Abstract
Transcription factors and signaling pathways that regulate stem cells and specialized hormone-producing cells in the pituitary gland have been the subject of intense study and have yielded a ...mechanistic understanding of pituitary organogenesis and disease. However, the regulation of stem cell proliferation and differentiation, the heterogeneity among specialized hormone-producing cells, and the role of nonendocrine cells in the gland remain important, unanswered questions. Recent advances in single-cell RNA sequencing (scRNAseq) technologies provide new avenues to address these questions. We performed scRNAseq on ∼13,663 cells pooled from six whole pituitary glands of 7-week-old C57BL/6 male mice. We identified pituitary endocrine and stem cells in silico, as well as other support cell types such as endothelia, connective tissue, and red and white blood cells. Differential gene expression analyses identify known and novel markers of pituitary endocrine and stem cell populations. We demonstrate the value of scRNAseq by in vivo validation of a novel gonadotrope-enriched marker, Foxp2. We present novel scRNAseq data of in vivo pituitary tissue, including data from agnostic clustering algorithms that suggest the presence of a somatotrope subpopulation enriched in sterol/cholesterol synthesis genes. Additionally, we show that incomplete transcriptome annotation can cause false negatives on some scRNAseq platforms that only generate 3′ transcript end sequences, and we use in vivo data to recover reads of the pituitary transcription factor Prop1. Ultimately, scRNAseq technologies represent a significant opportunity to address long-standing questions regarding the development and function of the different populations of the pituitary gland throughout life.
The genetic basis for combined pituitary hormone deficiency (CPHD) is complex, involving 30 genes in a variety of syndromic and nonsyndromic presentations. Molecular diagnosis of this disorder is ...valuable for predicting disease progression, avoiding unnecessary surgery, and family planning. We expect that the application of high throughput sequencing will uncover additional contributing genes and eventually become a valuable tool for molecular diagnosis. For example, in the last 3 years, six new genes have been implicated in CPHD using whole-exome sequencing. In this review, we present a historical perspective on gene discovery for CPHD and predict approaches that may facilitate future gene identification projects conducted by clinicians and basic scientists. Guidelines for systematic reporting of genetic variants and assigning causality are emerging. We apply these guidelines retrospectively to reports of the genetic basis of CPHD and summarize modes of inheritance and penetrance for each of the known genes. In recent years, there have been great improvements in databases of genetic information for diverse populations. Some issues remain that make molecular diagnosis challenging in some cases. These include the inherent genetic complexity of this disorder, technical challenges like uneven coverage, differing results from variant calling and interpretation pipelines, the number of tolerated genetic alterations, and imperfect methods for predicting pathogenicity. We discuss approaches for future research in the genetics of CPHD.
Instant blood mediated inflammatory reaction (IBMIR) occurs when islets are exposed to blood and manifests clinically as portal vein thrombosis and graft failure. The aim of this study was to ...determine the impact of recombinant human activated protein C (rhAPC) and platelet inhibition on IBMIR in order to develop a better targeted treatment for this condition. Five thousand human islet cell equivalents (IEQ) were mixed in a PVC loop system with 7 mL of ABO compatible human blood and incubated with rhAPC, either alone or in combination with tirofiban. Admixing human islets and blood caused rapid clot formation, consumption of platelets, leukocytes, fibrinogen, coagulation factors and raised d‐dimers. Islets were encased in a fibrin and platelet clot heavily infiltrated with neutrophils. Tirofiban monotherapy was ineffective, whereas rhAPC monotherapy prevented IBMIR in a dose‐dependent manner, preserving islet integrity while maintaining platelet and leukocyte counts, fibrinogen and coagulation factor levels, and reducing d‐dimer formation. The combination of tirofiban and low‐dose rhAPC inhibited IBMIR synergistically with an efficacy equal to high dose rhAPC. Tirofiban and rhAPC worked synergistically to preserve islets, suggesting that co‐inhibition of the platelet and coagulation pathways’ contribution to thrombin generation is required for the optimal anti‐IBMIR effect.
Targeting the platelet and coagulation pathways with tirofiban and rhAPC was found to be more effective at preserving human islets than either agent used alone in an in vitro assay simulating clinical IBMIR.
Abstract
Mammalian genomes contain thousands of genes for long non-coding RNA (lncRNAs) that affect the expression of other genes through diverse mechanisms. The lncRNA transcripts are longer than ...200 nucleotides and are often capped, spliced and polyadenylated, but they are not translated into proteins. Nuclear lncRNAs modify chromatin structure and transcription in trans or cis by interacting with the DNA, forming R-loops, and recruiting regulatory proteins. Not much is known about the role of lncRNA in pituitary gland differentiation and function, but there are several examples that provide a strong premise for investigating this class of transcripts more deeply. For example, the lncRNA MEG3 is a tumor suppressor that is silenced in non-functioning pituitary adenomas; and lncRNAs have been implicated in regulating the function of key transcription factors such as POU1F1, PITX1, and PITX2. To develop a research resource for the study of lncRNA, we used cell-type specific cre transgenes to tag individual adult pituitary cell types with fluorescent markers and enriched for thyrotropes, gonadotropes and somatotropes using fluorescence activated cell sorting. We determined the transcriptome of each cell population using RNA sequencing and mined the data for lncRNA. We detected hundreds of lncRNA in adult pituitary cells, and some were cell-type specific, and located in immediate proximity of genes that encode pituitary hormones or lineage-specific transcription factors. We discovered that the lncRNA Nr5a1os is enriched in gonadotropes, while Foxl2os is enriched in gonadotropes and thyrotropes. The lncRNA Platr9 is located on the opposite strand near Cga, which encodes the alpha subunit of thyrotropin and gonadotropin. We also mined transcriptome data from pituitary glands collected at embryonic days 12.5 and 14.5 and identified 351 unique lncRNAs, many of which are exclusively expressed during development. Among these is Six3os1, which is located upstream of Six3, a gene associated with holoprosencephaly and hypopituitarism. The location of many of these lncRNAs suggests that they may have a cis-acting regulatory role in pituitary development or physiological function. This research resource sets the foundation for examining the actions of lncRNAs on expression of nearby genes during development and in response to physiological demand. Future research will elucidate their mechanisms of action, determining whether they recruit regulatory proteins to the chromatin and/or form R-loops in these regions, and how this affects expression of the target gene(s). We expect these studies to further our understanding of pituitary biology.
Presentation: Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.
We used Northern blot analysis in order to investigate the ontogeny of the murine joining (J)‐chain gene. No J‐chain expression was detected in embryonic tissues, including liver, spleen and ...intestine, but an expression of µ heavy chain was detected in foetal liver at day 17. J‐chain expression was detected in the spleen at day 9 and in the intestine at day 15 after birth. Western blot analysis was carried out in order to compare the protein levels of J and µ heavy chains in serum from day 8 to day 24 after birth, using antihuman J chain and antimouse µ chain antibodies. Although µ chain protein could be detected in serum from day 8, J‐chain protein was detectable only at day 24. These results suggest that the expression of J chain is a later event than the µ chain in the mouse, which thus differs in embryogenesis from humans.
The aly/aly mouse has a severe immunodeficiency, because it lacks peripheral lymph nodes as well as IgA and IgG immunoglobulin synthesis. In the present study, we performed histopathological and ...immunohistological examinations to clarify histological disorders of various immune organs in these mice. Carbon CH40 injections into the apex of the tongue confirmed the absence of submandibular lymph nodes in aly/aly mice. The thymus had a poorly constructed cortex and medulla, and the number of lymphoid follicles was clearly decreased in the spleen. No IgG-or IgA- producing cells were found in any immune organs, including the mucosal immune sites, though several IgM-producing cells were identified. Other characteristic findings included perivascular lymphocytes accumulation in the salivary glands, lungs, liver and pancreas, which caused tissues damage. These results demonstrated that the various lymphoid tissues disorders and organ-specific lymphocyte infiltration cause immuno-deficiency in the aly/aly mouse. (J. Oral Sci. 43, 91-96, 2001)
Renal impairment occurs in neonates receiving indomethacin for treatment of patent ductus arteriosus. Inhibition of cyclooxygenase within the neonatal kidney results in decreased prostaglandin ...synthesis and consequent reduction in renal perfusion. Indomethacin has been reported to cause short-term reduction in glomerular filtration that resolves after cessation of the drug. There is little information on the long-term effects of postnatal exposure to indomethacin. The aim of this study was to determine the incidence of renal impairment in infants treated with indomethacin in a single center, to determine whether there is evidence of renal impairment on day 30 or at discharge, and to identify risk factors for renal impairment. In a retrospective study, infants of less than 30 weeks completed gestation who received indomethacin to close the ductus arteriosus were matched with infants of the same gestation, birth weight, and severity of illness. Serum creatinine and glomerular filtration rates (GFR) were obtained prior to commencing indomethacin and on days 2, 7, and 30 following indomethacin administration. Acute renal failure was defined as an increase in creatinine of greater than 25%. Of those infants who were less than 30 weeks completed gestation, 24% had acute renal failure following indomethacin administration. There was a significant elevation in serum creatinine on day 2 and day 7 ( P<0.0001, P=0.002) and a decrease in GFR on day 2 and day 7 ( P<0.0001, P=0.01) following administration of indomethacin. Renal function had normalized by day 30 or discharge. The incidence of acute renal failure in neonates treated with indomethacin is clinically significant. Renal function returns to normal by day 30. Linear regression found no statistical significance for gestational age, day of indomethacin dosing, Clinical Risk Index for Babies (CRIB) score, and presence of an umbilical artery catheter to confound the effect of indomethacin on renal function.
Experimental results indicate that superconductivity in Sr2RuO4 is described by the p-wave Eu representation of the D4h point group. Results on vortex lattice structures for this representation are ...presented. Theoretical results are compared with experiment. 15 refs.
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