Real-Time Fluorometric Isothermal LAMP Assay for Detection of Chlamydia pecorum in Rapidly Processed Ovine Abortion Samples: A Veterinary Practitioner’s Perspective

Traditional methods of detecting Chlamydia pecorum in tissue samples such as polymerase chain reaction or cell culture are laborious and costly. We evaluated the use of a previously developed C. pecorum LAMP assay using minimally processed ovine samples. Cotyledon (n = 16), foetal liver (n = 22), foetal lung (n = 2), and vaginal (n = 6) swabs, in addition to cotyledon (n = 6) and foetal liver (n = 8) tissue samples, were rapidly processed and used for LAMP testing without DNA extraction. Overall, LAMP test results were highly congruent with the in-house reference qPCR, with 80.43% (37/46; 72.73% positive agreement (PA); 84.75% negative agreement (NA)) overall agreeance for swab samples, and 85.71% (12/14; 80% PA; 88.89% NA) overall agreeance for tissue samples. Out of the 11 total discrepant results, discrepancy was mainly observed in samples (n = 10) with less than 100 copies/µL C. pecorum DNA. While sensitivity could be improved, the simplicity, low cost, and accuracy of detection makes this test amenable for use at point-of-care for detecting C. pecorum in sheep.

Tissue-resident macrophages regulate lymphatic vessel growth and patterning in the developing heart

ABSTRACTMacrophages are components of the innate immune system with key roles in tissue inflammation and repair. It is now evident that macrophages also support organogenesis, but few studies have characterized their identity, ontogeny and function during heart development. Here, we show that the distribution and prevalence of resident macrophages in the subepicardial compartment of the developing heart coincides with the emergence of new lymphatics, and that macrophages interact closely with the nascent lymphatic capillaries. Consequently, global macrophage deficiency led to extensive vessel disruption, with mutant hearts exhibiting shortened and mis-patterned lymphatics. The origin of cardiac macrophages was linked to the yolk sac and foetal liver. Moreover, the Cx3cr1+ myeloid lineage was found to play essential functions in the remodelling of the lymphatic endothelium. Mechanistically, macrophage hyaluronan was required for lymphatic sprouting by mediating direct macrophage-lymphatic endothelial cell interactions. Together, these findings reveal insight into the role of macrophages as indispensable mediators of lymphatic growth during the development of the mammalian cardiac vasculature.

Integrative Single-cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Haematopoiesis

Regulation of haematopoiesis during human development remains poorly defined. Here, we applied single-cell (sc)RNA-Seq and scATAC-Seq analysis to over 8,000 human immunophenotypic blood cells from foetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream from haematopoietic stem cell/multipotent progenitors (HSC/MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSC/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSC/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental haematopoiesis in the context of blood pathologies and regenerative medicine.

Correlation of Maternal BMI with Foetal Liver Blood Flow and Neonatal Adiposity in Normal Pregnancies and Pregnancies Complicated by Gestational Diabetes Mellitus and Foetal Growth Restriction

Introduction: The foetus exhibits a wide array of structural and functional adaptations in response to intrauterine conditions, towards protection of vital organs and maintaining the supply of essential nutrients. When oxygen is limited, foetal adaptations prioritise brain growth, irrespective of whether other essential nutrients are limited or not. The hypothesis for this study was that fatty acid synthesis occurs in foetal liver therefore if adaptive changes occur in the hepatic and umbilical flow it will affect fat deposition which will manifest as neonatal adiposity. Aim: Correlation of maternal Basal Metabolic Index (BMI) with Foetal liver blood flow and neonatal adiposity in normal pregnancies and pregnancies complicated by Gestational Diabetes Mellitus (GDM) and Foetal Growth Restriction (FGR) .Materials and Methods: An observational pilot study was carried out in a tertiary care referral hospital of Northern India. Antenatal women were recruited in three groups of singleton uncomplicated pregnancies (40), Women with GDM (31) and women with FGR (29). Maternal characteristics including pre-pregnancy BMI and obstetric ultrasound doppler study were recorded at 35 weeks gestation. The time-averaged maximum velocity (TAMX) was calculated for Umbilical Vein (UV) and Ductus Venosus (DV) as (Vmax) UV and (Vmax) DV. Blood flow (Q) was calculated as Q=h×(D/2)2×p×TAMX. The neonatal biometry and Skin Fold Thickness (SFT) was measured. Statistical techniques used were t-tests for analyses of dichotomous outcomes, Pearson’s correlation (r) and multivariate regression. Results: In mothers with higher DV shunting neonatal adiposity was significantly lower in the FGR group. In the uncomplicated group about 46% of variation in adiposity was explained by all the study variables and overall regression equation was statistically significant (p=0.004). Conclusion: Mothers with low BMI and normal umbilical and middle cerebral doppler flow had higher foetal hepatic flow to improve substrate deposition. DV shunting was significantly higher in hypoxic foetuses with reduced hepatic flow.

Disruption of the mouse Shmt2 gene confers embryonic anaemia via foetal liver-specific metabolomic disorders

In a previous study, we proposed that age-related mitochondrial respiration defects observed in elderly subjects are partially due to age-associated downregulation of nuclear-encoded genes, including serine hydroxymethyltransferase 2 (SHMT2), which is involved in mitochondrial one-carbon (1C) metabolism. This assertion is supported by evidence that the disruption of mouse Shmt2 induces mitochondrial respiration defects in mouse embryonic fibroblasts generated from Shmt2-knockout E13.5 embryos experiencing anaemia and lethality. Here, we elucidated the potential mechanisms by which the disruption of this gene induces mitochondrial respiration defects and embryonic anaemia using Shmt2-knockout E13.5 embryos. The livers but not the brains of Shmt2-knockout E13.5 embryos presented mitochondrial respiration defects and growth retardation. Metabolomic profiling revealed that Shmt2 deficiency induced foetal liver-specific downregulation of 1C-metabolic pathways that create taurine and nucleotides required for mitochondrial respiratory function and cell division, respectively, resulting in the manifestation of mitochondrial respiration defects and growth retardation. Given that foetal livers function to produce erythroblasts in mouse embryos, growth retardation in foetal livers directly induced depletion of erythroblasts. By contrast, mitochondrial respiration defects in foetal livers also induced depletion of erythroblasts as a consequence of the inhibition of erythroblast differentiation, resulting in the manifestation of anaemia in Shmt2-knockout E13.5 embryos.

Functions and the Emerging Role of the Foetal Liver into Regenerative Medicine

During foetal life, the liver plays the important roles of connection and transient hematopoietic function. Foetal liver cells develop in an environment called a hematopoietic stem cell niche composed of several cell types, where stem cells can proliferate and give rise to mature blood cells. Embryologically, at about the third week of gestation, the liver appears, and it grows rapidly from the fifth to 10th week under WNT/β-Catenin signaling pathway stimulation, which induces hepatic progenitor cells proliferation and differentiation into hepatocytes. Development of new strategies and identification of new cell sources should represent the main aim in liver regenerative medicine and cell therapy. Cells isolated from organs with endodermal origin, like the liver, bile ducts, and pancreas, could be preferable cell sources. Furthermore, stem cells isolated from these organs could be more susceptible to differentiate into mature liver cells after transplantation with respect to stem cells isolated from organs or tissues with a different embryological origin. The foetal liver possesses unique features given the co-existence of cells having endodermal and mesenchymal origin, and it could be highly available source candidate for regenerative medicine in both the liver and pancreas. Taking into account these advantages, the foetal liver can be the highest potential and available cell source for cell therapy regarding liver diseases and diabetes.