Bridging the Technology Divide in the COVID-19 Era: Using Virtual Outreach to Expose Middle and High School Students to Imaging Technology.

Increasing the diversity of students choosing careers in science, technology, engineering, and mathematics (STEM) fields is an area of intense focus across the USA, especially in kindergarten through 12th grade (K-12)-focused pipeline programs in medical schools. A diverse STEM workforce contributes to better problem-solving and equity in health care. Two of the many major barriers for rural students are the lack of sufficient STEM role models and limited access to technology in the classroom. Medical schools often serve as an important resource for students in the local community who can easily gain access to STEM professionals and modern technology through on-campus, sponsored events and STEM outreach to the local classrooms. However, underrepresented minority (URM) students often live in socioeconomically distressed parts of rural states such as Arkansas, where access to STEM role models and technology is limited. Virtual learning in the COVID-19 era has proven that the imaging technology resources of a medical school can be harnessed to reach a wider audience, especially students living in rural areas far from the medical school campus.

Association of Gnrhr mRNA With the Stem Cell Determinant Musashi: A Mechanism for Leptin-Mediated Modulation of GnRHR Expression.

The cyclic expression of pituitary gonadotropin-releasing hormone receptors (GnRHRs) may be an important checkpoint for leptin regulatory signals. Gonadotrope Lepr-null mice have reduced GnRHR levels, suggesting these receptors may be leptin targets. To determine if leptin stimulated GnRHR directly, primary pituitary cultures or pieces were exposed to 1 to 100 nM leptin. Leptin increased GnRHR protein levels and the percentages of gonadotropes that bound biotinylated analogs of gonadotropin-releasing hormone (bio-GnRH) but had no effect on Gnrhr messenger RNA (mRNA). An in silico analysis revealed three consensus Musashi (MSI) binding elements (MBEs) for this translational control protein in the 3' untranslated region (UTR) of Gnrhr mRNA. Several experiments determined that these Gnrhr mRNA MBE were active: (1) RNA electrophoretic mobility shift assay analyses showed that MSI1 specifically bound Gnrhr mRNA 3'-UTR; (2) RNA immunoprecipitation of pituitary fractions with MSI1 antibody pulled down a complex enriched in endogenous MSI protein and endogenous Gnrhr mRNA; and (3) fluorescence reporter assays showed that MSI1 repressed translation of the reporter coupled to the Gnrhr 3'-UTR. In vitro, leptin stimulation of pituitary pieces reduced Msi1 mRNA in female pituitaries, and leptin stimulation of pituitary cultures reduced MSI1 proteins selectively in gonadotropes identified by binding to bio-GnRH. These findings show that leptin's direct stimulatory actions on gonadotrope GnRHR correlate with a direct inhibition of expression of the posttranscriptional regulator MSI1. We also show MSI1 interaction with the 3'-UTR of Gnrhr mRNA. These findings now open the door to future studies of leptin-modulated posttranscriptional pathways.

Leptin Regulation of Gonadotrope Gonadotropin-Releasing Hormone Receptors As a Metabolic Checkpoint and Gateway to Reproductive Competence.

The adipokine leptin signals the body's nutritional status to the brain, and particularly, the hypothalamus. However, leptin receptors (LEPRs) can be found all throughout the body and brain, including the pituitary. It is known that leptin is permissive for reproduction, and mice that cannot produce leptin (Lep/Lep) are infertile. Many studies have pinpointed leptin's regulation of reproduction to the hypothalamus. However, LEPRs exist at all levels of the hypothalamic-pituitary-gonadal axis. We have previously shown that deleting the signaling portion of the LEPR specifically in gonadotropes impairs fertility in female mice. Our recent studies have targeted this regulation to the control of gonadotropin releasing hormone receptor (GnRHR) expression. The hypotheses presented here are twofold: (1) cyclic regulation of pituitary GnRHR levels sets up a target metabolic checkpoint for control of the reproductive axis and (2) multiple checkpoints are required for the metabolic signaling that regulates the reproductive axis. Here, we emphasize and explore the relationship between the hypothalamus and the pituitary with regard to the regulation of GnRHR. The original data we present strengthen these hypotheses and build on our previous studies. We show that we can cause infertility in 70% of female mice by deleting all isoforms of LEPR specifically in gonadotropes. Our findings implicate activin subunit (InhBa) mRNA as a potential leptin target in gonadotropes. We further show gonadotrope-specific upregulation of GnRHR protein (but not mRNA levels) following leptin stimulation. In order to try and understand this post-transcriptional regulation, we tested candidate miRNAs (identified with in silico analysis) that may be binding the Gnrhr mRNA. We show significant upregulation of one of these miRNAs in our gonadotrope-Lepr-null females. The evidence provided here, combined with our previous work, lay the foundation for metabolically regulated post-transcriptional control of the gonadotrope. We discuss possible mechanisms, including miRNA regulation and the involvement of the RNA binding protein, Musashi. We also demonstrate how this regulation may be vital for the dynamic remodeling of gonadotropes in the cycling female. Finally, we propose that the leptin receptivity of both the hypothalamus and the pituitary are vital for the body's ability to delay or slow reproduction during periods of low nutrition.

A Sex-Dependent, Tropic Role for Leptin in the Somatotrope as a Regulator of POU1F1 and POU1F1-Dependent Hormones.

Pituitary somatotropes perform the key function of coordinating organismic growth and body composition with metabolic signals. However, the mechanism by which they sense and respond to metabolic signals via the adipokine leptin is unknown. The complex interplay between the heterogeneous cell types of the pituitary confounds the identification of somatotrope-specific mechanisms. Somatotropes represent 30%-40% of the anterior pituitary population and are derived from a lineage of cells that are activated by the Pit-Oct-Unc domain family domain class 1 transcription factor 1 (POU1F1) to produce GH, prolactin (PRL). and TSH. To determine the mechanism by which leptin controls somatotrope function, we used Cre-LoxP technology and fluorescence-activated cell sorting to purify and study control or leptin receptor-deleted (Lepr null) somatotropes. We report that Lepr-null somatotropes show significant reductions in GH protein (GH) and Gh mRNA. By contrast, enzyme immunoassays detected no changes in ACTH, LH, and FSH levels in mutants, indicating that the control of these hormones is independent of leptin signaling to somatotropes. Reduced TSH and PRL levels were also observed, but interestingly, this reduction occurred only in in Lepr-null somatotropes from mutant females and not from males. Consistent with the sex-specific reduction in Gh mRNA, TSH, and PRL, enzyme immunoassays detected a sex-specific reduction in POU1F1 protein levels in adult female Lepr-null somatotropes. Collectively, this study of purified Lepr-null somatotropes has uncovered an unexpected tropic role for leptin in the control of POU1F1 and all POU1F1-dependent hormones. This supports a broader role for somatotropes as metabolic sensors including sex-specific responses to leptin.

Adipocyte Versus Somatotrope Leptin: Regulation of Metabolic Functions in the Mouse.

Leptin regulates food intake and energy expenditure (EE) and is produced in adipocytes, the pituitary, and several other tissues. Animals that are leptin or leptin receptor deficient have major metabolic complications, including obesity. This study tests the hypothesis that the pituitary somatotrope may contribute a source of leptin that maintains some of these metabolic functions. We created 2 different tissue-specific leptin knockout animals: a Somatotrope-Lep-null model and an Adipocyte-Lep-null model. Metabolic analysis of both models, along with a global deletion model, was performed. The Somatotrope-Lep-null animals had fewer somatotropes, and females had a 76% decrease in serum prolactin. During the dark (feeding) phase, females had a 35% increase in ambulation coupled with a 4% increase in EE. Mutants showed no change in food intake or weight gain and EE was unchanged in males. During the light (sleep) phase, Somatotrope-Lep-null mutant males had lower EE and females continued to have higher EE. The respiratory quotients (RQs) of mutants and littermate controls were decreased in males and increased in females; all were within the range that indicates predominant carbohydrate burning. The massively obese Adipocyte-Lep-null animals, however, had significant increases in food intake, sleep, and increased EE, with decreased activity. Changes in RQ were sexually dimorphic, with female mutants having higher RQ and males having decreased RQ. We conclude that both adipocyte and somatotrope leptin contribute to the metabolic homeostasis of the mouse, and that extraadipocyte sources of leptin cannot overcome the major metabolic challenges seen in these animals.

Adipocyte versus pituitary leptin in the regulation of pituitary hormones: somatotropes develop normally in the absence of circulating leptin.

Leptin is a cytokine produced by white fat cells, skeletal muscle, the placenta, and the pituitary gland among other tissues. Best known for its role in regulating appetite and energy expenditure, leptin is produced largely by and in proportion to white fat cells. Leptin is also important to the maintenance and function of the GH cells of the pituitary. This was shown when the deletion of leptin receptors on somatotropes caused decreased numbers of GH cells, decreased circulating GH, and adult-onset obesity. To determine the source of leptin most vital to GH cells and other pituitary cell types, we compared two different leptin knockout models with Cre-lox technology. The global Lep-null model is like the ob/ob mouse, whereby only the entire exon 3 is deleted. The selective adipocyte-Lep-null model lacks adipocyte leptin but retains pituitary leptin, allowing us to investigate the pituitary as a potential source of circulating leptin. Male and female mice lacking adipocyte leptin (Adipocyte-lep-null) did not produce any detectable circulating leptin and were infertile, suggesting that the pituitary does not contribute to serum levels. In the presence of only pituitary leptin, however, these same mutants were able to maintain somatotrope numbers and GH mRNA levels. Serum GH trended low, but values were not significant. However, hypothalamic GHRH mRNA was significantly reduced in these animals. Other serum hormone and pituitary mRNA differences were observed, some of which varied from previous results reported in ob/ob animals. Whereas pituitary leptin is capable of maintaining somatotrope numbers and GH mRNA production, the decreased hypothalamic GHRH mRNA and low (but not significant) serum GH levels indicate an important role for adipocyte leptin in the regulation of GH secretion in the mouse. Thus, normal GH secretion may require the coordinated actions of both adipocyte and pituitary leptin.

Selective deletion of leptin receptors in gonadotropes reveals activin and GnRH-binding sites as leptin targets in support of fertility.

The adipokine, leptin (LEP), is a hormonal gateway, signaling energy stores to appetite-regulatory neurons, permitting reproduction when stores are sufficient. Dual-labeling for LEP receptors (LEPRs) and gonadotropins or GH revealed a 2-fold increase in LEPR during proestrus, some of which was seen in LH gonadotropes. We therefore investigated LEPR functions in gonadotropes with Cre-LoxP technology, deleting the signaling domain of the LEPR (Lepr-exon 17) with Cre-recombinase driven by the rat LH-ß promoter (Lhß-cre). Selectivity of the deletion was validated by organ genotyping and lack of LEPR and responses to LEP by mutant gonadotropes. The mutation had no impact on growth, body weight, the timing of puberty, or pregnancy. Mutant females took 36% longer to produce their first litter and had 50% fewer pups/litter. When the broad impact of the loss of gonadotrope LEPR on all pituitary hormones was studied, mutant diestrous females had reduced serum levels of LH (40%), FSH (70%), and GH (54%) and mRNA levels of Fshß (59%) and inhibin/activin ß A and ß B (25%). Mutant males had reduced serum levels of GH (74%), TSH (31%), and prolactin (69%) and mRNA levels of Gh (31%), Ghrhr (30%), Fshß (22%), and glycoprotein α-subunit (Cga) (22%). Serum levels of LEP and ACTH and mRNA levels of Gnrhr were unchanged. However, binding to GnRH receptors was reduced in LEPR-null LH or FSH gonadotropes by 82% or 89%, respectively, in females (P < .0001) and 27% or 53%, respectively, in males (P < .03). This correlated with reductions in GnRH receptor protein immunolabeling, suggesting that LEP's actions may be posttranscriptional. Collectively, these studies highlight the importance of LEP to gonadotropes with GnRH-binding sites and activin as potential targets. LEP may modulate population growth, adjusting the number of offspring to the availability of food supplies.

Ghrelin restoration of function in vitro in somatotropes from male mice lacking the Janus kinase (JAK)-binding site of the leptin receptor.

Deletion of the signaling domain of leptin receptors selectively in somatotropes, with Cre-loxP technology, reduced the percentage of immunolabeled GH cells and serum GH. We hypothesized that the deficit occurred when leptin's postnatal surge failed to stimulate an expansion in the cell population. To learn more about the deficiency in GH cells, we tested their expression of GHRH receptors and GH mRNA and the restorative potential of secretagogue stimulation in vitro. In freshly plated dissociated pituitary cells from control male mice, GHRH alone (0.3 nM) increased the percentage of immunolabeled GH cells from 27 ± 0.05% (vehicle) to 42 ± 1.8% (P < .002) and the secretion of GH 1.8-3×. Deletion mutant pituitary cells showed a 40% reduction in percentages of immunolabeled GH cells (16.7 ± 0.4%), which correlated with a 47% reduction in basal GH levels (50 ng/mL control; 26.7 ng/mL mutants P = .01). A 50% reduction in the percentage of mutant cells expressing GHRH receptors (to 12%) correlated with no or reduced responses to GHRH. Ghrelin alone (10 nM) stimulated more GH cells in mutants (from 16.7-23%). When added with 1-3 nM GHRH, ghrelin restored GH cell percentages and GH secretion to levels similar to those of stimulated controls. Counts of somatotropes labeled for GH mRNA confirmed normal percentages of somatotropes in the population. These discoveries suggest that leptin may optimize somatotrope function by facilitating expression of membrane GHRH receptors and the production or maintenance of GH stores.

Ablation of leptin signaling to somatotropes: changes in metabolic factors that cause obesity.

Mice with somatotrope-specific deletion of the Janus kinase binding site in leptin receptors are GH deficient as young adults and become obese by 6 months of age. This study focused on the metabolic status of young (3-4.5 month old) preobese mutant mice. These mutants had normal body weights, lean body mass, serum leptin, glucose, and triglycerides. Mutant males and females showed significantly higher respiratory quotients (RQ) and lower energy output, resulting from a higher volume of CO(2) output and lower volume of O(2) consumption. Deletion mutant females were significantly less active than controls; they had higher levels of total serum ghrelin and ate more food. Mutant females also had lower serum insulin and higher glucagon. In contrast, deletion mutant males were not hyperphagic, but they were more active and spent less time sleeping. Adiponectin and resistin, both products of adipocytes, were increased in male and female mutant mice. In addition, mutant males showed an increase in circulating levels of the potent lipogenic hormone, glucose-dependent insulinotropic peptide. Taken together, these results indicate that mutant mice may become obese due to a reduction in lipid oxidation and energy expenditure. This may stem from GH deficiency. Reduced fat oxidation and enhanced insulin sensitivity (in females) are directly related to GH deficiency in mutant mice because GH has been shown by others to increase insulin sensitivity and fat oxidation and reduce carbohydrate oxidation. Gender-dependent alterations in metabolic signals may further exacerbate the future obese phenotype and affect the timing of its onset. Females show a delay in onset of obesity, perhaps because of their low serum insulin, which is lipogenic, whereas young males already have higher levels of the lipogenic hormone, glucose-dependent insulinotropic peptide. These findings signify that leptin signals to somatotropes are vital for the normal metabolic activity needed to optimize body composition.

The somatotrope as a metabolic sensor: deletion of leptin receptors causes obesity.

Leptin, the product of the Lep gene, reports levels of adiposity to the hypothalamus and other regulatory cells, including pituitary somatotropes, which secrete GH. Leptin deficiency is associated with a decline in somatotrope numbers and function, suggesting that leptin may be important in their maintenance. This hypothesis was tested in a new animal model in which exon 17 of the leptin receptor (Lepr) protein was selectively deleted in somatotropes by Cre-loxP technology. Organ genotyping confirmed the recombination of the floxed LepR allele only in the pituitary. Deletion mutant mice showed a 72% reduction in pituitary cells bearing leptin receptor (LEPR)-b, a 43% reduction in LEPR proteins and a 60% reduction in percentages of immunopositive GH cells, which correlated with reduced serum GH. In mutants, LEPR expression by other pituitary cells was like that of normal animals. Leptin stimulated phosphorylated Signal transducer and activator of transcription 3 expression in somatotropes from normal animals but not from mutants. Pituitary weights, cell numbers, IGF-I, and the timing of puberty were not different from control values. Growth curves were normal during the first 3 months. Deletion mutant mice became approximately 30-46% heavier than controls with age, which was attributed to an increase in fat mass. Serum leptin levels were either normal in younger animals or reflected the level of obesity in older animals. The specific ablation of the Lepr exon 17 gene in somatotropes resulted in GH deficiency with a consequential reduction in lipolytic activity normally maintained by GH and increased adiposity.

Fasting and glucose effects on pituitary leptin expression: is leptin a local signal for nutrient status?

Leptin, a potent anorexigenic hormone, is found in the anterior pituitary (AP). The aim of this study was to determine whether and how pituitary leptin-bearing cells are regulated by nutritional status. Male rats showed 64% reductions in pituitary leptin mRNA 24 hr after fasting, accompanied by significant (30-50%) reductions in growth hormone (GH), prolactin, and luteinizing hormone (LH), and 70-80% reductions in target cells for gonadotropin-releasing hormone or growth hormone-releasing hormone. There was a 2-fold increase in corticotropes. Subsets (22%) of pituitary cells coexpressed leptin and GH, and <5% coexpressed leptin and LH, prolactin, thyroid-stimulating hormone, or adrenocorticotropic hormone. Fasting resulted in significant (55-75%) losses in cells with leptin proteins or mRNA, and GH or LH. To determine whether restoration of serum glucose could rescue leptin, LH, and GH, additional fasted rats were given 10% glucose water for 24 hr. Restoring serum glucose in fasted rats resulted in pituitary cell populations with normal levels of leptin and GH and LH cells. Similarly, LH and GH cells were restored in vitro after populations from fasted rats were treated for as little as 1 hr in 10-100 pg/ml leptin. These correlative changes in pituitary leptin, LH, and GH, coupled with leptin's rapid restoration of GH and LH in vitro, suggest that pituitary leptin may signal nutritional changes. Collectively, the findings suggest that pituitary leptin expression could be coupled to glucose sensors like glucokinase to facilitate rapid responses by the neuroendocrine system to nutritional cues.

Anterior pituitary leptin expression changes in different reproductive states: in vitro stimulation by gonadotropin-releasing hormone.

This study was designed to learn more about the changes in expression of rat anterior pituitary (AP) leptin during the estrous cycle. QRT-PCR assays of cycling rat AP leptin mRNA showed 2-fold increases from metestrus to diestrus followed by an 86% decrease on the morning of proestrus. Percentages of leptin cells increased in proestrus and pregnancy to 55-60% of AP cells. Dual labeling for leptin proteins and growth hormone (GH) or gonadotropins showed that the rise in leptin protein-bearing cells from diestrus to proestrus was mainly in GH cells. Only 10-20% of leptin cells in male or cycling female rats coexpress gonadotropins. In contrast, 50-73% of leptin cells from pregnant or lactating females coexpress gonadotropins and only 19% coexpress GH, indicating plasticity in the distribution of leptin. Leptin cells expressed GnRH receptors, and estrogen and GnRH together increased the coexpression of leptin mRNA and gonadotropins. GnRH increased cellular leptin proteins three to four times and mRNA 9.8 times in proestrous rats and stimulated leptin secretion in cultures from diestrous, proestrous, and pregnant rats. These regulatory influences, and the high expression of AP leptin during proestrus and pregnancy, suggest a supportive role for leptin during key events involved with reproduction.

Bipotential effects of estrogen on growth hormone synthesis and storage in vitro.

Increased pulses of serum GH coincide with rising estrogens during the reproductive cycle, suggesting estrogen regulation. However, there is lack of agreement about estrogen's direct effects on the pituitary. Pituitaries from cycling female rats were dispersed and plated for 24 h in defined media containing vehicle or 0.001-250 nm 17beta-estradiol. Estrogen (0.01-10 nm) increased the percentages of GH antigen-bearing cells in the anterior pituitary significantly (1.3- to 1.6-fold) and 0.01-1 nm concentrations also stimulated significant increases in GH mRNA-bearing cells and in the integrated OD for GH mRNA. However, 100-250 nm either had no effect or, inhibitory effects on the area of label for GH mRNA. To test estrogen's effects on expression of GHRH receptors, cultures were stimulated with biotinylated analogs of GHRH and target cells detected by affinity cytochemistry. Estrogen increased GHRH target cells in populations from rats in all stages of the cycle tested. Basal expression of GHRH target cells declined at metestrus. Cultures treated with 0-1 nm estrogen were then dual labeled for bio-GHRH followed by immunolabeling for GH with the antirabbit IgG-ImmPRESS peroxidase polymer. Over 98% of GH cells bound GHRH and 90-96% of GHRH-bound cells contained GH in all treatment groups. Thus, low concentrations of estrogen may stimulate expression of more cells with GH proteins, biotinylated GHRH binding sites, and GH mRNA, whereas high concentrations have no effect, or may reduce GH mRNA. These bipotential effects may help explain the different findings reported in the literature.

Regulation of leptin mRNA and protein expression in pituitary somatotropes.

Leptin, the ob protein, regulates food intake and satiety and can be found in the anterior pituitary. Leptin antigens and mRNA were studied in the anterior pituitary (AP) cells of male and female rats to learn more about its regulation. Leptin antigens were found in over 40% of cells in diestrous or proestrous female rats and in male rats. Lower percentages of AP cells were seen in the estrous population (21 +/- 7%). During peak expression of antigens, co-expression of leptin and growth hormone (GH) was found in 27 +/- 4% of AP cells. Affinity cytochemistry studies detected 24 +/- 3% of AP cells with leptin proteins and growth hormone releasing hormone (GHRH) receptors. These data suggested that somatotropes were a significant source of leptin. To test regulatory factors, estrous and diestrous AP populations were treated with estrogen (100 pM) and/or GHRH (2 nM) to learn if either would increase leptin expression in GH cells. To rule out the possibility that the immunoreactive leptin was bound to receptors in somatotropes, leptin mRNA was also detected by non-radioactive in situ hybridization in this group of cells. In estrous female rats, 39 +/- 0.9% of AP cells expressed leptin mRNA, indicating that the potential for leptin production was greater than predicted from the immunolabeling. Estrogen and GHRH together (but not alone) increased percentages of cells with leptin protein (41 +/- 9%) or mRNA (57 +/- 5%). Estrogen and GHRH also increased the percentages of AP cells that co-express leptin mRNA and GH antigens from 20 +/- 2% of AP cells to 37 +/- 5%. Although the significance of leptin in GH cells is not understood, it is clearly increased after stimulation with GHRH and estrogen. Because GH cells also have leptin receptors, this AP leptin may be an autocrine or paracrine regulator of pituitary cell function.

Comparison of single dose and three dose antibiotic prophylaxis with cefotaxime sodium in cholecystectomy.

BACKGROUND: This study was conducted in Surgical Unit-I at Pakistan Institute of Medical Sciences (PIMS), Islamabad from October 2000 to March 2002. The objective of the study was to compare the results of single dose versus three-dose prophylaxis by cefotaxime sodium in patients undergoing elective cholecystectomy. METHODS: Intravenous Cefotaxime sodium as a prophylaxis was used in 150 patients who underwent elective cholecystectomy. Half of the patients were given single dose one hour before surgery (Group A) while the other half (Group B) was given three doses, first one hour before surgery, second and third were given at 8 hour interval after surgery. Postoperative hospital stay of all but two patients was not more than three days. Evidence of wound infection was observed for 4 weeks post-operative in surgical out patient department in both groups. RESULTS: Three patients in group A and four in group B got wound infection. The difference was not statistically significant. CONCLUSION: Single preoperative dose can be recommended in cholecystectomy as it is less costly and has the same prophylactic benefits as of single dose.