Deciphering "Immaturity-Stemness" in Human Epidermal Stem Cells at the Levels of Protein-Coding and Non-Coding Genomes: A Prospective Computational Approach.

The epidermis hosts populations of epithelial stem cells endowed with well-documented renewal and regenerative functions. This tissue thus constitutes a model for exploring the molecular characteristics of stem cells, which remain to date partially characterized at the molecular level in human skin. Our group has investigated the regulatory functions of the KLF4/TGFB1 and the MAD4/MAX/MYC signaling pathways in the control of the immaturity-stemness versus differentiation fate of keratinocyte stem and precursor cells from human interfollicular epidermis. We described that down-modulation of either KLF4 or MXD4/MAD4 using RNA interference tools promoted an augmented stemness cellular status; an effect which was associated with significant transcriptional changes, as assessed by RNA-sequencing. Here, we have implemented a computational approach aimed at integrating the level of the coding genome, comprising the transcripts encoding conventional proteins, and the non-coding genome, with a focus on long non-coding RNAs (lncRNAs). In addition, datasets of micro-RNAs (miRNAs) with validated functions were interrogated in view of identifying miRNAs that could make the link between protein-coding and non-coding transcripts. Putative regulons comprising both coding and long non-coding transcripts were built, which are expected to contain original pro-stemness candidate effectors available for functional validation approaches. In summary, interpretation of our basic functional data together with in silico biomodeling gave rise to a prospective picture of the complex constellation of transcripts regulating the keratinocyte stemness status.

In vivo quantitative MRI: T1 and T2 measurements of the human brain at 0.064 T.

OBJECTIVE: To measure healthy brain [Formula: see text] and [Formula: see text] relaxation times at 0.064 T. MATERIALS AND METHODS: [Formula: see text] and [Formula: see text] relaxation times were measured in vivo for 10 healthy volunteers using a 0.064 T magnetic resonance imaging (MRI) system and for 10 test samples on both the MRI and a separate 0.064 T nuclear magnetic resonance (NMR) system. In vivo [Formula: see text] and [Formula: see text] values are reported for white matter (WM), gray matter (GM), and cerebrospinal fluid (CSF) for automatic segmentation regions and manual regions of interest (ROIs). RESULTS: [Formula: see text] sample measurements on the MRI system were within 10% of the NMR measurement for 9 samples, and one sample was within 11%. Eight [Formula: see text] sample MRI measurements were within 25% of the NMR measurement, and the two longest [Formula: see text] samples had more than 25% variation. Automatic segmentations generally resulted in larger [Formula: see text] and [Formula: see text] estimates than manual ROIs. DISCUSSION: [Formula: see text] and [Formula: see text] times for brain tissue were measured at 0.064 T. Test samples demonstrated accuracy in WM and GM ranges of values but underestimated long [Formula: see text] in the CSF range. This work contributes to measuring quantitative MRI properties of the human body at a range of field strengths.

Relaxation measurements of an MRI system phantom at low magnetic field strengths.

OBJECTIVE: Temperature controlled T1 and T2 relaxation times are measured on NiCl2 and MnCl2 solutions from the ISMRM/NIST system phantom at low magnetic field strengths of 6.5 mT, 64 mT and 550 mT. MATERIALS AND METHODS: The T1 and T2 were measured of five samples with increasing concentrations of NiCl2 and five samples with increasing concentrations of MnCl2. All samples were scanned at 6.5 mT, 64 mT and 550 mT, at sample temperatures ranging from 10 °C to 37 °C. RESULTS: The NiCl2 solutions showed little change in T1 and T2 with magnetic field strength, and both relaxation times decreased with increasing temperature. The MnCl2 solutions showed an increase in T1 and a decrease in T2 with increasing magnetic field strength, and both T1 and T2 increased with increasing temperature. DISCUSSION: The low field relaxation rates of the NiCl2 and MnCl2 arrays in the ISMRM/NIST system phantom are investigated and compared to results from clinical field strengths of 1.5 T and 3.0 T. The measurements can be used as a benchmark for MRI system functionality and stability, especially when MRI systems are taken out of the radiology suite or laboratory and into less traditional environments.

Heterozygote Dopamine Transporter Knockout Rats Display Enhanced Cocaine Locomotion in Adolescent Females.

Cocaine is a powerful psychostimulant that is one of the most widely used illicit addictive. The dopamine transporter (DAT) plays a major role in mediating cocaine's reward effect. Decreases in DAT expression increase rates of drug abuse and vulnerability to comorbid psychiatric disorders. We used the novel DAT transgenic rat model to study the effects of cocaine on locomotor behaviors in adolescent rats, with an emphasis on sex. Female rats showed higher response rates to cocaine at lower acute and chronic doses, highlighting a higher vulnerability and perceived gender effects. In contrast, locomotor responses to an acute high dose of cocaine were more marked and sustained in male DAT heterozygous (HET) adolescents. The results demonstrate the augmented effects of chronic cocaine in HET DAT adolescent female rats. Knockout (KO) DAT led to a level of hyperdopaminergia which caused a marked basal hyperactivity that was unchanged, consistent with a possible ceiling effect. We suggest a role of alpha synuclein (α-syn) and PICK 1 protein expressions to the increased vulnerability in female rats. These proteins showed a lower expression in female HET and KO rats. This study highlights gender differences associated with mutations which affect DAT expression and can increase susceptibility to cocaine abuse in adolescence.

A standard system phantom for magnetic resonance imaging.

PURPOSE: A standard MRI system phantom has been designed and fabricated to assess scanner performance, stability, comparability and assess the accuracy of quantitative relaxation time imaging. The phantom is unique in having traceability to the International System of Units, a high level of precision, and monitoring by a national metrology institute. Here, we describe the phantom design, construction, imaging protocols, and measurement of geometric distortion, resolution, slice profile, signal-to-noise ratio (SNR), proton-spin relaxation times, image uniformity and proton density. METHODS: The system phantom, designed by the International Society of Magnetic Resonance in Medicine ad hoc committee on Standards for Quantitative MR, is a 200 mm spherical structure that contains a 57-element fiducial array; two relaxation time arrays; a proton density/SNR array; resolution and slice-profile insets. Standard imaging protocols are presented, which provide rapid assessment of geometric distortion, image uniformity, T1 and T2 mapping, image resolution, slice profile, and SNR. RESULTS: Fiducial array analysis gives assessment of intrinsic geometric distortions, which can vary considerably between scanners and correction techniques. This analysis also measures scanner/coil image uniformity, spatial calibration accuracy, and local volume distortion. An advanced resolution analysis gives both scanner and protocol contributions. SNR analysis gives both temporal and spatial contributions. CONCLUSIONS: A standard system phantom is useful for characterization of scanner performance, monitoring a scanner over time, and to compare different scanners. This type of calibration structure is useful for quality assurance, benchmarking quantitative MRI protocols, and to transition MRI from a qualitative imaging technique to a precise metrology with documented accuracy and uncertainty.

Gestational diabetes - Major problems with post-partum testing.

BACKGROUND: Post-partum follow up testing of women with gestational diabetes mellitus (GDM) is important. All women, and their family doctors, receive written reminders. There are no recent major Australian reviews of the efficacy and compliance with this advice conducted in an ethnically representative population and using the current diagnostic criteria. AIM: The aim was to examine a cohort of women with recently diagnosed GDM and a completed pregnancy to determine what proportion had been tested and what were the difficulties in having testing carried out. METHODS: Women who were diagnosed with gestational diabetes and attended the Diabetes Service in 2017 were followed up in 2019. Attempted contact was made using an unidentified land line, an identifiable mobile phone and a postal survey. Compliance with testing advice was the major parameter considered. RESULTS: There were 714 women with GDM, 75 were excluded: 64 after pass one and 11 after pass two. In total, only 339/639 (53.1%) could be contacted. Of these women, 334 agreed to be surveyed; 207 (62.0%) had a post-partum test. Of the 127 women who had not had a test, 113 agreed to have an HbA1c. Only 13/113 (11.5%) had this done within a month. CONCLUSION: Contacting women, even within a short time after the pregnancy, is difficult. The number of post-partum tests carried out is suboptimal. Written advice to all women and their doctors does not appear to be working. A review of the cost effectiveness of this approach and development of new methods may be worthwhile.

Magnetic resonance fingerprinting Part 1: Potential uses, current challenges, and recommendations.

Magnetic resonance fingerprinting (MRF) is a powerful quantitative MRI technique capable of acquiring multiple property maps simultaneously in a short timeframe. The MRF framework has been adapted to a wide variety of clinical applications, but faces challenges in technical development, and to date has only demonstrated repeatability and reproducibility in small studies. In this review, we discuss the current implementations of MRF and their use in a clinical setting. Based on this analysis, we highlight areas of need that must be addressed before MRF can be fully adopted into the clinic and make recommendations to the MRF community on standardization and validation strategies of MRF techniques. Level of Evidence: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2020;51:675-692.

(2) H and (139) La†NMR Spectroscopy in Aqueous Solutions at Geochemical Pressures.

Nuclear spin relaxation rates of (2) H and (139) La in LaCl3 +(2) H2 O and La(ClO4 )3 +(2) H2 O solutions were determined as a function of pressure in order to demonstrate a new NMR probe designed for solution spectroscopy at geochemical pressures. The (2) H longitudinal relaxation rates (T1 ) vary linearly to 1.6 GPa, consistent with previous work at lower pressures. The (139) La T1 values vary both with solution chemistry and pressure, but converge with pressure, suggesting that the combined effects of increased viscosity and enhanced rates of ligand exchange control relaxation. This simple NMR probe design allows experiments on aqueous solutions to pressures corresponding roughly to those at the base of the Earth's continental crust.

MXD4/MAD4 Regulates Human Keratinocyte Precursor Fate.

Deciphering the pathways that regulate human epidermal precursor cell fate is necessary for future developments in skin repair and graft bioengineering. Among them, characterization of pathways regulating the keratinocyte (KC) precursor immaturity versus differentiation balance is required for improving the efficiency of KC precursor ex vivo expansion. In this study, we show that the transcription factor MXD4/MAD4 is expressed at a higher level in quiescent KC stem/progenitor cells located in the basal layer of human epidermis than in cycling progenitors. In holoclone KCs, stable short hairpin-RNA‒mediated decreased expression of MXD4/MAD4 increases MYC expression, whose modulation increases the proliferation of KC precursors and maintenance of their clonogenic potential and preserves the functionality of these precursors in three-dimensional epidermis organoid generation. Altogether, these results characterize MXD4/MAD4 as a major piece of the stemness puzzle in the human epidermis KC lineage and pinpoint an original avenue for ex vivo expansion of human KC precursors.

Immunosuppressive Properties of Epidermal Keratinocytes Differ According to Their Immaturity Status.

Preservation of a functional keratinocyte stem cell pool is essential to ensure the long-term maintenance of epidermis integrity, through continuous physiological renewal and regeneration in case of injury. Protecting stem cells from inflammation and immune reactions is thus a critical issue that needs to be explored. Here, we show that the immature CD49fhigh precursor cell fraction from interfollicular epidermis keratinocytes, comprising stem cells and progenitors, is able to inhibit CD4 + T-cell proliferation. Of note, both the stem cell-enriched CD49fhigh/EGFRlow subpopulation and the less immature CD49fhigh/EGFRhigh progenitors ensure this effect. Moreover, we show that HLA-G and PD-L1 immune checkpoints are overexpressed in CD49fhigh precursors, as compared to CD49flow differentiated keratinocytes. This potency may limit immune reactions against immature precursors including stem cells, and protect them from exacerbated inflammation. Further exploring this correlation between immuno-modulation and immaturity may open perspectives in allogenic cell therapies.

Fibroblast growth factor type 2 signaling is critical for DNA repair in human keratinocyte stem cells.

Tissue stem cells must be endowed with superior maintenance and repair systems to ensure genomic stability over multiple generations, which would be less necessary in more differentiated cells. We previously reported that human keratinocyte stem cells were more resistant to ionizing radiation toxicity than their direct progeny, the keratinocyte progenitor cells. In the present study we addressed the mechanisms underlying this difference. Investigations of DNA repair showed that both single and double DNA strand breaks were repaired more rapidly and more efficiently in stem cells than in progenitors. As cell signaling is a key regulatory step in the management of DNA damage, a gene profiling study was performed. Data revealed that several genes of the fibroblast growth factor type 2 (FGF2) signaling pathway were induced by DNA damage in stem cells and not in progenitors. Furthermore, an increased content of the FGF2 protein was found in irradiated stem cells, both for the secreted and the cellular forms of the protein. To examine the role of endogenous FGF2 in DNA repair, stem cells were exposed to FGF2 pathway inhibitors. Blocking the FGF2 receptor (FGF receptor 1) or the kinase (Ras-mitogen-activated protein kinase 1) resulted in a inhibition of single and double DNA strand-break repair in the keratinocyte stem cells. Moreover, supplementing the progenitor cells with exogenous FGF2 activated their DNA repair. We propose that, apart from its well-known role as a strong mitogen and prosurvival factor, FGF2 helps to maintain genomic integrity in stem cells by activating stress-induced DNA repair.

Investigating human keratinocyte stem cell identity.

Studies of the regulatory networks controlling intrinsic properties and fate of adult stem cells are in a large part performed in animal models. Epidermis is one of the most accessible human tissues for researchers, which is a critical parameter for conducting programs dedicated to this knowledge in human stem cell systems. Keratinocyte stem cells constitute a particularly valuable model, because of this practical aspect, but more importantly because their existence is for decades validated by the clinical demonstration of their impressive capacity for epidermis regeneration. For the fundamentalist, human keratinocyte stem cells represent a unique system to dissect the genetic and epigenetic controls of "stemness" and self-renewal. For this purpose, a highly limiting point is our current inability of obtaining a cellular material corresponding to keratinocyte stem cells with homogeneous phenotypic and functional characteristics. The search for tools suitable for the prospective selection of keratinocyte stem cells will benefit from studies conducted at the broad level of the global stem cell field, as well as from more specifically targeted approaches. Advances in that direction are tightly linked to the development of functional assays allowing reliable assessment and modeling of the different stem cell-associated functional characteristics.

Cellular adhesion on collagen: a simple method to select human basal keratinocytes which preserves their high growth capacity.

The regenerative capacity of human interfollicular epidermis is closely linked to the potential of immature keratinocytes present within its basal layer. The availability of selection methods and culture systems allowing precise assessment of basal keratinocyte characteristics is critical for increasing our knowledge of this cellular compartment. This report presents a multi-parametric comparative study of basal keratinocytes selected according to two different principles: 1) high adhesion capacity on a type-I collagen-coated substrate [Adh⁺⁺⁺], 2) high cell-surface expression of α6-integrin [Itg-α6 (high)]. Importantly, analysis performed at the single-cell level revealed similar primary clone-forming efficiency values of 45.5% ±â€Š6.7% [Itg-α6(high)] and 43.7% ±â€Š7.4% [Adh⁺⁺⁺], which were markedly higher than those previously reported. In addition, both methods selected keratinocytes exhibiting an extensive long-term growth potential exceeding 100 cell doublings and the capacity for generating a pluristratified epidermis. Our study also included a global transcriptome comparison. Genome-wide profiling indicated a strong similarity between [Adh⁺⁺⁺] and [Itg-α6(high)] keratinocytes, and revealed a common basal-associated transcriptional signature. In summary, cross-analysis of [Adh⁺⁺⁺] and [Itg-α6(high)] keratinocyte characteristics showed that these criteria identified highly equivalent cellular populations, both characterized by unexpectedly high growth capacities. These results may have broad impacts in the tissue engineering and cell therapy fields.

Skin Immunity and Tolerance: Focus on Epidermal Keratinocytes Expressing HLA-G.

Although the role of epidermal cells in skin regeneration has been extensively documented, their functions in immunity and tolerance mechanisms are largely underestimated. The aim of the present review was to outline the state of knowledge on resident immune cells of hematopoietic origin hosted in the epidermis, and then to focus on the involvement of keratinocytes in the complex skin immune networks acting in homeostasis and regeneration conditions. Based on this knowledge, the mechanisms of immune tolerance are reviewed. In particular, strategies based on immunosuppression mediated by HLA-G are highlighted, as recent advances in this field open up perspectives in epidermis-substitute bioengineering for temporary and permanent skin replacement strategies.

Human Keratinocytes Inhibit CD4+ T-Cell Proliferation through TGFB1 Secretion and Surface Expression of HLA-G1 and PD-L1 Immune Checkpoints.

Human skin protects the body against infection and injury. This protection involves immune and epithelial cells, but their interactions remain largely unknown. Here, we show that cultured epidermal keratinocytes inhibit allogenic CD4+ T-cell proliferation under both normal and inflammatory conditions. Inhibition occurs through the secretion of soluble factors, including TGFB1 and the cell-surface expression of HLA-G1 and PD-L1 immune checkpoints. For the first time, we here describe the expression of the HLA-G1 protein in healthy human skin and its role in keratinocyte-driven tissue immunomodulation. The overexpression of HLA-G1 with an inducible vector increased the immunosuppressive properties of keratinocytes, opening up perspectives for their use in allogeneic settings for cell therapy.

The ADIPS Pilot National Diabetes in Pregnancy Benchmarking Programme.

BACKGROUND: To test the feasibility of benchmarking the care of women with pregnancies complicated by hyperglycaemia. METHODS: A retrospective audit of volunteer diabetes services in Australia and New Zealand involving singleton pregnancies resulting in live births between 2014 and 2020. Ranges are shown and compared across services. RESULTS: The audit included 10,144 pregnancies (gestational diabetes mellitus (GDM) = 8696; type 1 diabetes (T1D) = 435; type 2 diabetes (T2D) = 1013) from 11 diabetes services. Among women with GDM, diet alone was used in 39.4% (ranging among centres from 28.8-57.3%), metformin alone in 18.8% (0.4-43.7%), and metformin and insulin in 10.1% (1.5-23.4%); when compared between sites, all p < 0.001. Birth was by elective caesarean in 12.1% (3.6-23.7%) or emergency caesarean in 9.5% (3.5-21.2%) (all p < 0.001). Preterm births (<37 weeks) ranged from 3.7% to 9.4% (p < 0.05), large for gestational age 10.3-26.7% (p < 0.001), admission to special care nursery 16.7-25.0% (p < 0.001), and neonatal hypoglycaemia (<2.6 mmol/L) 6.0-27.0% (p < 0.001). Many women with T1D and T2D had limited pregnancy planning including first trimester hyperglycaemia (HbA1c > 6.5% (48 mmol/mol)), 78.4% and 54.6%, respectively (p < 0.001). CONCLUSION: Management of maternal hyperglycaemia and pregnancy outcomes varied significantly. The maintenance and extension of this benchmarking service provides opportunities to identify policy and clinical approaches to improve pregnancy outcomes among women with hyperglycaemia in pregnancy.

Density of tumour stroma is correlated to outcome after adoptive transfer of CD4+ and CD8+ T cells in a murine mammary carcinoma model.

Adoptive immunotherapy shows promise for the treatment of cancer; however, partial or mixed responses remain common outcomes due to the heterogeneity of tumours. We studied three murine mammary tumour lines that express an ovalbumin-tagged version of HER-2/neu and reproducibly undergo complete regression (CR), partial regression (PR), or progressive disease (PD) after adoptive transfer of ovalbumin-specific CD8(+) (OT-I) and CD4(+) (OT-II) T cells. The three tumour lines were implanted in immunocompetent C57Bl/6 host mice, and established tumours were treated by adoptive transfer of naive OT-I and OT-II T cells. Tumours of the CR and PR classes triggered almost indistinguishable T cell responses in terms of activation, proliferation, trafficking to the tumour site, infiltration of tumour stroma, and intratumoural T cell proliferation; however, tumours of the PR class showed reduced infiltration of tumour epithelium by donor T cells. PD responses were associated with early impairment of T cell activation and proliferation in draining lymph node, followed by negligible infiltration of tumour tissue by donor T cells. Histopathological determinants of outcome were investigated through an unsupervised analysis of 64 untreated tumours representing the three response classes. Tumours of the CR class had proportionately more stroma, which had a looser, more collagen-rich histological appearance. Thus, the amount and composition of tumour stroma distinguished successfully (CR) from unsuccessful (PR or PD) outcomes after adoptive T cell transfer, a finding that might facilitate the design of immunotherapy trials for human breast cancer.