Racial/ethnic minority females smoke more cigarettes after social interaction with others who smoke.

The present study investigated the effects of social interaction with others who smoke on daily cigarette use among diverse females via ecological momentary assessment methods. Ninety-eight premenopausal females (29.6% White, 70.4% racial/ethnic minority) who smoke daily reported their social interactions and cigarette use over 35-days. Greater than usual levels of social interaction with others who smoke was associated with increased cigarette use that day among racial/ethnic minority females. Future smoking cessation interventions targeting racial/ethnic minority females should consider the impact of social environments on smoking behaviors, such as the frequency of peer interactions with others who smoke.

Iron deficiency in healthy, term infants aged five months, in a pediatric outpatient clinic: a prospective study.

BACKGROUND: Iron deficiency (ID) is prevalent in Malaysian children. The incidence of ID in infants under 6 months of age is unknown. Our aim was to determine the prevalence of iron deficiency (ID) and iron deficiency anemia (IDA) in healthy, term infants aged below 6 months in our hospital population. METHODS: A prospective longitudinal pilot study of mother-infant pairs was conducted on infants receiving routine immunizations in a mother and child clinic at a university hospital, in Kuala Lumpur, Malaysia. Mothers completed standardized questionnaires at 3- and 5-month postnatal visits. Maternal and infant full blood count, ferritin, and C-reactive protein (CRP) levels were measured at 3 months and for the infants repeated at 5 months. Infant anthropometric measurements were obtained at both visits. We conducted a univariate analysis to identify factors associated with ID and IDA. RESULTS: Altogether, 91 mother-infant pairs were enrolled, with 88 completing the study. No infant had ID or IDA at 3 months; the lowest ferritin level was 16.6 µg/L. At 5 months, 5.9% (5/85) of infants had ID, and 2.4% (2/85) had IDA. Median (interquartile range) infant ferritin levels significantly declined from 113.4 (65.0-183.6) µg/L at 3 months to 50.9 (29.2-70.4) µg/L at 5 months, p < 0.001. Exclusive breastfeeding until 3 or 5 months was significantly associated with ID at 5 months (p = 0.020, and p = 0.008, respectively) on univariate analysis. The drop in ferritin between 3-5 months was significantly associated with weight and length gains between 0-3 months (p = 0.018, p = 0.009, respectively). Altogether, 14.3% of infants exclusively breastfed until 5 months developed ID. At 5 months, 3.4% of infants were underweight, 1.1% stunted, and 10.2% wasted. CONCLUSIONS: In exclusively breastfed term infants, ID occurred by 5 months. Early introduction of iron-rich foods should be considered in exclusively breastfed babies. A high prevalence of wasting suggests a calorie deficit in this population and will lead to stunting if not addressed.

A Two-Phased Telehealth Model to Treat Post-Traumatic Stress Disorder in a Health Care Worker due to the COVID-19 Pandemic: A Case Report.

Background: We report a case describing the use of a two-step telehealth intervention to treat symptoms of post-traumatic stress disorder (PTSD) that developed in a frontline health care worker (HCW) during the COVID-19 pandemic. HCWs are at increased risk of adverse psychological outcomes, including PTSD, due to the nature of their work, which has been exacerbated by the global pandemic. Methods: This case represents the first successfully completed participant in a larger ongoing trial to address psychological distress, PTSD, and comorbidities in HCWs consequent to the COVID-19 pandemic. Following a two-step intervention of self-directed narrative writing delivered entirely online followed by prolonged exposure therapy using videoconferencing, the HCW displayed significant improvement in symptoms of PTSD, depression, anxiety, and substance use. Results: The treatment model described here offers preliminary support for a two-step remote delivery approach to meet the need for scalable self-directed distance technology-based mental health interventions for HCWs. This study is registered on clinicaltrials.gov (NCT04626050).

Can a propensity score matching method be applied to assessing efficacy from single-arm proof-of-concept trials in oncology?

As a result of the escalating number of new cancer treatments being developed and competition among pharmaceutical companies, decisions regarding how to proceed with phase III trials are frequently based on findings from either single-arm phase I expansion cohorts or phase II studies that compare the efficacy of the study drug to a standard-of-care benchmark derived from historical data. However, even when eligibility criteria are matched, differences in the distribution of baseline patient features may influence the outcome of single-arm trials in real-world scenarios. Therefore, novel methods are needed to enhance the accuracy of efficacy prediction from current cohorts relative to historical data. In this study, we demonstrated the feasibility of using the propensity score matching (PSM) method to improve decision making by matching relevant baseline features between current and historical cohorts. According to our findings, utilizing the PSM method may provide a less biased means of comparing outcomes between current and historical cohorts relative to a naïve approach, which relies solely on differences in average outcomes between the cohorts.

Culturally Responsive Cognitive Behavioral Therapy for Ethnically Diverse Populations.

Cognitive behavioral therapy (CBT) is often referred to as the "gold standard" treatment for mental health problems, given the large body of evidence supporting its efficacy. However, there are persistent questions about the generalizability of CBTs to culturally diverse populations and whether culturally sensitive approaches are warranted. In this review, we synthesize the literature on CBT for ethnic minorities, with an emphasis on randomized trials that address cultural sensitivity within the context of CBT. In general, we find that CBT is effective for ethnic minorities with diverse mental health problems, although nonsignificant trends suggest that CBT effects may be somewhat weaker for ethnic minorities compared to Whites. We find mixed support for the cultural adaptation of CBTs, but evidence for cultural sensitivity training of CBT clinicians is lacking, given a dearth of relevant trials. Based on the limited evidence thus far, we summarize three broad models for addressing cultural issues when providing CBT to diverse populations.

A Randomized Pilot Trial of Micronutrient Supplementation for Under-5 Children in an Urban Low-Cost Flat Community in Malaysia: A Framework for Community-Based Research Integration.

Early childhood nutritional deficiency has detrimental consequences on physical and cognitive development. We conducted a single-center, single-blind, two-arm pilot randomized no-treatment controlled trial (the Child of Urban Poverty Iron Project (CUPIP); NCT03819530) in a people's housing project locale in Selangor, Malaysia, between September 2019 and February 2020, to assess the trial's general feasibility and preliminary benefits of daily micronutrient supplementation for iron storage and anthropometric outcomes in under-5 children. Those with history of premature births, congenital abnormalities, or baseline hemoglobin <70 g/L were excluded. Participants received baseline deworming and were simply randomized in a 1:1 ratio to either micronutrient (4-month daily micronutrient packets) or control (no micronutrient supplementation) groups. Information on anthropometric, erythrocytic, and iron storage endpoints were collected. Overall, 45 (25 micronutrient and 20 controls) participants were enrolled and completed 4-month endpoint assessments. Micronutrient recipients demonstrated higher median mean corpuscular volume, serum ferritin level with no significant differences in all anthropometric endpoints. In conclusion, this pilot trial was implementable, demonstrating that micronutrient supplementation significantly improved hematological, but not anthropometric, endpoints, of under-5-year-old children living in an underprivileged environment. A definitive well-designed trial with larger sample sizes and greater attrition control should be contemplated in the future.

The Immigrant Paradox in the Problem Behaviors of Youth in the United States: A Meta-analysis.

This meta-analysis synthesizes the empirical data on problem behaviors among foreign- (G1) and U.S-born (G2+) youth and explores the effects of immigrant status on youth internalizing and externalizing problems. A random effects meta-regression with robust variance estimates summarized effect sizes for internalizing and externalizing problems across 91 studies (N = 179,315, Mage  = 13.98). Results indicated that G1 youth reported significantly more internalizing problems (g = .06), and fewer externalizing problems than G2+ youth (g = -.06). Gender and sample type moderated the effects. The findings provide a first-step toward reconciling mixed support for the immigrant paradox by identifying for whom and under what conditions the immigrant experience serves as a risk or protective factor for youth.

Synchronous Bilateral Primary Testicular Tumors With Discordant Histopathology.

Concomitant presentation of histologically distinct bilateral testicular tumors is exceedingly rare. Here we report the case of a 20-year-old male who presented with a left testicular mass. He was found to have bilateral testicular masses on ultrasound and underwent bilateral orchiectomy. Left testicular pathology revealed a mixed germ cell tumor consisting of teratoma, seminoma, and germ cell neoplasia in situ; right testicular pathology revealed two foci of pure seminomas. He is currently on active surveillance and remains in remission at 18-month follow-up. Our case demonstrates the rare occurrence of bilateral primary synchronous testicular tumors with different histopathology in each testis. Despite the rarity of this condition, its treatment is based on standard management of unilateral testicular carcinoma, with the added element of prioritization of one tumor over the other. It is important for clinicians to tailor management for bilateral testicular germ cell tumors according to the most aggressive component.

Assessing dehydration status in dengue patients using urine colourimetry and mobile phone technology.

BACKGROUND: Dengue is a systemic and dynamic disease with symptoms ranging from undifferentiated fever to dengue shock syndrome. Assessment of patients' severity of dehydration is integral to appropriate care and management. Urine colour has been shown to have a high correlation with overall assessment of hydration status. This study tests the feasibility of measuring dehydration severity in dengue fever patients by comparing urine colour captured by mobile phone cameras to established laboratory parameters. METHODOLOGY/PRINCIPAL FINDINGS: Photos of urine samples were taken in a customized photo booth, then processed using Adobe Photoshop to index urine colour into the red, green, and blue (RGB) colour space and assigned a unique RGB value. The RGB values were then correlated with patients' clinical and laboratory hydration indices using Pearson's correlation and multiple linear regression. There were strong correlations between urine osmolality and the RGB of urine colour, with r = -0.701 (red), r = -0.741 (green), and r = -0.761 (blue) (all p-value <0.05). There were strong correlations between urine specific gravity and the RGB of urine colour, with r = -0.759 (red), r = -0.785 (green), and r = -0.820 (blue) (all p-value <0.05). The blue component had the highest correlations with urine specific gravity and urine osmolality. There were moderate correlations between RGB components and serum urea, at r = -0.338 (red), -0.329 (green), -0.360 (blue). In terms of urine biochemical parameters linked to dehydration, multiple linear regression studies showed that the green colourimetry code was predictive of urine osmolality (ß coefficient -0.082, p-value <0.001) while the blue colourimetry code was predictive of urine specific gravity (ß coefficient -2,946.255, p-value 0.007). CONCLUSIONS/SIGNIFICANCE: Urine colourimetry using mobile phones was highly correlated with the hydration status of dengue patients, making it a potentially useful hydration status tool.

Case based simulation in MRI for suspected appendicitis in children.

PURPOSE: To establish the effect on diagnostic confidence of a simulation setting, in which radiologists re-interpret anonymized pediatric MRI cases. MATERIALS: In this IRB-approved study, participants completed surveys rating confidence before and after interpreting 10 MRI cases for suspected appendicitis in children. RESULTS: 18 radiologists (4 faculty, 5 fellows, and 9 residents) correctly identified an average of 7.44 cases (range 5-9). Self-described confidence regarding technique and interpretation increased from 2.0 (SD 0.77) and 2.33 (SD 0.69) to 2.83 (SD 0.71) and 2.94 (SD 0.73), respectively. CONCLUSION: Simulated interpretation of pediatric MRI in suspected appendicitis results in increased self-describe confidence without requiring additional capital/equipment expenses.

Microfluidics Cell Loading-Dock System: Ordered Cellular Array for Dynamic Lymphocyte-Communication Study.

It remains a great challenge to establish a high-throughput platform that can explore the interactions among multiple lymphocytes (>2 cells) and retrieve the interested cells for downstream analysis. This study demonstrates a microfluidics cell loading-dock system (Cell-Dock) to enclose multiple cells in 1D, 2D, and 3D chambers with high throughput and efficiency and single-cell accuracy. The loading efficiencies of 95%, 85%, and 74% for one-, three-, and five-cell systems are achieved, respectively. The Cell-Dock system provides precise and dynamic cell packing models to facilitate lymphocyte-interaction studies. The results demonstrate that individual natural killer (NK) cells may function independently rather than cooperate to lyse target cells in the defined microenvironment. Furthermore, the strong/weak NK cells are retrieved based on their on-chip cytotoxicity and mRNA sequencing is conducted to find the possible mechanisms for "serial killing," an important but unsolved issue. This study finds that the stronger NK cells overexpress multiple genes involved in cytotoxicity and adhesion molecules (including the well-known ICAM1 and seldom reported B4GALT1) might play important roles in the regulation of NK cytolysis.

Interferon Stimulated Gene Expression in HIV/HCV Coinfected Patients Treated with Nitazoxanide/Peginterferon-Alfa-2a and Ribavirin.

A combination of nitazoxanide (NTZ), peginterferon (PegIFN), and ribavirin (RBV) may result in higher sustained virologic response (SVR) rates in hepatitis C virus (HCV) monoinfected patients. This study evaluated the effect of NTZ on interferon-stimulated gene (ISG) expression in vitro and in vivo among HIV/HCV genotype-1 (GT-1) treatment-naive patients. The ability of NTZ to enhance host response to interferon (IFN) signaling using the HCV cell culture system was initially evaluated. Second, ISG expression in 53 patients with treatment outcomes [21 SVR and 32 nonresponders (NR)] in the ACTG A5269 trial, a phase-II study (4-week lead in of NTZ 500 mg daily followed by 48 weeks of NTZ, PegIFN, and weight-based RBV), was assessed. The relative expression of 48 ISGs in peripheral blood mononuclear cells (PBMCs) was measured at baseline, week 4, and week 8 of treatment in a blinded manner. In vitro NTZ produced a direct and additive antiviral effect with IFN-alfa, with pretreatment of NTZ resulting in maximal HCV suppression. NTZ augmented IFN-mediated ISG induction in PBMCs from relapsers and SVRs (p < 0.05), but not NR. In ACTG A5269, baseline expression of most ISGs was similar between NR and SVR. NTZ minimally induced 17 genes in NR and 13 genes in SVR after 4 weeks of therapy. However, after initiation of PegIFN and RBV, ISG induction was predominantly observed in the SVR group and not NR group. NTZ treatment facilitates IFN-induced suppression of HCV replication. Inability to achieve SVR with IFN-based therapy in this clinical trial is associated with diminished ISG response to therapy that is refractory to NTZ.

International Collaborative Research Partnerships: Blending Science with Management and Diplomacy.

As globalization progressively connects and impacts the health of people across the world, collaborative research partnerships provide mutual advantages by sharing knowledge and resources to address locally and globally relevant scientific and public health questions. Partnerships undertaken for scientific research are similar to business collaborations in that they require attention to partner systems, whether local, international, political, academic, or non-academic. Scientists, like diplomats or entrepreneurs, are representatives of their field, culture, and country and become obligatory agents in health diplomacy. This role significantly influences current and future collaborations with not only the immediate partner but with other in country partners as well. Research partnerships need continuous evaluation of the collaboration's productivity, perspectives of all partners, and desired outcomes for success to avoid engaging in "research tourism", particularly in developing regions. International engagement is a cornerstone in addressing the impact of infectious diseases globally. Global partnerships are strategically aligned with national, partner and global health priorities and may be based on specific requests for assistance from the partnering country governments. Here we share experiences from select research collaborations to highlight principles that we have found key in building long-term relationships with collaborators and in meeting the aim to address scientific questions relevant to the host country and strategic global health initiatives.

High interferon-stimulated gene ISG-15 expression affects HCV treatment outcome in patients co-infected with HIV and HCV.

Increased baseline expression and lack of induction of interferon-stimulated genes (ISG) are strong negative predictors of therapeutic response to PegIFN/RBV in patients co-infected with HIV and hepatitis C virus (HCV). This study specifically addressed whether ISG-15 expression influences therapeutic responses in 20 HIV/HCV genotype-1 subjects undergoing HCV treatment. Non-responders had significantly higher baseline expression and selective induction of ISG-15 after IFN-α treatment relative to participants with sustained virological response. High baseline levels of ISG-15 were also associated with less induction of ISG with treatment. These results support a role for ISG-15 as a prognostic indicator and resistance factor to IFN-α.

Molecular characterization of prolactin receptor (cPRLR) gene in chickens: gene structure, tissue expression, promoter analysis, and its interaction with chicken prolactin (cPRL) and prolactin-like protein (cPRL-L).

In this study, gene structure, tissue expression, and promoter usage of prolactin receptor (PRLR) and its interaction with prolactin (PRL) and the newly identified prolactin-like protein (PRL-L) were investigated in chickens. The results showed that (1) PRLR gene was found to consist of at least 25 exons by 5'-RACE and RT-PCR assays; (2) multiple PRLR 5'-UTR sequences different in exon composition were isolated from chicken liver or intestine by 5'-RACE and could be subdivided into type I and type II transcripts according to the first exon used (exon 1G or exon 1A); (3) PRLR Type I transcripts with exon 1G were detected to be predominantly expressed in adult kidney and small intestine by RT-PCR, implying their expression is likely controlled by a tissue-specific promoter (P1). By contrast, PRLR type II transcripts containing exon 1A are widely expressed in adult and embryonic tissues examined and their expression is controlled by a generic promoter (P2) near exon 1A, which was demonstrated to display promoter activities in cultured DF-1, HEK293 and LoVo cells by the dual-luciferase reporter assay; (4) Using a 5×STAT5-luciferase reporter system, cPRLR expressed in HepG2 cells was shown to be activated by recombinant cPRL and cPRL-L via interaction with PRLR membrane-proximal ligand-binding domain, suggesting that like cPRL, cPRL-L is also a functional ligand of cPRLR. Collectively, characterization of cPRLR gene helps to elucidate the roles of PRLR and its ligands in birds and provides insights into the regulatory mechanisms of PRLR expression conserved in birds and mammals.

Identification of the receptors for prolactin-releasing peptide (PrRP) and Carassius RFamide peptide (C-RFa) in chickens.

Prolactin-releasing peptide (PrRP) and its structurally related peptide, Carassius Arg-Phe-amide peptide (C-RFa), have been reported to play similar roles in regulating food intake and pituitary functions in vertebrates. However, the identity, functionality, and expression of the receptor(s) for PrRP and C-RFa remain largely unknown in nonmammalian vertebrates, including birds. In this study, three receptors homologous to mammalian PrRP receptor (PrRPR), named cPrRPR1, cPrRPR2, and cC-RFaR, respectively, were cloned from chicken brain by RT-PCR. Using a pGL3-NFAT-RE-luciferase reporter system, we demonstrated that cPrRPR1 and cPrRPR2 expressed in Chinese hamster ovarian cells could be activated by cPrRP20 and cC-RFa20 potently, whereas cC-RFaR could only be activated effectively by cC-RFa20 (EC50, 0.11 nM), indicating that cPrRPR1 and cPrRPR2 can function as common receptors for PrRP and C-RFa, whereas cC-RFaR is a receptor specific to C-RFa. Using a pGL3-CRE-luciferase reporter system, cPrRPR1, cPrRPR2, and cC-RFaR expressed in Chinese hamster ovarian cells were also shown to activate intracellular protein kinase A signaling pathway upon cC-RFa20 treatment (100 nM). Moreover, RT-PCR assay revealed that cPrRPR1, cPrRPR2, and cC-RFaR were widely expressed in most adult chicken tissues examined, including various regions of brain. These findings, together with evidence of PrRP and C-RFa encoded by separate genes in chicken, Xenopus, and zebrafish, and the differential expression of PrRP and C-RFa genes in chicken tissues, strongly suggest that PrRP and C-RFa may play similar yet distinctive roles in nonmammalian vertebrates, including chicken, and their actions are mediated by common receptor(s) or a specific C-RFa receptor.

Molecular cloning and characterization of chicken prostaglandin E receptor subtypes 2 and 4 (EP2 and EP4).

Prostaglandin E(2) (PGE(2)) is an important chemical mediator responsible for regulation of many vital physiological processes. Four receptor subtypes have been identified to mediate its biological actions. Among these subtypes, prostaglandin E receptor subtypes 2 and 4 (EP(2) and EP(4)), both coupled to cAMP-protein kinase A (cAMP-PKA) signaling pathway, are proposed to play crucial roles under both physiological and pathological conditions. Though both receptors were extensively studied in mammals, little is known about their functionality and expression in non-mammalian species including chicken. In present study, the full-length cDNAs for chicken EP(2) and EP(4) receptors were first cloned from adult chicken ovary and testis, respectively. Chicken EP(2) is 356 amino acids in length and shows high amino acid identity to that of human (61%), mouse (63%), and rat (61%). On the other hand, the full-length cDNA of EP(4) gene encodes a precursor of 475 amino acids with a high degree of amino acid identity to that of mammals, including human (87%), mouse (86%), rat (84%), dog (85%), and cattle (83%), and a comparatively lower sequence identity to zebrafish (52%). RT-PCR assays revealed that EP(2) mRNA was expressed in all tissues examined including the oviduct, while EP(4) expression was detected only in a few tissues. Using the pGL3-CRE-luciferase reporter system, we also demonstrated that PGE(2) could induce luciferase activity in DF-1 cells expressing EP(2) and EP(4) in dose-dependent manners (EC(50): <1 nM), confirming that both receptors could be activated by PGE(2) and functionally coupled to the cAMP-PKA signaling pathway. Together, our study establishes a molecular basis to understand the physiological roles of PGE(2) in target tissues of chicken.

Identification of two novel chicken GHRH receptor splice variants: implications for the roles of aspartate 56 in the receptor activation and direct ligand receptor interaction.

In this study, two novel GHRHR receptor splice variants, named chicken GHRHR-v1 (cGHRHR-v1) and cGHRHR-v2 respectively, were identified from chicken pituitary using RT-PCR assay. cGHRHR-v1 is characterized by an N-terminal deletion of 36 amino acid residues, including an aspartate at position 56 (Asp(56)) conserved in G protein-coupled receptor B-I subfamily. cGHRHR-v2 is a carboxyl-terminal truncated receptor variant with four putative transmembrane domains, which arose from alternative use of a splice acceptor site on intron 8. Using the pGL3-CRE-luciferase reporter system, the functionality of the two variants was examined in Chinese hamster ovary cells. cGHRHR-v1 was shown to be capable of transmitting signal upon agonist stimulation, but cGHRHR-v2 could not. Both GHRH and pituitary adenylate cyclase-activating peptide (PACAP) could activate cGHRHR-v1 at high dosages (GHRH >/=10(-8) M; PACAP >/=10(-6) M) and GHRH was much more potent than PACAP, suggesting that cGHRHR-v1 is a functional membrane-spanning receptor with an impairment in high-affinity ligand binding, rather than in receptor activation and ligand-binding specificity. This finding also points out the possibility that Asp(56) is not a critical determinant for receptor activation and direct ligand-receptor interaction. To substantiate this hypothesis, using site-directed mutagenesis, two receptor mutants with replacement of Asp(56) by Ala or Gly were generated. Expectedly, chicken or human GHRH could still activate both receptor mutants with reduced potencies (about 2- to 14-fold less potent). Taken together, our findings not only suggest that cGHRHR variants may play a role in controlling normal pituitary functions, but also support that Asp(56) is nonessential for receptor activation and direct ligand-receptor interaction.

Epidermal growth factor (EGF) receptor ligands in the chicken ovary: I. Evidence for heparin-binding EGF-like growth factor (HB-EGF) as a potential oocyte-derived signal to control granulosa cell proliferation and HB-EGF and kit ligand expression.

There is increasing evidence that epidermal growth factor (EGF) receptor (EGFR) ligand and Kit ligand (KL) play critical roles in controlling follicular development in mammals. Because little is known about their expressions in the ovary of nonmammalian vertebrate, our study aimed to examine the expression, hormonal regulation, and interaction of HB-EGF and KL in the chicken ovary. Using semiquantitative RT-PCR, we demonstrated that ovarian HB-EGF expression increased dramatically with the posthatching ovarian growth. In line with this finding, HB-EGF was shown to be produced primarily by the growing oocytes and capable of stimulating the proliferation of granulosa cells in prehierarchal (3 mm) and preovulatory follicles (F5 and F1). Although HB-EGF expression is mainly restricted to the oocytes, its expression in cultured granulosa cells could be transiently yet strongly induced by HB-EGF and other EGFR ligands including EGF and TGF-alpha. And the inducing effect of HB-EGF was completely abolished by AG1478 (10 microM) or PD98059 (100 microM), indicating that the action of HB-EGF is mediated by EGFR and intracellular MAPK/ERK signaling pathway. Unlike mammals, only KL-1, not the other three isoforms identified (KL-2, -3, and -4), was detected to be predominantly expressed in the chicken ovary. Interestingly, KL expression in undifferentiated and differentiated granulosa cells could be transiently down-regulated by HB-EGF, implying an intrafollicular communication between growing oocyte and surrounding granulosa cells through the interplay of EGFR ligand and KL. Collectively, our data suggest that HB-EGF is likely a paracrine signal from the oocyte to regulate granulosa cell proliferation and HB-EGF and KL expression during ovarian follicular development.

Identification of the endogenous ligands for chicken growth hormone-releasing hormone (GHRH) receptor: evidence for a separate gene encoding GHRH in submammalian vertebrates.

It is generally believed that hypothalamic GHRH activates GHRH receptor (GHRHR) to stimulate GH synthesis and release in the pituitary of mammals. However, the identity of the endogenous ligand of GHRHR is still unresolved in submammalian vertebrates including birds. In this study, we have successfully identified the chicken GHRH (cGHRH) gene, which consists of seven exons including two exons (exons 4 and 5) coding for the predicted mature GHRH peptide of 47 amino acids. Interestingly, the differential usage of splice donor sites at exon 6 results in the generation of two prepro-GHRHs (172 and 188 amino acids in length) with different C-terminal tails. Similar to mammals, cGHRH was detected to be predominantly expressed in the hypothalamus by RT-PCR assay. Using the pGL3-CRE-luciferase reporter system, we further demonstrated that both the synthetic cGHRH peptides (cGHRH(1-47) and cGHRH(1-31)) and conditioned medium from CHO cells expressing cGHRH could strongly induce luciferase activity via activation of cGHRHR, indicating that cGHRH could bind cGHRHR and activate downstream cAMP-protein kinase A signaling pathway. Using the same system, cGHRH-like peptide was also shown to be capable of activating cGHRHR in vitro. As in chicken, a conserved GHRH gene was identified in the genomes of lower vertebrate species including zebrafish, fugu, tetraodon, and Xenopus by synteny analysis. Collectively, our data suggest that GHRH, perhaps together with GHRH-like peptide (chicken/carp-like), may function as the authentic endogenous ligands of GHRHR in chicken as well as in other lower vertebrate species.