein-coated microcrystals of Pseudomonas aeruginosa PseA lipase.

Highly active Pseudomonas aeruginosa lipase protein-coated microcrystals (PAL PCMC) have been prepared by immobilization of protein onto K(2)SO(4) as excipient solid support carrier and n-propanol as precipitating solvent. Transmission electron micrographs confirmed the formation of PAL PCMC. These PCMC were found to be a catalytically more active and stable preparation for p-nitrophenyl palmitate hydrolysis in n-heptane, compared to free lipase. The V (max), K (m), and temperature optimum for PAL PCMC increased from 0.49 to 5.66 nmol min(-1) mg(-1), 589 to 679.8 mmol, and 40 degrees C to 45 degrees C, respectively. These were thermally more stable with 4.65, 2.56, and 1.24-fold improvement in half lives at 45 degrees C, 55 degrees C, and 60 degrees C compared to free P. aeruginosa PseA lipase. Their catalytic efficiency was enhanced by tenfold over that of free enzyme. PAL PCMC offer a simple and effective technique for obtaining stable and efficient lipase preparation for biocatalysis in nonaqueous medium.

The creation of linear contiguous lesions in the atria with an expandable loop catheter.

OBJECTIVES: This article describes a catheter system designed to create linear atrial lesions and identifies electrophysiologic markers that are associated with the creation of linear lesions. BACKGROUND: Atrial fibrillation (Afib) is the most common arrhythmia in humans and causes a significant morbidity. The success of surgical interventions has provided the impetus for the development of a catheter-based approach for the ablation of Afib. METHODS: We tested a catheter system with 24 4-mm ring electrodes that can create loops in the atria. The electrodes can be used to record electrical activity and deliver radiofrequency power for ablation. In 33 dogs, 82 linear lesions were generated using three power titration protocols: fixed levels, manual titration guided by local electrogram activity and temperature control. Bipolar activity was recorded from the 24 electrodes before, during and after lesion generation. Data were gathered regarding lesion contiguity, transmurality and dimensions; the changes in local electrical activity amplitude; the incidence rate of rapid impedance rises and desiccation or char formation; and rhythm outcomes. RESULTS: Catheter deployment usually requires <60 s. Linear lesions (12 to 16 cm in length and 6 +/- 2 mm wide) can be generated in 24 to 48 min without moving the catheter. Effective lesion formation can be predicted by a decrease of greater than 50% in the amplitude of bipolar recordings. Splitting or fragmentation of the electrogram and increasing pacing threshold (3.1 +/- 3.3 mV to 7.1 +/- 3.8 mV, p < 0.01) are indicative of effective lesion formation. Impedance rises and char formation occurred at 91 +/- 12 degrees C. Linear lesion creation does not result in the initiation of Afib. However, atrial flutter was recorded after the completion of the final lesion in 3/12 hearts. When using temperature control, no char was noted in the left atrium, whereas 8% of the right atrium burns had char. CONCLUSIONS: This adjustable loop catheter forces the atrial tissue to conform around the catheter and is capable of producing linear, contiguous lesions up to 16 cm long with minimal effort and radiation exposure. Pacing thresholds and electrogram amplitude and character are markers of effective lesion formation. Although Afib could not be induced after lesion set completion, sustained atrial flutter could be induced in 25% of the hearts.

Subcutaneous bioabsorbable pellets of norethindrone for contraception in women: Phase I. Clinical study.

Ten healthy, normally menstruating female volunteers participated in a 1-year phase I clinical study in which subcutaneous pellet implants of norethindrone (NET) were employed as a low-dose and long-acting potential contraceptive. Two NET pellets were implanted subcutaneously by the aid of a trocar in the forearm of each volunteer on the fifth day after the start of menstrual bleeding. Serum levels of NET, follicle-stimulating hormone, luteinizing hormone, 17 beta-estradiol, and progesterone were determined weekly by radioimmunoassay. The daily NET release from the pellets, based on mean values (+/- standard error of the mean) in five subjects was 79.4 +/- 7.6 micrograms. The mean serum NET level was initially 1.0 +/- 0.34 ng/ml; thereafter, it gradually lowered during the 343 days of the study period to the level of 0.43 +/- 0.09 ng/ml. The ovarian response, days of bleeding, and cycle lengths with continuously sustained release of NET from the pellets were similar to those observed in women taking the daily oral "minipill" of 300 micrograms NET. The results of the phase I study suggest that NET pellet implants may provide a simple and acceptable approach to long-term contraception in women.

Release of norethisterone from a bioabsorbable implant in female bonnet monkeys (Macaca radiata).

Bioabsorbable implants prepared by fusion of 85% norethisterone (NET) and 15% cholesterol were inserted subdermally in four cycling bonnet monkeys (Macaca radiata). No skin reaction or inflammation was observed at the site of implantation. Plasma concentration of norethisterone (NET) measured by radioimmunoassay were monitored for 14 to 16 months at monthly intervals. In the first month weekly samples were analysed. NET was released into circulation within 24 hours after insertion of the implant. In all the monkeys, except one, a sharp rise in NET (1.9 to 20 ng/ml) occurred immediately after insertion. Levels then remained between 1.7 and 0.6 ng/ml for about 4 months. Thereafter they remained steady up to about 9 months in two monkeys and gradually declined to about 0.4 ng/ml in the remaining two. In all except one, there was a sudden burst of NET release between the 10th and 11th month. The hormone almost cleared out of circulation by the 14th to 16th month. Initial menstrual cycles after pellet insertion were disturbed, leading to spotting and irregular bleeding. Regular cycles appeared from 5 to 8 months after the insertion of the implant. These cycles were ovulatory, as determined by the progesterone levels.

Subdermal norethindrone pellets -- a method for contraception?

The mode of action of compressed pellets containing 85 per cent norethindrone (NET) and 15 per cent cholesterol was studied. Four pellets were inserted subcutaneously, in each of five healthy volunteers and left in place for 200--229 days. The NET content of the pellets varied between 23.9 mg and 25.6 mg; and the cholesterol content between 4.2 mg and 4.5 mg. Plasma levels of NET, estradiol and progesterone were determined by radioimmunoassays. Plasma levels of NET varied mostly between 1--2 ng/ml the first month after insertion. After two months plasma levels of NET ranged between 0.5 ng/ml and 1 ng/ml in all volunteers and there was a gradual decrease of the plasma NET levels throughout treatment. Pronounced day-to-day variations in plasma NET levels were recorded. The release rates of NET was calculated to be between 187 micrograms/day and 243 micrograms/day among the five volunteers. Ovulations occurred in four out of five subjects during treatment. This study indicates that the release of gestagen from four NET pellets was only initially high enough to completely inhibit ovulation and that to accomplish full contraceptive efficacy, a higher dose, i.e. more pellets, would have to be inserted.

PIP: Pellets containing norethindrone (NET)-cholesterol (85%:15%, weight to weight) used for long-term subdermal implant for contraception were studied to elucidate their mode of action. Plasma levels of NET, estradiol, and progesterone were measured by radioimmunoassay; bleeding pattern was recorded; and daily release of NET was calculated after pellet removal from 5 healthy volunteers, each of whom had 4 subdermal pellets implanted for 200-229 days. NET content of pellets varied from 23.9 mg-25.6 mg; cholesterol content varied from 4.2-4.5 mg. Plasma levels of NET varied mostly between 1 and 2 ng/ml the 1st month after insertion. After 2 months, plasma levels of NET ranged from .5-1 ng/ml in all volunteers; there was a gradual decrease of the plasma NET levels throughout treatment. Pronounced day-to-day variations in plasma NET levels were recorded. Release rates of NET were calculated as between 187 and 243 mcg/day among the 5 volunteers. Ovulation occurred in 4/5 subjects, as determiend by hormonal profiles, during treatment. Overall, the release of NET was only high enough during the initial phase to completely inhibit ovulation, and therefore, to accomplish full contraceptive efficacy, a higher dose, i.e., more pellets, must be inserted subdermally.