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author:("Singh, harmed")
1.  Activity-Modulating Monoclonal Antibodies to the Human Serine Protease HtrA3 Provide Novel Insights into Regulating HtrA Proteolytic Activities 
PLoS ONE  2014;9(9):e108235.
Mammalian HtrA (high temperature requirement factor A) proteases, comprising 4 multi-domain members HtrA1-4, play important roles in a number of normal cellular processes as well as pathological conditions such as cancer, arthritis, neurodegenerative diseases and pregnancy disorders. However, how HtrA activities are regulated is not well understood, and to date no inhibitors specific to individual HtrA proteins have been identified. Here we investigated five HtrA3 monoclonal antibodies (mAbs) that we have previously produced, and demonstrated that two of them regulated HtrA3 activity in an opposing fashion: one inhibited while the other stimulated. The inhibitory mAb also blocked HtrA3 activity in trophoblast cells and enhanced migration and invasion, confirming its potential in vivo utility. To understand how the binding of these mAbs modulated HtrA3 protease activity, their epitopes were visualized in relation to a 3-dimensional HtrA3 homology model. This model suggests that the inhibitory HtrA3 mAb blocks substrate access to the protease catalytic site, whereas the stimulatory mAb may bind to the PDZ domain alone or in combination with the N-terminal and protease domains. Since HtrA1, HtrA3 and HtrA4 share identical domain organization, our results establish important foundations for developing potential therapeutics to target these HtrA proteins specifically for the treatment of a number of diseases, including cancer and pregnancy disorders.
PMCID: PMC4172569  PMID: 25248123
2.  Small Molecule Proprotein Convertase Inhibitors for Inhibition of Embryo Implantation 
PLoS ONE  2013;8(12):e81380.
Uterine proprotein convertase (PC) 6 plays a critical role in embryo implantation and is pivotal for pregnancy establishment. Inhibition of PC6 may provide a novel approach for the development of non-hormonal and female-controlled contraceptives. We investigated a class of five synthetic non-peptidic small molecule compounds that were previously reported as potent inhibitors of furin, another PC member. We examined (i) the potency of these compounds in inhibiting PC6 activity in vitro; (ii) their binding modes in the PC6 active site in silico; (iii) their efficacy in inhibiting PC6-dependent cellular processes essential for embryo implantation using human cell-based models. All five compounds showed potent inhibition of PC6 activity in vitro, and in silico docking demonstrated that these inhibitors could adopt a similar binding mode in the PC6 active site. However, when these compounds were tested for their inhibition of decidualization of primary human endometrial stromal cells, a PC6-dependent cellular process critical for embryo implantation, only one (compound 1o) showed potent inhibition. The lack of activity in the cell-based assay may reflect the inability of the compounds to penetrate the cell membrane. Because compound's lipophilicity is linked to cell penetration, a measurement of lipophilicity (logP) was calculated for each compound. Compound 1o is unique as it appears the most lipophilic among the five compounds. Compound 1o also inhibited another crucial PC6-dependent process, the attachment of human trophoblast spheroids to endometrial epithelial cells (a model for human embryo attachment). We thus identified compound 1o as a potent small molecule PC6 inhibitor with pharmaceutical potential to inhibit embryo implantation. Our findings also highlight that human cell-based functional models are vital to complement the biochemical and in silico analyses in the selection of promising drug candidates. Further investigations for compound 1o are warranted in animal models to test its utility as an implantation-inhibiting contraceptive drug.
PMCID: PMC3852413  PMID: 24324690
3.  Endometrial Exosomes/Microvesicles in the Uterine Microenvironment: A New Paradigm for Embryo-Endometrial Cross Talk at Implantation 
PLoS ONE  2013;8(3):e58502.
Exosomes are nanoparticles (∼100 nm diameter) released from cells, which can transfer small RNAs and mRNA via the extracellular environment to cells at distant sites. We hypothesised that exosomes or the slightly larger microvesicles (100–300 nm) are released from the endometrial epithelium into the uterine cavity, and that these contain specific micro (mi)RNA that could be transferred to either the trophectodermal cells of the blastocyst or to endometrial epithelial cells, to promote implantation. The aim of this study was to specifically identify and characterise exosomes/microvesicles (mv) released from endometrial epithelial cells and to determine whether exosomes/mv are present in uterine fluid. Immunostaining demonstrated that the tetraspanins, CD9 and CD63 used as cell surface markers of exosomes are present on the apical surfaces of endometrial epithelial cells in tissue sections taken across the menstrual cycle: CD63 showed cyclical regulation. Exosome/mv pellets were prepared from culture medium of endometrial epithelial cell (ECC1 cells) and from uterine fluid and its associated mucus by sequential ultracentifugation. Exosomes/mv were positively identified in all preparations by FACS and immunofluorescence staining following exosome binding to beads. Size particle analysis confirmed the predominance of particles of 50–150 nm in each of these fluids. MiRNA analysis of the ECC1 cells and their exosomes/mv demonstrated sorting of miRNA into exosomes/mv: 13 of the 227 miRNA were specific to exosomes/mv, while a further 5 were not present in these. The most abundant miRNA in exosomes/mv were hsa-miR-200c, hsa-miR-17 and hsa-miR-106a. Bioinformatic analysis showed that the exosome/mv-specific miRNAs have potential targets in biological pathways highly relevant for embryo implantation. Thus exosomes/mv containing specific miRNA are present in the microenvironment in which embryo implantation occurs and may contribute to the endometrial-embryo cross talk essential for this process.
PMCID: PMC3596344  PMID: 23516492
4.  HtrA3 Is Downregulated in Cancer Cell Lines and Significantly Reduced in Primary Serous and Granulosa Cell Ovarian Tumors 
Journal of Cancer  2013;4(2):152-164.
Objective. The high temperature requirement factor A3 (HtrA3) is a serine protease homologous to bacterial HtrA. Four human HtrAs have been identified. HtrA1 and HtrA3 share a high degree of domain organization and are downregulated in a number of cancers, suggesting a widespread loss of these proteases in cancer. This study examined how extensively the HtrA (HtrA1-3) proteins are downregulated in commonly used cancer cell lines and primary ovarian tumors.
Methods. RT-PCR was applied to various cancer cell lines (n=17) derived from the ovary, endometrium, testes, breast, prostate, and colon, and different subtypes of primary ovarian tumors [granulosa cell tumors (n=19), mucinous cystadenocarcinomas (n=6), serous cystadenocarcinomas (n=8)] and normal ovary (n = 9). HtrA3 protein was localized by immunohistochemistry.
Results. HtrA3 was extensively downregulated in the cancer cell lines examined including the granulosa cell tumor-derived cell lines. In primary ovarian tumors, the HtrA3 was significantly lower in serous cystadenocarcinoma and granulosa cell tumors. In contrast, HtrA1 and HtrA2 were expressed in all samples with no significant differences between the control and tumors. In normal postmenopausal ovary, HtrA3 protein was localized to lutenizing stromal cells and corpus albicans. In serous cystadenocarcinoma, HtrA3 protein was absent in the papillae but detected in the mesenchymal cyst wall.
Conclusion. HtrA3 is more extensively downregulated than HtrA1-2 in cancer cell lines. HtrA3, but not HtrA1 or HtrA2, was decreased in primary ovarian serous cystadenocarcinoma and granulosa cell tumors. This study provides evidence that HtrA3 may be the most relevant HtrA associated with ovarian malignancy.
PMCID: PMC3572407  PMID: 23412729
HtrA3; ovarian cancer; protease; GCT; Serous cystadenocarcinoma.
5.  Primary Small Cell Carcinoma of the Larynx: Report of a Rare Tumor 
Primary small cell carcinoma of larynx is a rare tumor representing less than 0.5% of all laryngeal cancers. This is one of the most lethal of all malignancies associated with frequent early widespread metastases and dismal prognosis. We report a case of a 60-year-old patient with small cell carcinoma of the subglottic larynx, who presented with hepatic metastasis.
PMCID: PMC3350277  PMID: 22606450
6.  Non-union scaphoid: Four-corner fusion of the wrist 
Indian Journal of Orthopaedics  2010;44(2):208-211.
Four-corner fusion of the wrist is an option for management of non-union scaphoid with painful arthritis of the wrist. Various surgical techniques have been devised for four-corner fusion, with inconsistent results. We present our experience of four-corner fusion achieved using a standard H-plate, designed originally for anterior cervical plating.
Materials and Methods:
The study is a retrospective analysis of six cases of painful wrist arthritis resulting from nonunion of scaphoid treated by four-corner fusion, between 1996 and 2004. The average duration of follow-up was 5.8 years. Each patient was evaluated clinically according to the rating scales described by Bach.
The mean grip-strength calculated as a percentage of the uninvolved side was 47% pre-operatively, and 74% post- operatively at the final follow-up. The difference between the preoperative and postoperative ‘pain ratings’ and ‘activity ratings’ was found to be statistically significant (P<0.001). Mean time to fusion was 16.1 weeks. Dorsal impingement was the most common associated problem.
H-plate, used for four-corner fusion, provides rigid fixation, ensures fusion, and is a good alternative to the available options.
PMCID: PMC2856398  PMID: 20419010
Four corner fusion; Cervical H-plate; nonunion scaphoid
7.  Results of operative treatment of acetabular fractures from the Third World—how local factors affect the outcome 
International Orthopaedics  2007;33(2):347-352.
The objective of this study was to assess the outcome of operations on acetabular fractures from a developing country in the presence of locally available facilities. Sixty-three acetabular fractures were assessed at an average follow up of 52.94 months after operation. Twenty-six patients operated upon in the first three years and 37 operated thereafter were separately studied to discover the effect of the learning curve. Regarding the fractures, 47 of 63 (74.6%) had excellent/good results (Harris Hip Score>80). The complications included broken drill bit in eight patients (12.69%), deep infection and heterotopic ossification in five patients (7.93%), avascular necrosis and sciatic nerve palsy in two patients (3.17%) and implant failure in one patient (1.58%). The results collected during the learning curve were inferior in the complex fractures (p value<0.001). Complications were common in patients opting for local implants and in those operated after over 2 weeks delay.
PMCID: PMC2899060  PMID: 17940767
8.  Irritant contact dermatitis complicated by deep-seated staphylococcal infection caused by a hair relaxer. 
Chemical hair relaxers are used by many women to straighten their hair. We describe a case of a deep soft tissue staphylococcal abscess that complicated an irritant contact dermatitis from a hair relaxer treatment.
PMCID: PMC2594120  PMID: 11853045

Results 1-8 (8)