Molecular Cloning and Expression Analysis of fushi tarazu Factor 1 in the Brain of Air-Breathing Catfish, Clarias gariepinus

BACKGROUND: Fushi tarazu factor 1 (FTZ-F1) encodes an orphan nuclear receptor belonging to the nuclear receptor family 5A (NR5A) which includes adrenal 4-binding protein or steroidogenic factor-1 (Ad4BP/SF-1) and liver receptor homologue 1 (LRH-1) and plays a pivotal role in the regulation of aromatases. METHODOLOGY/PRINCIPAL FINDINGS: Present study was aimed to understand the importance of FTZ-F1 in relation to brain aromatase (cyp19a1b) during development, recrudescence and after human chorionic gonadotropin (hCG) induction. Initially, we cloned FTZ-F1 from the brain of air-breathing catfish, Clarias gariepinus through degenerate primer RT-PCR and RACE. Its sequence analysis revealed high homology with other NR5A1 group members Ad4BP/SF-1 and LRH-1, and also analogous to the spatial expression pattern of the latter. In order to draw functional correlation of cyp19a1b and FTZ-F1, we analyzed the expression pattern of the latter in brain during gonadal ontogeny, which revealed early expression during gonadal differentiation. The tissue distribution both at transcript and protein levels revealed its prominent expression in brain along with liver, kidney and testis. The expression pattern of brain FTZ-F1 during reproductive cycle and after hCG induction, in vivo was analogous to that of cyp19a1b shown in our earlier study indicating its involvement in recrudescence. CONCLUSIONS/SIGNIFICANCE: Based on our previous results on cyp19a1b and the present data, it is plausible to implicate potential roles for brain FTZ-F1 in ovarian differentiation and recrudescence process probably through regulation of cyp19a1b in teleosts. Nevertheless, these interactions would require primary coordinated response from ovarian aromatase and its related transcription factors

List of primers used for cloning and expression analysis of catfish brain <i>FTZ-F1</i>.

<p><b><i>Note:</i></b> The abbreviations (underlined) for the degenerate bases used in primers, 1 and 2 are <b><u>W</u></b>  =  A or T; <b><u>V</u></b>  =  A, C, or G; <b><u>R</u></b>  =  A or G; <b><u>S</u></b>  =  G or C; <b><u>K</u></b>  =  G or T; <b><u>Y</u></b>  =  C or T; <b><u>M</u></b>  =  A or C.</p><p><b><i>Other abbreviations:</i></b><b>De</b> =  Degenerate; <b>Fw</b> =  Forward; <b>Rv</b> =  Reverse; <b>P</b> = Primary; <b>N</b> = Nested; <b>RT</b> & <b>qRT-PCR</b> = Real time PCR; <b>RACE</b> =  Rapid Amplification of cDNA Ends.</p

Phylogenetic tree showing the evolutionary status of catfish FTZ-F1.

<p>Phylogenetic tree constructed using NJ method and bootstrap analysis with 1000 replicates was used to assess the strength of nodes in the tree. Phylogenetic analysis was done using ClustalW software of DNA data bank of Japan and supported by Tree view software. The scale bar represents 0.1 substitutions per amino acid site. GenBank accession numbers of the sequences used in the analysis are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028867#pone-0028867-g001" target="_blank">Fig. 1.</a></p

Expression of <i>FTZ-F1</i> in brain during different phases of catfish ovarian cycle.

<p>Quantitative analysis of brain <i>FTZ-F1</i> expression relative to <i>β-actin</i> expression during different phases of ovarian cycle was reported as fold change relative to preparatory phase calculated using 2^−ΔΔCT method. Values are mean ± SEM, n = 5. Means with different letters differ significantly and are compared group-wise (<i>P</i><0.05).</p

Peptide affinity purification profile of FTZ-F1 antibody and Western blot analysis.

<p>A) IgG fraction of FTZ-F1 antibody showing band at ∼55 kDa (heavy chain) and ∼25 kDa (light chain). B) No signal was seen in negative control. C) A protein band at ∼45 kDa corresponding to deduced FTZ-F1 protein detected in the female brain protein homogenate of preparatory phase catfish.</p

ClustalW alignment of deduced amino acid sequence of catfish FTZ-F1 with other vertebrate counter parts.

<p>The alignment was done using software ClustalW (EBI tools). Shaded region represents conserved amino acids and signature domains are represented by rectangle boxes. GenBank accession numbers of the sequences we used are as follows: <i>Clarias gariepinus</i>; JN859075, <i>Ictalurus punctatus</i>; DQ000612, <i>Oreochromis niloticus</i>; AB060814, <i>Oryzias latipes</i>; AB016834, <i>Oncorhynchus mykiss</i>; NM_001124537, <i>Danio rerio</i>; NM_131463, <i>Acanthopagrus schlegeli</i>; AY491379, <i>Taeniopygia guttata</i>; NM_001076692, <i>Pelteobagrus fulvidraco</i>; EU860284, <i>Xenopus laevis</i>; BC169770, <i>Gallus gallus</i>; NM_205077, <i>Mus musculus</i>; AF511594 and <i>Homo sapiens</i>; BC118571.</p

Strengthening Health System and Community Mobilization for Sickle Cell Disease Screening and Management among Tribal Populations in India: An Interventional Study

Sickle cell disease (SCD) affects 5% of the global population, with over 300,000 infants born yearly. In India, 73% of those with the sickle hemoglobin gene belong to indigenous tribes in remote regions lacking proper healthcare. Despite the prevalence of SCD, India lacked state-led public health programs until recently, leaving a gap in screening and comprehensive care. Hence, the Indian Council of Medical Research conducted implementation research to address this gap. This paper discusses the development and impact of the program, including screening and treatment coverage for SCD in tribal areas. With a quasi-experimental design, this study was conducted in six tribal-dominated districts in three phases – formative, intervention, and evaluation. The intervention included advocacy, partnership building, building the health system’s capacity and community mobilization, and enabling the health systems to screen and manage SCD patients. The capacity building included improving healthcare workers’ skills through training and infrastructure development of primary healthcare (PHC) facilities. The impact of the intervention is visible in terms of people’s participation (54%, 76% and 93% of the participants participated in some intervention activities, underwent symptomatic screening and demanded the continuity of the program, respectively), and improvement in SCD-related knowledge of the community and health workers (with more than 50% of net change in many of the knowledge-related outcomes). By developing screening and treatment models, this intervention model demonstrated the feasibility of SCD care at the PHC level in remote rural areas. This accessible approach allows the tribal population in India to routinely seek SCD care at their local PHCs, offering great convenience. Nevertheless, additional research employing rigorous methodology is required to fine-tune the model. National SCD program may adopt this model, specifically for community-level screening and management of SCD in remote and rural areas.</p