Preclinical Targeting of the PGRMC1-CK2 Axis with Silmitasertib: A Potential Strategy for Lung Adenocarcinoma Therapy.

Progesterone receptor membrane component 1 (PGRMC1) is a pleiotropic protein over-expressed in lung adenocarcinoma (LUAD). The precise molecular mechanisms underlying the signature motif of Casein kinase (CK2) presence in PGRMC1 and their role in LUAD remain unclear. X-ray crystallographic structure for CK2 and PGRMC1 from the PubChem database was obtained and subjected to protein-protein interaction (PPI) analysis to identify their interactions. In addition, the CK2 inhibitor - Silmitasertib was also utilised to understand the interaction between PGRMC1-CK2. The PPI complex (PGRMC1-CK2) and the PPI-ligand interaction analysis and their Molecular Dynamics (MD) studies revealed the stability of their interactions and critical amino acid contacts within the 5Ǻ vicinity of the CK2 signature motif "T/S-x-x-E/D". Moreover, in-vitro colony formation assay, migration assay, and gene expression analysis using quantitative Real-time PCR revealed that Silmitasertib (IC50-2.5 µM) was highly influential in suppressing the PGRMC1-CK2 expression axis. In conclusion, our study infers that PGRMC1-CK-2 axis inhibition could be a potential therapeutic option to limit the promotion and progression of lung cancer.

In vitro hemocompatibility studies on small-caliber stents for cardiovascular applications.

The doping of biologically meaningful ions into biphasic calcium phosphate (BCP) bioceramics, which exhibit biocompatibility with human body parts, has led to their effective use in biomedical applications in recent years. Doping with metal ions while changing the characteristics of the dopant ions, an arrangement of various ions in the Ca/P crystal structure. In our work, small-diameter vascular stents based on BCP and biologically appropriate ion substitute-BCP bioceramic materials were developed for cardiovascular applications. The small-diameter vascular stents were created using an extrusion process. FTIR, XRD, and FESEM were used to identify the functional groups, crystallinity, and morphology of the synthesized bioceramic materials. In addition, investigation of the blood compatibility of the 3D porous vascular stents was carried out via hemolysis. The outcomes indicate that the prepared grafts are appropriate for clinical requirements.

Fabrication and biological evaluation of three-dimensional (3D) Mg substituted bi-phasic calcium phosphate porous scaffolds for hard tissue engineering.

This work reports on the fabrication of three-dimensional (3D) magnesium substituted bi-phasic calcium phosphate (Mg-BCP) scaffolds by gel-casting, their structural and physico-chemical characterization, and on the assessment of their in vitro and in vivo performances. The crystalline phase assemblage, chemical functional groups and porous morphology features of the scaffolds were evaluated by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR) and field emission scanning electron microscopy (FE-SEM), respectively. The sintered scaffolds revealed an interconnected porosity with pore sizes ranging from 4.3 to 7.28 µm. The scaffolds exhibited good biomineralization activity upon immersion in simulated body fluid (SBF), while an in vitro study using MG-63 cell line cultures confirmed their improved biocompatibility, cell proliferation and bioactivity. Bone grafting of 3D scaffolds was performed in non-load bearing bone defects surgically created in tibia of rabbits, used as animal model. Histological and radiological observations indicated the successful restoration of bone defects. The overall results confirmed the suitability of the scaffolds to be further tested as synthetic bone grafts in bone regeneration surgeries and in bone tissue engineering applications.

Fabrication of three dimensional bioactive Sr2+ substituted apatite scaffolds by gel-casting technique for hard tissue regeneration.

This study aimed to fabricate three-dimensional (3D) bioactive Sr2+ -substituted apatite (Sr-HAP) scaffolds prepared by gel-casting with polymer sponge infiltration technique. 3D Sr-HAP scaffolds were prepared as engineering constructs with interconnected porous structure with a pore size of 200-600 µm ranging from a 10 × 10 × 6 mm size was designed. The characterization of X-ray diffraction, field emission scanning electron microscopy, and energy dispersion spectroscopy was utilized in order to evaluate the crystalline phase, structure, and morphology in the interconnected porous of the synthesized Sr-HAP scaffold. The bioactive and biocompatible of the resultant Sr-HAP scaffolds were analyzed by using simulated body fluid solution. Furthermore, the cytotoxicity and proliferation of MG-63 cell lines on the scaffolds were examined in 24 h culture. Furthermore, in vivo experiments demonstrated that the tibia bone defect with 4 mm diameter in rabbits was successfully healed by Sr-HAP porous scaffold after 45 days implantation. The histological images indicated the improved cell proliferation and new bone formation occurred in the porous scaffold treated group. The results indicated that the fabricated Sr-HAP scaffold is a promising capacity to infuse bone regeneration and promote in vivo tissue repair.

Identification of novel heterozygous Apex 1 gene variant (Glu87Gln) in patients with head and neck cancer of Indian origin.

Gene polymorphism among humans is one of the factors governing individual's susceptibility and resistance to various diseases including cancer. DNA repair enzymes play an important role in protecting our genome from various mutagens and preventing cancer. The role of DNA repair enzyme Apurinic/Apyrimidinic endodeoxyribonuclease 1 (Apex 1) in cancer has been very well documented. Using genomic DNA, Apex 1 coding region of 76 patients (n = 76) with head and neck cancer were amplified and sequenced to detect variations in the sequence. Of 76 patients, 1 patient with heterozygous novel Apex 1 variant (Glu87Gln) was identified. A comparative analysis of wild type and variant protein using in silico approach was performed to understand the difference in the structure and the function. This further revealed that the variant had a slight impact on the structure, which affected the stability and function of the protein. Using the state-of-the-art Molecular dynamic simulation analysis, we observed a loss in number of hydrogen bonds and salt bridge with a substitution of Gln for Glu at Position 87. This could be a possible reason behind the loss of stability/function of the protein. This study revealed a new variant of the Apex 1 gene; further studies will lead to the novel roles played by the variant Apex 1 protein in cause, disease progression, and response to the treatment in patients with cancer with Glu87Gln variant.

Expression, purification and immobilization of tannase from Staphylococcus lugdunensis MTCC 3614.

Enzymes find their applications in various industries, due to their error free conversion of substrate into product. Tannase is an enzyme used by various industries for degradation of tannin. Biochemical characterization of a specific enzyme from one organism to other is one of the ways to search for enzymes with better traits for industrial applications. Here, tannase encoding gene from Staphylococcus lugdunensis was cloned and suitability of the enzyme in various conditions was analysed to find its application in various industry. The recombinant protein was expressed with 6× His tag and purified using nickel affinity beads. The enzyme was purified up to homogeneity, with approximate molecular weight of 66 kDa. Purified tannase exhibited specific activity of about 716 U/mg. Optimum enzyme activity was found to be 40 °C at pH 7.0. Biochemical characterization revealed; metal ions such as Zn2+, Fe2+, Fe3+ and Mn2+ inhibited tannase activity, and SDS at lower concentration, increased tannase activity. Non polar organic solvents increased the tannase activity and polar solvents inhibited the tannase activity. Tannase immobilization studies show protection of the enzyme under wide range of pH and temperature. Also in this study we report a method for recovery and repeated use of the tannase.

Role of estrogen in regulation of cellular differentiation: a study using human placental and rat Leydig cells.

Estrogen classically is recognized as a growth-promoting hormone. Recent evidence suggests that estrogens are also involved in a wide variety of cellular and physiological functions involving the central nervous system, immune system, cardiovascular system and bone homeostasis. Our studies in cytotrophoblasts and BeWo cells, demonstrated that 17beta-estradiol induces terminal differentiation of placental trophoblasts directly and this differentiation is coupled with an increased production of TGFbeta1, which, in turn, affects telomerase activity and telomerase associated components at the level of hTERT. Furthermore, using rats treated in vivo with either EDS or estradiol and in vitro Leydig cell cultures, we proposed that 17beta-estradiol mediated down-regulation of collagen IV alpha4 expression could be one of the possible mechanisms for the inhibition of progenitor Leydig cell proliferation. In this review, we summarize the results from both the model systems, the human placental cytotrophoblast and rat Leydig cells to conclude that 17beta-estradiol has a unique stage-specific role in differentiation.

Comparison of target concentration intervention strategy with conventional dosing of digoxin.

Digoxin is a widely used drug in patients with congestive heart failure. The present study compared the quality of life of congestive heart failure patients on one year follow-up period with two different dosing of digoxin (5/7 therapy and 7/7 therapy in whom the target serum digoxin concentration is maintained). Quality of life significantly improved in intervention group thus emphasizing the need for continuous dosing of digoxin based on target concentration.

Collagen IV-mediated signalling is involved in progenitor Leydig cell proliferation.

In rats, during postnatal Leydig cell development, the progenitor Leydig cells (PLC) proliferate actively during days 14-21 of postnatal life. Luteinizing hormone (LH) is known to stimulate Leydig cell proliferation and oestradiol 17beta inhibits this process. In order to identify the molecules involved in Leydig cell proliferation, differentially expressed genes in proliferating and non-proliferating PLC isolated from vehicle and oestradiol 17beta-treated rats respectively, were analysed by differential display reverse transcription polymerase chain reaction (DD-RT-PCR). Results revealed that the expression of collagen IV alpha4 (Col IV alpha4), a subunit of extracellular matrix (ECM) protein collagen IV, was down regulated in PLC isolated from oestradiol 17beta-treated rats. Studies on stage specific expression of Col IV alpha4 during Leydig cell development revealed that this transcript is abundantly expressed at the stage where Leydig cell proliferation is maximal and the expression of this transcript decreased during differentiation of Leydig cells, which is associated with loss of proliferation. These observations suggest that Col IV alpha4 is important for PLC proliferation. Stimulation of PLC proliferation in vitro in the presence collagen IV provides additional support for the conclusion that collagen IV-mediated signalling is involved in PLC proliferation. Further studies revealed that active forms of focal adhesion kinase (FAK) and mitogen activated protein kinase 1/2 (MAPK 1/2), the intracellular signalling molecules that are known to mediate ECM protein signalling are present only in proliferating forms of Leydig cells and are absent in non-proliferating Leydig cells. These results suggest that collagen IV-mediated signalling is involved in PLC proliferation.

DD-RT-PCR identifies 7-dehydrocholesterol reductase as a key marker of early Leydig cell steroidogenesis.

Postnatal Leydig cell development in rat involves an initial phase of proliferation of progenitor Leydig cells (PLCs) and subsequent differentiation of these cells into immature Leydig cells (ILCs) and adult Leydig cells (ALCs). With an objective to identify the molecular changes associated with Leydig cell differentiation, the mRNA population in PLCs and ILCs were analyzed by the technique of differential display reverse transcription polymerase chain reaction (DD-RT-PCR). Results revealed differential expression of several transcripts in PLCs and ILCs. Of the several differentially expressed transcripts, the expression of transcripts corresponding to collagen IV alpha6 (Col IV alpha6) and ribosomal protein L 41 (RpL41) decreased during the differentiation of PLC to ILC. Also there was an increase in the expression of transcripts encoding enzymes such as microsomal glutathione-S-transferase (mGST 1) and 7-dehydrocholesterol reductase (7-DHCR) during this process. While Col IV alpha6 and RpL41 are known to be involved in cellular proliferation, mGST 1 and 7-DHCR are essential for normal Leydig cell steroidogenesis. A detailed study on 7-DHCR expression in Leydig cells revealed that this enzyme plays a crucial role in steroidogenesis. Interestingly expression of this enzyme is not under acute regulation by Luteinizing hormone (LH).

LCN6, a novel human epididymal lipocalin.

BACKGROUND: The lipocalin (LCN) family of structurally conserved hydrophobic ligand binding proteins is represented in all major taxonomic groups from prokaryotes to primates. The importance of lipocalins in reproduction and the similarity to known epididymal lipocalins prompted us to characterize the novel human epididymal LCN6. METHODS AND RESULTS: LCN6 cDNA was identified by database analysis in a comprehensive human library sequencing program. Macaca mulatta (rhesus monkey) cDNA was obtained from an epididymis cDNA library and is 93% homologous to the human. The gene is located on chromosome 9q34 adjacent LCN8 and LCN5. LCN6 amino acid sequence is most closely related to LCN5, but the LCN6 beta-barrel structure is best modeled on mouse major urinary protein 1, a pheromone binding protein. Northern blot analysis of RNAs isolated from 25 human tissues revealed predominant expression of a 1.0 kb mRNA in the epididymis. No other transcript was detected except for weak expression of a larger hybridizing mRNA in urinary bladder. Northern hybridization analysis of LCN6 mRNA expression in sham-operated, castrated and testosterone replaced rhesus monkeys suggests mRNA levels are little affected 6 days after castration. Immunohistochemical staining revealed that LCN6 protein is abundant in the caput epithelium and lumen. Immunofluorescent staining of human spermatozoa shows LCN6 located on the head and tail of spermatozoa with the highest concentration of LCN6 on the post-acrosomal region of the head, where it appeared aggregated into large patches. CONCLUSIONS: LCN6 is a novel lipocalin closely related to Lcn5 and Lcn8 and these three genes are likely products of gene duplication events that predate rodent-primate divergence. Predominant expression in the epididymis and location on sperm surface are consistent with a role for LCN6 in male fertility.

Isolation and characterization of Leydig cells from adult bonnet monkeys (Macaca radiata): evidence for low steroidogenic capacity in monkey Leydig cells in contrast to rat Leydig cells.

Most of the available information on Leydig cells has been obtained using a rodent model system. With an objective to extend the observations made with rat Leydig cells (RLCs) to primates, a method has been developed to isolate Leydig cells from monkey (Macaca radiata) testis. Enzymatic dissociation of monkey testis followed by Percoll-gradient fractionation of the interstitial cells resulted in the recovery of Leydig cells at densities corresponding to 1.064-1.070 g/ml. Purified (90-94%) monkey Leydig cells (MLCs) stained positive for the Leydig cell marker 3beta-hydroxysteroid dehydrogenase. The cells responded to in vitro addition of human chorionic gonadotropin (hCG) and produced testosterone. Comparison of the in vitro testosterone-producing ability of MLCs with RLCs revealed that MLCs have much less steroidogenic capacity compared with the RLCs. Analysis revealed that limitation in substrate availability to mitochondrial P(450) side chain cleavage enzyme and low mitochondrial and smooth endoplasmic reticulum content in MLCs could be the possible reasons for the low steroidogenic capacity of the MLCs.

Community leaders involvement in leprosy health education.

As an alternative approach, 310 community leaders consisting of village political leaders, school teachers, Govt. staff, members of socio-welfare and religious agencies, graduate students and traders from 21 villages of Chingleput district of Tamilnadu (India) were interviewed to explore the possibilities of their involvement in leprosy health education community. Though majority (76%) of respondents were not fully aware about various aspects of leprosy and showed negative reactions (51%) towards leprosy patients; almost all realised the importance of educating community about leprosy for its early control, for which a large majority (88%) of them had expressed their willingness to participate in leprosy health education and control programme by devoting an average of 4.4 +/- 5.4 hours per week. A good number (54%) of them had also been educating people about leprosy in one or the other way. The leaders who had been exposed to leprosy health education especially in recent past, were significantly better equipped with knowledge about leprosy and its control and were much more willing to participate in NLCP, than others. Study concluded that if the community leaders are approached, educate and motivated properly, they would certainly involve themselves to provide a valuable strength to our leprosy health education and control programme.

Community awareness about leprosy and participation in National Leprosy Control Programme.

To evaluate the health education component of our National Leprosy Control Programme (NLCP), 955 adult community members and 225 adult leprosy patients were interviewed with a view to assess their awareness about leprosy and participation in NLCP. The early signs/symptoms of leprosy were poorly perceived by the community. Majority of the community (81%) and patients (75%) were unaware or held superstitious ideas about causation of leprosy. The spread of disease through close contact with patient(s) was better known to the community (65%) than the patients (45%); but the role of open cases in spread was stressed by more patients (17%) than community (5.5%). About 31% community and 23% patients had no idea about the ways to prevent leprosy spread. As against 89% patients, only 62% community believed in curability of leprosy with early and regular treatment; but 20% of the community members did not know where to refer patients for treatment. The causation and prevention of deformities were poorly perceived by 71% patients, and likewise 62% of the patients did not take precaution(s) to prevent the deformities. About 32% respondents were unaware of the efforts being made to control leprosy; and their (79-84% respondents) participation in NLCP was very vague. About 44% community members showed prejudice towards leprosy. The NLCP infra-structure and mass media could not educate community effectively. The implications of the findings are discussed in this paper.

Socio-economic experiences of leprosy patients.

225 adult leprosy patients were interviewed to study their socio-economic experiences, about various aspects of their lives. It was observed that 17.34%, 14.33% and 45.78% of patients experienced negative reactions from their families, spouses and society members, respectively. Out of 79 unmarried patients, 53 (67.1%) attributed leprosy as the only reason for not getting a partner for marriage. Out of 146 married patients, 34 (23.3%) were not living with their spouses; this also included 9 (6.2%) patients, deserted by their partners. Leprosy uprooted 44 (13.55%) patients from their residences, of whom 27 settled in leprosy village/settlement. The occupational status of 104 (46.22%) patients was adversely affected due to leprosy, of whom 43 became dependents and 17 beggars. Monthly income of 115 (51.1%) patients reduced to the extent of 84%, after getting leprosy. The social prejudice and deformities due to leprosy, have played key roles in socio-economic deterioration of patients. The leprosy control programme (LCP) need to be implemented more efficiently and effectively, with active involvement of communities. The socio-medical units, if included in LCP, may be utilized more effectively to prevent the socio-economic dehabilitation of patients, as well to tackle the abnormal psycholygical behaviours of patients.

Utilization of medical agencies and treatment compliance by urban (Madras) leprosy patients.

The utilization of medical agencies and treatment compliance by 3880 leprosy patients registered with Govt. Leprosy Control Unit, Saidapet, Madras were studied. It was observed that 39% patients waited for 1.32 (+/- 1.75) years before medical consultation, for their negligence and unawareness of disease. About 16% and 4% patients consulted Gen. Hospitals and Private Practitioners, respectively. The leprosy clinics were most popular, 35% patients changed medical agencies. On an average, one patient had consulted 1.47 (+/- 0.51) medical agencies and 1.23 (+/- 0.52) leprosy clinics, for treatment of leprosy. Only 45% patient attended clinic regularly, others attended irregularly (22.5%) or discontinued (32.4%). The unsuitable clinic timing (morning) was an important factor for defaulting the clinics. Of the 2625 (67.66%) patients who attended last clinic, one patient had missed an average of 5.5 (+/- 8.3) DDS tablets in a month. The implications of findings and suggestions to improve the service utilization with good compliance by patients, are discussed in this communication.

Illness and service utilization behaviours of leprosy patients.

225 adult leprosy patients attending the CLTRI, were interviewed to study their illness and medical agency utilization behaviours. Almost all patients perceived their disease as leprosy but 71.50% did not know how they got it. 10-11% did not reveal the disease to their family for fear of rejection. The time-lag between first suspicion and medical consultation was 1 year or more in 48% of cases. For treatment of leprosy, 36-38% of patients consulted Private Practitioners and General Hospitals, at one or the other time. 42.6% of patients changed 3 or more medical agencies for treatment. On an average patient had taken 62.39% of expected treatment. 41% of patients were not aware of the name of drug (DDS) they were taking. 44% of patients had tried home remedies. Most of the patients preferred to take treatment at leprosy referral hospitals.