Potential and distribution of transplanted hematopoietic stem cells in a nonablated mouse model

Increasingly, allogeneic and even more often autologous bone marrow transplants are being done to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide an opportune means of delivering genes in transfected, engrafting stem cells. However, despite its widespread clinical use and promising gene therapy applications, relatively little is known about the mechanisms of engraftment in marrow transplant recipients. This is especially so in the nonablated recipient setting. Our data show that purified lineage negative rhodamine 123/Hoechst 33342 dull transplanted hematopoietic stem cells engraft into the marrow of nonablated syngeneic recipients. These cells have multilineage potential, and maintain a distinct subpopulation with stem cell characteristics. The data also suggests a spatial localization of stem cell niches to the endosteal surface, with all donor cells having a high spatial affinity to this area. However, the level of stem cell engraftment observed following a transplant of stem cells was significantly lower than that expected following a transplant of the same number of unseparated marrow cells from which the purified cells were derived, suggesting the existence of a nonstem cell facilitator population, which is required in a nonablated syngeneic transplant setting

Cells Capable of Bone Production Engraft from Whole Bone Marrow Transplants in Nonablated Mice

Allogeneic and autologous marrow transplants are routinely used to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide opportune means of delivering genes in transfected, engrafting stem cells. However, relatively little is known about the mechanisms of engraftment in transplant recipients, especially in the nonablated setting and with regard to cells not of hemopoietic origin. In particular, this includes stromal cells and progenitors of the osteoblastic lineage. We have demonstrated for the first time that a whole bone marrow transplant contains cells that engraft and become competent osteoblasts capable of producing bone matrix. This was done at the individual cell level in situ, with significant numbers of donor cells being detected by fluorescence in situ hybridization in whole femoral sections. Engrafted cells were functionally active as osteoblasts producing bone before being encapsulated within the bone lacunae and terminally differentiating into osteocytes. Transplanted cells were also detected as flattened bone lining cells on the periosteal bone surface

Observational two-country study of undergraduate nursing students’ self-perceptions of leadership behaviours in clinical practice

Aim Strengthening the future of the nursing workforce through nurturing leadership development in novice and newly qualified nurses through educational programmes is viewed as crucial internationally. Enabling and developing leadership skills is challenging, and nursing students require clinical and academic support throughout their degree programmes. This study aimed to measure undergraduate nursing students’ self-perceptions of clinical leadership behaviours between two countries. Methods: A cross-sectional observational study was completed with two cohorts of undergraduate nursing students in England and Israel following ethical approval. The Spanish version of Self-Assessment Leadership 40 item Instrument (SALI) ES-SALI measuring four leadership dimensions was used following translation into English and Hebrew. A web-based anonymous survey using Qualtrics online software was distributed from October 2021 to April 2022. Results: The overall response rate was 22.5% (n=138) [27% (Israel); 18% (England)]—Cronbach’s Alpha= 0.94 overall and >0.7 in each dimension. Demographic differences noted older aged students: (>32 years) in England 50.1% V Israel 6.6% p <0.001; and previous work experience: England 84.8% V Israel 44.3% p<0.001. Significant differences were identified in two leadership dimensions, with English students reporting higher scores: “Emotional Intelligence” England M= 3.22 (SD 0.54) V Israel M= 3.02 (SD 0.54) and “Impact and Influence” England M= 3.13 (SD 0.58) V Israeli M= 2.97 (SD 0.53). Year of study was consistent with higher leadership scores for both cohorts in the middle year of study. Conclusions: Previous evidence establishes the importance of emotional intelligence in leadership development and providing quality care. This study demonstrates differences in perceptions of leadership among nursing students in two countries with implications for the profession and workforce development. Nurse educators should consider enhanced leadership skill development in preparing nurses to provide quality, safe and person-centred care

A CX3CRI Reporter hESC Line Facilitates Integrative Analysis of In-Vitro-Derived Microglia and Improved Microglia Identity upon Neuron-Glia Co-culture

Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies.Peer reviewe

Glucose tolerance is associated with differential expression of microRNAs in skeletal muscle: results from studies of twins with and without type 2 diabetes.

AIMS/HYPOTHESIS: We aimed to identify microRNAs (miRNAs) associated with type 2 diabetes and risk of developing the disease in skeletal muscle biopsies from phenotypically well-characterised twins. METHODS: We measured muscle miRNA levels in monozygotic (MZ) twins discordant for type 2 diabetes using arrays. Further investigations of selected miRNAs included target prediction, pathway analysis, silencing in cells and association analyses in a separate cohort of 164 non-diabetic MZ and dizygotic twins. The effects of elevated glucose and insulin levels on miRNA expression were examined, and the effect of low birthweight (LBW) was studied in rats. RESULTS: We identified 20 miRNAs that were downregulated in MZ twins with diabetes compared with their non-diabetic co-twins. Differences for members of the miR-15 family (miR-15b and miR-16) were the most statistically significant, and these miRNAs were predicted to influence insulin signalling. Indeed, miR-15b and miR-16 levels were associated with levels of key insulin signalling proteins, miR-15b was associated with the insulin receptor in non-diabetic twins and knockdown of miR-15b/miR-16 in myocytes changed the levels of insulin signalling proteins. LBW in twins and undernutrition during pregnancy in rats were, in contrast to overt type 2 diabetes, associated with increased expression of miR-15b and/or miR-16. Elevated glucose and insulin suppressed miR-16 expression in vitro. CONCLUSIONS: Type 2 diabetes is associated with non-genetic downregulation of several miRNAs in skeletal muscle including miR-15b and miR-16, potentially targeting insulin signalling. The paradoxical findings in twins with overt diabetes and twins at increased risk of the disease underscore the complexity of the regulation of muscle insulin signalling in glucose homeostasis.JB-J was supported by a grant from the Danish PhD School for Molecular Metabolism. The study was supported by grants from the Danish Medical Research Council, the Danish Strategic Research Council. The Novo Nordisk Foundation, the Danish Ministry of Science, Technology and Innovation. DSF-T was supported by the Biotechnology and Biological Sciences Research Council project grant BB/F-15364/1. SEO is a British Heart Foundation Senior Fellow (FS/09/029/27902).This is the final version of the article. It was first published by Springer at http://link.springer.com/article/10.1007%2Fs00125-014-3434-

Platelet-rich plasma in Achilles tendon healing 2 (PATH-2) trial: statistical analysis plan for a multicentre, double-blinded, parallel-group, placebo-controlled randomised clinical trial.

BACKGROUND: There has been a recent steep growth in platelet-rich plasma (PRP) use for musculoskeletal conditions, but findings from high quality clinical trial data are lacking in the literature. Here, we describe the statistical analysis plan (SAP) for the Platelet-rich plasma in Achilles Tendon Healing 2 (PATH-2) trial. METHODS: PATH-2 is a pragmatic, parallel-group, multi-centre, double-blinded, randomised, placebo-controlled, superiority trial. The study aims to evaluate the clinical efficacy of PRP in acute Achilles tendon rupture in terms of muscle-tendon function. Patients are identified in the orthopaedic/trauma outpatient clinic. The primary outcome is muscle-tendon work capacity from the Heel Rise Endurance Test result, expressed as the Limb Symmetry Index (work, in joules), at 24 weeks post randomisation. Multivariate linear regression adjusting for the stratification factors (centre and age) and additional prognostic factors will be used to investigate the adjusted effect of the intervention. The analysis will be by modified intention-to-treat. Sensitivity analysis will assess the internal validity of the trial results by performing a per-protocol analysis. Safety will be summarised by treatment arm for all patients who started treatment. Secondary patient-reported outcome measures will be analysed using linear mixed effects models to allow all data collected at all follow-up points to be considered. Missing data will be summarised and reported by treatment arm. Missing data imputation will be performed, if appropriate. DISCUSSION: The PATH-2 trial will be reported in accordance with the CONSORT statement. This SAP publication will avoid bias arising from prior knowledge of the study results. Any changes or deviations from the current SAP will be described and justified in the final report. TRIAL REGISTRATION: ISRCTN registry: ISRCTN54992179 , assigned 12 January 2015. ClinicalTrials.gov: NCT02302664, received 18 November 2014. UK Clinical Research Network Study Portfolio Database: ID 17850

Identification of a Thymic Epithelial Cell Subset Sharing Expression of the Class Ib HLA-G Molecule with Fetal Trophoblasts

HLA-G is the only class I determinant of the major histocompatibility complex (MHC) expressed by the trophoblasts, the fetal cells invading the maternal decidua during pregnancy. A unique feature of this nonclassical HLA molecule is its low polymorphism, a property that has been postulated to play an important role in preventing local activation of maternal alloreactive T and natural killer cells against the fetus. Yet, the mechanisms by which fetal HLA-G can be recognized as a self-MHC molecule by the maternal immune system remain unclear. Here we report the novel observation that HLA-G is expressed in the human thymus. Expression is targeted to the cell surface of thymic medullary and subcapsular epithelium. Thymic epithelial cell lines were generated and shown to express three alternatively spliced HLA-G transcripts, previously identified in human trophoblasts. Sequencing of HLA-G1 transcripts revealed a few nucleotide changes resulting in amino acid substitutions, all clustered within exon 3 of HLA-G, encoding for the α2 domain of the molecule. Our findings raise the possibility that maternal unresponsiveness to HLA-G–expressing fetal tissues may be shaped in the thymus by a previously unrecognized central presentation of this MHC molecule on the medullary epithelium

The RNA-binding protein SRSF3 has an essential role in megakaryocyte maturation and platelet production

RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine–rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.publishedVersionPeer reviewe

Identification of Estrogen Receptor Dimer Selective Ligands Reveals Growth-Inhibitory Effects on Cells That Co-Express ERα and ERβ

Estrogens play essential roles in the progression of mammary and prostatic diseases. The transcriptional effects of estrogens are transduced by two estrogen receptors, ERα and ERβ, which elicit opposing roles in regulating proliferation: ERα is proliferative while ERβ is anti-proliferative. Exogenous expression of ERβ in ERα-positive cancer cell lines inhibits cell proliferation in response to estrogen and reduces xenografted tumor growth in vivo, suggesting that ERβ might oppose ERα's proliferative effects via formation of ERα/β heterodimers. Despite biochemical and cellular evidence of ERα/β heterodimer formation in cells co-expressing both receptors, the biological roles of the ERα/β heterodimer remain to be elucidated. Here we report the identification of two phytoestrogens that selectively activate ERα/β heterodimers at specific concentrations using a cell-based, two-step high throughput small molecule screen for ER transcriptional activity and ER dimer selectivity. Using ERα/β heterodimer-selective ligands at defined concentrations, we demonstrate that ERα/β heterodimers are growth inhibitory in breast and prostate cells which co-express the two ER isoforms. Furthermore, using Automated Quantitative Analysis (AQUA) to examine nuclear expression of ERα and ERβ in human breast tissue microarrays, we demonstrate that ERα and ERβ are co-expressed in the same cells in breast tumors. The co-expression of ERα and ERβ in the same cells supports the possibility of ERα/β heterodimer formation at physio- and pathological conditions, further suggesting that targeting ERα/β heterodimers might be a novel therapeutic approach to the treatment of cancers which co-express ERα and ERβ

Resurrection and redescription of Varestrongylus alces (Nematoda; Protostrongylidae), a lungworm of the Eurasian moose (Alces alces), with report on associated pathology

Varestrongylus alces, a lungworm in Eurasian moose from Europe has been considered a junior synonym of Varestrongylus capreoli, in European roe deer, due to a poorly detailed morphological description and the absence of a type-series. Methods Specimens used in the redescription were collected from lesions in the lungs of Eurasian moose, from Vestby, Norway. Specimens were described based on comparative morphology and integrated approaches. Molecular identification was based on PCR, cloning and sequencing of the ITS-2 region of the nuclear ribosomal DNA. Phylogenetic analysis compared V. alces ITS-2 sequences to these of other Varestrongylus species and other protostrongylids. Results Varestrongylus alces is resurrected for protostrongylid nematodes of Eurasian moose from Europe. Varestrongylus alces causes firm nodular lesions that are clearly differentiated from the adjacent lung tissue. Histologically, lesions are restricted to the parenchyma with adult, egg and larval parasites surrounded by multinucleated giant cells, macrophages, eosinophilic granulocytes, lymphocytes. The species is valid and distinct from others referred to Varestrongylus, and should be separated from V. capreoli. Morphologically, V. alces can be distinguished from other species by characters in the males that include a distally bifurcated gubernaculum, arched denticulate crura, spicules that are equal in length and relatively short, and a dorsal ray that is elongate and bifurcated. Females have a well-developed provagina, and are very similar to those of V. capreoli. Morphometrics of first-stage larvae largely overlap with those of other Varestrongylus. Sequences of the ITS-2 region strongly support mutual independence of V. alces, V. cf. capreoli, and the yet undescribed species of Varestrongylus from North American ungulates. These three taxa form a well-supported crown-clade as the putative sister of V. alpenae. The association of V. alces and Alces or its ancestors is discussed in light of host and parasite phylogeny and host historical biogeography. Varestrongylus alces is a valid species, and should be considered distinct from V. capreoli. Phylogenetic relationships among Varestrongylus spp. from Eurasia and North America are complex and consistent with faunal assembly involving recurrent events of geographic expansion, host switching and subsequent speciation. Cervidae, Cryptic species, Historical biogeography, ITS-2, Metastrongyloidea, Parasite biodiversity, Varestrongylinae, Varestrongylus capreoli, Verminous pneumoniapublishedVersio