Coronary artery disease (CAD) is known as a life threatening disease, worldwide. In this study the role of HTLV-1 infection was evaluated on cardiac involvement in an endemic region of northeastern Iran.Serologic and molecular tests for HTLV-1 infection were carried out in subjects who had coronary angiography. A real-time PCR, TaqMan method, to quantify HTLV-1 proviral load (PVL), and routine hematological and biochemical tests were performed for study subjects. P=0.003), and LDL (P=0.007) levels, and monocyte count (P=0.05) had meaningful differences. The mean HTLV-1 PVL in HTLV-1+CAD+ subjects was 992.62±120 which was higher compared with HTLV-1+CAD- group (406.54±302 copies/104 PBMCs). Moreover, HTLV-1 PVL in males (833±108) was lower compared with females (1218±141 copies/104 PBMCs) (P=0.05). Patients with HTLV-1-PVL of more than 500 copies/104 had more diffused atherosclerosis plaque than patients with less than 500 . Furthermore, patients with diffused coronary atherosclerosis had significantly higher levels of HTLV-1 PVL than patients with middle, proximal, and normal location of coronary sclerotic lesions (P<0.05). Twenty nine patients were HTLV-1+CAD+ and 13 cases were HTLV-1+CAD-. Although, there were no significant differences for risk factors like FBS, HDL, triglyceride, systolic and diastolic blood pressure , waist circumference (WC), hip circumference (WL), cholesterol (Taken together, in endemic area, HTLV-1 infection, more likely is a facilitating factor for heart complications and the high HTLV-1 PVL might affect CAD manifestations.  Cardiovascular diseases in developing countries are the most common cause of death . AccordiThe associations of some infections have been recently confirmed in the pathogenesis of atherosclerotic plaques. Furthermore, inflammation was involved as one of the definite probable causes in this process . On the Previous studies have investigated the role of inflammatory mediators, cytokines, and some vasoactive substances as important risk factors of atherosclerosis, including TNF-α, IL-1, IL-6, IFN-γ, and C-reactive protein (CRP). High blood levels of these cytokines have been associated with atherosclerosis, autoimmune disease, chronic inflammatory diseases, and cancers -13.  Patients suffering from acute myocardial infarction exhibit high concentrations of pro-inflammatory cytokines like TNF-α and IL-6 . Larger  Chlamydia pneumonia, Porphromonus gingivalis and Helicobacter pylori , herpes simplex virus (HSV-1), hepatitis virus, and bacteria liker pylori . National Population and Housing Census 2011), as an endemic area for HTLV-1, in this study HTLV-1 proviral load as a major determinant of HTLV-I infection in patients with CAD along with some other biochemical and hematological factors were investigated. To have a close look, the possible effect of HTLV-1 infection on CAD, the type of coronary involvements, the location of sclerotic lesions, and the diffusion of atherosclerotic plaques were also considered.Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that allows the majority of HTLV-I-infected individuals remain healthy carriers, whereas less than 5% of the infected individuals develop acute T Cell leukemia/lymphoma (ATLL) or HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) -23. NortEthics and study population 4 PBMCS group and less than 500 group. Healthy carrier was attributed to patients with positive HTLV-1 patients and negative CAD. The Research Ethics Committee of Mashhad University of Medical Sciences approved this study (IR.MUMS.REC.900177) and a written informed consent form was obtained from each participant. Among 73 HTLV-1 infected patients who referred for coronary artery angiography, 42 subjects obtained the appropriate criteria to include in this study. Then, HTLV-1 proviral load was measured and coronary artery involvements were determined based on the angiography reports. According to the HTLV-1 proviral load, patients were divided into two groups: more than 500 HTLV-1 proviral copies/10Data collection procedures A blood sample (10 ml) was collected from each subject for evaluation of risk factors in cardiovascular disease. Biochemical parameters including low density lipid (LDL), high density lipid (HDL), cholesterol (Ch), triglycerides, and fasting blood sugar (FBS) levels as common cardiovascular disease risk factors, as well as cell blood count (CBC), systolic and diastolic blood pressure , waist circumference (WC), and hip circumference (WL) were measured. A cardiologist examined the patients and an echocardiogram was performed to determine the coronary lesions and the last angiography film was inspected by two specialists separately to distinguish involvement in coronary arteries. All participants completed a check list including demographic information and medical history. Serological and molecular assays°C until further analysis. Genomic DNA was extracted on the same day from PBMCs using DNeasy Kit  according to the manufacturer’s instructions. To confirm HTLV-1 infection, conventional PCR was carried out for Tax and the long terminal repeat (LTR) using the following primers: Tax forward primer (5’- AGGGTTTGGACAGAGTCTT-3’), Tax reverse primer (5’-AAGGACCTTGAGGGTCTTA), LTR forward primer (5’-ATAAGCTCAGACCTCCGGG-3’), and LTR reverse primer (5’-GGATGGCGGCCTCAGGTAGG-3’). The PCR products were analyzed on 1.5% agarose gel, stained with ethidium bromide, and then visualized under UV light  and then confirmed by the PCR technique. Peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll–Hypaque  density centrifugation. Cells were washed twice by Phosphate buffer slain (PBS)  and PBMCs were kept at –20 UV light .HTLV-1 proviral load measurementTax DNA of HTLV-1 and human albumin DNA concentrations were calculated from two five-point standard curves. The normalized value of the HTLV-1 proviral load was calculated as the ratio of  × 104 and expressed as the number of HTLV-1 provirus per 104 PBMCs  was carried out using a commercial absolute quantification kit  by Rotor Gene 6000 software . The test was performed with the Universal Master Mix  to measure HTLV-1 proviral load in PBMCs with the Taqman method. The HTLV-1 proviral copies number was reported as an actual amount of cellular DNA by means of the quantification of the albumin gene as the reference gene. Then 04 PBMCs . Statistical analysist-test was used to compare HTLV-1 proviral load between HTLV-1+CAD+ and HTLV-1-CAD-) patients. One-way ANOVA test was performed to analyze the association between CAD and type of coronary vessel involved with HTLV-1 proviral load in patients with CAD. Data were statistically analyzed using SPSS software version 16.0 . Descriptive data were summarized as mean±standard deviation (SD), standard error of mean (SEM), and percent. Normality of the data was checked prior to data analysis by the Kolmogorov-Smirnov test. Independent samples t-test was applied for analyzing the association between diffusion of atherosclerotic involvement and HTLV-1 proviral load in the HTLV-1+CAD+ group. Chi-square or Fisher’s exact test was used for determining association between confounding variables and coronary artery disease. The odd ratios (ORs) and their 95% confidence interval (95% CI) were estimated. The results were considered statistically significant if P≤0.05. The independent samples Patients’ characteristics+CAD+ and 13 cases were HTLV-1+CAD- (+ (65±8 years) and CAD- (62±10 years) patients. + and CAD- groups. This table represents that there are not any significant differences in demographic variables between the two studied groups. However, mild pulmonary illness was detected in CAD+ group not in CAD-. Furthermore, significant differences in risk factors for cardiovascular diseases such as FBS, HDL, WC, WL, Cbp, Dbp, and just (Ch) (P=0.003) and LDL (P=0.007) were meaningful. Comparing the mean of CBC results indicates that there was only significant difference for monocytes (P=0.05) between CAD+ and CAD- groups. Clinical and laboratory findings of HTLV-1+CAD+ and HTLV-1+CAD- are summarized in Among 48 patients who participate in this study, 29 patients were HTLV-1V-1+CAD- . There wCoronary artery involvementThe number and types of involved coronary arteries, the location of sclerotic lesions, and the diffusion of atherosclerotic involvement were determined . AccordiLocalization of sclerotic lesions P=0.02) and LAD and RCA lesions (P=0.001). Generally, 14 patients (48.3%) had diffused coronary atherosclerosis; however, non-diffuse coronary artery disease was observed in 15 (51.7%) of patients.Coronary artery involvement was investigated at the beginning, end, and middle of the RCA, LCX, and LAD . The invHTLV-1 proviral load in heart complications +CAD+ and HTLV-1+CAD- were 992.62±12 and 406.54±602 copies/104 PBMCs, respectively, in which the difference was statistically different (P=0.045). The mean HTLV-1 proviral load in HTLV-1+CAD+ subjects in males (833±108copies/104 PBMCs) was significantly lower than females (1218±141) (P=0.05). While in HTLV-1+CAD- patients, the males (760±812 copies/104 PBMCs) had significantly higher proviral load compared to females (249±453) (P=0.034). The proviral load of HTLV-1P=0.027, R=0.03). The patients with three-vessel involvement had a mean HTLV-1 proviral load of 937.77±189.26 copies/104 PBMCs, which is significantly higher than those with one vessel involvement (767.23±300.71) or two vessels involved (409.98±174.93). Besides, the findings showed that statistically significant correlations were observed between HTLV-1 proviral load of coronary artery involvement of the RCA vessel, in the beginning of the RCA vessel (P=0.002), and diffuse coronary atherosclerosis status (P=0.031). Furthermore, the statistically significant correlation between the mean of HTLV-1 proviral load and coronary artery involvement was (4 PBMCs (L group) and equal or more than 500 copies (M group). Coronary artery disease patients who had a proviral load of more than 500 copies/104 PBMCs had higher estimated risk of diffuse coronary artery disease than patients with less than 500 copies  were 6.87 times more in risk of diffused atherosclerosis plaque formation than patients with less than 500 copies or HTLV-1 healthy carrier. HTLV-1 proviral load association with coronary artery involvement is depicted in HTLV-1 infected patients were put in two groups according to the proviral load , patients with less than 500 copies/10Strikingly, statistical analysis findings demonstrated that there are significant differences in HTLV-1 proviral load in patients with different localization of sclerotic lesions in involvement vessels . Figure et al. reported a 60-year-old woman with cardiac failure due to aortic and mitral regurgitations from our study region . Assessing her samples by PCR test showed that she was positive for HTLV-1. It was the first report of an isolated lymphomatous cardiac valve involvement without the other cardiac abnormalities . Furthermore, the findings showed that there were significant differences in HTLV-1 proviral load among various sclerotic plaque localizations. This was due to higher levels of HTLV-1 proviral load in diffused localization of plaque in LCX, LAD, and RCA vessel than patients with middle, proximal, and normal coronary sclerotic involvement lesions, respectively (P<0.05).In the present study, the proviral load of HTLV-1 was assessed in HTLV-1 positive patients with normal or CADet al. , towards HAM/TSP manifestations. The recent studies had more emphasis on endothelial cells and HTLV-1 infected cells interactions. For example, the changes in CCR1, CCR2, CXCR5, CXCR6, CXCL9, and CXCL10 showed the tendency of HTLV-1 infected cells to the vessels, particularly, BBB . Another+ or unique infected T cell subset of CCR4+ CD4+ CD25+ with CAD are suggested. Moreover, repeated cross-sectional studies are recommended to understand the dynamics of HTLV-1 epidemic in high prevalence areas. Finding such risk factors, for example viruses, should allow us suitable intervention strategies for prevention or therapy. Of importance, due to the previous documentation on association of HTLV-1 genotype and risk of HAM/TSP, the authors suggest further studies could concentrate on correlation between the incidence of CAD or its severity and HTLV-1 genotype or with other associated diseases. Taken together, these findings showed that HTLV-1 infected T-cells have a very close interaction with endothelial cells and by inducing inflammatory reactions in the surface of the particular vessels might facilitate CAD. Therefore, evaluating the impact of HTLV-1-TCD4-IFN-γ.It is more likely that infecting particular endothelial cells with HTLV-1 or cell-cell interactions between endothelial cells and HTLV-1 infected TCD4+ disrupt endothelium integrity, which can induce inflammatory reactions toward inflammatory diseases such as HAM/TSP  or CAD m


Drosophila larva. We show that GABAB-receptor and insulin-receptor play important roles during OSN modulation. Using an OSN-specific gene expression analysis, we explore downstream targets of insulin signaling in OSNs. Our results suggest that insulin and GABA signaling pathways interact within OSNs and modulate OSN function by impacting olfactory information processing. We further show that manipulating these signaling pathways specifically in the OSNs impact larval feeding behavior and its body weight. These results challenge the prevailing model of OSN modulation and highlight opportunities to better understand OSN modulation mechanisms and their relationship to animal physiology.Starvation enhances olfactory sensitivity that encourage animals to search for food. The molecular mechanisms that enable sensory neurons to remain flexible and adapt to a particular internal state remain poorly understood. Here, we study the roles of GABA and insulin signaling in starvation-dependent modulation of olfactory sensory neuron (OSN) function in the  However, the mechanisms by which an animal’s starved state modulates sensory neuron function remain poorly understood. Our understanding of these mechanisms significantly improved in the last decade or so from studies that showed how neuromodulators enable changes in the gain of peripheral sensory inputs10. The prevailing mechanistic model for olfactory sensory neuron (OSN) modulation by the animal’s starved state is that during the animal’s starved-state, lower insulin signaling frees production of the short neuropeptide F receptor (sNPFR1), which increases sNPF signaling. Higher sNPF signaling increases presynaptic facilitation of OSNs, which leads to enhanced responses to odors8. Interestingly, insulin and neuropeptide Y  signaling also feature in the vertebrate olfactory bulb13.Starvation increases olfactory sensitivity that enhances an animal’s search for food. This has been shown in insects, worms, and mammals including humans16. The model also does not account for interactions between GABA and insulin signaling pathways that are known to impact neuromodulation in both fly and mammalian systems: For instance, GABAB-Receptor (GABABR) mediates a GABA signal from fly brain interneurons, which may be involved in the inhibitory control of Drosophila insulin like peptide (DILP) production17; In mammalian CNS neurons, insulin increases the expression of GABAAR on the postsynaptic and dendritic membranes18; GABA administration to humans resulted in a significant increase in circulating Insulin levels under both fasting and fed conditions19. Finally, the model does not account for the ultimate targets of insulin/GABA/sNPF signaling that alter OSN sensitivity to odors and its function.However, the above model is incomplete and several questions remain. For instance, the model does not account for the role of GABA signaling, which plays important roles during both starvation and olfactory behavior in flies and mammals20.The above questions are significant because the mechanisms driving neural circuit modulations are fundamental to our understanding of how neural circuits support animal cognition and behavior. If we better understood these mechanisms, we could learn how flexibility and the ability to adapt to a particular internal state are built into the sensory circuit. Understanding the mechanisms by which the starved state of an animal modulates its olfactory sensitivity and thereby controls its food-search behavior is important for both olfactory and appetite research. Finally, we cannot ignore this connection in light of the obesity epidemic and the demonstration that obese adults have reduced olfactory sensitivityDrosophila larval system to demonstrate evidence in support of our argument. Using larval behavior assays, we show that GABABR and insulin receptor (InR) are required for starvation dependent increases in larval olfactory behavior. Using a novel OSN-specific gene expression analysis, we show that insulin and GABA signaling pathways interact within OSNs and modulate OSN function by impacting odor reception, olfactory information processing, and neurotransmission. Finally, we show that manipulating these signaling pathways specifically in the OSNs impact larval feeding behavior and its body weight.Here, we build upon the prevailing model and argue that GABA and insulin signaling pathways interact within OSNs to mediate starvation-dependent modulation of its function and that defects in these signaling pathways impact larval food-search and feeding behaviors, which in turn impact weight gain. We use the convenient 7. We asked whether starvation alters olfactory behavior in the Drosophila larva. We used the simple two-choice assay to measure larval behavior responses to an odor source 24. We noted that starved third-instar larvae were more attracted to odors than non-starved larvae. However, this increase in attraction was statistically significant in only three of the seven odors tested  vs. 0.33 (non-starved), p < 0.001; pentyl acetate: RI = 0.56 (Starved) vs. 0.48 (non-starved), p < 0.05; 4,5-dimethylthiazole: RI = 0.35 (Starved) vs. 0.21 (non-starved), p < 0.001)  Fig. . The Sha25. Insulin and GABA signals are transduced via corresponding receptors (InR and GABAB-R respectively) expressed on neuronal membranes. While InR and GABAB-R expression have been clearly demonstrated at terminals of adult fruit fly OSNs26, we wanted to confirm their expression at the terminals of larval OSNs. We used an anti-InR antibody to label InR in dissected third-instar larval preparations. We found that InR was expressed at the terminals as well as along the axonal projections of larval OSNs  to characterize the distribution of GABAB-receptors in larval OSNs. We found that GABAB-R1 also localized to the terminals as well as axonal projections of larval OSNs  or in a single pair of OSNs . InR levels were altered in OSNs by driving either a UAS-InR-RNAi (↓ InR levels) (Vienna Drosophila RNAi (VDRC) Stock Center) or a constitutively active version of InR: UAS-InRCA (↑ InR activity)2. GABABR levels were reduced in OSNs by driving a UAS-GABABR2-RNAi16. Since OSN::47a responds strongly to pentyl acetate24, we measured larval attraction to pentyl acetate (10−2 vol:vol) in the two choice assay. We also chose pentyl acetate due to its ecological relevance for the fruit-fly larva; pentyl acetate is present in many fruit aromas, especially bananas. The data are presented in Fig. p > 0.05). Increasing InR activity by driving the expression of UAS-InRCA impacted the starvation-dependent modulation of olfactory behavior. Starved larvae in which InR activity was increased in all OSNs showed a significantly reduced attraction to the pentyl acetate (RI = 0.28) compared to non-starved larvae . Even more dramatic effects were observed in both starved and non-starved larvae in which InR activity was increased in only OSN::47a. Both non-starved larvae (RI = −0.15) and starved larvae (RI = −0.10) were repulsed by the odor. Reducing GABABR levels also impacted the starvation-dependent modulation of olfactory behavior. While the overall attraction toward pentyl acetate was lower in all cases, reducing GABABR levels in all OSNs eliminated the starvation-dependent increase in attraction observed in control animals (p > 0.05). A more dramatic effect was observed in starved larvae when GABABR levels were reduced in only OSN::47a. In this case, the non-starved larvae were attracted to the odor (RI = 0.15) at similar strengths compared to the corresponding non-starved parental control lines (p > 0.05), while the starved larvae were repulsed by the odor . For the data presented in Fig. Next, we studied the roles of InR and GABAThese results suggest that both insulin signaling and GABA signaling are required for starvation-dependent modulation of OSN function. Consistent with the prevailing model of OSN modulation, our results suggest that reducing insulin signaling in OSN::47a mimics starved conditions, leading to an overall increase in attraction to odors in both the non-starved and starved states. On the other hand, increasing insulin signaling artificially mimics non-starved conditions and thereby reduces attraction to odor. The role of GABA signaling in this mechanism was previously unexplored. We note that overall attraction to odor is reduced upon decreasing GABA signaling. However, these preliminary results suggest that GABA signaling possibly plays an opposite role compared to insulin signaling.BR levels in OSNs impacted starvation-dependent modulation of OSN function, we wondered whether their expression levels in larval CNS are sensitive to the animal’s starved state. To test this, we dissected heads of starved and non-starved larval samples, extracted mRNA from the dissected heads and carried out gene expression analyses using RT-qPCR  (four shown here)29. Consistent with the prevailing model of OSN modulation, we noted that starvation decreased expression levels of several insulin signaling components including InR , DILP-2 , and DILP-6   but increased the expression of GABAAR   Fig. . For gen05) Fig. . The RT-31. Using this technique, we successfully isolated larval OSN nuclei and carried out OSN-specific gene expression analyses. Briefly, OSN nuclei were genetically tagged using a UAS-eGFP-Msp300KASH construct   and Or94b  and eGFP  but not genes such as Syt1 and Act42a that are commonly expressed in most neuron types support the effectiveness of OSN isolation using this technique  Fig. . GFP-tagne) Fig. . RNA extque Fig. . Success18, it was not clear whether these signaling pathways interact within OSNs to mediate OSN function. To test any potential interaction between the signaling pathways, we evaluated gene expression in OSNs using the method implemented in our lab  or UAS-InRCA (↑ InR activity) in all OSNs. We enriched GFP-tagged OSN nuclei from control larvae and the two experimental larval strains. We extracted RNA from the isolated OSN nuclei and quantified expression levels of InR and the three GABABR subunit genes  and GABABR2  subunits while increasing InR levels significantly increased expression levels of GABABR2  and GABABR3  subunits  , Rutabaga (Rut) , and Synaptotagmin (Syt1)  compared to wild type controls. Orco is the co-receptor for odor receptors expressed in OSNs35. Rut encodes adenylyl cyclase, a component of olfactory signal transduction36. Syt1 plays a role in neurotransmission37. However, increasing or decreasing insulin signaling had no impact on other OSN genes tested including two other components of olfactory signal transduction, Gαi and Golf39. While this is not an exhaustive list of OSN genes, our results so far suggest that downstream targets of insulin signaling in OSNs potentially play important roles in odor detection, olfactory information processing, and neurotransmission.We noted that decreasing insulin signaling in OSNs led to ~50% increase in 05) Fig. . This renes Fig. . We note40.Since an inability to regulate sensitivity to food odors at appropriate times could influence irregular foraging habits, which in turn could impact weight gain, we wondered whether altering insulin and GABA signaling specifically in the OSNs would affect the animal’s overall body weight. In support of this warrant, recent studies have shown that genetically obese rats have low levels of insulin in the brain including the olfactory bulb and imbalanced insulin signaling via insulin receptors is associated with obesity phenotypesDrosophila larval body weight in genotypes expressing high or low levels of InR or GABABR in the OSNs. We found that altering GABABR or InR levels in OSNs led to significant increases in larval body-weight compared to parental control  assay to test food consumption in larval genotypes in which GABABR or InR levels were altered in OSNs . A previous study demonstrated that larvae engage in appetitive cue-driven feeding behavior42. When the assay was conducted in the presence of a food odor like pentyl acetate, we found that larvae expressing GABABR-RNAi in all OSNs had 70% lower levels of food intake compared to other genotypes (p < 0.001) . Similar feeding defects were observed in larvae expressing GABABR-RNAi only in OSN::47a. However, in this case, feeding defects were observed both in the presence and absence of odor (p < 0.05)  Fig. . ANOVA w05) Fig.  but alsoOrco), olfactory information processing (Rut), and/or neurotransmission (Syt1)  Fig. . Defectst1) Fig.  and 7. T25. While GABA signaling in different regions of the animal brain is known to mediate starvation-dependent behavior, its role in specific olfactory neurons during starvation is unclear25. Similarly, insulin has long been considered as an important mediator of state dependent modulation of feeding behavior. However, its precise role in olfactory neurons during starvation is controversial. According to the prevailing model, insulin signaling decreases upon starvation8. However, a previous study showed that there is a three-fold increase in DILP-6 (Drosophila Insulin like Peptide) mRNA expression in larval tissue including fat bodies upon starvation, which is inconsistent with the model43. While the significance of DILP-6 increase in larval tissue during starvation is as yet unclear, consistent with the prevailing model, we show that InR and DILP-6 expression in larval head samples decrease upon starvation . Other examples show how GABA signaling might influence insulin signaling. For instance, in flies, GABA signaling from interneurons has been shown to affect insulin signaling by regulating DILP production ; In humans, GABA administration significantly increases circulating insulin levels under both fasting and fed conditions ; In diabetic rodent models, combined oral administration of GABA and an anti-diabetic drug (Sitagliptin) promoted beta cell regeneration and reduced blood glucose levels . Overall, our study adds to this growing body of literature and strongly suggests that GABA and insulin signaling pathways interact within larval OSNs to mediate OSN modulation.We also show that higher insulin signaling increases expression levels of GABASNs Fig. . This re44 and nonfood-related odors45. While similar results have also been shown in humans, the findings regarding the relevance of odor to feeding are rather mixed6. This study along with previous studies from our lab and others raise the possibility that starvation differentially modulates individual OSNs. Indeed, individual OSNs exhibit functional diversity that may lend them to differential modulation by the animal’s internal state48. This diversity may stem from heterogeneous GABABR levels on the terminals of individual OSNs that determine differential presynaptic gain control16. It is reasonable to speculate that heterogeneous GABABR and/or InR levels in individual OSNs could contribute to differential modulation of OSNs by the animal’s starved state, which in turn impacts behavior toward only a subset of odors.We note that starvation enhanced larval attraction toward only a subset of the odors tested Fig. . A relatsNPF in Drosophila and NPY injection in the hypothalamus of rats leads to increased food-intake and bigger and heavier phenotypes50. Genetically obese rats have low levels of insulin in the brain including in the olfactory bulb and imbalanced insulin signaling via insulin receptors is associated with obesity phenotypes40. Adenylyl cyclase (rut) deficient mice were found to be obese51 and both Adenylyl cyclase3 and Synaptotagmin4 have been targeted for anti-obesity drug development53. These studies provide added significance to our observation that manipulating mechanisms mediating starvation-dependent modulation of OSNs impact feeding behavior and weight gain in larvae.An inability to regulate sensitivity to food odors at appropriate times leads to irregular feeding habits, which in turn leads to weight gain. Obesity researchers will readily acknowledge that while several obvious risk factors for obesity  have been heavily researched, the relationship between nutrient sensing/sensory behavior and obesity remains grossly understudied. The present study sets the stage to further explore this relationship. Interestingly, several of the signaling molecules described in this study that play a role in OSN modulation have also been implicated in hyperphagia and obesity phenotypes. For instance, overexpression of 42. Our studies show that manipulating GABABR signaling in first-order OSNs impact appetitive cue-driven feeding behavior in larvae. While it remains to be seen whether parallel regulations during different stages of olfactory information processing impact feeding behavior, further studies are needed to reveal the mechanistic relationship between GABABR/InR signaling in OSNs, feeding behavior, and changes in body-weight.Indeed, food odors can be powerful appetitive cues. A previous study showed that larvae engage in appetitive cue-driven feeding behavior and that this behavior required NPF signaling within dopaminergic neurons in higher-order olfactory processing centersBR impacts OSN function either at the level of odor reception by affecting the expression of Orco or at the level of olfactory signal transduction by affecting the expression of Rut or at the level of neurotransmission by affecting the expression of Syt1 and sNPFR1. We acknowledge that more exhaustive gene expression analyses are required to identify other molecular players downstream of InR and GABABR. It would also be valuable to investigate the relationship between InR expression levels on the terminals of individual OSNs and the sensitivity of individual OSNs to modulation by the animal’s starved state.Based on the evidence so far, we propose a motivating model for future investigations Fig. . In this57. Another possibility to be considered is that changes in OSN sensitivity, food-search and/or feeding behaviors are independently regulated. For instance, Yu and colleagues noted that starvation-induced hyperactivity in adult Drosophila was independently regulated from food consumption behavior in the flies. They showed that blocking octopamine signaling in a small group of octopaminergic neurons located in the subesophageal zone (SEZ) of the fly brain neurons eliminated starvation induced hyperactivity but not the increase in food consumption58. While we cannot rule out such a possibility, the evidence presented in this study support the argument that starvation induced-changes in OSN function is related to the observed changes in food search and feeding behaviors. We acknowledge that other studies have opted to keep larvae on sucrose with the intention of starving them of amino acids and other nutrients59. So, the non-starved conditions in the present study actually represents partial starvation of macronutrients other than sugar. We did so to control the nutrient intake in the non-starved state with the intention of measuring the impact of individual macronutrients on OSN modulation in future studies. Finally, while we tested the hypothesis that increases in body-weight of mutant genotypes are due to altered food consumption, we have not yet tested alternate hypotheses that body-weight increases may be due to altered metabolism or increased fat accumulation.A valid concern in this study is that an innate attraction of larvae toward an odorant does not necessarily equate to food-search behavior. However, we argue that attractiveness toward an odor source is a reliable measure of food-search behavior because an animal’s ability to efficiently smell and move toward an odor source necessarily predicates most forms of such behaviorOverall, our study conducted in a simple, tractable, and highly conserved model system builds upon the prevailing model of starved-state dependent modulation of OSN function. It highlights and offers unique opportunities that are now possible to address our inadequate understanding of OSN modulation mechanisms at the resolution of single neurons, which in turn would help us better understand how flexibility and the ability to adapt to a particular internal state are built into the sensory circuit.Flies were reared on standard cornmeal-dextrose agar food  at 25 °C and 60% humidity.Canton-S (CS) and w118 lines were used as wild type lines in behavioral experiments. The following OSN-Gal4 drivers were used: Orco-Gal4 and O47a-Gal4 (from Dr. John Carlson). The following UAS-lines were used: UAS-InRCA (BDSC #8262), UAS-InR-RNAi (VDRC #992), and UAS-GABABR2-RNAi (VDRC #1784). The following UAS-line was used for the OSN-nuclei isolation experiments: UAS-eGFP-MSP300KASH (from Dr. Vikki Weake).Drosophila larvae (~96 hours after egg laying). The larvae were extracted from food using a high-density (15%) sucrose (Sigma Aldrich Inc.) solution. Larvae that float to the surface of the sucrose solution were separated into a 1000 ml glass beaker and washed four times with distilled water.Behavioral experiments utilized third-instar 2O added to a piece of Kim wipe (starved condition) or 350 µL of 0.2 M sucrose (Acros Organics #177140050) added to a piece of Kim wipe (non-starved condition). In pilot experiments we noted that a 2 hour starvation period was ideal for larval performance as well as for eliciting differences in behavior responses between starved and non-starved animals.Washed larvae were allowed to roam freely for 2 hours at RT in a 6 cm petri-dish  containing either 350 µL dH22 , calculated as Eq. . The temperature of the behavior room was maintained between 22 °C and 23 °C and between 45% and 50% relative humidity.41 . Images were analyzed using Motic Images Plus 3.0 ML. The blue colored area in each larva corresponding to dye intake was measured and normalized against the total area of the larva.The assay was adapted from41 Fig. . Larvae 60 and28. Larvae were dissected in Phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min at RT. Fixed larval samples were washed three times in PBS and treated with PBS-T (PBS + 0.2% TritonX). Samples were incubated with primary antibodies diluted in 5% BSA for 16–18 hours at 4 °C. Following three washes in PBS, samples were incubated with secondary antibodies diluted in 0.4% BSA for 4 hours at RT. Samples were washed again three times, mounted in Vectashield (Vector Laboratories #H-1000) on glass slides and analyzed with a Leica TCS SP8 Confocal Microscope.Third-instar larval dissection and antibody staining methods were adapted fromB-Receptor in larval OSNs, we generated a rabbit anti-GABABR1 antibody. To do so, we custom synthesized a 15 amino acid peptide (TVAEAAKMWNLIVLC) specific to the GABAB-R1 subunit. This peptide spanning amino acids 121–135 in GABAB-R1 was selected because it is a conserved motif across insect species. We used this peptide as an antigen to generate a rabbit polyclonal antibody (Pocono Rabbit Farm & Laboratory Inc.). GABABR was stained using a (1:125) dilution of the rabbit anti-GABABR1 polyclonal antibody, which was visualized with a (1:125) dilution of goat anti-rabbit IgG coupled to Alexa Fluor Plus 647 . GFP ectopically expressed in OSNs was stained using a (1:125) dilution of chicken anti-GFP polyclonal antibody , which was visualized with a (1:125) dilution of goat anti-chicken IgY coupled to Alexa Fluor 488 .InR was stained using a (1:65) dilution of rabbit anti-InR polyclonal antibody , which was visualized with a (1:65) dilution of goat anti-rabbit IgG coupled to Alexa Fluor Plus 647 . To visualize the GABAStarved or non-starved third-instar larvae were used for this preparation. 15 larval heads were dissected for each condition using 3 mm surgical scissors and stored in RNAlater (Invitrogen #AM7020). Samples were homogenized using a mortar and pestle in RLT lysis buffer (Qiagen #74134) before subjecting each sample to RNA extraction protocol.31 ). Samples were homogenized using a mortar and pestle. Pre-isolation samples were collected to determine nuclear yield, nuclei integrity, and to determine transcript levels of target genes prior to nuclei isolation. Affinity based isolation of nuclei was performed as described in31. Briefly, GFP-Msp300KASH tagged OSN nuclei were pulled down using a Chicken anti-GFP antibody (Invitrogen #PA1–9533) bound to magnetic Dynabeads Protein G (Invitrogen #10003D). Antibody-bound beads and homogenate were placed in a magnetic rack, MagRack6 (GE #28948964) for 2 minutes to allow the magnetic beads to bind to the magnet. Homogenate containing the unbound nuclei fraction was removed . An additional gDNA digestion was conducted using the TURBO DNA-free kit (Invitrogen #AM1907)  or with RNase-Free DNase Set  . OSN samples were eluted into a total of 60 µL RNase-free water and 1 µL RNAsecure RNase Inactivation Reagent (Invitrogen #AM7005). RNA Clean & Concentrator-5 (Zymo Research # R1013) was performed according to manufacturer’s protocol and RNA was eluted into 12 µL of RNase-free water. RNA quantification was performed using a Nanodrop 1000 spectrophotometer (Thermo Scientific). Larval head cDNA library was constructed from 1 μg of the total RNA using Superscript VILO Master Mix (Invitrogen # 11755050). Larval OSN nuclei cDNA library was constructed from 100–200 ng RNA with Superscript IV VILO Master Mix (Invitrogen #11754050). All cDNA samples were diluted to the equivalent of 1 ng/μl RNA.Drosophila larvae. The MIQE guidelines were followed as far as possible61. Primer sequences for individual genes were derived from FlyPrimerBank (flyrnai.org), designed using PrimerBlast, or obtained from literature  was performed to compare gene expression differences in head Fig.  and OSN-A 20 μL RT-qPCR reaction included cDNA template synthesized from 1 ng RNA, 0.4 μM of each primer and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad # 1725270). The RT-qPCR was performed on a CFX96 C1000 Touch Real-Time PCR detection system (Bio-Rad) with thermal cycling conditions as follows: an initial denaturation of 95 °C for 30 sec, followed by 40 cycles of 95 °C for 10 sec and 60 °C for 30 sec. Each reaction was conducted in triplicates. Occasional reactions within the triplicates with standard deviation >0.3 were omitted from analysis as PCR outliers.APPL and Nrv2 for normalization. Nrv2, Act42a, TBP, and EF1 were used as reference genes. This combination was picked because it yielded the lowest stability values among prospective reference genes, including ELAV, Orco, and eGFP. The BIO-RAD CFX software measured the collective reference gene expression stability yielding a mean coefficient variance <0.250 and a mean M value, a measure of reference gene expression stability <0.5. For larval heads samples , washed and dried the larvae, and carefully measured their combined weight on a Mettler-Toledo Precision balance. We repeated this process 10 times for each genotype considered.To quantify larval body-weight, we collected exactly 100 third-instar Supplementary information.


HTLV-1 was the first described human retrovirus and was soon found to be associated with severe clinical diseases, including a devastating lymphoma/leukemia and other inflammatory diseases. Although HTLV-2 is not usually pathogenic, it is widely distributed among native Indian populations in Brazil, particularly in the Amazon region of the country. Presently, HTLV spreads mainly by the sexual route and from mother to child, and virus persistence is an active biological factor aiding its transmission. Recently, the use of illicit drugs has been shown to be an additional risk factor, showing the influence of new habits on the epidemiology of HTLV in the region. Despite the detection of the virus in several different populations in the Amazon region of Brazil for almost 30 years, the exact prevalence of HTLV-1/2 is not well defined. The original biases in sampling and the selection of epidemiologically unsuitable populations were commonly repeated in most prevalence studies, generating unreliable and conflicting figures that do not represent the actual prevalence of HTLV. The improvements in clinical and laboratory facilities have resulted in the description of several clinical manifestations that were previously unknown in the region. The extent of the spread of the virus must be defined in this region, which is the largest geographical area of the country. As prophylaxis advances toward the use of vaccines against HTLV-1, it is important to determine who is at risk of being infected and developing a disease to successfully implement preventive measures, particularly as proposals are made to eradicate the virus among humans. Retroviridae, genus Deltaretrovirus [Human T cell lymphotropic viruses 1 and 2 (HTLV-1 and HTLV-2) are medium-sized virus particles 80–120 nm) belonging to the family trovirus  and fourtrovirus –8. Two o0–120 nm trovirus  as examptrovirus , 11.Retroviruses share similar biological and replicative properties, including the evolutionary aspect of viral and cell nucleic acid integration, viral persistence, viral latency and vertical transmission to the offspring. HTLV integrates the transcribed RNA as a DNA provirus into the cell nucleic acid –16, and HTLV is an ancient infection in humans and alternates between persistence and productive cycles, which favors an effective mechanism involving vertical and horizontal transmission. According to the geographical environment and behavioral risk factors, the increased risk of transmission of the virus increases the prevalence and incidence of infection and disease , 28–32.Viral dispersion in the human body leads to the infection of several biological fluids, including the blood, semen, vaginal fluid, and milk and results in its vertical transmission from mother to child , the injection of drugs, the transfusion of blood and its components, the transplantation of organs and the engagement in sexual relations , 33–38. Vertical transmission is common and is probably the most important route for the maintenance of HTLV within epidemiologically closed communities, as seen with HTLV-2c among Indians communities in the Amazon area of Brazil and in urban areas , 42–44. The geographical distribution of the virus is influenced by the transmission route used. In North America, HTLV-2 was probably spread from American Indians to injection drug users (IDUs), which resulted in the transmission of the virus to other IDU communities in Europe and was the most likely route that carried HTLV to Vietnam, during the war in 1960–1970 –49. In tSexual transmission is certainly the most important transmission route for HTLV-1 and HTLV-2 and serves as an efficient mechanism for the spread of HTLV-2c among native Indian groups , 28, 56.The description of HTLV-1 and HTLV-2 soon led to seroepidemiological studies based on the detection of antibodies against HTLV, which is the usual approach to determine the initial prevalence rates of HTLV according to geographical locations, age and sex, among other variables. Few studies have been conducted strictly with controlled populations to verify the published information from the 1980s and 1990s. Consequently, there have been few attempts to establish the trends in prevalence and incidence rates and the spread of HTLV in Brazil and, particularly in the Amazon region of the country.HTLV-1 was described in 1980, and HTLV-2 was described in 1982 –62. SoonHTLV-2, however, showed a distribution limited to intravenous drug users in the USA, Europe, Southeast Asia and among American Indians from North America to South America as well as in the Pygmy tribes in Central Africa , 66–72. In the Amazon region of Brazil, HTLV-1 and HTLV-2 infections were primarily described , 84–86, HTLV human infections in the Amazon region of Brazil have been recorded by several studies that considered widely different populations, including blood donors, pregnant women, urban familial aggregates and native indigenous people. These investigations comprise the epidemiological picture in the North region of the country and will be presented and discussed further in the following sections.2, which represents 60% of the country but is inhabited by approximately 15% of the Brazilian population.The Amazon is a large geographical area involving six countries, and Brazil holds the largest area in the system. The Amazon region of Brazil (ARB) involves nine federative States and an area of 5.1 million kmThere is a large difference in demographic, social, cultural and development between the ARB and the rest of the country, which is evidenced in some commonly used markers related to the health and education of the population and its level of welfare and development. Historically, the ARB was always left behind by policies given that the population of the region represents only 13% of the gross internal revenue of the country, has a higher rate of illiteracy (12.9% vs. 10.2%), a slightly higher infant mortality rate (18.6 vs. 15.9 per 1000) and a lower life expectancy (72 vs. 76 years) in comparison to the other areas of Brazil .On the other hand, the ARB is a unique geographical area, considering the diversity of humans and other living species. Approximately half of the so far undescribed living organisms on earth  reside within the ARB .The history of HTLV is a fascinating one regarding its possible origin in the African continent and its spread in different directions according to human migration routes , and theHuman infections by HTLV in the Amazon region of Brazil have been recorded by several studies involving blood donors , 98–102,The isolation of HTLV-1 and HTLV-2 led to the manufacturing of serological assays to detect human antibodies against the viruses, and several studies were immediately performed describing prevalence rates around the world. South American countries reported quite different figures with large variations, including in Argentina (0.07%), Chile (0.73%), Venezuela (6.8%), Colombia, where the prevalence was higher in low geographical areas (4.3%) than in higher areas (0.73%) of the country, and in French Guiana (6.7–13.1%) –110. It In a second round of prevalence studies, a more comprehensive investigation was conducted with national public blood banks , and theThe main issue of concern presented is the large variation obtained by the different investigations conducted in the same geographical area. The data include the states in the North region of the country, but the results are not different from those in the rest of the country. The discrepancy is shown in Table Plasmodium sp. [In Rio Branco (Acre), the investigation of HTLV among blood donors showed that using two enzyme immunoassays, the results were significantly different ; the Western blot results confirmed the presence of antibodies in 8 samples for HTLV-1 and 2 for HTLV-2. PCR results confirmed one HTLV-1 and one HTLV-2 reaction. The study shows the nature of conflicting results when comparing those with later results: 0.09%  and 0.5%dium sp. . Even thIn Manaus (Amazonas State), the results are also conflicting. The initial prevalence was set at 0.08%  and was The presence of the virus in different areas and populations in the State of Amapa has been investigated a few times, and low prevalence rates of infection (0.71%) have been found among blood donors . The virIn Sao Luiz, the capital city of the State of Maranhao, the highest prevalence rate (1%) among blood banks in Brazil was identified , but theThe State of Para has been the site of the majority of epidemiological studies intending to define prevalence rates of antibodies against HTLV-1/2 with a variety of different populations, and several conflicting results have been obtained. The initial figures indicated that the prevalence rates measured by immunoenzymatic assays ranged from 3.6% to more than 30% for HTLV-1, mostly among Indians populations , 85, thaTwo different studies dealt with HTLV/HIV-1 co-infections. The first approach found a prevalence rate of 8% (12/149) for individuals, mostly males (n = 10), positive for HTLV-1 (n = 4) and HTLV-2 (n = 7) . A laterThe initial approach of Japanese immigrants did not result in HTLV reactivity , but latFairly rapid changes in the epidemiology of HTLV-1/2 are occurring in the ARB. A recent and extensive study was performed in the State of Para, which detected an intermediate prevalence of 4.3% with almost the same frequencies of HTLV-1 (n = 25) and HTLV-2 (n = 19) among drug users . Both viEpidemiological studies in the ARB, as in other areas of the country and abroad, have been plagued by imprecise sampling (“grab samples” was common), which generated inadequate and conflicting results among several studies. Groups of sub-populations were consistently chosen, with the equivocal assumption of a general figure for most of the region, leading to a selection bias that rendered untrustworthy results. The selection of blood donors, the selection of diseased population subgroups, and the retrospective collection of data are some of the examples that should be avoided in future prevalence studies. Relevant population groups should be selected and preferably using multiple centers of study working together to increase sample size. It is relevant to stress that confirmatory tests should be always used to make sure the information is not equivocal and the absence of correct information should be put ahead to avoid misinterpretations. Urban populations should be the optimal target to answer important questions such as the following: who is mostly affected by this neglected infection? Who should receive future preventive measures such as vaccines? Which risk factors are relevant for the transmission of the virus?HTLV-1 is an important human pathogen and the only human retrovirus associated with a large array of diseases, including lymphoma/leukemia. However, in the possible event of the development of a vaccine in the near future, health authorities will not be able to immediately define the population at risk who should receive it, either in the ARB or in other areas of the country. New and authoritative epidemiological information should be gathered to gauge the actual need for such a product. Australian aboriginal people are examples of persons at high risk for morbidity and mortality associated with the wide dissemination and high prevalence of HTLV-1. Respiratory diseases recently also described in the ARB  are new Small epidemiologically closed or semi-closed human aggregates are easier with regard to the implementation of preventive measures, although transmission in such communities is further enhanced by the mother to child transmission route in utero, during birth and during breast feeding both perinatal and after birth, which corroborates the formation of familial aggregates. The most recent report from our laboratory has shown that Asurini and Arawete Indian tribes have remained free from HTLV-1/2 infections due to cultural and social isolation from the infected neighboring tribes and villages . Indian It is reasonable to infer that the general prevalence of the virus should be decreasing because of the general policies regarding testing blood donations and some efforts to disseminate general knowledge of the virus, but so far this has not been substantiated, as conflicting prevalence results generate confusion regarding the adequate delivery of information. National associations of HTLV-infected persons are active and give full support to the elimination of the virus, but their messages are not always disseminated through the appropriate channels. Although the existing preventive campaigns are not aggressive, they should receive full official support to attain success in the future, particularly as proposals are being made with regard to virus eradication .


The hedgehog (HH) signaling pathway regulates normal cell growth and differentiation. As a consequence of improper control, aberrant HH signaling results in tumorigenesis and supports aggressive phenotypes of human cancers, such as neoplastic transformation, tumor progression, metastasis, and drug resistance. Canonical activation of HH signaling occurs through binding of HH ligands to the transmembrane receptor Patched 1 (PTCH1), which derepresses the transmembrane G protein-coupled receptor Smoothened (SMO). Consequently, the glioma-associated oncogene homolog 1 (GLI1) zinc-finger transcription factors, the terminal effectors of the HH pathway, are released from suppressor of fused (SUFU)-mediated cytoplasmic sequestration, permitting nuclear translocation and activation of target genes. Aberrant activation of this pathway has been implicated in several cancer types, including medulloblastoma, rhabdomyosarcoma, basal cell carcinoma, glioblastoma, and cancers of lung, colon, stomach, pancreas, ovarian, and breast. Therefore, several components of the HH pathway are under investigation for targeted cancer therapy, particularly GLI1 and SMO. GLI1 transcripts are reported to undergo alternative splicing to produce truncated variants: loss-of-function GLI1ΔN and gain-of-function truncated GLI1 (tGLI1). This review covers the biochemical steps necessary for propagation of the HH activating signal and the involvement of aberrant HH signaling in human cancers, with a highlight on the tumor-specific gain-of-function tGLI1 isoform. Drosophila melanogaster that disrupted the larval body plan . SM. SM20]. boratory ,24. Reseboratory . In starboratory ,26,27. Fboratory ,31,32,33The canonical mammalian pathway includes several key components including the hedgehog glycoproteins Sonic hedgehog (SHH), Desert hedgehog (DHH), and Indian hedgehog (IHH); the 12-transmembrane protein Patched1 (PTCH1), the 7-transmembrane protein Smoothened (SMO), suppressor of fused (SUFU), and the GLI zinc-finger transcription factors, which act as the terminal effectors of the pathway. As previously stated, the family of GLI transcription factors consists of three separate proteins—GLI1 primarily functions as a transcriptional activator, GLI2 as either an activator or repressor, and GLI3 as a transcriptional repressor . The expIn the absence of SHH ligand, PTCH1 constitutively represses SMO activity . CurrentPTCH1 and GLI1 target genes [Binding of SHH to the PTCH1 receptor derepresses SMO, ultimately freeing GLI1 transcription factors from cytoplasmic retention. However, the understanding of how the activation signal is transmitted from ciliary SMO to the cytoplasmic GLI1 effectors remains incomplete . As mentet genes . This noet genes ,62,63,64Once SMO accumulates in the primary cilia, a protein complex containing kinesin protein (Kif7) and SUFU bound to GLI1 transcription factors is dynamically trafficked to the activated SMO in the primary cilia ,66,67. HThe notion of noncanonical SHH signaling refers to the cellular and tissue responses to HH isoforms that are not mediated by GLI1 isoform transcriptional activity. There are three classifications of noncanonical HH signaling: Type I acts through PTCH1 via functions distinct from its inhibition of SMO, Type II is mediated through GLI1-independent activities of SMO such as activation of small GTPases, and Type III refers to all other mechanisms of GLI1 transcription factor activation occurring independently of upstream PTCH1-SMO signaling ,75.Bcl-2) via GLI1-dependent transcription; activation of Akt, a pro-survival kinase; and the Type I noncanonical pathway [Canonical SHH signaling combats apoptosis through several mechanisms: upregulation of pro-survival genes , demonstrates selectivity toward heterotrimeric Gproteins . GPCRs aproteins . These sproteins . In the proteins . In thistivation . Despitetivation . Additiory cilia . Kif3a-dry cilia .Type III noncanonical hedgehog signaling encompasses all other mechanisms of GLI1 activation that occur independently of the noncanonical PTCH1 and SMO signaling activities described above. This form of GLI1 activation primarily arises through post-translational modification of GLI1 proteins and crosstalk with other oncogenic pathways that promote transcriptional activation of GLI1 genes. GLI1 proteins are extensively modified post-translationally, and these modifications largely determine intracellular trafficking and transcriptional output of the SHH pathway. Known post-translational modifications (PTMs) of GLI1 include phosphorylation, ubiquitination/proteasomal truncation, sumoylation, acetylation, and O-GlcNAcylation ,96. PhosUbiquitination, a process that targets substrate proteins for proteolytic degradation via the ubiquitin-proteasome system, is an important GLI1 PTM that balances the repressor and active forms of GLI1 to keep the pathway in the off state and acts as a negative feedback look in the on state . SeveralSumoylation is another repressive modification of many transcription factors that is catalyzed by enzymes similar to ubiquitin ligases. Pias1 small ubiquitin-related modifier (SUMO) ligase was found to decrease GLI1 ubiquitination, thereby increasing GLI1 transcriptional activity in vitro . AlternaGLI1 gene was identified as a putative oncogene in 1987 by Kinzler et al. after observing the amplification of a region within chromosome 12 [The human osome 12 . The genosome 12 ,125. Usiosome 12 . Only thosome 12 . Zinc-fiosome 12 . To ensuosome 12 ,130. Furosome 12 ,132,133.osome 12 ,135,136.Nearly 20 years later, the GLI1 splice variant, GLI1ΔN, was discovered in 2008 by Shimokawa et al. . SkippinThe GLI1 isoforms C’ΔGLI1 and N’ΔGLI1 were observed following transfection of U251 glioma cells with an N-terminal myc-tagged GLI1 expression plasmid . Using aThe 130 kD N’ΔGLI1 isoform, distinct from GLI1ΔN, was not discovered until repeat sample analysis using an antibody that targeted a domain immediately C-terminal to the zinc-fingers of the full-length GLI1 protein . The N-tOur laboratory discovered a second novel GLI1 isoform, named truncated GLI1 (tGLI1), in 2009 . AlternaPTCH1 mutations (Type I); ligand-dependent autocrine or juxtacrine signaling via overexpression of the SHH ligand observed in colorectal, ovarian, and pancreatic cancers (Type II); and ligand-dependent paracrine signaling via SHH ligand overexpression induced by tumor–stroma interactions commonly found in colon, pancreatic, and prostate cancers (Type III) [SHH signaling is tightly linked to many fundamental processes, including embryonic development and tissue homeostasis ,3. Emergype III) ,17,18.PTCH1 mutations on chromosome 9, leading to SMO hyperactivity [PTCH1 or more rarely by activating mutations in SMO [GLI1, but not SHH or GLI3 [SMO mutations or inactivating SUFU mutations [PTCH1 somatic mutations and loss of heterozygosity have been described in transitional cell carcinoma (TCC) of the bladder and esophageal squamous cell carcinoma [Though unknown at the time, aberrant SHH signaling was first linked to cancer when Robert J. Gorlin described Gorlin Syndrome  in 1960 . Charactactivity ,182,183.s in SMO ,185. The or GLI3 . This suutations ,15,186. utations . Similararcinoma ,189,190.PTCH1 and GLI1 expression.A second mechanism of uncontrolled activation of SHH signaling may arise through increased secretion of SHH ligand by tumor cells into the immediate surrounding area. Since the SHH pathway is activated in a cell-autonomous manner, SHH ligand is produced by and taken up by the same or immediately surrounding tumor cells. This ligand-dependent autocrine/juxtacrine signaling has been described in several cancers including colorectal, prostate, liver, breast, ovarian, brain, and melanoma ,192,193.GLI1 and PTCH1 mRNA levels in the stroma and not the tumor compartment, indicating that the SHH ligands activate SHH signaling in the surrounding stroma instead of the tumor epithelium [GLI1 and PTCH1 was found in both the tumor and stroma [SHH expression was detected in the tumor epithelium of prostate cancer samples while GLI1 expression, was upregulated in the stromal compartment, again providing evidence of paracrine ligand-dependent signaling to facilitate cancer development [In contrast to Type I and Type II, paracrine ligand-dependent signaling co-opts the stromal cells within the tumor microenvironment to facilitate formation of a favorable tumoral niche. Paracrine SHH ,194 signithelium . In the ithelium ,199,200.ithelium . For exaInterestingly, a “reverse paracrine” signaling model has been observed. In this instance, stromal SHH is believed to provide a suitable microenvironment for promoting tumor growth. Although this phenomenon has yet to be observed in solid tumors, it has been observed in several hematological malignancies including multiple myeloma, lymphoma, and leukemia ,202. AcuThat said, findings by Becher et al. (2008) suggest that PDGF-induced gliomas may employ paracrine SHH signaling . Using aDuring tumor development, SHH signaling has three major roles: driving tumor development, promoting tumor growth, and regulating residual cancer cells after therapy . MetastaPTCH1 and SUFU) or gain-of-function changes to promotors of the pathway . Patients afflicted by Gorlin syndrome present with loss of PTCH1 heterozygosity, increasing the likelihood of developing a distinct set of solid tumors: BCC, medulloblastoma, and rhabdomyosarcoma [PTCH1 mutations leading to SMO hyperactivity can be leveraged for therapeutic efficacy and currently represent the most effective therapeutic strategy. Indeed, the SMO inhibitors Vismodegib and Sonidegib are FDA-approved to treat SMO hyperactivity in BCC and have shown promising efficacy in SHH-dependent preclinical models of medulloblastoma and others [Tightly controlled SHH signaling is necessary for the proper embryonic development and maintenance of differentiated tissues. However, a plethora of studies have linked dysregulated SHH signaling to numerous diseases from insufficient pathway activation, resulting in tissue degradation to aberrant signaling, leading to tumorigenesis. Genetic alterations in this pathway are commonly loss-of-function changes to suppressors of the pathway  is the most common and most lethal brain tumor in adults, accounting for 15% of all brain tumors . Given tiagnosis . Recent iagnosis ,236,237.iagnosis . Gene exoma CSCs . Gliomasutations ,241,242.utations ,244. GLIGLI1 mRNA transcript undergoes alternative splicing in which 41 amino acids are deleted, corresponding to the loss of exon III and part of exon IV [CD24, CD44, HPSE, TEM7, VEGF-A, VEGF-C, and VEGFR2, thus promoting cancer cell growth, migration, invasion, and angiogenesis [CD24 expression was found to increase GBM invasion [While attempting to gain further insight into the GLI1 structure, our laboratory discovered the novel tGLI1 isoform while attempting to clone GLI1 cDNA. To produce the tGLI1 variant, the  exon IV . This co exon IV ,33. Our  exon IV ,26,27,29 exon IV . This maogenesis ,28,30,31invasion ,246. Usiinvasion . EvidencSHH, PTCH1, and GLI1 mutations are exceedingly rare in breast cancer which argues against the potential involvement of mutational activation of the SHH pathway in breast cancer [Breast cancer is the second leading cause of cancer-related mortality in women and metastasis to distant organs result in 90% of breast cancer deaths . Metastat cancer ,254,255.t cancer ,257. Addt cancer . GLI1 knt cancer .R-Ras2, Cep70, and UPF3A  has been approved by the FDA ,265. TheCurrently, no tGLI1 inhibitors have been developed to date. However, several tGLI1 target genes can be targeted using previously developed agents including HPSE, VEGF-A, and VEGFR2. Antiangiogenic therapies are an attractive focus for cancer drug development because heightened tumor vascularization is canonical phenotype of aggressive cancers . HPSE hap = 0.047). When combined with paclitaxel, ramucirumab significantly increased overall and progression-free survival in patients with metastatic gastric cancer [VEGF-A and VEGFRs are well-characterized targets for antiangiogenic therapeutics and these drugs have shown a varying range of effectiveness across tumor types . Monocloc cancer . In our c cancer .In addition to inhibiting tGLI1 target genes, inhibition of tGLI1 synthesis and transcriptional co-regulators could lead to tumor cell kill in tGLI1-high tumors. However, further studies are needed to delineate the splicing mechanisms responsible for the in-frame deletion of the wild-type GLI1 gene transcript that results in tGLI1 synthesis. Furthermore, whether tGLI1 requires transcription co-factors to activate target gene expression remains unknown. TGLI1 may also be subject to regulation by noncanonical pathways. Addressing these knowledge gaps will aid development of novel strategies to target tGLI1-driven tumors.GLI1 transcript creates a more potent oncogenic protein. Going forward, it will be important to determine the precise mechanisms by which tGLI1 expression is regulated including the splicing machinery that influences tGLI1 expression levels. Additionally, whether tGLI1 is regulated by any noncanonical pathways should be investigated. Such interactions could allow tGLI1 to serve as an oncogenic signaling hub similar to STAT3 that permits convergence of several signaling pathways to create a novel mechanism of tumor progression. Finally, elucidating the mechanisms by which tGLI1 activates its target genes is critical to identifying other novel targets of tGLI1 that could contribute to the aggressive phenotypes of tGLI1-positive tumors. Given the established role of aberrant SHH signaling in tumorigenesis of several human cancers, understanding the fundamental mechanisms of SHH pathway regulation and the impact of alternative GLI1 splice variants will aid the development of novel therapeutics and achieve improved therapeutic outcomes for these patients.The SHH pathway is evolutionarily conserved and essential for the regulation of normal development and differentiation in vertebrates. However, aberrant SHH pathway activation by either mutation or ligand overexpression is closely correlated with several cancers. Although the link between the SHH signaling pathway and tumorigenesis varies by cancer type, it is clear that the aberrant activation of SHH signaling leads to the growth, proliferation, and invasion of tumor cells. Furthermore, data indicate that the GLI transcription factors are regulated by several oncogenic signaling pathways in addition to canonical PTCH-SMO signaling. Thus, targeting the SHH pathway may provide therapeutic options. In fact, the truncated GLI1 (tGLI1) splice variant presents a unique opportunity to validate a novel actionable target for gliomas and breast cancer brain metastases due to its tumor-specific expression and potent oncogenic effects. The alternative splicing of the


Some major research and clinical questions about human T-cell leukemia virus type 1 (HTLV-1) infection and rheumatic diseases remain: (1) Does HTLV-1 infection cause rheumatic diseases? (2) Do patients with rheumatic diseases display different responses to treatment with anti-rheumatic agents when they are HTLV-1 carriers? (3) Is adult T-cell leukemia/lymphoma (ATL) or HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) more prevalent in HTLV-1 carriers with rheumatic diseases who are treated with anti-rheumatic agents? These questions are important because increasing numbers of patients with rheumatic diseases are currently receiving treatment with aggressive medicines such as immunosuppressants and biologics. Studies on HTLV-1 gene-transgenic mice have shown manifestations resembling rheumatic diseases. Epidemiological studies have shown a high incidence of HTLV-1 infection in patients with rheumatic diseases including rheumatoid arthritis (RA), Sjogren’s syndrome, and polymyositis. HTLV-1-positive and HTLV-1-negative patients with RA have displayed similar immunological features including the seroprevalence of anti-citrullinated peptide antibodies. Conversely, attenuated effectiveness of tumor necrosis factor inhibitors for HTLV-1-positive patients with RA in Japan has been reported. Therefore, although no direct evidence has shown that HTLV-1 infection alone causes rheumatic diseases, HTLV-1 may affect the inflammation of RA. Although the incidence of ATL or HAM/TSP among patients with rheumatic diseases has not been investigated in large-scale studies, ATL or HAM/TSP has developed among HTLV-1-positive patients with rheumatic diseases. HTLV-1 infection may affect the clinical course of patients with rheumatic diseases, particularly after receiving anti-rheumatic agents. Because studies on these issues are limited, further investigation with large sample sizes is necessary. Human T-cell leukemia virus type 1 (HTLV-1) is a human retrovirus that is a causative agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) . There hThe major research and clinical questions about HTLV-1 infection and rheumatic diseases evaluated in this article are as follows: (1) Does HTLV-1 infection cause rheumatic diseases? (2) Do patients with rheumatic diseases display a different response to treatment with anti-rheumatic agents or immunosuppressive agents when they are HTLV-1 carriers? (3) Are ATL or HAM/TSP more prevalent in HTLV-1 carriers with rheumatic diseases who are treated with anti-rheumatic agents? We reviewed the studies on these topics (mainly the relationship between HTLV-1 infection and RA) and described what we know and what areas of HTLV-1 research must be addressed in the future.Tax and HBZ genes  and polyclonal expansion of HTLV-1-infected cells. Peripheral blood mononuclear cells isolated from patients with HAM/TSP showed autonomously produced inflammatory cytokines such as interferon (IFN)-gamma, IL-6, and TNF-alpha . HTLV-1  HAM/TSP . Chemoki HAM/TSP . CD4 + C HAM/TSP . This po HAM/TSP .Ex vivo cultures of lymphocytes from HTLV-1 carriers show spontaneous proliferation  compared with HTLV-1-negative patients . Two retIt is unknown whether the association of rheumatic diseases and treatment against such diseases affects the natural history of HTLV-1 infection, particularly in view of the development of HTLV-1-associated diseases such as ATL and HAM/TSP.Patients with RA have reported higher incidence rates of malignant lymphoma, although the surface phenotype of lymphoma found in RA is B-cell predominant . For ATLin vivo; however, it is clear that these medicines cause various levels of immune suppression. HTLV-1 carriers with mother-to-child infection were shown to have higher HTLV-1 PVLs in the peripheral blood than those with infection between spouses , is well known as the anchor drug for RA with immunosuppressive effects. Another DMARD, tacrolimus, specifically suppresses T-cell function by inhibiting calcineurin. Several biological DMARDs mainly inhibit the function of proinflammatory cytokines such as TNF and IL-6. Recently, novel DMARDs were developed, which inhibit Janus kinase for signal transduction from the receptors of proinflammatory cytokines. It is unknown whether there are any direct effects of these medicines on HTLV-1-infected cells  spouses . The clo spouses . A clone spouses . An HTLV spouses . The fav spouses . TherefoIn addition, the incidence of lymphoproliferative disorders (LPDs) including lymphoma has been reported to increase in RA patients who were treated with MTX . LPDs thIt is important to recognize that two major routes exist in natural HTLV-1 infection: mother-to-child transmission in infancy and transmission between spouses in adulthood . HTLV-1 Immune status in patients with RA is considered Th1 and Th17 dominant. The immune status of HAM/TSP is also considered Th1 dominant. Therefore, whether RA affects the etiology and clinical condition of HAM/TSP is an important question. We must also consider the effect of the DMARDs that patients with RA are receiving. Indeed, worsening symptoms of both HAM/TSP and HTLV-1-associated uveitis were reported in an HTLV-1-positive patient with RA who received treatment with a soluble IL-6 receptor inhibitor . More reIt is important to understand the relationships among rheumatic diseases, treatment using immune modulating or immunosuppressive drugs, and chronic viral infection such as HTLV-1 ; howeverCurrently, the HTLV-1 screening test conducted in pregnant women for preventing HTLV-1 transmission to their child has been performed all over the country of Japan. Consequently, most pregnant women know if they are positive for HTLV-1. Because rheumatic diseases such as RA are generally more common in females than in males, more patients with rheumatic diseases and their doctors are assumed to be anxious about the effects of HTLV-1 infection on the disease condition and treatment using DMARDs than in the past. We previously performed a nationwide investigation in Japan using a questionnaire for rheumatologists that addressed these issues. Indeed, many doctors mentioned that they did not have enough knowledge about HTLV-1 infection and claimed the necessity of having guidelines on how to treat HTLV-1-positive patients with RA.The clinical presentation of ATL and HAM/TSP is different between Japanese and Caribbean populations. The onset age of ATL in the Caribbean population is earlier than that in the Japanese population . The freAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intravitreal injection of medications is one of the most common procedures performed in ophthalmology. Intravitreal anti-VEGF agents are currently the chosen treatment for ocular fundus diseases, including age-related macular degeneration and diabetic retinopathy. As an invasive procedure it involves risks. The most serious complication from intravitreal injection of anti-VEGF agents is endophthalmitis (EO). Although rare, EO can result in devastating loss of vision. This article evaluates whether the use of an ultra-clean air flow (UA) can be another useful tool in the prevention of EOs. Accordingly, the maintenance of asepsis of the surgical field of intravitreal injections was verified with and without the use of UA.The study was conducted in operating room of an ambulatory surgery center on four different surgical days when just intravitreal injections were scheduled. Two experiments using two Blood Agar and two Chocolate Agar plates  were carried out by positioning an UA directed to the surgical table and two other experiments  were carried out using similar plates without the use of the UA. All Blood Agar and four Chocolate Agar plates were positioned on the surgical table, close to the surgical filed. At the end of the day, after the conclusion of the intravitreous injections, the plates were sent for a biomolecular study that was carried out after 1 day of incubation at 37 °C.Micrococcus sp. with the use of the UA and without the the UA bacterias of the genera Bacillus sp, Staphylococcus haemolyticus, Staphylococcus aureus and Staphylococcus cohnii ssp urealyticus were found.The sixteen plates, eight Blood Agar and eight Chocolate Agar, were analyzed qualitatively for the growth or not of microorganism’s colonies and identification of their species. The biomolecular study demonstrated the growth of bacteria of the genus The use of UA close to the operating table prevented the growth of pathogenic bacteria and should be considered as an alternative tool to avoid the contamination of materials and drugs used for intravitreal injections. The development of targeted molecular therapy to inhibit vascular endothelial growth factor (VEGF) has revolutionized the treatment and visual prognosis of retinal diseases such as diabetic retinopathy and age-related macular degeneration, as well as macular edema and retinal vein occlusion through performing intravitreal injections (IVIs) of these drugs , 2. As aStaphylococcus epidermidis followed by Streptococcus viridans, which is especially related to contamination of the operating room when there is frequent conversation during the IVIs procedures [Staphylococcus aureus, Bacillus spss and Pseudomonas spss are also important microorganisms to be considered in the etiology of EO [Etiological agents that usually cause EO are fairly abundant in the conjunctival flora of the normal human eye. The most usual ones are coagulase-negative staphylococci (CONS), most commonly ocedures . Staphylgy of EO , 8.®, Toul Meditech), which produces a directed, non-turbulent ultra-clean airflow over the sterile instruments. The airflow speed is 0.4–0.5 m/s and has a capacity to clean the air of 400 m3/h and a protection area up to 120 cm. This system uses a HEPA filter that eliminates particles smaller than 0.3 µm from the air [In Canada and in USA, IVIs are mainly performed in the office , whereas the air , and, coThe study was conducted in the OR of the Ophthalmology sector at Hospital das Clínicas of the University Hospital, School of Medicine of Ribeirão Preto, University of São Paulo, a tertiary health care hospital.The study was carried out on four different surgical days. The analyzes were performed as follows: the surgical tables were always positioned in the same place. On each day of IVIs, two Blood Agar and two Chocolate Agar were placed in the same location, at a distance of 120 cm from the site where the UA system would be located and in the same place without the UA. Two days, 11/29/2018 and 12/6/2018 with the UA directed to the operating table. Two days, 12/12/2018 and 12/19/2018, without the use of the UA. A total of 16 culture plates were used. In total 99 IVIs were done with the UA and 102 without it.®, Toul Meditech) which produces a directed, non-turbulent ultra-clean airflow over the sterile instruments. According to the manufacturer’s specifications, this system uses a HEPA filter that eliminates particles smaller than 0.3 µm from the air. The air flow speed is 0.4–0.5 m/s, with a capacity to clean the air of 400 m3/h and a protection area of up to 120 cm , Staphylococcus aureus (98%) and Staphylococcus cohnii ssp urealyticus (96%). . Table , Fig. 3.In all four IVIs days, there were no cases of EO, with or without the use of UA.Antisepsis with iodine-povidone, eyelid retraction with speculum, use of face masks to prevent the spread of droplets by the medical team and patient and reduced speech in OR, are recommended measures that may contribute to the reduction in EO after IVIs , 10. On One of the reasons to recommend the performance of IVIs in a surgical environment and not in the office, is the OR air quality and the theory that air borne particles may cause infection. The first report of the efficacy of UA in reducing the rates of surgical infection was in orthopedic implant surgery . In oculMicrococcus sspp, a Gram-positive bacteria 0.3 to 3.5 µm in diameter, usually present in the normal flora of the skin, mucosa and oropharynx, and far known to have no virulence mechanisms [From our samples of microorganisms that showed growth in the culture plates, we observed that in the presence of UA, there was growth of chanisms .S. cohnii, S. haemolyticus (both bacteria of the CONS group) and S. aureus are known to be etiological agents of OE after intraocular procedures. In addition, S. cohnii is related to rare cases of endocarditis, pneumonias, urinary tract infections, brain abscesses, and septic arthritis, among another infections [S. cohnii shows a multiresistant profile. Finally, S. haemolyticus is the second species most frequently isolated species from human blood cultures and is highly resistant to antimicrobial agents [Among the microorganisms deposited on the culture plates in the surgical field without the UA, fections –20. WhenBacillus sspp that grew, also in the absence of UA, the majority is non-pathogenic, but for a complete analysis of the identified bacillus it would be necessary to apply the BAAR method, in order to rule out the possibility of being a pathogenic agent such as B. tuberculosis [As for the strain of rculosis .Regarding the maintenance of asepsis of the surgical field, the literature has emphasized the importance of maintaining it throughout the surgical period , 12, 13.The data presented in this work reveal that the use of UA directed to the surgical field can be a useful auxiliary tool in an attempt to ensure the maintenance of asepsis of instruments and needles to be used in IVIs, as well as ensuring asepsis during manipulation (aspiration of the medications) of IVIs.Our work has some limitations. The number of colonies for each isolated microorganism was not verified, and for this reason there was just a qualitative analysis, instead of a quantitative analysis with statistics. In addition, due to the reduced physical space, the UA was not placed so its effect would extend over the ocular surface, which is the objective of our next study. Finally, there was not a control group with a device providing airflow that was not “ultraclean”, to check if the absence of pathogenic bacteria growth may be due to rapid air flow and not the HEPA filtered air.The use of UA close to the operating table prevented the growth of pathogenic bacteria and should be considered as an alternative tool to avoid the contamination of materials and drugs used for intravitreal injections.


The intestine is extremely dynamic and the epithelial cells that line the intestine get replaced every 3–5 days by highly proliferative intestinal stem cells (ISCs). The instructions for ISCs to self-renew or to differentiate come as cues from their surrounding microenvironment or their niche. A small number of evolutionarily conserved signaling pathways act as a critical regulator of the stem cells in the adult intestine, and these pathways are well characterized. However, the mechanisms, nutritional, and environmental signals that help establish the stem cell niche in the neonatal intestine are less studied. Deciphering the key signaling pathways that regulate the development and maintenance of the stem cells is particularly important to understanding how the intestine regenerates from necrotizing enterocolitis, a devastating disease in newborn infants characterized by inflammation, tissues necrosis, and stem cell injury. In this review, we piece together current knowledge on morphogenetic and immune pathways that regulate intestinal stem cell in neonates and highlight how the cross talk among these pathways affect tissue regeneration. We further discuss how these key pathways are perturbed in NEC and review the scientific knowledge relating to options for stem cell therapy in NEC gleaned from pre-clinical experimental models of NEC. The small intestinal tract is a tube-like structure that is critical for the absorption and digestion of nutrients as well as excretion of waste. Besides being a metabolic organ, numerous epithelial and non-epithelial cells play an important role in host immunity acting as a barrier to a multitude of commensal and pathogenic microbiota. The intestine is composed primarily of three tissue layers. The outer smooth muscle layer together with the enteric nervous system is responsible for peristalsis. The middle submucosal layer is the space between the outer muscle and the inner mucosal layer that is filled with stromal cells, nerve fibers, and various cells of the immune system. The inner mucosal layer consists of a single layer of epithelium with the lamina propria and basement membrane . The sinThe intestinal epithelium is a continuously self-renewing tissue which serves a multitude of functions. Through its interaction with its surrounding mesenchyme, cells of the intestinal epithelium progress though repeated cycles to proliferate, terminally differentiate, and ultimately shed into the intestinal lumen. To fulfill this cellular demand, the small intestinal epithelium is organized into two spatially distinct units-the crypts of Lieberkühn which contain highly proliferative ISCs, and the long finger-like projections called villi which host terminally differentiated cells that are post-mitotic . The intLrig [Physical positioning of the epithelial cells within the crypt have identified two subtypes of ISCs—the first subtype is the active intestinal stem cell called crypt base columnar cells (CBCs) which frequently divide and thereby contribute to majority of the tissue regeneration. These cells are wedged between Paneth cells and are identified by the marker leucine rich repeat containing G protein coupled receptor 5 (Lgr5). The second subtype of ISCs are the quiescent or reserve intestinal stem cells, which are slow dividing ISCs present in the +4 to +6 position from the base of the crypt. These quiescent ISCs, though resistant to stress, can be activated when the CBC function is compromised. Several proposed markers for +4 ISCs include but are not limited to are Bmi, mTert, Hopx, Dclk1, and Lrig . LineageLrig . The ISCLrig . In brieLrig ,11. ThouIn the early embryonic stage, the primitive gut epithelium develops as an undifferentiated pseudostratified layer from the endoderm with surrounding mesenchymal tissues. During later stages in fetal ontogeny, the epithelium differentiates into a monolayer of columnar epithelial cells forming villi. Unlike the adult mouse intestine, neonate mouse lack crypts at birth and the intestine matures postnatally . Fully fAt the paracrine level, the cross talk between the surrounding mesenchyme and epithelium induce villus and crypt morphogenesis. The control of intestinal epithelial stem cell proliferation, differentiation and self-renewal are regulated by developmental pathways, which are evolutionarily conserved. These include the hedgehog (Hh), Wnt, BMP, and notch signaling pathways. These pathways overlap between organ morphogenesis and stem cell biology. Here, we describe the effect of evolutionarily conserved paracellular signaling on neonatal gut development.Hedgehog signaling involves the binding of Hh ligands like sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh) to their transmembrane receptor Patched 1 & 2 (Ptch1 & 2) to release the Smoothened (SMO) signal transducer from its Patched-dependent suppression. SMO stabilizes Gli, the effector of Hh signaling and leads to Gli-dependent transcriptional activation of target genes . During + mesenchymal cells but does not affect epithelial proliferation or differentiation [Another example of epithelial mesenchymal interaction leading to organogenesis is PDGF signaling. Expression of PDGF is predominantly in the intestinal epithelium, while its corresponding receptor PDGFR is in the mesenchyme. Loss of PDGFα ligand or its corresponding receptor affects villus morphogenesis and depletes surrounding PDGFRntiation . It is lAs members of the TGF-β superfamily, BMPs traffic signaling between mesenchyme and epithelium. BMP signaling is initiated by binding of BMPs to their corresponding receptors—the most prevalent of which are type I (Ia or Ib) and type II. Binding of ligand to its corresponding receptor leads to the phosphorylation and translocation of the SMAD family of transcription factors, a downstream mediator for both TGFs and BMPs, into the nucleus . These mSMAD4 and BMPR1a are frequently found in individuals with familial Juvenile Polyposis. This condition is characterized by intestinal hamartomatous polyps comprised of both stromal and epithelial tissue [noggin show an extensive loss of villi and ectopic crypt formation and hyperplasia of stroma similar to that of Juvenile Polyposis in humans [noggin leads to development of a large number of crypt-like structures on the sides of the villi as well as where the crypts are localized [Germline mutations in l tissue ,21. Misen humans ,22. Convocalized . Loss of2+-NFAT-IFN γ and the planar cell polarity (PCP) pathways [Wnt signaling is highly conserved across species and plays an important role in normal homeostasis and disease pathophysiology during development. Wnt ligands act as paracrine signaling molecules to initiate downstream signaling that include both canonical and non-canonical pathways. Canonical Wnt signaling is initiated by binding of Wnt ligands to their cognate membrane bound receptors the frizzled protein and co-receptor the LDL receptor-related proteins 5 and 6 (LRP5/6). Formation of this complex results in nuclear accumulation of β-catenin and subsequent activation of Transcription Factor 7 (TCF) target genes like cMyc. In non-canonical Wnt signaling, binding of Wnt ligands to the frizzled receptor stimulates downstream intracellular signaling independent of β-catenin, which influences various downstream pathways including the Capathways ,25,26.Canonical Wnt signaling ligands  as well as their corresponding receptors  are present only in the crypt epithelium, while members of non-canonical Wnt signaling are present in the Villus epithelium and the mesenchyme . In the Wnt signaling also controls segregation of villus from crypt cells through Ephrin and Ephrin receptors. Akin to notch ligands, Ephrin ligands and Ephrin receptors are membrane bound and communicate with adjacent cells. These ligands and receptors repel each other and naturally create boundaries. In the neonatal mouse, ephrin B1 is expressed by the epithelial cells of the villi, and EphB2 and EphB3 by cells of the intervillus pockets. Genetic deletion of the EphB2 and EphB3 results in the abnormal presence of Paneth cells in the villus instead of being localized in the crypt. Post-mitotic cells present in the villus start appearing at the crypt base . Thus, iNotch signaling is highly conserved and well recognized for its role in ISC proliferation and differentiation in the adult intestine . Notch sTaken together, notch signaling appears to have variable effects at different stages of development in the intestine. While it predominantly affects proliferation in the fetal stage, in the neonates, it affects crypt proliferation and differentiation of all cellular lineages. In the adult intestine, however, it favors the absorptive fate and affects ISC proliferation. Furthermore, the effects of notch on proliferation requires cooperation with Wnt signaling molecules likeTCF4, whereas its influence on intestinal differentiation appears independent of Wnt .Another layer of regulation for intestinal crypt formation is at the endocrine level. This is reflected in the steep increase in plasma thyroid hormone (T3) levels immediately after birth. In parallel, levels of the protein arginine methyltransferase 1 (PRMT1), a coactivator for the Thyroid hormone (TH)receptor, is upregulated strictly in the intervillus region. Lack of T3 or its receptor leads to a decrease in intestinal epithelial cells and abnormal intestinal morphology ,48. In aAt the molecular level, the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1), a downstream target of the BMP/SMAD pathway, regulates intestinal maturation. Blimp1, which is strongly expressed in the entire intestinal epithelium throughout the fetal stage, becomes restricted to the intervillus region in neonates, and this phenomenon coincides with the rise in TH level. This suggests that Blimp1 expression is reciprocally regulated by Thyroid hormone activity.Intestinal specific conditional deletion of Blimp1 led to enhanced formation of crypts, accelerated development of Paneth cells, and increased migration of enterocytes. Altogether, the conditional inactivation of Blimp1 compromises intestine function and survival ,50. ThisSox9, a transcription factor and a downstream target of Wnt signaling, is expressed throughout the duodenal epithelium as early as embryonic day 13.5 (E13.5) and subsequently restricted to the crypt of the adult intestine. Sox9 represses Cdx2 and Muc2, two genes involved in epithelial differentiation, implicating it in intestinal stem cell differentiation. Conditional loss of Sox9 in the adult intestinal epithelium also led to increased proliferation and Paneth cell loss, ,53. HoweAltogether, these studies illuminate the fact that a plethora of endocrine, paracrine, and transcription factors orchestrate intestinal morphogenesis from the neonatal stage to adult. The involvement of factors involved in the process during the neonatal period declines over time during maturation to adulthood, suggesting that adult stem cells have unique requirements of factors and cofactors for intestinal stem cell maintenance and function distinct from what roles that were predominant in neonates.Necrotizing enterocolitis (NEC) is the most common, life-threatening gastrointestinal emergency in the neonatal period. Mostly afflicting premature infants born at <36 weeks gestation, the incidence varies between 5 to 10%, with a mortality of 15–30% ,55. ClinThe premise for a dysmature intestinal immune response in NEC is founded on the fact that NEC is rare in full term neonates, and occurs in the setting of ischemia related to congenital heart disease or gastroschisis ,61. DataHuman studies on intestinal tissue obtained from infants with NEC show increased expression of TLR4 and related downstream cytokines and chemokines, while inhibitors of TLR signaling such as SIGIRR are decreased ,70,71,72While TLR4 activation is a major mechanism for NEC pathogenesis, investigators have identified other pathways that are relevant to NEC pathogenesis. Paneth cells lining the crypt of Lieberkuhn are key components of the innate immune system by secreting anti-microbial peptides ,79. PaneEnterobacteria, and decreases in Bacteroides and Bifidobacteria [Gammaproteobacteria, a class of Gram-negative bacteria that encompasses many known pathogens implicated in NEC and sepsis in preterm infants including Enterobacter, Klebsiella, E. coli, and Psuedomonas [Bifidobacteria and Bacteriodes [Gammaproteobacteria penetrating the immature intestinal barrier in preterm neonates and triggering exaggerated TLR signaling, which results in the intestinal inflammation and necrosis  ,85,86,87bacteria ,85,91,92edomonas ,88,93. Wteriodes ,94. The necrosis .Over the last decade, the contribution of genetic risk factors to NEC susceptibility has been investigated by several investigators. Genetic risk factors can seek to explain the inter-individual differences in NEC vulnerability noted in premature infants who have similar clinical risk factors. Genetic variants in innate immune genes, vascular tone/growth regulating genes, anti-oxidant response genes, and cytokine/chemokine genes have been major areas of research . Not sur+ ISCs [Under normal homeostatic conditions, the epithelial barrier is maintained by an intact crypt–villus axis. Transit amplifying cells arise from the ISC present in the crypt, migrating and differentiating along the villus. Finally, enterocytes undergo apoptosis and are shed into the lumen. This entire process takes 3–5 days. However, in conditions like NEC, the gut barrier function is impaired, and it is now increasingly recognized that the Toll like receptor, TLR4, plays a key role in the maintenance of epithelial barrier integrity. TLR4 is predominantly expressed in the crypt and was found to be colocalized with LGR5+ ISCs . In both+ ISCs ,103. Mec+ ISCs . Transcr+ ISCs .In patients with NEC as well as in the mouse model of NEC, a significant reduction in the formation of the goblet cell is observed . A lack Taken together, these studies demonstrate that immune signaling cooperates with morphogenetic pathways like Wnt and notch and contribute towards the development of NEC. These findings also illuminate differences in the role of TLR4 signaling in the neonate versus adult intestine . TLR4 isMesenchymal stem cells (MSCs) were first noted for their ability to regenerate infarcted myocardium , and areContrary to the initial belief, MSCs do not directly become new tissue, but instead secrete growth factors and cytokines, as well as packaged exosomes and microvesicles containing protein, lipid, and nucleic acids to mediate beneficial effects . ParacriMSCs for necrotizing enterocolitis (NEC) have been evaluated in animal models for the last decade, with multiple studies and a recent review . Besner Synergistic therapy with other novel interventions, such as exogenous heparin-binding EGF-like growth factor  and extrFinally, although there are no current clinical trials of MSCs or MSC-derived exosomes for NEC, an interesting case report for MSC therapy in NEC was published . A 22-daThe conserved morphogenetic pathways that regulate the maintenance of the stem cell in the adult intestine, and aid in tissue regeneration during disease are relatively well characterized compared to the neonatal intestine. Early studies suggest that the intestinal microbiota, IEC innate immune signaling, and metabolites in the neonatal intestine shape the development of the intestinal stem cell. Morphogenetic pathways including Wnt, BMP, Hh, and Notch may have distinct roles in the neonatal intestine, different from that described in the adult intestine. Understanding how the stem cell is generated, maintained, and disrupted in diseases such as NEC in the neonatal intestine will enable translating the power of stem cell therapy to improve outcomes for neonatal diseases.