Do children with Osteosarcoma benefit from Pulmonary Metastasectomy? - A Systematic Review Of Published Studies and 'Real world' Outcomes.

OBJECTIVE: To critically examine the evidence-base for survival benefit of Pulmonary metastasectomy (PM) for Osteosarcoma (OS) in the paediatric population. SUMMARY BACKGROUND DATA: PM for OS is recommended as standard of care in both paediatric and adult treatment protocols. Recent results from the PulMiCC trial demonstrate no survival benefit from Pulmonary Metastasectomy (PM) in Colorectal Cancer in adults. METHODS: A systematic review was undertaken according to PRISMA guidelines. Medline, Embase and 2 clinical trials registers were searched for ALL STUDIES detailing paediatric OS patients (<18 y) undergoing PM with a comparison cohort group that did not receive PM. RESULTS: Eleven studies met inclusion criteria dating from 1984 - 2017. All studies were retrospective and none directly compared PM versus No PM in paediatric patients as its main objective(s). Three-year survival rates ranged from 0-54% for PM and 0-16% for no PM. No PM patients were usually those with unresectable disease and/or considered to have a poor prognosis. All studies were at high risk of bias and there was marked heterogeneity in the patient selection. CONCLUSIONS: There is a weak evidence-base (Level IV) for a survival benefit of PM for OS in paediatric patients likely due to selection bias of 'favourable cases'. The included studies many of which detailed outdated treatment protocols were not designed in their reporting to specifically address the questions directly. A randomised controlled trial - whilst ethically challenging in a paediatric population - incorporating modern OS chemotherapy protocols is needed to crucially address any 'survival benefit'.

Steroid efficacy in Meralgia Paresthetica: A systematic review and meta-analysis.

Objective: To determine the efficacy of steroid injections for pain relief in patients with meralgia paresthetica (MP). Methods: All the literature published until March 2023 was explored from several databases, including EBSCO, PubMed, EMBASE, Cochrane Library, Google Scholar, and Scopus. Articles investigating the change in pain status of MP patients after steroid injection were included. The primary outcomes were complete pain relief, pain scores at 15 days and one month after intervention. When compared to the baseline, the secondary outcomes for the steroid group included pain scores at the end of treatment and quality of life, which were further evaluated by two factors, namely mental and physical health. Results: The analysis of the studies validated that steroids were significantly successful in providing complete pain relief (p-value = 0.00001), and in reducing the pain score of patients with meralgia paresthetica at 15 days (p-value = 0.02), but not at one month (p-value = 0.79) as compared to the control group. The analysis did not reveal any significant subgroup differences among various steroids (P = 0.52; CI: 0.01 - 0.10; RR: 0.04; I2 = 0%). Mental health (MD = 4.23; 95% CI = 0.42 to 8.03; p = 0.03, I2 = 0%) was significantly improved in the steroid group when compared with baseline. Conclusion: Steroids injections can play an important role in improving symptoms and complications of meralgia paresthetica, especially in the short term.

For the love of all that is holy, stop prescribing sodium valproate and carbamazepine together.

Sodium valproate and carbamazepine are two time-tested drugs for treatment of epilepsy. Individually, they are usually excellent choices in treating a broad spectrum of epileptic seizures. They are, however, not friendly with each other. Their co-administration affects the drug levels of each other by influencing the action of hepatic enzymes. This write up attempts to give an overview of the mechanism of this drug interaction, and informs readers why this combination is a bad choice. The author hopes that this will help in raising awareness among the physicians regarding the dangers of this prescription, and in putting a full stop to this practice.

Factors affecting prognosis of status epilepticus among patients presenting to a tertiary care hospital.

OBJECTIVES: This study aimed to evaluate the etiology, outcomes and prognostic factors associated with status epilepticus (SE) admissions in Neurology Department of a tertiary care hospital. METHODS: A retrospective review was performed on all SE admissions at Dr. Ruth K.M. Pfau Civil Hospital Karachi over a five-year period from July 2015 to June 2020. Demographic, clinical, and etiological factors were investigated for prognostic value. Statistical tests were applied to determine significant prognostic factors. A five percent significance level was used. RESULTS: A total of 176 patients were included in the study. Mortality was reported in 22 cases (12.5%) and morbidity at six months was observed in 44 cases (25.0%). Male gender, previous history of SE, prolonged seizure duration, and late presentation to hospital were significantly associated with mortality (p<0.05). De novo cases of SE tended to be older (p=0.048) and were associated with morbidity at follow-up (p=0.000). The most common causes of epilepsy in our patients with SE were CNS infections (n=54) and Idiopathic epilepsy (n=34). Non-compliance to medicines/under-dosing was the most common provocative factor (n=68). Acute symptomatic causes of SE were more likely to be associated with greater morbidity (p=0.000). Refractory and super-refractory SE were strongly associated with higher mortality (p=0.000). A longer duration of hospital stay was associated with higher morbidity (p=0.000). CONCLUSION: Male gender, poor control of seizures, CNS infections, prolonged seizures, delayed hospital arrival and refractory/super-refractory status epilepticus were key determinants of mortality in our setting. Previous history of status epilepticus, and acute and symptomatic etiologies were associated with higher morbidity.

Relationship between cerebrospinal fluid protein level and electrophysiologic abnormalities in the acute inflammatory demyelinating polyradiculoneuropathy variant of Guillain-Barré syndrome.

Objective: Guillain-Barré syndrome (GBS) is an autoimmune disease characterized by weakness in limbs or cranial nerve innervated muscles. Acute inflammatory demyelinating polyradiculoneuropathy (AIDP) is the most common variant. Electrophysiologic abnormalities and elevated cerebrospinal fluid (CSF) protein are frequently present in AIDP, but the relationship between these two parameters is not well known. We aimed to fill this gap by studying this relationship. Methods: This was a prospective cross-sectional study conducted for two years in the Department of Neurology, Dr. Ruth K. M. Pfau Civil Hospital, Karachi, Pakistan. All 90 adult patients with the AIDP variant of GBS were selected. Nerve conduction studies were performed to determine the degree of demyelination through the four electrophysiologic demyelination criteria. The CSF sample was sent to lab immediately after lumbar puncture. SPSS version 20.0 was used. The CSF protein level was measured with mean ±SD. Demyelination criteria were measured in frequency and percentages. Chi-square test was applied to a number of demyelination criteria and T-test/ANOVA was applied on mean CSF protein level. Results: We found a mean CSF protein of 37.41 mg/dl (±3.69) with one demyelination criterion, 81.87 mg/dl (±17.39) with two demyelination criteria, 119.75 mg/dl (±31.42) with three demyelination criteria, and 134.00 mg/dl (±42.87) with four demyelination criteria (P-value <0.001). Conclusion: This study demonstrates a significant relationship between CSF protein levels and degree of demyelination in the AIDP variant of GBS. This is an under-researched area in GBS and this study adds favorably to limited data in this regard.

UK paediatric surgical academic output (2005-2020): A cause for concern?

BACKGROUND: The publication record can be regarded as a key metric of the academic output of a craft surgical speciality with an almost exponential increase in the number of such publications worldwide over the past 20 years (Ashfaq et al. J Surg Res 2018;229:10-11). We aimed to examine and explore if this was the experience within UK paediatric surgery centres. METHODS: The academic search engine Scopus™ (Elsevier) was used to track every paediatric surgeon's (NHS or University) publication history between Jan. 2005 - Dec. 2020. This was validated by an algorithmic search of PubMed™. The h-index (citations/publication), considered a validated metric of career academic output, was also calculated for each individual surgeon. A Field-Weighted Citation Index (Scopus™) (FWCI) was used to assess impact of individual publications. Textbooks, book chapters, abstracts, duplications ("double dipping") and output attributed to UK BAPS-CASS national studies were excluded. Some output(s), not considered as relevant to "paediatric surgery", was edited. Data are quoted as median(range). RESULTS: During this 16-year period, there were 3838 publications identified from 26 centres with a "top ten" listing of those paediatric surgical units contributing over half the output (n = 2189, 57%). To look for evidence of trend(s) we analysed the output from these surgical centres in two 5-year periods (2005-9 and 2015-19) and showed an overall fall in output(s) - [730 (53.4%) to 645 (46.4%)] with 6/10 (60%) ' top ten ' centres here recording a reduction in publications. The median h-index of the 232 contributing paediatric surgical consultants was 12 (range 1-56). The best performing publication from the "top ten" centres had 96.5(51-442) citations with the FWCI being 4.5 (2.2 - 30.2). CONCLUSIONS: This study highlights current paediatric surgery publication output metrics in UK centres. There is evidence of a relative reduction in outputs overall which is a cause for concern for the future, although individual publications from the 10 most active units in the UK remain highly cited. These findings may serve purpose in several ways: (i) UK paediatric surgical centre rankings may be helpful for guiding residency / trainee application; (ii) surgical research funding for the top performing units may be better facilitated and finally (iii) UK centres showing a ' fertile ground ' for nurturing and training paediatric surgeons with academic aspirations could be useful for future workforce planning.

Does the Presence of a Hernia Sac Improve Survival in Newborns with Congenital Diaphragmatic Hernia? A United Kingdom Single-Center Experience.

INTRODUCTION: A developing body of literature suggests that the presence of a hernia sac in fetuses with congenital diaphragmatic hernia (CDH) may indicate improved prognosis. By examining a large cohort of CDH newborns admitted to a single United Kingdom specialist center, we aimed to establish if presence of hernia sac is a robust predictor of improved survival. MATERIALS AND METHODS: All CDH patients admitted to a single center were recruited. Postneonatal presentations and Morgagni hernias were excluded. Demographics, defect type, laterality, survival, and hernia recurrence were recorded. RESULTS: In this study, 192 CDH newborns were managed from 1997 to 2017; 39 were excluded (10 Morgagni and 29 postneonatal); 22 (14%) neonates had a hernia sac. Survival in patients with a hernia sac was 21/22 (95%) versus 107/124 (86%) in cases without hernia sac (p = 0.2). There was no difference in hernia sac proportion by gender (male:female 15 vs. 13.2%, p = 0.8). CONCLUSION: In contrast to studies showing a survival advantage, albeit with smaller patient numbers, we report a statistical nonsignificant benefit of hernia sac. Better survival outcomes at this specialist center with CDH patients without a hernia sac than reported in other published studies are likely responsible for the lack of statistical significance observed, despite a larger cohort. National and international CDH registries yielding "big data" may provide further answers on the utility of a CDH hernia sac as a new prognostic scoring tool.

Influence of birth weight on primary surgical management of newborns with esophageal atresia.

BACKGROUND: To determine if birth-weight (BW) influences primary surgical management of newborns undergoing operation for esophageal atresia and tracheo-esophageal fistula (EA-TEF). METHODS: Newborns undergoing repair of esophageal atresia at a single specialist centre between 1999 and 2017 were categorised into three groups based on BW; Group A < 1.5 kg, Group B <2.5 kg and Group C >2.5 kg. Outcome data analysed were (i) technical ability of the surgeon to perform primary esophageal anastomosis, (ii) anastomotic leak, (iii) anastomotic stricture, (iv) esophageal replacement, (v) need for other procedures notably fundoplication, aortopexy, tracheostomy and (vi) mortality. Statistical analysis was performed using a two-tailed Fisher's exact test and logistic regression. RESULTS: 198 patients underwent surgery for EA-TEF during the study period, Group A (n = 13), Group B (n = 73) and Group C (n = 112). Inability to perform a primary anastomosis was significantly higher in Group A vs Group B (p = 0.003) and Group C (p = 0.004). Birthweight was a significant variable in the ability to perform a primary esophageal anastomosis (OR 1.009, p = 0.004). Mortality rate was significantly higher in Group A vs Group C (P = 0.0158). CONCLUSIONS: Very low birth weight infants are less likely to achieve a definitive primary anastomosis during emergent repair of esophageal atresia, and have a higher mortality.

Association Between Hyperuricemia and Acute Ischemic Stroke in Patients at a Tertiary Care Hospital.

Introduction Stroke is the most debilitating of neurologic diseases. The rationale of the current study was to determine the association between hyperuricemia and ischemic stroke to establish a local perspective. Methods A total of 148 patients at a tertiary care hospital in Pakistan who fulfilled the inclusion criteria were enrolled in the study and then equally distributed into two study groups consisting of cases and controls (n = 74 in each group). In this study, there were 36 (48.6%) participants in the case group with hyperuricemia and ischemic stroke and 18 (24.3%) participants in the control group with hyperuricemia. The mean and standard deviations were computed for quantitative variables such as age, body mass index (BMI), and duration of stroke. Frequencies and percentages for the qualitative variables such as gender, hypertension, type 2 diabetes (T2D), dyslipidemia, smoking status, socioeconomic status, educational level, and hyperuricemia were calculated. The chi-square test was applied to compare both groups, with p ≤ 0.05 indicating significance. The odds ratio was also calculated to determine the association between case and control. Effect modifiers were controlled through stratification of age, gender, BMI, duration of stroke, hypertension, T2D, dyslipidemia, socioeconomic status, educational level, and smoking status to determine the effect of these on outcome variables. A post-stratification chi-square test was applied, with p ≤ 0.05 indicating statistical significance. Results In our study, stratification of hyperuricemia into cases and controls was performed for age, gender, T2D, hypertension, dyslipidemia, smoking status, socioeconomic status, and educational status. The maximum results were significant, with high strength of association between both groups. In the case group, the frequency of elevated uric acid was significantly higher than that of the control group. A comparison of hyperuricemia indicated p = 0.002, with an odds ratio of 2.95, which showed that elevated uric acid could be taken as a predictor of ischemic stroke. The uric acid level was significantly higher in men than in women. Additionally, hyperuricemia was associated with dyslipidemia. In patients with ischemic stroke, there was a significant association between serum uric acid level and T2D, hypertension, and smoking. Conclusions This study showed that the prevalence of hyperuricemia in patients with ischemic stroke was significantly higher as compared to the healthy population. Hyperuricemia can be considered as a risk factor for ischemic stroke because of its high prevalence in ischemic stroke patients. Our study explored the relationship between stroke and hyperuricemia and enabled increased understanding for caregivers so that effective management plans can be formulated for patients with ischemic stroke to prevent adverse outcomes.

Etiology and Types of Seizures in Patients Presenting to a Tertiary Care Hospital in Karachi: A Cross-Sectional Study.

Introduction Epilepsy is a burdensome disorder for affected individuals and community. There is limited data available on the epidemiological aspects of seizures in Pakistan and further research is necessary. We aimed to fill this gap by studying this information in epilepsy patients presenting to our neurology department. The purpose of this study is to evaluate the causes and types of seizures among the target population. Method This is a cross-sectional study conducted at the Department of Neurology, Dr. Ruth K.M. Pfau Civil Hospital Karachi. In this study we evaluated the causes and types of seizures among patients presenting to our department during the two-year study duration (January 2018-December 2019). Informed consent was taken. Detailed history was taken including features of seizure episodes, age at first seizure, family history and comorbid conditions. Relevant investigations were carried out. The data was compiled to deduce the relevant information using SPSS v20.0. T-test and Chi-square were used for analyzing the data. Results A total of 996 patients presented during the study duration. Primary seizures were found in 58% cases while secondary seizures were found in 42% cases. This distribution was more equal in children with 49.6% primary seizures and 50.4% secondary seizures; the gap widened in adults with 64.3% primary seizures and 35.7% secondary seizures. The most common cause of secondary seizures was neonatal encephalopathy which was present in 18.7% patients, followed by traumatic head injury in 18.2% patients. Central nervous system (CNS) infection was the cause in 17.9% patients, cerebral tumors in 14.1% patients, stroke in 11.5%, metabolic encephalopathy in 7.4%, febrile seizures in 6.5% and CNS malformations in 5.7% patients. The top three causes in children were neonatal encephalopathy (28.3%), CNS infections (19.3%) and febrile seizures (12.7%). Adults with secondary seizures were diagnosed most often with head trauma (25.2%), cerebral tumors (19.9%) and stroke (18.4%) as causative factors. The most common type of seizures was generalized onset tonic-clonic seizures which was found in 73.0% patients followed by focal to bilateral tonic-clonic seizures in 8.9% patients. Other types of seizures included focal aware seizures in 5.0%, mixed seizure types in 4.2%, focal impaired awareness seizures in 3.1%, absence seizures in 2.7%, myoclonic seizures in 2.0% and atonic seizures in 1.0% patients. Seizures in children were mostly generalized onset tonic-clonic seizures (75.4%), mixed seizure types (5.7%) and focal to bilateral tonic-clonic seizures (5.2%). In adults the three most common types corresponded to the overall result: generalized onset tonic-clonic seizures (71.2%), focal to bilateral tonic-clonic seizures (11.6%) and focal aware seizures (6.6%). Conclusion We found that the most common cause of seizures overall in our study population was primary seizures, though primary and secondary seizures were more evenly present in children. Among secondary causes neonatal encephalopathy stood out as the most common cause in children; head trauma was the predominant cause in adults. Most common type of seizures overall and in adults was generalized onset tonic-clonic seizures, followed by focal to bilateral tonic-clonic and focal aware seizure types. Pediatric patients presented most often with generalized onset tonic-clonic seizures, followed by mixed seizure types and focal to bilateral tonic-clonic seizures.

Iterative Single-Cell Analyses Define the Transcriptome of the First Functional Hematopoietic Stem Cells.

Whereas hundreds of cells in the mouse embryonic aorta transdifferentiate to hematopoietic cells, only very few establish hematopoietic stem cell (HSC) identity at a single time point. The Gata2 transcription factor is essential for HSC generation and function. In contrast to surface-marker-based cell isolation, Gata2-based enrichment provides a direct link to the internal HSC regulatory network. Here, we use iterations of index-sorting of Gata2-expressing intra-aortic hematopoietic cluster (IAHC) cells, single-cell transcriptomics, and functional analyses to connect HSC identity to specific gene expression. Gata2-expressing IAHC cells separate into 5 major transcriptomic clusters. Iterative analyses reveal refined CD31, cKit, and CD27 phenotypic parameters that associate specific molecular profiles in one cluster with distinct HSC and multipotent progenitor function. Thus, by iterations of single-cell approaches, we identify the transcriptome of the first functional HSCs as they emerge in the mouse embryo and localize them to aortic clusters containing 1-2 cells.

Nanog regulates Pou3f1 expression at the exit from pluripotency during gastrulation.

Pluripotency is regulated by a network of transcription factors that maintain early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development is its expression restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses genes involved in anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog Use of Nanog null ES cells further demonstrated a role for Nanog in repressing a subset of anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting neural regulatory networks by repressing Pou3f1 at the onset of gastrulation.This article has an associated First Person interview with the joint first authors of the paper.

Neuro-mesodermal progenitors (NMPs): a comparative study between pluripotent stem cells and embryo-derived populations.

The mammalian embryo's caudal lateral epiblast (CLE) harbours bipotent progenitors, called neural mesodermal progenitors (NMPs), that contribute to the spinal cord and the paraxial mesoderm throughout axial elongation. Here, we performed a single cell analysis of different in vitro NMP populations produced either from embryonic stem cells (ESCs) or epiblast stem cells (EpiSCs) and compared them with E8.25 CLE mouse embryos. In our analysis of this region, our findings challenge the notion that NMPs can be defined by the exclusive co-expression of Sox2 and T at mRNA level. We analyse the in vitro NMP-like populations using a purpose-built support vector machine (SVM) based on the embryo CLE and use it as a classification model to compare the in vivo and in vitro populations. Our results show that NMP differentiation from ESCs leads to heterogeneous progenitor populations with few NMP-like cells, as defined by the SVM algorithm, whereas starting with EpiSCs yields a high proportion of cells with the embryo NMP signature. We find that the population from which the Epi-NMPs are derived in culture contains a node-like population, which suggests that this population probably maintains the expression of T in vitro and thereby a source of NMPs. In conclusion, differentiation of EpiSCs into NMPs reproduces events in vivo and suggests a sequence of events for the emergence of the NMP population.

The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1.

Progenitors of the first hematopoietic cells in the mouse arise in the early embryo from Brachyury-positive multipotent cells in the posterior-proximal region of the epiblast, but the mechanisms that specify primitive blood cells are still largely unknown. Pluripotency factors maintain uncommitted cells of the blastocyst and embryonic stem cells in the pluripotent state. However, little is known about the role played by these factors during later development, despite being expressed in the postimplantation epiblast. Using a dual transgene system for controlled expression at postimplantation stages, we found that Nanog blocks primitive hematopoiesis in the gastrulating embryo, resulting in a loss of red blood cells and downregulation of erythropoietic genes. Accordingly, Nanog-deficient embryonic stem cells are prone to erythropoietic differentiation. Moreover, Nanog expression in adults prevents the maturation of erythroid cells. By analysis of previous data for NANOG binding during stem cell differentiation and CRISPR/Cas9 genome editing, we found that Tal1 is a direct NANOG target. Our results show that Nanog regulates primitive hematopoiesis by directly repressing critical erythroid lineage specifiers.

A single-cell molecular map of mouse gastrulation and early organogenesis.

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.

Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.

Capturing where and how multipotency is lost is crucial to understand how blood formation is controlled. Blood lineage specification is currently thought to occur downstream of multipotent haematopoietic stem cells (HSC). Here we show that, in human, the first lineage restriction events occur within the CD19-CD34+CD38-CD45RA-CD49f+CD90+ (49f+) HSC compartment to generate myelo-lymphoid committed cells with no erythroid differentiation capacity. At single-cell resolution, we observe a continuous but polarised organisation of the 49f+ compartment, where transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. CLEC9AhiCD34lo cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics, whereas CLEC9AloCD34hi cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. We thus propose that human HSCs gradually transition to a discrete lymphoid-primed state, distinct from lymphoid-primed multipotent progenitors, representing the earliest entry point into lymphoid commitment.

Defining murine organogenesis at single-cell resolution reveals a role for the leukotriene pathway in regulating blood progenitor formation.

During gastrulation, cell types from all three germ layers are specified and the basic body plan is established 1 . However, molecular analysis of this key developmental stage has been hampered by limited cell numbers and a paucity of markers. Single-cell RNA sequencing circumvents these problems, but has so far been limited to specific organ systems 2 . Here, we report single-cell transcriptomic characterization of >20,000 cells immediately following gastrulation at E8.25 of mouse development. We identify 20 major cell types, which frequently contain substructure, including three distinct signatures in early foregut cells. Pseudo-space ordering of somitic progenitor cells identifies dynamic waves of transcription and candidate regulators, which are validated by molecular characterization of spatially resolved regions of the embryo. Within the endothelial population, cells that transition from haemogenic endothelial to erythro-myeloid progenitors specifically express Alox5 and its co-factor Alox5ap, which control leukotriene production. Functional assays using mouse embryonic stem cells demonstrate that leukotrienes promote haematopoietic progenitor cell generation. Thus, this comprehensive single-cell map can be exploited to reveal previously unrecognized pathways that contribute to tissue development.

Single-cell RNA-sequencing reveals a distinct population of proglucagon-expressing cells specific to the mouse upper small intestine.

OBJECTIVES: To identify sub-populations of intestinal preproglucagon-expressing (PPG) cells producing Glucagon-like Peptide-1, and their associated expression profiles of sensory receptors, thereby enabling the discovery of therapeutic strategies that target these cell populations for the treatment of diabetes and obesity. METHODS: We performed single cell RNA sequencing of PPG-cells purified by flow cytometry from the upper small intestine of 3 GLU-Venus mice. Cells from 2 mice were sequenced at low depth, and from the third mouse at high depth. High quality sequencing data from 234 PPG-cells were used to identify clusters by tSNE analysis. qPCR was performed to compare the longitudinal and crypt/villus locations of cluster-specific genes. Immunofluorescence and mass spectrometry were used to confirm protein expression. RESULTS: PPG-cells formed 3 major clusters: a group with typical characteristics of classical L-cells, including high expression of Gcg and Pyy (comprising 51% of all PPG-cells); a cell type overlapping with Gip-expressing K-cells (14%); and a unique cluster expressing Tph1 and Pzp that was predominantly located in proximal small intestine villi and co-produced 5-HT (35%). Expression of G-protein coupled receptors differed between clusters, suggesting the cell types are differentially regulated and would be differentially targetable. CONCLUSIONS: Our findings support the emerging concept that many enteroendocrine cell populations are highly overlapping, with individual cells producing a range of peptides previously assigned to distinct cell types. Different receptor expression profiles across the clusters highlight potential drug targets to increase gut hormone secretion for the treatment of diabetes and obesity.