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OBJECTIVE: IBD is characterised by dysbiosis, but it remains unclear to what extent dysbiosis develops in unaffected at-risk individuals. To address this, we investigated age-related patterns of faecal and serum markers of dysbiosis in high-risk multiplex IBD families (two or more affected first-degree relatives). DESIGN: Faecal and serum samples were collected from multiplex IBD and control families (95 IBD, 292 unaffected, 51 controls). Findings were validated in independent cohorts of 616 and 1173 subjects including patients with IBD, infants born to mothers with IBD and controls. 16S rRNA gene sequencing and global untargeted metabolomics profiling of faeces and serum were performed. RESULTS: Microbial and metabolomic parameters of dysbiosis progressively decreased from infancy until age 8. This microbial maturation process was slower in infants born to mothers with IBD. After age 15, dysbiosis steadily increased in unaffected relatives throughout adulthood. Dysbiosis was accompanied by marked shifts in the faecal metabolome and, to a lesser extent, the serum metabolome. Faecal and serum metabolomics dysbiosis indices were validated in an independent cohort. Dysbiosis was associated with elevated antimicrobial serologies but not with faecal calprotectin. Dysbiosis metrics differentiated IBD from non-IBD comparably to serologies, with a model combining calprotectin, faecal metabolomics dysbiosis index and serology score demonstrating highest accuracy. CONCLUSION: These findings support that dysbiosis exists as a pre-disease state detectable by faecal and serum biomarkers for IBD risk prediction. Given the expansion of disease-modifying agents and non-invasive imaging, the indices developed here may facilitate earlier diagnoses and improved management in at-risk individuals.
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Introduction: Metabolic dysfunction-associated steatotic liver disease (MASLD) affects nearly 25% of the population and is the leading cause for liver-related mortality. Bariatric surgery is a well-known treatment for MASLD and obesity. Understanding the fundamental mechanisms by which bariatric surgery can alter MASLD can lead to new avenues of therapy and research. Previous studies have identified the microbiome's role in bariatric surgery and in inflammatory immune cell populations. The host innate immune system modulates hepatic inflammation and fibrosis, and thus the progression of MASLD. The precise role of immune cell types in the pathogenesis of MASLD remains an active area of investigation. The aim of this study was to understand the interplay between microbiota composition post-bariatric surgery and the immune system in MASLD. Methods: Eighteen morbidly obese females undergoing sleeve gastrectomy were followed pre-and post-surgery. Stool from four patients, showing resolved MASLD post-surgery with sustained weight loss, was transplanted into antibiotic treated mice. Mice received pre-or post-surgery stool and were fed a standard or high-fat diet. Bodyweight, food intake, and physiological parameters were tracked weekly. Metabolic parameters were measured post-study termination. Results: The human study revealed that bariatric surgery led to significant weight loss (p > 0.05), decreased inflammatory markers, and improved glucose levels six months post-surgery. Patients with weight loss of 20% or more showed distinct changes in blood metabolites and gut microbiome composition, notably an increase in Bacteroides. The mouse model confirmed surgery-induced microbiome changes to be a major factor in the reduction of markers and attenuation of MASLD progression. Mice receiving post-surgery fecal transplants had significantly less weight gain and liver steatosis compared to pre-surgery recipients. There was also a significant decrease in inflammatory cytokines interferon gamma, interleukin 2, interleukin 15, and mig. This was accompanied by alterations in liver immunophenotype, including an increase in natural killer T cells and reduction of Kupfer cells in the post-surgery transplant group. Discussion: Our findings suggest surgery induced microbial changes significantly reduce inflammatory markers and fatty liver progression. The results indicate a potential causal link between the microbiome and the host immune system, possibly mediated through modulation of liver NKT and Kupffer cells.
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Inflammatory bowel disease (IBD) is an immunologically complex disorder involving genetic, microbial, and environmental risk factors. Its global burden has continued to rise since industrialization, with epidemiological studies suggesting that ambient particulate matter (PM) in air pollution could be a contributing factor. Prior animal studies have shown that oral PM10 exposure promotes intestinal inflammation in a genetic IBD model and that PM2.5 inhalation exposure can increase intestinal levels of pro-inflammatory cytokines. PM10 and PM2.5 include ultrafine particles (UFP), which have an aerodynamic diameter of <0.10 µm and biophysical and biochemical properties that promote toxicity. UFP inhalation, however, has not been previously studied in the context of murine models of IBD. Here, we demonstrated that ambient PM is toxic to cultured Caco-2 intestinal epithelial cells and examined whether UFP inhalation affected acute colitis induced by dextran sodium sulfate and 2,4,6-trinitrobenzenesulfonic acid. C57BL/6J mice were exposed to filtered air (FA) or various types of ambient PM reaerosolized in the ultrafine size range at ~300 µg/m3, 6 h/day, 3-5 days/week, starting 7-10 days before disease induction. No differences in weight change, clinical disease activity, or histology were observed between the PM and FA-exposed groups. In conclusion, UFP inhalation exposure did not exacerbate intestinal inflammation in acute, chemically-induced colitis models.
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Colitis , Sulfato de Dextran , Ratones Endogámicos C57BL , Material Particulado , Ácido Trinitrobencenosulfónico , Material Particulado/toxicidad , Animales , Colitis/inducido químicamente , Colitis/patología , Ratones , Humanos , Sulfato de Dextran/toxicidad , Células CACO-2 , Ácido Trinitrobencenosulfónico/toxicidad , Ácido Trinitrobencenosulfónico/efectos adversos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Mucosa Intestinal/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Células Epiteliales/metabolismo , Modelos Animales de Enfermedad , Masculino , Tamaño de la PartículaRESUMEN
BACKGROUND AND AIMS: The gut microbiota contributes to aberrant inflammation in inflammatory bowel disease, but the bacterial factors causing or exacerbating inflammation are not fully understood. Further, the predictive or prognostic value of gut microbial biomarkers for remission in response to biologic therapy is unclear. METHODS: We perform whole metagenomic sequencing of 550 stool samples from 287 ulcerative colitis patients from a large phase 3 head-to-head study of infliximab and etrolizumab. RESULTS: We identify several bacterial species in baseline and/or post-treatment samples that associate with clinical remission. These include previously described associations (Faecalibacterium prausnitzii_F) as well as new associations with remission to biologic therapy (Flavonifractor plautii). We build multivariate models and find that gut microbial species are better predictors for remission than clinical variables alone. Finally, we describe patient groups that differ in microbiome composition and remission rate after induction therapy, suggesting the potential utility of microbiome-based endotyping. CONCLUSIONS: In this large study of ulcerative colitis patients, we show that few individual species associate strongly with clinical remission, but multivariate models including microbiome can predict clinical remission and have better predictive power compared to clinical data alone.
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Recent studies involving transplantation of feces from schizophrenia (SCZ) patients and their healthy controls into germ-free mice have demonstrated that the gut microbiome plays a critical role in mediating SCZ-linked physiology and behavior. To date, only one animal model (a metabotropic glutamate receptor 5 knockout) of SCZ has been reported to recapitulate SCZ-linked gut dysbiosis. Since human 22q11.2 microdeletion syndrome is associated with increased risk of SCZ, we investigated whether the 22q11.2 microdeletion ("Q22") mouse model of SCZ exhibits both SCZ-linked behaviors and intestinal dysbiosis. We demonstrated that Q22 mice display increased acoustic startle response and ileal (but not colonic) dysbiosis, which may be due to the role of the ileum as an intestinal region with high immune and neuroimmune activity. We additionally identified a negative correlation between the abundance of a Streptococcus species in the ilea of Q22 mice and their acoustic startle response, providing early evidence of a gut-brain relationship in these mice. Given the translational relevance of this mouse model, our work suggests that Q22 mice could have considerable utility in preclinical research probing the relationship between gut dysbiosis and the gut-brain axis in the pathogenesis of SCZ.
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Acústica , Deleción Cromosómica , Cromosomas Humanos Par 22 , Disbiosis , Microbioma Gastrointestinal , Esquizofrenia , Modelos Animales de Enfermedad , Esquizofrenia/complicaciones , Esquizofrenia/genética , Disbiosis/complicaciones , Disbiosis/genética , Íleon/microbiología , Reflejo de Sobresalto , Humanos , Animales , Ratones , Ratones Endogámicos C57BLRESUMEN
Bariatric surgery remains a potent therapy for nonalcoholic fatty liver disease (NAFLD), but its inherent risk and eligibility requirement limit its adoption. Therefore, understanding how bariatric surgery improves NAFLD is paramount to developing novel therapeutics. Here, we show that the microbiome changes induced by sleeve gastrectomy (SG) reduce glucose-dependent insulinotropic polypeptide (GIP) signaling and confer resistance against diet-induced obesity (DIO) and NAFLD. We examined a cohort of NALFD patients undergoing SG and evaluated their microbiome, serum metabolites, and GI hormones. We observed significant changes in Bacteroides, lipid-related metabolites, and reduction in GIP. To examine if the changes in the microbiome were causally related to NAFLD, we performed fecal microbial transplants in antibiotic-treated mice from patients before and after their surgery who had significant weight loss and improvement of their NAFLD. Mice transplanted with the microbiome of patients after bariatric surgery were more resistant to DIO and NAFLD development compared to mice transplanted with the microbiome of patients before surgery. This resistance to DIO and NAFLD was also associated with a reduction in GIP levels in mice with post-bariatric microbiome. We further show that the reduction in GIP was related to higher levels of Akkermansia and differing levels of indolepropionate, bacteria-derived tryptophan-related metabolite. Overall, this is one of the few studies showing that GIP signaling is altered by the gut microbiome, and it supports that the positive effect of bariatric surgery on NAFLD is in part due to microbiome changes.
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Cirugía Bariátrica , Microbioma Gastrointestinal , Enfermedad del Hígado Graso no Alcohólico , Obesidad Mórbida , Animales , Ratones , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Obesidad Mórbida/cirugía , Obesidad/cirugía , Obesidad/complicaciones , Receptores Acoplados a Proteínas G , Péptidos , GlucosaRESUMEN
Biotin is an essential vitamin and critical cofactor in several metabolic pathways, and its deficiency has been linked to several disorders including inflammatory bowel disease (IBD). We previously reported that biotin deficiency (BD) in mice, whether modeled through intestine-specific deletion of biotin transporter (SMVT-icKO) or through a biotin-deficient diet, resulted in intestinal inflammation consistent with an IBD-like phenotype. To assess whether the gut microbiome is associated with these BD-induced changes, we collected stool and intestinal samples from both of these mouse models and utilized them for 16S rRNA gene sequencing. We find that both diet-mediated and deletion-mediated BD result in the expansion of opportunistic microbes including Klebsiella, Enterobacter, and Helicobacter, at the expense of mucus-resident microbes including Akkermansia. Additionally, microbiome dysbiosis resulting from diet-mediated BD precedes the onset of the IBD-like phenotypic changes. Lastly, through the use of predictive metagenomics, we report that the resulting BD-linked microbiome perturbations exhibit increased biotin biosynthesis in addition to several other perturbed metabolic pathways. Altogether, these results demonstrate that biotin deficiency results in a specific microbiome composition, which may favor microbes capable of biotin synthesis and which may contribute to intestinal inflammation.
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Biotina , Enfermedades Inflamatorias del Intestino , Animales , Ratones , Disbiosis , ARN Ribosómico 16S/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Fenotipo , InflamaciónRESUMEN
Nearly 80% of patients with pancreatic ductal adenocarcinoma (PDAC) develop cachexia along their disease course. Cachexia is characterized by progressive weight loss, muscle wasting, and systemic inflammation and has been linked to poorer outcomes and impairments in quality of life. Management of PDAC cachexia has historically involved a multidisciplinary effort comprised of nutritional support, pancreatic enzyme replacement therapy, and/or pharmacologic interventions. Despite current interventions to mitigate PDAC cachexia, a significant proportion of patients continue to die from complications associated with cachexia underscoring the need for novel insights and treatments for this syndrome. We highlight the feasibility and effectiveness of a recent enteral feeding prospective trial at our institution to improve cachexia outcomes in patients with advanced PDAC. Additionally, we were among the first to characterize the stool microbiome composition in patients with advanced PDAC receiving enteral feeding for the treatment of cachexia. Novel insights into the relationship between enteral nutritional support, cachexia, and the gut microbiome are presented. These promising results are discussed in the context of a potential ability to modulate the stool microbiome as a new interventional strategy to mitigate PDAC cachexia.
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In this phase I dose-escalation trial, we assess the maximum tolerated dose (MTD) of Bermekimab in combination with Nanoliposomal Irinotecan (Nal-Iri) and 5-Fluorouracil/Folinic Acid (5-FU/FA). Secondarily, we investigate effects on weight, lean body mass, quality-of-life, the gut microbiome composition, inflammatory biomarkers, progression-free survival, and overall survival. This was a single-arm, open-label adaptive Bayesian dose-escalation study of Bermekimab combined with Nal-Iri and 5FU/FA in patients with advanced or locally advanced PDAC who failed gemcitabine-based chemotherapy. 22 patients enrolled between 2017 and 2019. 3 of 21 patients experienced dose-limiting toxicities attributable to the chemotherapy backbone. 58% (10/17) of patients exhibited weight stability. Physical performance status was preserved among all subjects. Patients reported improvements in quality-of-life metrics via QLQ-PAN26 questioner (-3.6, p = 0.18) and functional well-being (1.78, p = 0.02). Subjects exhibited a decrease in inflammatory cytokines, notably, vascular endothelial growth factor (-0.86, p = 0.017) with Bermekimab. Bermekimab treatment was associated with an increased abundance of gut health-promoting bacterial genera Akkermansia, with 3.82 Log2-fold change from baseline. In sum, Bermekimab is safe to be used in conjunction with Nal-Iri and 5-FU/FA chemotherapy. This benign toxicological profile warrants further Phase I/II investigation of Bermekimab in combinatorial strategies, and the impact of anti-IL-1α antibodies on the gut microbiome.Clinical trials registration: NCT03207724 05/07/2017.
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Adenocarcinoma , Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias Pancreáticas , Adenocarcinoma/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Teorema de Bayes , Fluorouracilo/uso terapéutico , Humanos , Irinotecán , Leucovorina/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Factor A de Crecimiento Endotelial Vascular , Neoplasias PancreáticasRESUMEN
Background: The microbiome has been shown in pre-clinical and epidemiological studies to be important in both the development and treatment of obesity and metabolic associated fatty liver disease (MAFLD). However, few studies have examined the role of the microbiome in the clinical response to calorie restriction. To explore this area, we performed a prospective study examining the association of the intestinal microbiome with weight loss and change in hepatic steatosis on a calorie-restricted diet. Methods: A prospective dietary intervention study of 80 overweight and obese participants was performed at the Greater West Los Angeles Veterans Affair Hospital. Patients were placed on a macronutrient standardized diet for 16 weeks, including 14 weeks of calorie restriction (500 calorie deficit). Body composition analysis by impedance, plasma lipid measurements, and ultrasound elastography to measure hepatic steatosis were performed at baseline and week 16. Intestinal microbiome composition was assessed using 16S rRNA gene sequencing. A per protocol analysis was performed on all subjects completing the trial (n = 46). Results: Study completers showed significant reduction in weight, body mass index, total cholesterol, low density lipoprotein, and triglyceride. Subjects who lost at least 5% of their body weight had significantly greater reduction in serum triglyceride and hepatic steatosis than those with <5% body weight loss. Enterococcus and Klebsiella were reduced at the end of the trial while Coprococcus and Collinsella were increased. There were also significant baseline microbiome differences between patients who had at least 5% weight loss as compared to those that did not. Lachnoclostridium was positively associated with hepatic steatosis and Actinomyces was positively associated with hepatic steatosis and weight. Baseline microbiome profiles were able to predict which patients lost at least 5% of their body weight with an AUROC of 0.80. Conclusion: Calorie restriction alters the intestinal microbiome and improves hepatic steatosis in those who experience significant weight loss. Baseline microbiome differences predict weight loss on a calorie-restricted diet and are associated with improvement in hepatic steatosis, suggesting a role of the gut microbiome in mediating the clinical response to calorie restriction.
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Aim: The microbiome has been shown to be pivotal in the development of metabolic associated fatty liver disease (MAFLD). Few have examined the relationship of the microbiome specifically with steatosis grade. Therefore, our aim was to characterize the association of the microbiome with MAFLD steatosis severity while adjusting for metabolic comorbidities including diabetes. Methods: We enrolled patients with MAFLD at the West Los Angeles Veterans Affair Hospital. All patients underwent ultrasound elastography, fasting serum collection, and fecal sampling for 16S sequencing. We examined the associations of microbial diversity and composition with advanced steatosis, defined as a CAP score of ≥ 300 dB/m, with or without the presence of metabolic comorbidities. Results: Seventy-five patients were enrolled. African American were less likely to have advanced steatosis than either Hispanics or Whites (P = 0.001). Patients with more advanced steatosis had higher fasting serum triglyceride (192.6 ± 157.1 mg/dL vs. 122.5 ± 57.4 mg/dL), HbA1c (6.7% ± 1.4% vs. 6.1% ± 0.8%), transaminases, and were more likely to have metabolic syndrome (52.4% vs. 24.2%, P = 0.02). Advanced steatosis and diabetes were associated with altered microbial composition. Bacteroides was negatively associated with advanced steatosis while Megasphaera was positively associated with steatosis. Akkermansia was negatively associated with diabetes, while Anaerostipes and Parabacteroides were positively associated with diabetes. Conclusion: Diabetes and metabolic syndrome are associated with hepatic steatosis severity in MAFLD patients and both advanced steatosis and comorbid diabetes are independently associated with microbiome changes. These results provide insight into the role of the gut microbiome in MAFLD associated with metabolic syndrome.
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PURPOSE/AIM: Thyroid hormone has been implicated in the normal growth and development of articular cartilage; however, its effect on a disease state, such as hypothyroidism, is unknown. The purpose of this investigation was to compare normal articular cartilage from proximal femurs of immature miniature swine to proximal femurs from hypothyroid-induced immature miniature swine. MATERIALS AND METHODS: Two 11-week-old male Sinclair miniature swine were made hypothyroid by administration of 6-propyl-2-thiouracil (PTU) in their drinking water; two control animals did not receive PTU. At 25 weeks of age, the animals were euthanized and their proximal femurs were fixed and decalcified. Samples were sectioned and analyzed by histology to define extracellular matrix (ECM) structure, immunohistochemistry (IHC) to identify types II and X collagen, and histomorphometry to assess articular cartilage mean total and localized height and cell density. Statistics included nested mixed-effects ANOVA with p ≤ 0.05 considered statistically significant. RESULTS: Compared to controls, hypothyroid articular cartilage demonstrated statistically significant quantitative differences in mean tissue height, mean cell density and type II collagen localized zone height. Qualitative differences in ECM proteoglycans and overall collagen types were also found. Type X collagen was not detected in either hypothyroid or control articular cartilage specimens. CONCLUSIONS: Significant changes in articular cartilage structure in hypothyroid compared to control immature miniature swine suggest that thyroid hormone is critical in the growth and development of articular cartilage. CLINICAL SIGNIFICANCE: Understanding articular cartilage development in immature animal models may provide insight into healing or repair of degenerative human articular cartilage.
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Cartílago Articular , Hipotiroidismo , Animales , Cartílago Articular/patología , Colágeno Tipo II/metabolismo , Colágeno Tipo X/metabolismo , Hipotiroidismo/metabolismo , Hipotiroidismo/patología , Masculino , Porcinos , Porcinos EnanosRESUMEN
BACKGROUND: High protein calorie restriction diets have shown clinical efficacy for obesity, but the mechanisms are not fully known. The intestinal microbiome is a mediator of obesity and preclinical data support an effect of high protein diet (HPD) on the gut microbiome of obesity, but there are few studies in humans. METHODS: To address this, we conducted a dietary intervention trial of 80 overweight and obese subjects who were randomized to a calorie-restricted high protein diet (HPD) (30% calorie intake) or calorie-restricted normal protein diet (NPD) (15%) for 8 weeks. Baseline dietary intake patterns were assessed by the Diet History Questionnaire III. Longitudinal fecal sampling was performed at baseline, week 1, week 2, week 4, week 6, and week 8, for a total of 365 samples. Intestinal microbiome composition was assessed by 16S rRNA gene sequencing. RESULTS: At baseline, microbial composition was associated with fiber and protein intake. Subjects on the HPD showed a significant increase in microbial diversity as measured by the Shannon index compared to those on the NPD. The HPD was also associated with significant differences in microbial composition after treatment compared to the NPD. Both diets induced taxonomic shifts compared to baseline, including enrichment of Akkermansia spp. and Bifidobacterium spp. and depletion of Prevotella spp. Conclusion: These findings provide evidence that weight loss diets alter the gut microbiome in obesity and suggest differential effects of HPDs compared to NPDs which may influence the clinical response to HPD.
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Restricción Calórica , Dieta Rica en Proteínas , Dieta Reductora , Microbioma Gastrointestinal , Obesidad/dietoterapia , Obesidad/microbiología , Adulto , Anciano , Carbohidratos de la Dieta/administración & dosificación , Fibras de la Dieta/administración & dosificación , Ingestión de Energía , Heces/microbiología , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVE: Microtia is a congenital partial or total loss of the external ear with current treatment approaches involving autologous construction from costal cartilage. Alternatively, tissue engineering provides possible use of normal or microtia auricular chondrocytes harvested from patients. This study investigated effects in vitro of basic fibroblast growth factor (FGF-2) and osteogenic protein 1 (OP-1) on human pediatric normal and microtia auricular chondrocytes and their potential proliferation and differentiation for cellular expansion. A working hypothesis was that FGF-2 promotes proliferation and OP-1 maintains an auricular phenotype of these cells. METHODS: Two patients, one undergoing otoplasty and one an ear construction, yielded normal and microtia auricular chondrocytes, respectively. The two donor sets of isolated chondrocytes were equally divided into four experimental cell groups. These were controls without added growth factors and cells supplemented with FGF-2, OP-1 or FGF-2/OP-1 combined. Cells were cultured 3, 5, 7, and 10 days (3 replicates/time point), counted and assayed by RT-qPCR to determine elastin and types II and III collagen gene expression. RESULTS: Compared to control counterparts, normal and microtia chondrocytes with OP-1 alone were similar in numbers and varied in elastin and types II and III collagen expression over all culture times. Compared to respective controls and chondrocyte groups with OP-1 alone, normal and microtia cell groups with FGF-2 had statistically significant (p<0.05) enhanced proliferation and statistically significant (p<0.05) decreased elastin and types II and III collagen expression over 10 days of culture. CONCLUSIONS: FGF-2 effects on normal and microtia chondrocytes support its use for increasing cell numbers while OP-1 maintains a chondrocyte phenotype, otherwise marked by increasing type III collagen expression and cellular dedifferentiation to fibroblasts in culture.
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Proteína Morfogenética Ósea 7/farmacología , Condrocitos/efectos de los fármacos , Anomalías Congénitas/cirugía , Oído/anomalías , Factor 2 de Crecimiento de Fibroblastos/farmacología , Procedimientos de Cirugía Plástica/métodos , Ingeniería de Tejidos/métodos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Niño , Condrocitos/fisiología , Anomalías Congénitas/diagnóstico , Microtia Congénita , Oído/cirugía , Cartílago Auricular/cirugía , Humanos , Masculino , Valores de Referencia , Donantes de TejidosRESUMEN
Drosophila RNP-4F is a highly conserved protein from yeast to human and functions as a spliceosome assembly factor during pre-mRNA splicing. Two major developmentally regulated rnp-4f mRNA isoforms have been described during fly development, designated "long" and "short," differing by a 177-nt tract in the 5'-UTR. This region potentially folds into a single long stable stem-loop by pairing of intron 0 and part of exon 2. Since the coding potential for the two isoforms is identical, the interesting question arises as to the functional significance of this evolutionarily-conserved 5'-UTR feature. Here we describe the effects of wild-type and mutated stem-loop on modulation of rnp-4f gene expression in embryos using a GFP reporter assay. In this work, a new GFP expression vector designated pUAS-Neostinger was constructed. The UAS-GAL4 system was utilized to trigger GFP expression using tissue-specific promoter driver fly lines. Fluorescence microscopy visualization, Western blotting and real-time qRT-PÇR were used to study and quantify GFP reporter protein and mRNA levels. A significant increase in GFP reporter protein expression due to presence of the wild-type stem-loop sequence/structure was unexpectedly observed with no concomitant increase in GFP reporter mRNA levels, showing that the 177-nt region enhancement acts posttranscriptionally. The effects of potential cis-acting elements within the stem-loop were evaluated using the reporter assay in two mutant constructs. Results of GFP reporter over-expression show that RNP-4F translational regulation is highly sensitive in the developing fly central nervous system. The potential molecular mechanism behind the observed translational enhancement is discussed.