Pig conceptuses utilize extracellular vesicles for IFNG-mediated paracrine communication with the endometrium.

Interferon-gamma (IFNG) is a pro-inflammatory cytokine secreted by the porcine conceptus (embryo and extra-embryonic membranes) during the peri-implantation period of pregnancy. IFNG modifies the endometrial inflammatory immune response and is required for the implantation and survival of the conceptus. It is not known how IFNG from the conceptus trophectoderm is transported across the endometrial luminal epithelium (LE). In the present study, immunofluorescence analyses detected immunoreactive IFNG protein in both the trophectoderm and endometrial LE on Day 15 of pregnancy, while our previous research localized IFNG mRNA only to conceptus trophectoderm. Using minced endometrial explants to disrupt the barrier posed by the intact endometrial LE, treatment with recombinant IFNG induced the expression of genes that were not induced when IFNG was infused into the uterine lumen in vivo by McLendon et al. (Biology of Reproduction. 2020;103(5):1018-1029). We hypothesized that during pregnancy extracellular vesicles (EVs) serve as intercellular signaling vehicles to transport conceptus-derived IFNG across the intact endometrial LE and into the stromal compartment of the uterus. Western blotting detected the presence of IFNG in EVs isolated from the uterine fluid of pregnant gilts, but not nonpregnant gilts. Real-time PCR demonstrated increased expression of IFNG-stimulated genes in EV-treated endometrial explants and EV-mediated IFNG transport was confirmed in whole uterine sections cultured with EVs from day 15 of pregnancy. These results suggest that EVs are involved in IFNG transport across the endometrial LE to enable paracrine communication between the conceptus and cells within the endometrial stroma.

Metabolic pathways for glucose and fructose: I synthesis and metabolism of fructose by ovine conceptuses.

Fructose, the most abundant hexose sugar in fetal fluids and blood of sheep and other ungulates and cetaceans, is synthesized from glucose via the polyol pathway in trophectoderm and chorion. However, the cell-specific and temporal expression of enzymes for the synthesis and metabolism of fructose in sheep conceptuses (embryo and placental membranes) and placentomes has not been characterized. This study characterized key enzymes involved in fructose synthesis and metabolism by ovine conceptuses throughout pregnancy. Day 17 conceptuses expressed mRNAs for the polyol pathway (SORD and AKR1B1) and glucose and fructose metabolism (HK1, HK2, G6PD, OGT, and FBP), but not those required for gluconeogenesis (G6Pase or PCK). Ovine placentomes also expressed mRNAs for SORD, AKR1B1, HK1, and OGT. Fructose can be metabolized via the ketohexokinase (KHK) pathway and isoforms, KHK-A and KHK-C, were expressed in ovine conceptuses from Day 16 of pregnancy and placentomes during pregnancy in a cell specific manner: KHK-A protein was more abundant in trophectoderm and cotyledons of placentomes, while KHK-C protein was more abundant in endoderm of Day 16 conceptuses and chorionic epithelium in placentomes. Expression of KHK mRNAs in placentomes was greatest at Day 30 of pregnancy (P < 0.05), but not different among days later in gestation. These results provide novel insights into the synthesis and metabolism of fructose via the uninhibited KHK pathway in ovine conceptuses to generate ATP via the TCA cycle, as well as substrates for the pentose cycle, hexosamine biosynthesis pathway and one-carbon metabolism required for conceptus development throughout pregnancy.

Metabolic pathways of glucose and fructose: II spatiotemporal expression of genes involved in synthesis and transport of lactate in ovine conceptuses.

Lactate, an abundant molecule in fetal fluids and blood of mammalian species is often overlooked as a metabolic waste product generated during pregnancy. Most of the glucose and fructose consumed by ovine conceptuses is converted to lactate, but proteins involved in lactate metabolism and transport have not been investigated. This study characterized total lactate produced by ovine conceptuses throughout gestation, as well as expression of mRNAs and proteins involved in lactate metabolism. Lactate increased in abundance in the uterine lumen during the preimplantation period and was more abundant than pyruvate. The abundance of lactate in allantoic and amniotic fluids increased with advancing days of gestation and most abundant on Day 125 of pregnancy (P < 0.05). Lactate dehydrogenase (LDH) subunits A (converts pyruvate to lactate) and B (converts lactate to pyruvate) were expressed by conceptuses throughout gestation. Lactate is transported via monocarboxylic acid transporters SLC16A1 and SLC16A3, both of which were expressed by the conceptus throughout gestation. Additionally, the interplacentomal chorioallantois from Day 126 expressed SLC16A1 and SLC16A3 and transported lactate across the tissue. Hydrocarboxylic acid receptor 1 (HCAR1), a receptor for lactate, was localized to the uterine luminal and superficial glandular epithelia of pregnant ewes throughout gestation, and conceptus trophectoderm during the peri-implantation period of gestation. These results provide novel insights into the spatiotemporal profiles of enzymes, transporters, and receptor for lactate by ovine conceptuses throughout pregnancy.

Implementation of an electroencephalogram-guided propofol anesthesia practice in a large academic pediatric hospital: A quality improvement project.

BACKGROUND: Propofol-based total intravenous anesthesia is gaining popularity in pediatric anesthesia. Electroencephalogram can be used to guide propofol dosing to the individual patient to mitigate against overdosing and adverse events. However, electroencephalogram interpretation and propofol pharmacokinetics are not sufficiently taught in training programs to confidently deploy electroencephalogram-guided total intravenous anesthesia. AIMS: We conducted a quality improvement project with the smart aim of increasing the percentage of electroencephalogram-guided total intravenous anesthesia cases in our main operating room from 0% to 80% over 18 months. Balancing measures were number of total intravenous anesthesia cases, emergence times, and perioperative emergency activations. METHODS: The project key drivers were education, equipment, and electronic health record modifications. Plan-Do-Study-Act cycles included: (1) providing journal articles, didactic lectures, intraoperative training, and teaching documents; (2) scheduling electroencephalogram-guided total intravenous anesthesia teachers to train faculty, staff, and fellows for specific cases and to assess case-based knowledge; (3) adding age-based propofol dosing tables and electroencephalogram parameters to the electronic health record (EPIC co, Verona, WI); (4) procuring electroencephalogram monitors (Sedline, Masimo Inc). Electroencephalogram-guided total intravenous anesthesia cases and balancing measures were identified from the electronic health record. The smart aim was evaluated by statistical process control chart. RESULTS: After the four Plan-Do-Study-Act cycles, electroencephalogram-guided total intravenous anesthesia increased from 5% to 75% and was sustained at 72% 9 months after project completion. Total intravenous anesthesia cases/mo and number of perioperative emergency activations did not change significantly from start to end of the project, while emergence time for electroencephalogram-guided total intravenous anesthesia was greater statistically but not clinically (total intravenous anesthesia without electroencephalogram [16 ± 10 min], total intravenous anesthesia with electroencephalogram [18 ± 9 min], sevoflurane [17 ± 9 min] p < .001). CONCLUSION: Quality improvement methods may be deployed to adopt electroencephalogram-guided total intravenous anesthesia in a large academic pediatric anesthesia practice. Keys to success include education, in operating room case training, scheduling teachers with learners, electronic health record modifications, and electroencephalogram devices and supplies.

Evaluation of neurological behavior alterations and metabolic changes in mice under chronic glyphosate exposure.

Glyphosate is a widely used active ingredient in agricultural herbicides, inhibiting the biosynthesis of aromatic amino acids in plants by targeting their shikimate pathway. Our gut microbiota also facilitates the shikimate pathway, making it a vulnerable target when encountering glyphosate. Dysbiosis in the gut microbiota may impair the gut-brain axis, bringing neurological outcomes. To evaluate the neurotoxicity and biochemical changes attributed to glyphosate, we exposed mice with the reference dose (RfD) set by the U.S. EPA (1.75 mg/Kg-BW/day) and its hundred-time-equivalence (175 mg/Kg-BW/day) chronically via drinking water, then compared a series of neurobehaviors and their fecal/serum metabolomic profile against the non-exposed vehicles (n = 10/dosing group). There was little alteration in the neurobehavior, including motor activities, social approach, and conditioned fear, under glyphosate exposure. Metabolomic differences attributed to glyphosate were observed in the feces, corresponding to 68 and 29 identified metabolites with dysregulation in the higher and lower dose groups, respectively, compared to the vehicle-control. There were less alterations observed in the serum metabolome. Under 175 mg/Kg-BW/day of glyphosate exposure, the aromatic amino acids (phenylalanine, tryptophan, and tyrosine) were reduced in the feces but not in the serum of mice. We further focused on how tryptophan metabolism was dysregulated based on the pathway analysis, and identified the indole-derivatives were more altered compared to the serotonin and kynurenine derivatives. Together, we obtained a three-dimensional data set that records neurobehavioral, fecal metabolic, and serum biomolecular dynamics caused by glyphosate exposure at two different doses. Our data showed that even under the high dose of glyphosate irrelevant to human exposure, there were little evidence that supported the impairment of the gut-brain axis.

Understanding placentation in ruminants: a review focusing on cows and sheep.

Mammals differ regarding their placentae, but in all species placental trophoblasts interact intimately with the uterine endometrium to mediate the transfer of nutrients from the mother to the embryo/fetus through the closely juxtaposed microcirculatory systems of the uterus and placenta. Placentation in ruminants is intermediate between the non-invasive type, as observed in the epitheliochorial placenta of pigs, and the invasive type, as observed in the haemochorial placentae of mice and humans. In ruminants, placental trophoblast cells invade uterine endometrial tissue, but invasion is believed to be limited to the endometrial luminal epithelium (LE). In the LE there are varying degrees of syncytialisation among species, with syncytialisation being more extensive in sheep than cows. The hallmarks of placentation in ruminants include: (1) an extended period in which conceptuses (embryos and associated placental membranes) elongate and must be supported by secretions (histotroph) from the uterus; (2) a cascade involving an array of adhesion molecules that includes integrin-mediated attachment of the conceptus trophoblast to the endometrial LE for implantation; (3) syncytialisation of the developing early placenta, a process for which there is currently limited understanding; and (4) development of placentomes that define the cotyledonary placentae of cows and sheep, and provide haemotrophic support of fetal development.

Immunohistochemical examination of the utero-placental interface of cows on days 21, 31, 40, and 67 of gestation.

What we understand about early stages of placentation in cattle is based on an elegant series of electron microscopic images that provide exquisite detail, but limited appreciation for the microanatomy across the utero-placental interface. In order to achieve a global perspective on the histology of bovine placentation during critical early stages of gestation, i.e., days 21, 31, 40, and 67, we performed immunohistochemistry to detect cell-specific expression of pregnancy-associated glycoprotein (PAG), cytokeratin, epithelial (E)-cadherin, and serine hydroxymethyltransferase 2 (SHMT2) at the intact utero-placental interface. Key findings from the immunohistochemical analyses are that there are: (1) PAG-positive cells with a single nucleus within the uterine luminal epithelial (LE) cells; (2) PAG-positive cells with two nuclei in the LE; (3) PAG-positive syncytial cells with more than three nuclei in the LE; (4) LE cells that are dissociated from one another and dissociated from the basement membrane in regions of syncytialization within the LE layer; (5) replacement of the mononuclear LE with a multi-layer thick population of PAG-positive cells invading into the uterine stroma of caruncles, but not into the stroma of intercaruncular endometrium; and (6) PAG-, E-cadherin- and SHMT2-positive mononuclear cells at the leading edge of developing cotyledonary villi that eventually represent the majority of the epithelial surface separating caruncular stroma from cotyledonary stroma. Finally, the utero-placental interface of ruminants is not always uniform across a single cross-section of a site of placentation which allows different conclusions to be made depending on the part of the utero-placental interface being examined.

Synthesis of glycine from 4-hydroxyproline in tissues of neonatal pigs with intrauterine growth restriction.

This study tested the hypothesis that the synthesis of glycine from 4-hydroxyproline (an abundant amino acid in milk and neonatal blood) was impaired in tissues of piglets with intrauterine growth restriction (IUGR), thereby contributing to a severe glycine deficiency in these compromised neonates. At 0, 7, 14, and 21 days of age, IUGR piglets were euthanized, and tissues (liver, small intestine, kidney, pancreas, stomach, skeletal muscle, and heart) were obtained for metabolic studies, as well as the determination of enzymatic activities, cell-specific localization, and expression of mRNAs for glycine-synthetic enzymes. The results indicated relatively low enzymatic activities for 4-hydroxyproline oxidase (OH-POX), proline oxidase, serine hydroxymethyltransferase, threonine dehydrogenase (TDH), alanine: glyoxylate transaminase, and 4-hydroxy-2-oxoglutarate aldolase in the kidneys and liver from 0- to 21-day-old IUGR pigs, in the pancreas of 7- to 21-day-old IUGR pigs, and in the small intestine and skeletal muscle (except TDH) of 21-day-old IUGR pigs. Accordingly, the rates of conversion of 4-hydroxyproline into glycine were relatively low in tissues of IUGR piglets. The expression of mRNAs for glycine-synthetic enzymes followed the patterns of enzymatic activities and was also low. Immunohistochemical analyses revealed the relatively low abundance of OH-POX protein in the liver, kidney, and small intestine of IUGR piglets, and the lack of OH-POX zonation in their livers. These novel results provide a metabolic basis to explain why the endogenous synthesis of glycine is insufficient for optimum growth of IUGR piglets and have important implications for improving the nutrition and health of other mammalian neonates including humans with IUGR.

Integrins and their potential roles in mammalian pregnancy.

Integrins are a highly complex family of receptors that, when expressed on the surface of cells, can mediate reciprocal cell-to-cell and cell-to-extracellular matrix (ECM) interactions leading to assembly of integrin adhesion complexes (IACs) that initiate many signaling functions both at the membrane and deeper within the cytoplasm to coordinate processes including cell adhesion, migration, proliferation, survival, differentiation, and metabolism. All metazoan organisms possess integrins, and it is generally agreed that integrins were associated with the evolution of multicellularity, being essential for the association of cells with their neighbors and surroundings, during embryonic development and many aspects of cellular and molecular biology. Integrins have important roles in many aspects of embryonic development, normal physiology, and disease processes with a multitude of functions discovered and elucidated for integrins that directly influence many areas of biology and medicine, including mammalian pregnancy, in particular implantation of the blastocyst to the uterine wall, subsequent placentation and conceptus (embryo/fetus and associated placental membranes) development. This review provides a succinct overview of integrin structure, ligand binding, and signaling followed with a concise overview of embryonic development, implantation, and early placentation in pigs, sheep, humans, and mice as an example for rodents. A brief timeline of the initial localization of integrin subunits to the uterine luminal epithelium (LE) and conceptus trophoblast is then presented, followed by sequential summaries of integrin expression and function during gestation in pigs, sheep, humans, and rodents. As appropriate for this journal, summaries of integrin expression and function during gestation in pigs and sheep are in depth, whereas summaries for humans and rodents are brief. Because similar models to those illustrated in Fig. 1, 2, 3, 4, 5 and 6 are present throughout the scientific literature, the illustrations in this manuscript are drafted as Viking imagery for entertainment purposes.

Synthesis of glycine from 4-hydroxyproline in tissues of neonatal pigs.

Glycine from sow's milk only meets 20% of the requirement of suckling piglets. However, how glycine is synthesized endogenously in neonates is not known. This study determined glycine synthesis from 4-hydroxyproline (an abundant amino acid in milk and neonatal blood) in tissues of sow-reared piglets with normal birth weights. Piglets were euthanized at 0, 7, 14 and 21 days of age, and their tissues were used to determine glycine synthesis from 0 to 5 mM 4-hydroxyproline, activities and mRNA expression of key glycine-synthetic enzymes, and their cell-specific localization. Activities of 4-hydroxyproline oxidase (OH-POX), proline oxidase (POX), serine hydroxymethyltransferase (SHMT), threonine dehydrogenase (TDH), alanine:glyoxylate transaminase (AGT), and 4-hydroxy-2-oxoglutarate aldolase (HOA) occurred in the kidneys and liver from all age groups of piglets, and in the pancreas of 7- to 21-day-old piglets. Activities of OH-POX and HOA were absent from the small intestine of newborn pigs but present in the small intestine of 7- to 21-day-old piglets and in the skeletal muscle of 14- to 21-day-old piglets. Between days 0 and 21 of age, the enzymatic activities of OH-POX, AGT, and HOA decreased in the liver and kidneys but increased in the pancreas and small intestine with age. The mRNA levels of these three enzymes changed in a manner similar to their enzymatic activities. In contrast to OH-POX, AGT, and HOA, the enzymatic activities of POX, SHMT, and TDH were present in the kidneys, liver, and intestine of all age groups of piglets. Glycine was synthesized from 0.1 to 5 mM 4-hydroxyproline in the liver and kidney from 0- to 21-day-old piglets, as well as the pancreas, small intestine, and skeletal muscle from 14- to 21-day-old piglets in a concentration-dependent manner. Collectively, our findings indicate that 4-hydroxyproline is used for the synthesis of glycine in tissues of piglets to compensate for the deficiency of glycine in milk.

Outcomes of thoracic aortic interventions in Marfan syndrome in the state of Texas over 11 years.

OBJECTIVES: Marfan syndrome is a heritable connective tissue disorder with significant aortopathy and conveys substantial cardiovascular morbidity. This study characterizes the mortality and morbidities of thoracic aortic interventions (TAI) in the Marfan syndrome population in the state of Texas from 2009 to 2019. METHODS: A retrospective review of the Texas Inpatient Discharge Dataset from 1 January 2009 to 31 December 2019. Discharges from acute care hospitals with a Marfan syndrome diagnosis by the International Classification of Diseases 9/10 codes and a procedure code for TAI were analysed utilizing descriptive, univariate and multivariable regression statistics. RESULTS: There were 4641 Marfan syndrome discharges identified, of whom 644 (13.9%) underwent TAI. Thoracic or thoraco-abdominal aortic dissection or rupture was noted in 223 (34.6%). Thirty-three (5.1%) had a concomitant coronary artery intervention. There were 30 (4.7%) in-hospital mortalities, 126 (19.6%) diagnoses of acute renal failure (ARF), 52 (8.1%) had mechanical ventilation >96 h and the median length of stay was 10 [interquartile range (IQR) 7-16] days. After adjustment, concomitant coronary artery intervention was associated with in-hospital mortality [odds ratio (OR) 3.69 [IQR 1.15-11.90], P = 0.029] and ARF (OR 2.66 [IQR 1.19-5.94], P = 0.017). Aortic dissections/ruptures were associated with ARF (OR 1.73 [IQR 1.14-2.63], P = 0.010), ventilation >96 h (OR 2.19 [IQR 1.21-3.97], P = 0.010), and 15% longer length of stay (95% confidence interval 2.4-29.1%, P = 0.038). CONCLUSIONS: TAI are frequent among the hospitalized Marfan Syndrome population. Concomitant coronary intervention is associated with increased risk of death and aortic dissections/ruptures are associated with increased morbidity. The high prevalence of aortic dissections/ruptures points to a potential target for improving imaging surveillance, adherence to treatment guidelines and preventative management of Marfan syndrome aortopathy.

Atroposelective Brønsted Acid-Catalyzed Photocyclization to Access Chiral N-Aryl Quinolones with Low Rotational Barriers.

Herein, a novel route to atropisomeric N-aryl quinolones with low rotational barriers is demonstrated, leveraging a dual photochemical/organocatalytic approach to the required ring closure in up to 94% yield and up to >99% ee. The use of a continuous flow system allows for impurity suppression and enables rapid scale-up to a decagram scale.

Bruton's tyrosine kinase inhibition reduces disease severity in a model of secondary progressive autoimmune demyelination.

Bruton's tyrosine kinase (BTK) is an emerging target in multiple sclerosis (MS). Alongside its role in B cell receptor signaling and B cell development, BTK regulates myeloid cell activation and inflammatory responses. Here we demonstrate efficacy of BTK inhibition in a model of secondary progressive autoimmune demyelination in Biozzi mice with experimental autoimmune encephalomyelitis (EAE). We show that late in the course of disease, EAE severity could not be reduced with a potent relapse inhibitor, FTY720 (fingolimod), indicating that disease was relapse-independent. During this same phase of disease, treatment with a BTK inhibitor reduced both EAE severity and demyelination compared to vehicle treatment. Compared to vehicle treatment, late therapeutic BTK inhibition resulted in fewer spinal cord-infiltrating myeloid cells, with lower expression of CD86, pro-IL-1ß, CD206, and Iba1, and higher expression of Arg1, in both tissue-resident and infiltrating myeloid cells, suggesting a less inflammatory myeloid cell milieu. These changes were accompanied by decreased spinal cord axonal damage. We show similar efficacy with two small molecule inhibitors, including a novel, highly selective, central nervous system-penetrant BTK inhibitor, GB7208. These results suggest that through lymphoid and myeloid cell regulation, BTK inhibition reduced neurodegeneration and disease progression during secondary progressive EAE.

Creatine metabolism at the uterine-placental interface throughout gestation in sheep†.

The placenta requires high levels of adenosine triphosphate to maintain a metabolically active state throughout gestation. The creatine-creatine kinase-phosphocreatine system is known to buffer adenosine triphosphate levels; however, the role(s) creatine-creatine kinase-phosphocreatine system plays in uterine and placental metabolism throughout gestation is poorly understood. In this study, Suffolk ewes were ovariohysterectomized on Days 30, 50, 70, 90, 110 and 125 of gestation (n = 3-5 ewes/per day, except n = 2 on Day 50) and uterine and placental tissues subjected to analyses to measure metabolites, mRNAs, and proteins related to the creatine-creatine kinase-phosphocreatine system. Day of gestation affected concentrations and total amounts of guanidinoacetate and creatine in maternal plasma, amniotic fluid and allantoic fluid (P < 0.05). Expression of mRNAs for arginine:glycine amidinotransferase, guanidinoacetate methyltransferase, creatine kinase B, and solute carrier 16A12 in endometria and for arginine:glycine amidinotransferase and creatine kinase B in placentomes changed significantly across days of gestation (P < 0.05). The arginine:glycine amidinotransferase protein was more abundant in uterine luminal epithelium on Days 90 and 125 compared to Days 30 and 50 (P < 0.01). The chorionic epithelium of placentomes expressed guanidinoacetate methyltransferase and solute carrier 6A13 throughout gestation. Creatine transporter (solute carrier 6A8) was expressed by the uterine luminal epithelium and trophectoderm of placentomes throughout gestation. Creatine kinase (creatine kinase B and CKMT1) proteins were localized primarily to the uterine luminal epithelium and to the placental chorionic epithelium of placentomes throughout gestation. Collectively, these results demonstrate cell-specific and temporal regulation of components of the creatine-creatine kinase-phosphocreatine system that likely influence energy homeostasis for fetal-placental development.

Dietary supplementation with L-citrulline improves placental angiogenesis and embryonic survival in gilts.

This study was conducted with gilts as an animal model to test the hypothesis that dietary supplementation with L-citrulline (Cit) improves placental angiogenesis and embryonic survival. Between Days 14 and 25 of gestation, each gilt was fed a corn- and soybean-meal-based diet (2 kg/day) supplemented with 0.4% Cit or an isonitrogenous amount of L-alanine (Control). On Day 25 of gestation, gilts were hysterectomized to obtain conceptuses. Amniotic and allantoic fluids and placentae were analyzed for NOx [stable oxidation products of nitric oxide (NO)], polyamines, and amino acids (AAs). Placentae were also analyzed for syntheses of NO and polyamines; concentrations of AAs and related metabolites; and the expression of angiogenic factors and aquaporins (AQPs). Compared to the control group, Cit supplementation increased (P < 0.01) the number of viable fetuses by 2.0 per litter, the number and diameter of placental blood vessels (21% and 24%, respectively), placental weight (15%), and total allantoic and amniotic fluid volumes (20% and 47%, respectively). Cit supplementation also increased (P < 0.01) enzymatic activities of GTP-cyclohydrolase-1 (32%) and ornithine decarboxylase (27%) in placentae; syntheses of NO (29%) and polyamines (26%); concentrations of NOx (19%), tetrahydrobiopterin (28%), polyamines (22%), cAMP (26%), and cGMP (24%) in placentae; total amounts of NOx (22-40%), polyamines (23-40%), AAs (16-255%), glucose (22-44%), and fructose (22-43%) in allantoic and amniotic fluids. Furthermore, Cit supplementation increased (P < 0.05) placental mRNA levels for angiogenic factors (eNOS [84%], GTP-CH1 [55%], PGF [61%], VEGFA120 [26%], and VEGFR2 [137%], as well as AQPs - AQP1 [105%], AQP3 [53%], AQP5 [77%], AQP8 [57%], and AQP9 [31%]). Collectively, dietary Cit supplementation enhanced placental NO and polyamine syntheses as well as angiogenesis to improve conceptus development and survival.

Integrated intracellular organization and its variations in human iPS cells.

Understanding how a subset of expressed genes dictates cellular phenotype is a considerable challenge owing to the large numbers of molecules involved, their combinatorics and the plethora of cellular behaviours that they determine1,2. Here we reduced this complexity by focusing on cellular organization-a key readout and driver of cell behaviour3,4-at the level of major cellular structures that represent distinct organelles and functional machines, and generated the WTC-11 hiPSC Single-Cell Image Dataset v1, which contains more than 200,000 live cells in 3D, spanning 25 key cellular structures. The scale and quality of this dataset permitted the creation of a generalizable analysis framework to convert raw image data of cells and their structures into dimensionally reduced, quantitative measurements that can be interpreted by humans, and to facilitate data exploration. This framework embraces the vast cell-to-cell variability that is observed within a normal population, facilitates the integration of cell-by-cell structural data and allows quantitative analyses of distinct, separable aspects of organization within and across different cell populations. We found that the integrated intracellular organization of interphase cells was robust to the wide range of variation in cell shape in the population; that the average locations of some structures became polarized in cells at the edges of colonies while maintaining the 'wiring' of their interactions with other structures; and that, by contrast, changes in the location of structures during early mitotic reorganization were accompanied by changes in their wiring.

Experiences and insights from partners of individuals with single-ventricle CHD: a pilot qualitative research study.

INTRODUCTION: With advances in care, an increasing number of individuals with single-ventricle CHD are surviving into adulthood. Partners of individuals with chronic illness have unique experiences and challenges. The goal of this pilot qualitative research study was to explore the lived experiences of partners of individuals with single-ventricle CHD. METHODS: Partners of patients ≥18 years with single-ventricle CHD were recruited and participated in Experience Group sessions and 1:1 interviews. Experience Group sessions are lightly moderated groups that bring together individuals with similar circumstances to discuss their lived experiences, centreing them as the experts. Formal inductive qualitative coding was performed to identify salient themes. RESULTS: Six partners of patients participated. Of these, four were males and four were married; all were partners of someone of the opposite sex. Themes identified included uncertainty about their partners' future health and mortality, becoming a lay CHD specialist, balancing multiple roles, and providing positivity and optimism. Over time, they took on a role as advocates for their partners and as repositories of medical history to help navigate the health system. Despite the uncertainties, participants described championing positivity and optimism for the future. CONCLUSIONS: In this first-of-its-kind pilot study, partners of individuals with single-ventricle CHD expressed unique challenges and experiences in their lives. There is a tacit need to design strategies to help partners cope with those challenges. Further larger-scale research is required to better understand the experiences of this unique population.

The ovine conceptus utilizes extracellular serine, glucose, and fructose to generate formate via the one carbon metabolism pathway.

Highly proliferative cells rely on one carbon (1C) metabolism for production of formate required for synthesis of purines and thymidine for nucleic acid synthesis. This study was to determine if extracellular serine and/or glucose and fructose contribute the production of formate in ovine conceptuses. Suffolk ewes (n = 8) were synchronized to estrus, bred to fertile rams, and conceptuses were collected on Day 17 of gestation. Conceptuses were either snap frozen in liquid nitrogen (n = 3) or placed in culture in medium (n = 5) containing either: 1) 4 mM D-glucose + 2 mM [U-13C]serine; 2) 6 mM glycine + 4 mM D-glucose + 2 mM [U-13C]serine; 3) 4 mM D-fructose + 2 mM [U-13C]serine; 4) 6 mM glycine + 4 mM D-fructose + 2 mM [U-13C]serine; 5) 4 mM D-glucose + 4 mM D-fructose + 2 mM [U-13C]serine; or 6) 6 mM glycine + 4 mM D-glucose + 4 mM D-fructose + 2 mM [U-13C]serine. After 2 h incubation, conceptuses in their respective culture medium were homogenized and the supernatant analyzed for 12C- and 13C-formate by gas chromatography and amino acids by high performance liquid chromatography. Ovine conceptuses produced both 13C- and 12C-formate, indicating that the [U-13C]serine, glucose, and fructose were utilized to generate formate, respectively. Greater amounts of 12C-formate than 13C-formate were produced, indicating that the ovine conceptus utilized more glucose and fructose than serine to produce formate. This study is the first to demonstrate that both 1C metabolism and serinogenesis are active metabolic pathways in ovine conceptuses during the peri-implantation period of pregnancy, and that hexose sugars are the preferred substrate for generating formate required for nucleotide synthesis for proliferating trophectoderm cells.

Hospitalisations of children and adults with Hypoplastic Left Heart Syndrome in Texas from 2009 to 2019.

INTRODUCTION: Hypoplastic Left Heart Syndrome accounts for a significant proportion of CHD morbidity and mortality, despite improvements in care and improved survival. This study evaluates number of, reasons for, and trends in discharges of patients with hypoplastic left heart syndrome over 11 years in Texas. METHODS: The Texas Inpatient Discharge Dataset Public Use File captures almost all discharges in Texas and was reviewed from 2009 to 2019. Discharges of patients ≥5 years of age and diagnosis codes for Hypoplastic Left Heart Syndrome were included. The admitting and principle diagnoses were categorised and all discharges were evaluated for procedures performed. Descriptive and univariate statistical analyses were performed. RESULTS: A total of 1024 discharges were identified with a 16.9% annual increase over the study period. Median length of stay was 4 [IQR: 2-8] and there were 17 (1.7%) in-hospital mortalities with no differences across age groups. Seven (17.1%) discharges of patients 25+ years were uninsured, higher than other age groups (p < 0.001). The most common admitting diagnosis was CHD and 224 (21.9%) of discharges included a procedure, including 23 heart transplants. Discharges occurred from 67 different hospitals with 4 (6.0%) representing 71.4% of all discharges. CONCLUSIONS: Discharges of Hypoplastic Left Heart Syndrome have increased rapidly, particularly in the older age groups and were spread over a large number of hospitals. Further work is needed to understand the interplay between Hypoplastic Left Heart Syndrome and other conditions and care experiences that occur within the general population, which will become more common as this population ages and grows.

Metabolic pathways utilized by the porcine conceptus, uterus, and placenta.

Conceptus elongation and early placentation involve growth and remodeling that requires proliferation and migration of cells. This demands conceptuses expend energy before establishment of a placenta connection and when they are dependent upon components of histotroph secreted or transported into the uterine lumen from the uterus. Glucose and fructose, as well as many amino acids (including arginine, aspartate, glutamine, glutamate, glycine, methionine, and serine), increase in the uterine lumen during the peri-implantation period. Glucose and fructose enter cells via their transporters, SLC2A, SLC2A3, and SLC2A8, and amino acids enter the cells via specific transporters that are expressed by the conceptus trophectoderm. However, porcine conceptuses develop rapidly through extensive cellular proliferation and migration as they elongate and attach to the uterine wall resulting in increased metabolic demands. Therefore, coordination of multiple metabolic biosynthetic pathways is an essential aspect of conceptus development. Oxidative metabolism primarily occurs through the tricarboxylic acid (TCA) cycle and the electron transport chain, but proliferating and migrating cells, like the trophectoderm of pigs, enhance aerobic glycolysis. The glycolytic intermediates from glucose can then be shunted into the pentose phosphate pathway and one-carbon metabolism for the de novo synthesis of nucleotides. A result of aerobic glycolysis is limited availability of pyruvate for maintaining the TCA cycle, and trophectoderm cells likely replenish TCA cycle metabolites primarily through glutaminolysis to convert glutamine into TCA cycle intermediates. The synthesis of ATP, nucleotides, amino acids, and fatty acids through these biosynthetic pathways is essential to support elongation, migration, hormone synthesis, implantation, and early placental development of conceptuses.