Transcriptomic Profiling of Lactotroph Pituitary Neuroendocrine Tumors via RNA Sequencing and Ingenuity Pathway Analysis.

INTRODUCTION: Lactotroph pituitary neuroendocrine tumors (PitNETs) are common pituitary tumors, but their underlying molecular mechanisms remain unclear. This study aimed to investigate the transcriptomic landscape of lactotroph PitNETs and identify potential molecular mechanisms and therapeutic targets through RNA sequencing and ingenuity pathway analysis (IPA). METHODS: Lactotroph PitNET tissues from five surgical cases without dopamine agonist treatment underwent RNA sequencing. Normal pituitary tissues from 3 patients served as controls. Differentially expressed genes (DEGs) were identified, and the functional pathways and gene networks were explored by IPA. RESULTS: Transcriptome analysis revealed that lactotroph PitNETs had gene expression patterns that were distinct from normal pituitary tissues. We identified 1,172 upregulated DEGs, including nine long intergenic noncoding RNAs (lincRNAs) belonging to the top 30 DEGs. IPA of the upregulated DEGs showed that the estrogen receptor signaling, oxidative phosphorylation signaling, and EIF signaling were activated. In gene network analysis, key upstream regulators, such as EGR1, PRKACA, PITX2, CREB1, and JUND, may play critical roles in lactotroph PitNETs. CONCLUSION: This study provides a comprehensive transcriptomic profile of lactotroph PitNETs and highlights the potential involvement of lincRNAs and specific signaling pathways in tumor pathogenesis. The identified upstream regulators may be potential therapeutic targets for future investigations.

Endothelial cells regulate alveolar morphogenesis by constructing basement membranes acting as a scaffold for myofibroblasts.

Alveologenesis is a spatially coordinated morphogenetic event, during which alveolar myofibroblasts surround the terminal sacs constructed by epithelial cells and endothelial cells (ECs), then contract to form secondary septa to generate alveoli in the lungs. Recent studies have demonstrated the important role of alveolar ECs in this morphogenetic event. However, the mechanisms underlying EC-mediated alveologenesis remain unknown. Herein, we show that ECs regulate alveologenesis by constructing basement membranes (BMs) acting as a scaffold for myofibroblasts to induce septa formation through activating mechanical signaling. Rap1, a small GTPase of the Ras superfamily, is known to stimulate integrin-mediated cell adhesions. EC-specific Rap1-deficient (Rap1iECKO) mice exhibit impaired septa formation and hypo-alveolarization due to the decreased mechanical signaling in myofibroblasts. In Rap1iECKO mice, ECs fail to stimulate integrin ß1 to recruit Collagen type IV (Col-4) into BMs required for myofibroblast-mediated septa formation. Consistently, EC-specific integrin ß1-deficient mice show hypo-alveolarization, defective mechanical signaling in myofibroblasts, and disorganized BMs. These data demonstrate that alveolar ECs promote integrin ß1-mediated Col-4 recruitment in a Rap1-dependent manner, thereby constructing BMs acting as a scaffold for myofibroblasts to induce mechanical signal-mediated alveologenesis. Thus, this study unveils a mechanism of organ morphogenesis mediated by ECs through intrinsic functions.

Chronic estradiol exposure suppresses luteinizing hormone surge without affecting kisspeptin neurons and estrogen receptor alpha in anteroventral periventricular nucleus†.

Mammalian ovulation is induced by a luteinizing hormone surge, which is triggered by elevated plasma estrogen levels; however, chronic exposure to high levels of estradiol is known to inhibit luteinizing hormone secretion. In the present study, we hypothesized that the inhibition of the luteinizing hormone surge by chronic estradiol exposure is due to the downregulation of the estrogen receptor alpha in kisspeptin neurons at hypothalamic anteroventral periventricular nucleus, which is known as the gonadotropin-releasing hormone/luteinizing hormone surge generator. Animals exposed to estradiol for 2 days showed an luteinizing hormone surge, whereas those exposed for 14 days showed a significant suppression of luteinizing hormone. Chronic estradiol exposure did not affect the number of kisspeptin neurons and the percentage of kisspeptin neurons with estrogen receptor alpha or c-Fos in anteroventral periventricular nucleus, but it did affect the number of kisspeptin neurons in arcuate nucleus. Furthermore, chronic estradiol exposure did not affect gonadotropin-releasing hormone neurons. In the pituitary, 14-day estradiol exposure significantly reduced the expression of Lhb mRNA and LHß-immunoreactive areas. Gonadotropin-releasing hormone-induced luteinizing hormone release was also reduced significantly by 14-day estradiol exposure. We revealed that the suppression of an luteinizing hormone surge by chronic estradiol exposure was induced in association with the significant reduction in kisspeptin neurons in arcuate nucleus, luteinizing hormone expression in the pituitary, and pituitary responsiveness to gonadotropin-releasing hormone, and this was not caused by changes in the estrogen receptor alpha-expressing kisspeptin neurons in anteroventral periventricular nucleus and gonadotropin-releasing hormone neurons, which are responsible for estradiol positive feedback.

Sex and interspecies differences in ESR2-expressing cell distributions in mouse and rat brains.

BACKGROUND: ESR2, a nuclear estrogen receptor also known as estrogen receptor ß, is expressed in the brain and contributes to the actions of estrogen in various physiological phenomena. However, its expression profiles in the brain have long been debated because of difficulties in detecting ESR2-expressing cells. In the present study, we aimed to determine the distribution of ESR2 in rodent brains, as well as its sex and interspecies differences, using immunohistochemical detection with a well-validated anti-ESR2 antibody (PPZ0506). METHODS: To determine the expression profiles of ESR2 protein in rodent brains, whole brain sections from mice and rats of both sexes were subjected to immunostaining for ESR2. In addition, to evaluate the effects of circulating estrogen on ESR2 expression profiles, ovariectomized female mice and rats were treated with low or high doses of estrogen, and the resulting numbers of ESR2-immunopositive cells were analyzed. Welch's t-test was used for comparisons between two groups for sex differences, and one-way analysis of variance followed by the Tukey-Kramer test were used for comparisons among multiple groups with different estrogen treatments. RESULTS: ESR2-immunopositive cells were observed in several subregions of mouse and rat brains, including the preoptic area, extended amygdala, hypothalamus, mesencephalon, and cerebral cortex. Their distribution profiles exhibited sex and interspecies differences. In addition, low-dose estrogen treatment in ovariectomized female mice and rats tended to increase the numbers of ESR2-immunopositive cells, whereas high-dose estrogen treatment tended to decrease these numbers. CONCLUSIONS: Immunohistochemistry using the well-validated PPZ0506 antibody revealed a more localized expression of ESR2 protein in rodent brains than has previously been reported. Furthermore, there were marked sex and interspecies differences in its distribution. Our histological analyses also revealed estrogen-dependent changes in ESR2 expression levels in female brains. These findings will be helpful for understanding the ESR2-mediated actions of estrogen in the brain.

Although the brain is a major target organ of estrogens, the distribution of estrogen receptors in the brain is not fully understood. ESR2, also known as estrogen receptor ß, is an estrogen receptor subtype; its localization in the brain has long been controversial because it has traditionally been difficult to detect. In the present study, we analyzed the expression sites of ESR2 in mouse and rat brains using immunohistochemistry with a well-validated antibody, PPZ0506. The immunohistochemical analysis revealed a more localized expression of ESR2 protein in brain subregions than has previously been reported. Additionally, there were clear sex and interspecies differences in the distribution of this protein. We also observed changes in ESR2 expression in the female brain in response to circulating estrogen levels. Our results, which show the precise expression profiles of ESR2 protein in rodent brains, will be helpful for understanding the ESR2-mediated actions of estrogen.

Estrogen Receptor α Isoforms Generated by Alternative Use of Cryptic Exons.

Estrogen receptor α (ERα) regulates several physiological functions. In pathophysiological conditions, ERα is involved in the development and progression of estrogen-sensitive tumors. The ERα gene contains multiple 5'-untranslated exons and eight conventional coding exons and presents multiple isoforms generated by alternative promoter usage and alternative splicing. This gene also possesses non-conventional exons in the 3'- and intronic regions, and alternative use of cryptic exons contributes to further diversity of ERα mRNAs and proteins. Recently, the genomic organization of ERα genes and the splicing profiles of their transcripts were comparatively analyzed in humans, mice, and rats, and multiple ERα isoforms with distinct structures and functions were identified. These transcripts contain cryptic sequences that encode insertion-containing or truncated ERα proteins. In particular, alternative cryptic exons with in-frame stop codons yield transcripts encoding C-terminally-truncated ERα proteins. The C-terminally-truncated ERα isoforms lack part or all of the ligand-binding domain but possess an isoform-specific sequence. Some of these isoforms exhibit constitutive transactivation and resistance to estrogen receptor antagonists. Although numerous studies have reported conflicting results regarding their functions, the critical determinant for their gain-of-function has been identified structurally. Here we review recent progress in ERα variant research concerning the genomic organization of ERα genes, splicing profiles of ERα transcripts, and transactivation properties of ERα isoforms.

Recent Advances in High-sensitivity In Situ Hybridization and Costs and Benefits to Consider When Employing These Methods.

In situ hybridization (ISH), which visualizes nucleic acids in tissues and cells, is a powerful tool in histology and pathology. Over 50 years since its invention, multiple attempts have been made to increase the sensitivity and simplicity of these methods. Therefore, several highly sensitive in situ hybridization methods have been developed that offer researchers a wide range of options. When selecting these in situ hybridization variants, their signal-amplification principles and characteristics must be understood. In addition, from a practical point of view, a method with good monetary and time-cost performance must be chosen. This review introduces recent high-sensitivity in situ hybridization variants and presents their principles, characteristics, and costs.

Central injection of neuropeptide B induces luteinizing hormone release in male and female rats.

Neuropeptide B (NPB) has been identified as an endogenous peptide ligand for the orphan receptor NPBWR1. However, the effect of NPB on the central regulatory mechanisms of reproductive functions remains unclear. Our findings indicated the presence of Npb, Npw (which is another ligand for NPBWR1), and Npbwr1 mRNA in the hypothalamus of male and female rats at each stage of the estrous cycle. Npb mRNA expression was found to be significantly higher in diestrus compared to estrus. The expression of Npw mRNA was one order of magnitude lower than that of Npb mRNA, and Npw mRNA expression in diestrus was significantly higher than that in the other stages of the estrous cycle. Furthermore, Npbwr1 mRNA expression was found to be significantly higher in diestrus compared to the other stages of the estrous cycle and intact males. Notably, estrogen did not alter the expression of Npb, Npw, and Npbwr1 mRNAs in the hypothalamus of females. Central injection of NPB increased plasma luteinizing hormone (LH) levels in both intact males and estrogen-primed ovariectomized females but not in ovariectomized females. These results suggest that NPB-NPBWR1 signaling would be a facilitatory regulatory mechanism in the reproductive function of male and female rats. To the best of our knowledge, this study is the first report to describe the central role of NPB-NPBWR1 signaling in LH regulation in mammals.

Short-term depletion of plasma estrogen affects hypothalamic kisspeptin-neurokinin B-dynorphin A neurons, gonadotrophs, and pulsatile luteinizing hormone secretion in female rats.

Kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the arcuate nucleus (ARC) regulate pulsatile luteinizing hormone (LH) secretion. These neurons express estrogen receptors and are negatively regulated by estrogen. This study aimed to determine whether estrogen supplementation after short-term ovariectomy-induced estrogen depletion has different effects on KNDy neurons depending on the timing of the supplementation. To decrease endogenous estradiol (E2) for a short time, adult female rats received a tube filled with E2 one week after ovariectomy and utilized it one week later (O1w + E). From the results of immunohistochemistry, the response to E2 was attenuated in KNDy neurons of O1w + E rats. Enlarged LH-secreting cells in the anterior pituitary were found in O1w + E rats; however, such enlarged LH cells were not found in ones without previous short-term E2 depletion. From the analysis of LH pulses, plasma LH levels were increased in O1w + E rats relative to ones without previous short-term E2 depletion. These results suggested that once endogenous sex steroids were depleted, the response to E2 in hypothalamic KNDy neurons did not fully recover in one week. Thus, short-term sex steroid depletion due to gonadectomy could alter the response to the sex steroids in KNDy neurons even though the period without sex steroids is only one week, and the alteration is likely to affect plasma hormone levels.

Long-term effects of prenatal undernutrition on female rat hypothalamic KNDy neurons.

The nutritional environment during development periods induces metabolic programming, leading to metabolic disorders and detrimental influences on human reproductive health. This study aimed to determine the long-term adverse effect of intrauterine malnutrition on the reproductive center kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus (ARC) of female offspring. Twelve pregnant rats were divided into ad-lib-fed (control, n = 6) and 50% undernutrition (UN, n = 6) groups. The UN group was restricted to 50% daily food intake of the control dams from gestation day 9 until term delivery. Differences between the two groups in terms of various maternal parameters, including body weight (BW), pregnancy duration, and litter size, as well as birth weight, puberty onset, estrous cyclicity, pulsatile luteinizing hormone (LH) secretion, and hypothalamic gene expression of offspring, were determined. Female offspring of UN dams exhibited low BW from birth to 3 weeks, whereas UN offspring showed signs of precocious puberty; hypothalamic Tac3 (a neurokinin B gene) expression was increased in prepubertal UN offspring, and the BW at the virginal opening was lower in UN offspring than that in the control group. Interestingly, the UN offspring showed significant decreases in the number of KNDy gene-expressing cells after 29 weeks of age, but the number of ARC kisspeptin-immunoreactive cells, pulsatile LH secretions, and estrous cyclicity were comparable between the groups. In conclusion, intrauterine undernutrition induced various changes in KNDy gene expression depending on the life stage. Thus, intrauterine undernutrition affected hypothalamic developmental programming in female rats.

Optimized Mouse-on-mouse Immunohistochemical Detection of Mouse ESR2 Proteins with PPZ0506 Monoclonal Antibody.

Despite the physiological significance of ESR2, a lack of well-validated detection systems for ESR2 proteins has hindered progress in ESR2 research. Thus, recent identification of a specific anti-human ESR2 monoclonal antibody (PPZ0506) and its specific cross-reactivity against mouse and rat ESR2 proteins heightened momenta toward development of appropriate immunohistochemical detection systems for rodent ESR2 proteins. Building upon our previous optimization of ESR2 immunohistochemical detection in rats using PPZ0506, in this study, we further aimed to optimize mouse-on-mouse immunohistochemical detection using PPZ0506. Our assessment of several staining conditions using paraffin-embedded ovary sections revealed that intense heat-induced antigen retrieval, appropriate blocking, and appropriate antibody dilutions were necessary for optimization of mouse-on-mouse immunohistochemistry. Subsequently, we applied the optimized immunostaining method to determine expression profiles of mouse ESR2 proteins in peripheral tissues and brain subregions. Our analyses revealed more localized distribution of mouse ESR2 proteins than previously assumed. Moreover, comparison of these results with those obtained in humans and rats using PPZ0506 revealed interspecies differences in ESR2 expression. We expect that our optimized methodology for immunohistochemical staining of mouse ESR2 proteins will help researchers to solve multiple lines of controversial evidence concerning ESR2 expression.

Optimization of immunohistochemical detection of rat ESR2 proteins with well-validated monoclonal antibody PPZ0506.

Although there are few well-validated antibodies against ESR2 proteins, a recent validation assessment identified a specific monoclonal antibody against human ESR2 proteins (PPZ0506). Furthermore, our previous study confirmed its cross-reactivity and specificity against rodent ESR2 proteins, enabling the determination of true ESR2 distribution profiles in rodents. Therefore, we aimed to determine optimal conditions for ESR2 detection by PPZ0506 immunostaining and analyze ESR2 distribution in rats. We evaluated several staining conditions using paraffin-embedded and frozen ovary sections. Immunohistochemical staining with PPZ0506 antibody required strong antigen retrieval and appropriate antibody dilution. Subsequent immunohistochemical analysis in multiple tissues under optimized conditions revealed that rat ESR2 proteins are expressed in a more localized manner than previously assumed. Our results suggest that previous immunohistochemical studies using inadequately validated antibodies against ESR2 proteins overestimated their distribution profiles. We expect that optimized immunohistochemical detection with PPZ0506 antibody can help researchers solve several conflicting problems in ESR2 research.

Identification of Novel C-Terminally Truncated Estrogen Receptor ß Variant Transcripts and Their Distribution in Humans.

BACKGROUND: The nuclear receptor genes, including estrogen receptor ß (ERß), contain non-conventional internal and terminal exons, and alternative choice of the exons yields multiple mRNA and protein variants with unique structures and functions. However, the genomic structure of the intronic and 3'-downstream regions of the human ERß gene and the presence of novel ERß variants with non-conventional sequences have not been re-examined in about 20 years. Therefore, we attempted to re-characterize the structure of the human ERß gene and identify novel non-conventional exons and distinct splice variants. METHODS: Rapid amplification of cDNA 3'-end and RT-PCR cloning were used to isolate human ERß mRNA variants from the testis. The identified cDNA sequences were mapped on the human genome assembly. Expression profiles of the variants were assessed by RT-PCR analysis. RESULTS: We cloned multiple ERß mRNA variants with novel nucleotide sequences from the testis and identified several alternative splice sites, 3'-elongation of conventional coding exons, and novel terminal exons in the human ERß gene. The variants encode C-terminally truncated ERß proteins termed ERß6, ERß7, ERßEx. 4L, and ERßEx. 6L. Furthermore, we identified exon 7-defective forms of ERß2/ßcx, ERß4, ERß6, and ERß7. Subsequently, we noted distinct expression patterns of the variants in human peripheral organs and brain subregions. CONCLUSION: This study clarified complicated genomic organization and splicing patterns of the human ERß gene that contribute to the distinct heterogeneity of human ERß mRNAs and proteins.

Quantitative expression data of human estrogen receptor α variants in non-functioning pituitary adenomas obtained by reverse transcription-digital polymerase chain reaction analysis.

Expression profiles of gonadal steroid receptor variants have been reportedly associated with malignancy in breast and prostate cancers [1,2]. However, such associations with pituitary tumors remain unclear. Therefore, the expression levels of the wild-type ESR1 (ERα66) and the ESR1 variants (ERαi34, ERαi45c, and ERαΔ5) transcripts encoding constitutively active ERα proteins with C-terminal truncation in non-functioning pituitary adenomas (NFPAs) were evaluated using reverse transcription-digital polymerase chain reaction. The results revealed that the expression levels of the variants were approximately two orders of magnitude lower than that of ERα66 in NFPAs. These data were based on our previous article entitled "Accurate assessment of estrogen receptor profiles in non-functioning pituitary adenomas using RT-digital PCR and immunohistochemistry" [3].

Accurate assessment of estrogen receptor profiles in non-functioning pituitary adenomas using RT-digital PCR and immunohistochemistry.

BACKGROUND: Non-functioning pituitary adenomas (NFPAs) are common pituitary tumors, and surgery is generally the only treatment option. Few attempts have been made to explore target molecules for the development of NFPA pharmacological treatments. METHOD: We quantitatively assessed the expression profiles of estrogen receptor (ER) transcripts and proteins in NFPA samples, using reverse transcription-digital polymerase chain reaction (RT-dPCR) and immunohistochemistry, and further investigated the correlations between the expression levels of ER and those of downstream responsive genes. All patients had undergone surgery at the same high-volume hospital. A total of 20 patients with NFPAs were included. All patients were new-onset, and none were diagnosed with intratumoral hemorrhages or cysts. RESULTS: NFPA samples exhibited a bimodal ESR1 expression pattern and were categorized into significantly different high- and low-ESR1 expression level groups (P < 0.05). In contrast, expression levels of ESR1 variants and ESR2 could barely be detected. Similar results were obtained through the immunohistochemical staining of NFPAs, using well-validated antibodies against ERs. The expression levels of ESR1 positively correlated with those of GREB1, an estrogen-responsive gene [correlation coefficient (r) = 0.623, P = 0.003]. CONCLUSIONS: ESR1 expression levels in NFPAs exhibited a bimodal pattern and were positively correlated with GREB1 expression levels. The accurate assessment of ER expression levels may further advance future NFPA-related research.

GnRH(1-5), a metabolite of gonadotropin-releasing hormone, enhances luteinizing hormone release via activation of kisspeptin neurons in female rats.

Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC), which coexpress neurokinin B and dynorphin, are involved in gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulse generation, while the anteroventral periventricular nucleus (AVPV) kisspeptin neurons are responsible for GnRH/LH surge generation. The present study aims to examine whether GnRH(1-5), a GnRH metabolite, regulates LH release via kisspeptin neurons. GnRH(1-5) was intracerebroventricularly injected to ovariectomized and estrogen-treated Wistar-Imamichi female rats. Immediately after the central GnRH(1-5) administration at 2 nmol, plasma LH concentration increased, resulting in significantly higher levels of the area under the curve and baseline of plasma LH concentrations compared to vehicle-injected controls. On the other hand, in Kiss1 knockout rats, GnRH(1-5) administration failed to affect LH secretion, suggesting that the facilitatory effect of GnRH(1-5) on LH release is mediated by kisspeptin neurons. Double in situ hybridization (ISH) for Kiss1 and Gpr101, a GnRH(1-5) receptor gene, revealed that few Kiss1-expressing cells coexpress Gpr101 in both ARC and AVPV. On the other hand, double ISH for Gpr101 and Slc17a6, a glutamatergic marker gene, revealed that 29.2% of ARC Gpr101-expressing cells coexpress Slc17a6. Further, most of the AVPV and ARC Kiss1-expressing cells coexpress Grin1, a gene encoding a subunit of NMDA receptor. Taken together, these results suggest that the GnRH(1-5)-GPR101 signaling facilitates LH release via indirect activation of kisspeptin neurons and that glutamatergic neurons may mediate the signaling. This provides a new aspect of kisspeptin- and GnRH-neuronal communication with the presence of stimulation from GnRH to kisspeptin neurons in female rats.

Identification of a novel C-terminally truncated estrogen receptor α variant (ERαi34) with constitutive transactivation and estrogen receptor antagonist resistance.

Constitutively active estrogen receptor α (ERα) variants with C-terminal truncation are candidate molecules for gain of both endocrine- and chemotherapy-resistance in estrogen-sensitive tumors. Our previous reports using artificially truncated ERα constructs demonstrated that ERα variants encoded in 1-2-3-cryptic exon- and 1-2-3-4-cryptic exon-types of transcripts have potentials to display constitutive transactivation of an estrogen response element reporter. However, naturally occurring 1-2-3-cryptic exon-type ERα variants have not been cloned yet. Therefore, the present study was designed to identify naturally occurring ERα variants encoded in 1-2-3-cryptic exon-type ERα transcripts. We cloned a novel C-terminally truncated ERα variant (ERαi34) encoded in a 1-2-3-i34 transcript from MCF-7 cells and characterized its constitutive and ER antagonist-resistant transactivation in transfected cells. Stable transfection of the variant into MCF-7 cells augmented basal cell proliferation insensitive to fulvestrant. Collectively, we validated the structure-based mechanisms underlying constitutive and ER antagonist-resistant transactivation by C-terminally truncated ERα variants.

Applicability of Anti-Human Estrogen Receptor ß Antibody PPZ0506 for the Immunodetection of Rodent Estrogen Receptor ß Proteins.

Several lines of controversial evidence concerning estrogen receptor ß (ERß) remain to be solved because of the unavailability of specific antibodies against ERß. The recent validation analysis identified a monoclonal antibody (PPZ0506) with sufficient specificity against human ERß. However, the specificity and cross-reactivity of PPZ0506 antibody against ERß proteins from laboratory animals have not been confirmed. In the present study, we aimed to validate the applicability of PPZ0506 to rodent studies. The antibody exhibited specific cross-reactivity against mouse and rat ERß proteins in immunoblot and immunocytochemical experiments using transfected cells. In immunohistochemistry for rat tissue sections, PPZ0506 showed immunoreactive signals in the ovary, prostate, and brain. These immunohistochemical profiles of rat ERß proteins in rat tissues accord well with its mRNA expression patterns. Although the antibody was reported to show the moderate signals in human testis, no immunoreactive signals were observed in rat testis. Subsequent RT-PCR analysis revealed that this species difference in ERß expression resulted from different expression profiles related to the alternative promoter usage between humans and rats. In conclusion, we confirmed applicability of PPZ0506 for rodent ERß studies, and our results provide a fundamental basis for further examination of ERß functions.

Low-noise 750 MHz spaced ytterbium fiber frequency combs.

We demonstrate two low-noise 750 MHz ytterbium fiber frequency combs that are independently stabilized to a continuous-wave laser. A bulk electro-optic modulator and a single-stack piezo-electric transducer are employed as fast actuators for stabilizing the respective cavity length to heterodyne beat notes. Both combs exhibit in-loop fractional frequency instabilities of ∼10-18 at 1 s. To the best of our knowledge, this is the first demonstration of tightly phase-locked (<1 rad root mean square phase noise integrated from 0.1 Hz to 10 MHz) fiber frequency combs with 750 MHz fundamental repetition rate.

Implementing a standardised discharge analgesia guideline to reduce paediatric post tonsillectomy pain.

OBJECTIVES: To reduce readmission for pain control post-paediatric tonsillectomy. INTRODUCTION: Paediatric tonsillectomy is a common procedure in the UK. Uncontrolled pain at home is a common reason for re-admission and therefore adequate analgesic control following paediatric tonsillectomy is vital for a smooth post-operative recovery. Analgesic regimens at a district general hospital in England were audited and a standardised protocol was subsequently implemented. METHODS: A retrospective audit from September 2014 to August 2015 was completed. Discharge analgesic regimens and readmission rates post-tonsillectomy for recurrent tonsillitis in 2-17 year-old children were studied in a large general hospital in the United Kingdom. A standardised weight-based algorithm was used to dose scheduled regular paracetamol for 2 weeks. Second cycle prospective audit ran from December 2015 to November 2016. RESULTS: In cycle 1, 151 children (mean age, 7.9 years) underwent tonsillectomy for tonsillitis, 25 (16.6%) of whom were readmitted. 12 (7.9%) experienced postoperative haemorrhage, 13 (8.6%) required pain control, and one (1.2%) had infection. The discharging analgesic regimen varied widely and often included purchase of over-the-counter ibuprofen and paracetamol. In cycle 2, 118 children (mean age, 8.8 years) underwent tonsillectomy, 17 (14.4%) were readmitted; 12 (10.2%) had post-operative haemorrhage, 0 needed pain control, 5 (4.2%) had other problems. There was a significant reduction in readmission for pain control (p = 0.0027) from 7.3% to 0% in the study. There was no significant change in overall readmission rate (16.6%-14.4%) or postoperative haemorrhage rate (8.9% overall). DISCUSSION: Analgesia prescription post tonsillectomy varies widely and over the counter prescriptions of ibuprofen and paracetamol is based on age rather than weight with patients receiving inadequate analgesic doses. A readily available standardised postoperative analgesic protocol can significantly reduce readmission rates for pain control following paediatric tonsillectomy.