Machinability Analysis and Optimization in Wire EDM of Medical Grade NiTiNOL Memory Alloy.

NiTiNOL (Nickel-Titanium) shape memory alloys (SMAs) are ideal replacements for titanium alloys used in bio-medical applications because of their superior properties like shape memory and super elasticity. The machining of NiTiNOL alloy is challenging, as it is a difficult to cut material. Hence, in the current research the experimental studies on machinability aspects of medical grade NiTiNOL SMA during wire electric discharge machining (WEDM) using zinc coated brass wire as electrode material have been carried out. Pulse time (Ton), pause time (Toff), wire feed (WF), and servo voltage (SV) are chosen as varying input process variables and the effects of their combinational values on output responses such as surface roughness (SR), material removal rate (MRR), and tool wear rate (TWR) are studied through response surface methodology (RSM) based developed models. Modified differential evolution (MDE) optimization technique has been developed and the convergence curve of the same has been compared with the results of differential evolution (DE) technique. Scanning electron microscopy (SEM) and energy dispersive X-ray spectrography (EDS) analysis are carried out to study the surface morphology of the machined alloy. SV is found to be more influential process parameter for achieving better MRR with minimal SR and TWR, followed by Ton, Toff, and WF. The WF has good impact on reduced SR and TWR responses and found to be least significant in maximizing MRR.

Angiotensin II-forming activity in a reconstructed ancestral chymase.

The current model of serine protease diversity theorizes that the earliest protease molecules were simple digestive enzymes that gained complex regulatory functions and restricted substrate specificities through evolution. Among the chymase group of serine proteases are enzymes that convert angiotensin I to angiotensin II, as well as others that simply degrade angiotensins. An ancestral chymase reconstructed with the use of phylogenetic inference, total gene synthesis, and protein expression had efficient and specific angiotensin II-forming activity (turnover number, about 700 per second). Thus, angiotensin II-forming activity is the more primitive state for chymases, and the loss of such activity occurred later in the evolution of some of these serine proteases.

A Study about Co-Infection of Fungal Pathogens in Active Tuberculosis Patients.

The diagnosis of mycotic lung infection in pulmonary TB patients remains misdiagnosed because of its non-specific clinical manifestations which mimics the symptoms of TB. Physicians have to rely on the investigation but as radiology and pathology cannot probe the appropriate diagnosis, conventional microbiology or PCR testing continue as an essential mode for the diagnosis. In developing country like India PCR is not cost effective. Thus, Direct microscopy by KOH (10%), Gram's staining & Culture remains only option for identification. A three-year cross-sectional study was carried out in the Department of Microbiology, Maharishi Markandeshwar Institute of Medical Science & Research, Mullana, India from August 2015 to August 2018. On 300 LED positive sputum samples collected from previously treated cases of pulmonary TB. Early morning sputum was collected and subjected to KOH 10%, Gram's staining afterwards cultured on Sabouraud Dextrose Agar and species identification was done by LPCB preparation. In 300 LED smear positive samples, the dominant pathogens were C. albicans (43.3%), followed by C. non-albicans (26.7%), A. fumigatus (21.7%) etc. ATT administration for 5-8 months' duration of illness showed highest fungal infection (45%) and maximum growth of fungus was seen in the Autumn season (45%). The co-occurrence of fungi with tubercle bacteria adds fatal consequences thus routine screening is recommended for proper diagnosis and early treatment of mycotic infection in the patients of Pulmonary TB on ATT.

Atrial natriuretic peptide inhibits mineralocorticoid receptor function in rat colonic surface cells.

Atrial natriuretic peptide (ANP) inhibits and aldosterone (ALDO) stimulates Na conductive transport. Therefore, the effects of ANP and its second messenger cGMP on mineralocorticoid receptor (MR) function in rat colon surface and crypt cells were examined. 100 nM 8-Br-cGMP decreased surface [3H]ALDO binding by 42 +/- 4% but increased crypt [3HvALDO binding by 52+/-16%. ANP decreased surface [3H]ALDO binding by approximately 50% after a 2.5-h lag period but had no effect on crypt ALDO binding. ANP and cGMP rapidly (< 15 min) inhibited surface cell ALDO-induced MR nuclear translocation but did not affect crypt MR nuclear translocation. Inhibition of cGMP-dependent protein kinase with KT5823 blocked the inhibitory effects of ANP and 8-Br-cGMP on surface cell ALDO binding and MR nuclear translocation. In crypt, KT5823 increased baseline [3H]ALDO binding but did not inhibit the stimulatory effect of exogenous cGMP. DEAE-cellulose chromatography and gel mobility shift assay showed that ANP did not inhibit surface MR activation. ANP inhibited ALDO stimulated short circuit current in distal colon. These data demonstrate cell-specific regulation of MR function. In surface cells, ANP rapidly inhibits MR nuclear translocation and ALDO-induced short circuit current. ANP inhibition of MR function may be an additional mechanism of ANP antagonism of Na reabsorption.

Demonstration of nuclear translocation of the mineralocorticoid receptor (MR) using an anti-MR antibody and confocal laser scanning microscopy.

Using a synthetic peptide that corresponds to a unique region of the N-terminal domain of the human mineralocorticoid receptor (MR), amino acids 87-96, we have generated a polyclonal antibody, human (h) MRsN. This sequence shares no homology with the corresponding sequences of the glucocorticoid receptor or other steroid/thyroid hormone receptor superfamily members. Antibody hMRsN cross-reacts with MR from human, rat, and mouse cells and recognizes denatured MR from either crude preparations or partially purified rat kidney cytosol, rat colon, or recombinant hMR overexpressed in baculovirus-infected Sf9 cells. Immunoprecipitation of the native MR from either partially purified or crude preparations of rat kidney cytosol with hMRsN, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stain, demonstrated a major protein band with a mol wt of 116 kilodaltons. In addition, using confocal laser scanning microscopy and digital image analysis, the immunocytochemical localization of the recombinant hMR over-expressed in Sf9 cells 24 h post-transfection was determined. In the absence of ligand, the MR was detected solely in the cytoplasm, after a 30-min exposure to 100 nM aldosterone the MR was perinuclear, and after 60 min, the MR was predominantly nuclear. To ascertain that this phenomenon was not unique to insect cells, aldosterone induced MR nuclear translocation in mouse macrophage cells was also demonstrated immunocytochemically, clearly indicating a role for nuclear translocation in MR function.

Semaphorin 3A upregulates FOXO 3a-dependent MelCAM expression leading to attenuation of breast tumor growth and angiogenesis.

Semaphorin 3A (Sema 3A), a member of semaphorin family, serves as a guidance clue during embryonic development and is known as a candidate tumor suppressor that attenuates breast tumor progression by binding with its co-receptor, neuropilin-1 (NRP-1). However, the underlying mechanism by which Sema 3A suppresses breast tumor growth is still unexplored. In this study, we report that Sema 3A regulates phosphorylation and nuclear translocation of phosphatase and tensin homolog (PTEN) and FOXO 3a. Moreover, Sema 3A controls NRP-1-mediated PTEN-dependent FOXO 3a activation. Overexpression of PTEN and FOXO 3a enhances Sema 3A-induced attenuation of breast cancer cell migration. Chromatin immunoprecipitation and electrophoretic mobility shift assay data revealed that FOXO 3a regulates MelCAM at the transcriptional level. Furthermore, Sema 3A induces NRP-1-mediated MelCAM expression through PTEN and FOXO 3a. The data also showed that vascular endothelial growth factor-induced angiogenesis is inhibited by Sema 3A. Loss of or gain in function study revealed that Sema 3A modulates phosphorylation of PTEN and FOXO 3a and expression of MelCAM, leading to suppression of tumor growth and angiogenesis using in vivo mice model. Clinical specimen analysis revealed that reduced expression of Sema 3A and p-PTEN are correlated with enhanced breast cancer progression, further strengthening our in vitro and in vivo findings. Correlation of relapse-free survival of breast cancer patients (n=2878) with expression levels of Sema 3A, NRP-1, FOXO 3a and MelCAM were studied by Kaplan-Meier analysis. Statistical analysis revealed a close association between reduced expression of Sema 3A and MelCAM with that of poor patient's survival. Our study demonstrated a novel mechanism of regulation of tumor suppression by Sema 3A in coordination with a chain of tumor-suppressor genes, which in turn inhibits breast cancer cell migration, tumor growth and angiogenesis.

Biochemical characterization of recombinant factor IX.

Mature human factor IX is a 55,000-d glycoprotein with a modular domain structure and numerous posttranslational modifications. A recombinant form of human factor IX (rFIX) has been produced from a Chinese hamster ovary cell line that was engineered for high-level protein processing and expression. To ensure that the recombinant molecule contains the requisite structural and functional features of the plasma-derived form, rFIX was subjected to detailed biochemical and biophysical characterization. The laboratory studies showed that the posttranslational modifications and primary, secondary, and tertiary structures of rFIX were similar to those of plasma-derived factor IX (pdFIX). In addition, rFIX displayed a high degree of purity and a product release specification for specific activity that is > or = 200 IU/mg.

Reversible inactivation of AT(2) angiotensin II receptor from cysteine-disulfide bond exchange.

Dithiothreitol (DTT) treatment of angiotensin II (Ang II) type 2 (AT(2)) receptor potentiates ligand binding, but the underlying mechanism is not known. Two disulfide bonds proposed in the extracellular domain were examined in this report. Based on the analysis of ligand affinity of cysteine (Cys, C) to alanine (Ala, A) substitution mutants, we provide evidence that Cys(35)-Cys(290) and Cys(117)-Cys(195) disulfide bonds are formed in the wild-type AT(2) receptor. Disruption of the highly conserved Cys(117)-Cys(195) disulfide bond linking the second and third extracellular segments leads to inactivation of the receptor. The Cys(35)-Cys(290) bond is highly sensitive to DTT. Its breakage results in an increased binding affinity for both Ang II and the AT(2) receptor-specific antagonist PD123319. Surprisingly, in the single Cys mutants, C35A and C290A, a labile population of receptors is produced which can be re-folded to high-affinity state by DTT treatment. These results suggest that the free -SH group of Cys(35) or Cys(290) competes with the disulfide bond formation between Cys(117) and Cys(195). This Cys-disulfide bond exchange results in production of the inactive population of the mutant receptors through formation of a non-native disulfide bond.

Angiotensin II type 1 receptor-function affected by mutations in cytoplasmic loop CD.

To explore peptide hormone-induced conformational changes, we attempted to engineer a metal-ion binding site between the cytoplasmic loops CD and EF in the angiotensin II type 1 (AT(1)) receptor. We constructed 12 double and six triple histidine mutant receptors, and tested the ability of each mutant and the wild-type to activate inositol phosphate (IP) production with and without ZnCl(2). Inhibition by ZnCl(2) in the double and triple His mutant receptors was not significant, but these mutations directly decreased the IP production. Systematic analysis of single His mutants demonstrated that the loop CD-mutants displayed 52-74% inhibition of IP production, whereas the loop EF-mutants did not affect IP production. These results indicate that the cytoplasmic loop CD-segment from Tyr(127) to Ile(130) is important for G(q/11) activation by the AT(1) receptor.

Angiotensin II type 1 and type 2 receptors bind angiotensin II through different types of epitope recognition.

OBJECTIVE: This study was designed to demonstrate that the principle of molecular recognition underlying high-affinity binding of angiotensin II to the type 2 (AT2) receptor is distinct from that of the type 1 (AT1) receptor. In general, the same functional pharmacophores in hormones are used to bind and activate different subtypes of cell surface receptors. However, the binding of angiotensin II to the AT2 receptor is distinct from that of the AT1 receptor. DESIGN AND METHODS: To systematically evaluate the effect of modification of angiotensin II side chains on binding to both the receptors, several analogs of angiotensin II were synthesized. Rat AT1 or AT2 receptors expressed in COS1 cell membranes were used to determine the affinity of analogs using radioligand competition binding experiments under equilibrium conditions. RESULTS: Modifications of all angiotensin II side chains affected binding to the AT2 receptor to nearly similar extents. In contrast, binding to the AT1 receptor was significantly affected by modifications at side chain positions 2, 4, 6 and 7. In accordance with previous observations that Tyr4- or Phe8-modified angiotensin II analogs antagonized vasoconstriction mediated exclusively by the AT1 receptor, binding to the AT1 receptor was significantly dependent on Tyr4 or Phe8 of angiotensin II whereas binding to the AT2 receptor was not. Rather surprisingly, the affinity profile of several angiotensin II analogs towards the AT2 receptor was similar to the measured affinity of the constitutively active N111G mutant AT1 receptor. CONCLUSIONS: These results suggest that the AT2-receptor pharmacophore is very distinct from that of the AT1 receptor. The AT1 receptor is in a constrained conformation and is activated only when bound to angiotensin II. In contrast, the AT2 receptor is 'relaxed' in that no single interaction is critical for binding, like the N111G mutant AT1 receptor, which is constitutively active.

Cell surface blood group antigens in prostatic carcinoma.

Surface blood group antigens are present to some degree in most epithelia. These antigens frequently are lost during neoplastic transformation. The authors looked for the presence or absence of surface blood group antigens in 52 cases of prostatic carcinoma of various histologic grades using the specific red blood cell adherence test. The normal prostatic tissue showed a 2+ reaction in patients with type A or B blood and 0-1+ in type O. The hyperplastic areas were 4+ for red blood cell adherence in patients with type A or B blood and 0-4+ in type O. In contrast, all malignant foci were negative for blood group antigens, no matter what the histologic grade or blood type.

Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry.

Presented is a method for analyzing sulfated peptides, and differentiating the post-translational modification (PTM) from its isobaric counterpart phosphorylation, using quadrupole time-of-flight (Qq/TOF) mass spectrometry (MS) and positive ion nanoelectrospray MS/MS. A set of commercially available sulfo- and phosphopeptide standards was analyzed via in-source dissociation and MS/MS to generate fragmentation signatures that were used to characterize and differentiate the two modifications. All of the phosphorylated peptides retained their +80 Da modifications under collision-induced decomposition (CID) conditions and peptide backbone fragmentation allowed for the site-specific identification of the modification. In sharp contrast, sulfated peptides lost SO3 from the precursor as the collision energy (CE) was increased until only the non-sulfated form of the peptide was observed. The number of 80 Da losses indicated the number of sulfated sites. By continuing to ramp the CE further, it was possible to fragment the non-sulfated peptides and obtain detailed sequence information. It was not possible to obtain site-specific information on the location of the sulfate moieties using positive ion MS/MS as none of the original precursor ions were present at the time of peptide backbone fragmentation. This method was applied to the analysis of recombinant human B-domain deleted factor VIII (BDDrFVIII), which has six well-documented sulfation sites and several potential phosphorylation sites located in two of the sulfated regions of the protein. Seven peptides with single and multiple +80 Da modifications were isolated and analyzed for their respective PTMs. The fragmentation patterns obtained from the BDDrFVIII peptides were compared with those obtained for the standard peptides; and in all cases the peptides were sulfated. None of the potential phosphorylation sites were found to be occupied, and these results are consistent with the literature.

Molecular determinants of peptide and non-peptide binding to the AT1 receptor.

1. Several residues critically involved in AT(1) receptor ligand-binding and activation have now been identified based on mutational and biochemical studies. 2. Asp(281) and Lys(199) of the rat AT(1) receptor ion-pair with Arg(2) and the Phe(3) α-COOH of angiotensin II (AngII), respectively, and the Asp(281) /Arg(2) interaction is critical for full agonist activity. 3. Agonist activity of AngII also requires an interaction of the Phe(2) side chain with His(256), which is achieved by docking of the α-COOH with Lys(199). Non-peptide agonists interact with Lys(199) and His(256) in a similar fashion. 4. The crucial acid pharmacophores of AngII and the non-peptide antagonist, Iosartan, appear to occupy the same space within the receptor pocket. Binding of the tetrazole anion moiety of losartan involves multiple contacts, such as Lys(199) and His(256). However, this interaction does not involve a conventional salt bridge, but rather an unusual lysine-aromatic interaction. 5. Asp(1) of AngII forms an ion-pair with His(183), which stabilizes the receptor-bound conformation of AngII but is not critical for receptor activation. 6. These interactions and the involvement of other residues in stabilizing the wild-type receptor conformation or in receptor/G-protein coupling are considered here. 7. Despite these insights, considerable effort is still needed to elucidate how ligand binding induces receptor activation, what determines the specificity of AT(1) receptor coupling to multiple G-proteins and the in vivo role of receptor down-regulation.

Renal parameters during infancy.

In a prospective study we estimated common renal parameters in 48 full term normal neonates, of which 15 were also tested at 6 months and 12 months of age. The mean levels of serum creatinine, were high at birth (0.73 mg/dl) but normal for age at 6 and 12 months; uric acid followed a similar trend. The blood pH and bicarbonate were low at birth (7.28 and 20.36 mEq/L, respectively) reached normal adult values by 12 months; chloride levels were high at birth (110 +/- 5 mEq/L) and normal at 6 months. The plasma renin activity was higher than normal all throughout the first year (27.1, 416.8, 64.8 ng/ml/hr by RIA). Plasma aldosterone values were high at birth (1387.5 pg/ml) and reached normal level (301.6) at 12 months. Renal length and volume as assessed by ultrasonography compared well with American standards. Urinary constituents were variable due to breast feeding up to 6 months and varied diet during the weaning period. This study shows that mild metabolic acidosis and hyperchloremia due to immaturity of renal acidification mechanism and high renin and aldosterone levels due to partial nonresponsiveness of distal tubules are normal variables in babies from birth to 6 months. The levels of serum creatinine and uric acid are high at birth and in assessing renal functions this should be borne in mind.

Preparation of lyophilised lipid rich quality control serum by isolating lipids using large scale chromatography technology.

The plasma fractionation is a technology to separate therapeutic plasma proteins. The fractionation is carried out either by solvent precipitation (ethanol) or recently by chromatography. During the process of chromatography lipids were found in waste fraction. A new lipid rich lyophilised quality control (Q.C.) sera was prepared inexpensively using this waste plasma fraction. This is probably the first attempt to prepare the Q.C. sera by large scale chromatographic method in India.

Hypoxia-driven osteopontin contributes to breast tumor growth through modulation of HIF1α-mediated VEGF-dependent angiogenesis.

Hypoxia is a salient feature of most solid tumors, and hypoxic adaptation of cancer cells has crucial implications in propagation of malignant clonal cell population. Osteopontin (OPN) has been identified as a hypoxia-responsive gene, but the mechanistic and regulatory role of OPN under hypoxia is less characterized. The present study identifies the existence of a positive inter-regulatory loop between hypoxia and OPN. We have shown that hypoxia induces OPN expression in breast cancer cells; however, the expression was found to be HIF1α independent. OPN enabled transcriptional upregulation of HIF1α expression both under normoxia and hypoxia, whereas stability of HIF1α protein in breast cancer cells remained unaffected. Moreover, we have shown that OPN induces integrin-linked kinase (ILK)/Akt-mediated nuclear factor (NF)-κB p65 activation leading to HIF1α-dependent vascular endothelial growth factor (VEGF) expression and angiogenesis in response to hypoxia. These in vitro data are biologically important as OPN expressing cells induce greater tumor growth and angiogenesis through enhanced expressions of proangiogenic molecules as compared with control. Immunohistochemical analysis of human breast cancer specimens revealed significant correlation between OPN and HIF1α but not HIF2α. Elevated expression of HIF1α and OPN was observed in pre-neoplastic and early stage infiltrating ductal carcinoma implicating the role of these proteins in neoplastic progression of breast cancer. Together, our results substantiate the prime role of OPN in cellular adaptation through ILK and NF-κB-mediated HIF1α-dependent VEGF expression in response to hypoxia that ultimately controls breast cancer progression and angiogenesis. Our study reinforces the fact that targeting OPN and its regulated signaling network hold important therapeutic implications.

Facial wrigglies: live extralymphatic filarial infestation in subcutaneous tissues of the head and neck.

We report a rare case of a 32-year-old male with live extralymphatic filarial infestation presenting as a facial subcutaneous soft-tissue swelling. To the best of our knowledge these imaging findings have not been previously reported in the head and neck region in the existing English language literature. Real-time high-resolution ultrasonography revealed a solitary well-defined subcutaneous cystic lesion over the right zygomatic arch. It showed multiple linear, echogenic, undulating structures exhibiting a persistent twirling motion during the examination. This typical ultrasonographic appearance was consistent with the filarial dance sign (FDS) of live adult filarial worms. Subsequent MRI confirmed the cystic and solitary nature of the lesion. Complete excision of the cyst was performed, which revealed intracystic straw-coloured fluid and multiple white-coloured adult worms within the lesion. Histopathological examination confirmed multiple adult filarial worms with surrounding reactive inflammatory changes. In an endemic region, identification of the FDS in any normal anatomical structure or abnormal swelling, however remote or unusual the location within the body, should strongly suggest the diagnosis of live active filarial infestation. In view of the increasing migratory trends in the global population, it is imperative for radiologists in all countries to be aware of the typical imaging findings of this disease to arrive at the correct diagnosis.