Mapping key regions of the RXFP2 low-density lipoprotein class-A module that are involved in signal activation.

The peptide hormone INSL3 and its receptor, RXFP2, have co-evolved alongside relaxin and its receptor, RXFP1. Both RXFP1 and RXFP2 are G protein-coupled receptors (GPCRs) containing the hallmark seven transmembrane helices in addition to a distinct ectodomain of leucine-rich repeats (LRRs) and a single low-density lipoprotein class-A (LDLa) module at the N-terminus. RXFP1 and RXFP2 are the only mammalian GPCRs known to contain an LDLa, and its removal does not perturb primary ligand binding to the LRRs; however, signaling is abolished. This presents a general mechanism whereby ligand binding induces a conformational change in the receptor to position the LDLa to elicit a signal response. Although the LDLa interaction site has not been identified, the residues important to the action have been mapped within the RXFP1 LDLa module. In this study, we comprehensively study the RXFP2 LDLa module. We determine its structure using nuclear magnetic resonance (NMR) and concurrently investigate the signaling of an RXFP2 with the LDLa removed (RXFP2-short), confirming that the LDLa is essential to signaling. We then replaced the LDLa with the second ligand binding module from the LDL receptor, LB2, creating the RXFP2-LB2 chimera. Unlike that in the equivalent RXFP1-LB2 chimera, signaling is rescued albeit modestly. Guided by the NMR structure, we dissected regions of the RXFP2 LDLa to identify specific residues that are important to signal activation. We determine that although the module is important to the activation of RXFP2, unlike the RXFP1 receptor, specific residues in the N-terminus of the domain are not involved in signal activation.

The relaxin receptor (RXFP1) utilizes hydrophobic moieties on a signaling surface of its N-terminal low density lipoprotein class A module to mediate receptor activation.

The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true "ligand" of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation.

Highly fluorescent reticulocyte count is the early predictor of haematopoietic recovery on remission induction phase.

Highly fluorescent reticulocyte count a new routine parameter in the hematology analyzer can give the idea of the earliest morphologic change of bone marrow recovery before other test become positive after chemotherapy. A prospective study was carried out in the Department of Clinical Pathology in collaboration with Paediatric Haematology and Oncology, Bangabandhu Sheikh Mujib Medical University (BSMMU) during a period of one year starting from October 2009 to September 2010 to evaluate the bone marrow recovery in children with acute lymphoblastic leukemia (ALL) by automated reticulocyte analysis. Total fifty patients were enrolled in this study on remission induction phase. All patients were between 8 months to 15 years age range with a mean age of 5.5±3.2. At the end of the study highly fluroscent reticulocyte count recovery occurred earlier than the current practice of absolute neutrophil count (ANC) recovery.

Early diagnosis of neonatal septicemia by hematologic scoring system, C-reactive protein and serum haptoglobin.

Neonatal septicemia is one of the major health problems throughout the world. Infections are frequent and important cause of morbidity and mortality in neonatal period. The objective of the present study was to find out the role of hematologic scoring system (HSS), C-reactive protein (CRP) and haptoglobin in the early diagnosis of neonatal septicemia. This is a descriptive consisted of 100 neonates admitted at neonatal ICU, BSMMU, who were clinically suspected sepsis. The hematological parameter, C-reactive protein and haptoglobin were measured in all cases. Blood culture was done as the gold standard for diagnosis of neonatal septicemia. There were 12 out of 100 neonates (12%) who had culture proven sepsis and they were predominantly preterm and very low birth weight. On evaluation of various hematological parameters total leukocyte count, total neutrophil count, immature to total neutrophil ratio (>0.2), immature to mature neutrophil ratio (>0.3), total immature count, platelet count were found to have optimal sensitivities and negative predictive values. Using these values hematologic scoring system (HSS) was formulated according to Rodwell et al. Score ≥4 was found sensitivity of 100%, specificity of 60%. C-reactive protein (CRP) had sensitivity of 75%, specificity of 74%. Haptoglobin was not found significant (p<0.05) with sepsis and sensitivity was very low. But Combination of score ≥4 and CRP showed sensitivity of 75%, specificity 85%, positive predictive value (PPV) 41% and negative positive value (NPV) 96%. HSS and CRP are useful test to differentiate the septicemic from non septicemic neonates and also provide a effective guideline to make decisions regarding judicious use of antibiotic therapy. But haptoglobin level was not found useful for screening of sepsis.

Transcriptomic analysis of choroidal neovascularization reveals dysregulation of immune and fibrosis pathways that are attenuated by a novel anti-fibrotic treatment.

Neovascular AMD (nAMD) leads to vision loss and is a leading cause of visual impairment in the industrialised world. Current treatments that target blood vessel growth have not been able to treat subretinal fibrosis and nAMD patients continue to lose vision. The molecular mechanisms involved in the development of fibrotic lesions in nAMD are not well understood. The aim of this study was to further understand subretinal fibrosis in the laser photocoagulation model of choroidal neovascularization (CNV) by studying the whole transcriptome of the RPE/choroid following CNV and the application of an anti-fibrotic following CNV. Seven days after laser induced CNV, RPE and choroid tissue was separated and underwent RNAseq. Differential expression analysis and pathway analysis revealed an over representation of immune signalling and fibrotic associated pathways in CNV compared to control RPE/choroid tissue. Comparisons between the mouse CNV model to human CNV revealed an overlap in upregulated expression for immune genes (Ccl2, Ccl8 and Cxcl9) and extracellular matrix remodeling genes (Comp, Lrcc15, Fndc1 and Thbs2). Comparisons between the CNV model and other fibrosis models showed an overlap of over 60% of genes upregulated in either lung or kidney mouse models of fibrosis. Treatment of CNV using a novel cinnamoyl anthranilate anti-fibrotic (OCX063) in the laser induced CNV model was selected as this class of drugs have previously been shown to target fibrosis. CNV lesion leakage and fibrosis was found to be reduced using OCX063 and gene expression of genes within the TGF-beta signalling pathway. Our findings show the presence of fibrosis gene expression pathways present in the laser induced CNV mouse model and that anti-fibrotic treatments offer the potential to reduce subretinal fibrosis in AMD.

Inhibitory Effects of 3',4'-Dihydroxyflavonol in a Mouse Model of Glaucoma Filtration Surgery and TGFß1-Induced Responses in Human Tenon's Fibroblasts.

Purpose: Cytotoxic agents such as mitomycin C (MMC) are part of the mainstay treatment for limiting subconjunctival scarring following glaucoma filtration surgery (GFS). However, a safer antifibrotic therapy is clinically needed. The anti-scarring properties of 3',4'-dihydroxyflavonol (DiOHF) were evaluated in a mouse model of GFS and in cultured human Tenon's fibroblasts (HTFs). Methods: GFS was performed in C57BL/6 mice receiving daily intraperitoneal injections of DiOHF or vehicle or a single intraoperative injection of MMC. Eyes were harvested on day 14 for assessment of collagen deposition, expression of alpha-smooth muscle actin (α-SMA), cluster of differentiation 31 (CD31), and 4-hydroxy-2-nonenal (4HNE) in the conjunctiva/Tenon's layer. The inhibitory effects of DiOHF on transforming growth factor ß (TGFß)-induced responses were also assessed in HTFs. Results: Treatment with DiOHF demonstrated a reduction in collagen deposition at the GFS site compared to vehicle-treated mice. The degree of 4HNE-positive fluorescence was significantly reduced in DiOHF-treated eyes compared to the other groups, indicating a decrease in oxidative stress. A reduction in expression of α-SMA and CD31 was seen in DiOHF-treated conjunctiva compared to those treated with vehicle. Concordant results were demonstrated in cultured HTFs in vitro. Furthermore, treatment of cultured HTFs with DiOHF also displayed a reduction in the proliferation, migration, and contractility of HTFs. Conclusions: Treatment with DiOHF reduces scarring and angiogenesis in the conjunctiva of mice with GFS at a level comparable to MMC. The reduction in oxidative stress suggests that DiOHF may suppress scarring via different mechanisms from MMC. Translational Relevance: DiOHF may be a safer and superior wound modulating agent than conventional antifibrotic therapy in GFS.

Immature reticulocyte fraction as a predictor of bone marrow recovery in children with acute lymphoblastic leukaemia on remission induction phase.

Immature reticulocyte fraction a new routine parameter in the hematology analyzer can give the idea of the earliest morphologic change of bone marrow recovery before other test become positive after chemotherapy. A prospective observational study was carried out in the Department of Clinical Pathology in collaboration with Paediatric Haematology and Oncology, Bangabandhu Sheikh Mujib Medical University during a period of one year starting from October 2009 to September 2010 to evaluate the bone marrow recovery in children with acute lymphoblastic leukaemia by automated reticulocyte analysis. Total fifty patients were enrolled in this study on remission induction phase. All patients were between 8 months to 15 years age range with a mean age of 5.5 +/- 3.2. At the end of the study out of 50 cases, 52% patients showed early immature reticulocyte fraction recovery and concluded that the immature reticulocyte fraction parameter showed earlier haematopoietic recovery than the current practice of absolute neutrophil count recovery.

Usefulness of light emitting diode (LED) fluorescent microscopy as a tool for rapid and effective method for the diagnosis of pulmonary tuberculosis.

BACKGROUND: Tuberculosis remains world's leading cause of death from a single infectious agent. Fluorescence microscopy offers well-described benefits, comparing with brightfield microscopy, for the evaluation sputum smear samples for tuberculosis. We evaluated the diagnostic performance of fluorescence microscopy, using novel Light Emitting Diode (LED) technology as an alternative to the conventional fluorescence microscopy by Auramine stain as well as brightfield microscopy by Ziehl-Neelsen (ZN) stain. OBJECTIVES: The objective of the study was to see the usefulness of LED fluorescent microscopy in the diagnosis of pulmonary tuberculosis. METHODS: This is a prospective study consisted of 150 sputum samples from the patients of NIDCH, Mohakhali. All samples were stained by auramine and ZN stain at BSMMU and culture was done in Lowenstein-Jensen (L-J) media as gold standard at NTRL, Mohakhali. RESULTS: In this study total 66 (44%) out of 150 sputum specimens were positive for Mycobacterium Tuberculosis by culture. Sensitivity and specificity documented for the different modalities were 95.38% and 94.11%, respectively, for the LED assessment; 68.18% and 90.47%, respectively, for the CFM assessment; and 56.06% and 97.61%, respectively, for brightfield microscopy by ZN stain. The difference in their case detection rate was statistically significant (chi2=119.38, p<0.001). CONCLUSION: Fluorescence Microscopy (FM) is more sensitive than ZN for diagnosis of pulmonary tuberculosis. However, since FM is more sensitive and rapid, using this method (LED) in clinical laboratories with large specimen numbers is recommended.

Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules.

Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism.

Membrane receptors: structure and function of the relaxin family peptide receptors.

The receptors for members of the relaxin peptide family have only recently been discovered and are G-protein-coupled receptors (GPCRs). Relaxin and insulin-like peptide 3 (INSL3) interact with the leucine-rich-repeat-containing GPCRs (LGRs) LGR7 and LGR8, respectively. These receptors show closest similarity to the glycoprotein hormone receptors and contain large ectodomains with 10 leucine-rich repeats (LRRs) but are unique members of the LGR family (class C) as they have an LDL class A (LDLa) module at their N-terminus. In contrast, relaxin-3 and INSL5 interact with another class of type I GPCRs which lack a large ectodomain, the peptide receptors GPCR135 and GPCR142, respectively. These receptors are now classified as relaxin family peptide (RXFP) receptors, RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 (GPCR135) and RXFP4 (GPCR142). This review outlines the identification of the peptides and receptors, their expression profiles and physiological roles and the functional interactions of the peptides with their unique receptors.

A review of the history of prameha and diabetes mellitus.

Several diseases of polyuric nature were described in Ayurveda. Collectively called Prameha, this group includes an entity called Madhumeha which is the equivalent of diabetes mellitus. The medical history of the two diseases is described in the article.

A case of disseminated hydatidosis.

A young lady initially found to have hydatid cysts in the lung only, subsequently within a very short period was found to develop cysts in the liver. Soon after, she developed cysts in the subcutaneous tissue over the anterolateral chest wall, which, on investigation, revealed hepatic cysts herniating through the chest wall defects caused by previous operations. The unique features of this case include the degree of dissemination, the multiplicity of sites and the peculiar nature of herniation of the hepatic cysts into the parietes.