Conversion of adult endothelium to immunocompetent haematopoietic stem cells.

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1+ FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFß and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.

Erratum: Discovery of a neuromuscular syndrome caused by biallelic variants in ASCC3.

[This corrects the article DOI: 10.1016/j.xhgg.2021.100024.].

Discovery of a neuromuscular syndrome caused by biallelic variants in ASCC3.

Activating Signal Cointegrator 1 Complex, Subunit 3 (ASCC3) is part of the four-part ASC-1 transcriptional cointegrator complex. This complex includes ASCC1 (associated with spinal muscular atrophy with congenital bone fractures 2), TRIP4 (associated with spinal muscular atrophy with congenital bone fractures 1), and ASCC2 (not yet associated with human disease.) ASCC3 encodes a DNA helicase responsible for generating single-stranded DNA as part of the DNA damage response. Interestingly, ASCC3 expresses coding and non-coding isoforms, which act in opposition to balance the recovery of gene transcription after UV-induced DNA damage. Here we report the discovery of ASCC3 as the cause of a neuromuscular syndrome in seven unreported individuals from six unrelated families and updates on the one previously reported family. All the individuals share a neurologic phenotype that ranges from severe developmental delay to muscle fatigue. There appears to be genotype-phenotype correlation, as the most mildly affected individual is homozygous for a rare missense variant, while the more severely affected individuals are compound heterozygotes for a missense and a presumed loss-of-function (LOF) variant. There are no individuals with biallelic presumed LOF variants in our cohort or in gnomAD, as this genotype may not be compatible with life. In summary we report a syndrome in these eleven individuals from seven families with biallelic variants in ASCC3.

Breast Reconstruction Combining Lipofilling and Prepectoral Prosthesis after Radiotherapy.

Prosthetic reconstruction in previously irradiated breasts has been associated with a higher risk of complications. Here we describe the surgical and cosmetic outcome of our breast reconstruction process based on primary fat grafting combined with prosthetic placement. METHODS: In this multicenter retrospective study, 136 patients who underwent mastectomy and external chest wall radiotherapy between 2014 and 2018 were benefited from chest wall lipofilling and silicone implant placement were chosen. Patients were assessed for skin trophicity, thickness, and mobility and were allowed to undergo several lipofilling sessions before implant placement, if required. No patient had >3 lipofilling sessions. Cosmetic outcome was evaluated by the patient, surgeon, and nurse, using a Likert-type ordinal scale. RESULTS: We included 136 patients: 79 patients (58%) received only 1 session of lipofilling before implant placement, 33 (24.6%) had 2 sessions, and 24 (17.4%) had 3 sessions. The volume of the third lipofilling was significantly higher and the volume of the prosthesis of these patients was significantly lower than those of patients undergoing 1 or 2 lipofillings. Reconstruction failure rate was 2.2% (3 patients had explantation); however, all benefited from prosthesis reconstruction a year after the initial procedures. The average satisfaction score was 4.7 out of 5 as evaluated by patients, 4.8 out of 5 by surgeons, and 4.8 out of 5 by nurses. CONCLUSIONS: Primary lipofilling combined with prosthesis placement after radiotherapy is a reconstructive method that yields a satisfactory cosmetic outcome with a low complication rate. Such minimally invasive breast reconstruction approach can be an alternative to flap-based reconstruction.

Template-directed dye-terminator incorporation with fluorescence polarization detection for analysis of single nucleotide polymorphisms implicated in sepsis.

Sepsis continues to be a common source of morbidity and mortality in critically ill patients. Single nucleotide polymorphisms (SNPs) present in genes encoding inflammatory mediators have been associated with predisposition and outcome in this syndrome. The use of high throughput SNP analysis in large epidemiological studies is necessary to more fully understand the genetic underpinnings of this disease. We adapted template-directed dye-terminator incorporation with fluorescence polarization detection (TDI-FP) to the analysis of eight SNPs implicated in mediating the sepsis syndrome: TNF-alpha (-308), TNF-alpha (-238), TNF-beta (+250), IL-1beta (+3953), IL-6 (-174), IL-10 (-592), plasminogen activator inhibitor-1 (PAI-1 (-675)), and TLR4 299 (+1032). Optimization of PCR, amplicon purification, and template-directed dye-terminator incorporation reactions were necessary to achieve acceptable performance characteristics for these assays. Sequence validated samples served as controls. Using this method we were able to assign genotype in 99.3% of assays and identified 64 unique genotypes in samples obtained from 90 individuals. TDI-FP is a flexible and robust method of SNP detection that can be optimized in a systematic fashion. This method has potential advantages compared with other high throughput genotyping techniques and appears well suited to clinical situations requiring analysis of large numbers of samples.

The frequency and effects of cytochrome P450 (CYP) 2C9 polymorphisms in patients receiving warfarin.

BACKGROUND: Warfarin sodium (warfarin) is commonly prescribed in surgical practice. Warfarin use is complicated by an unpredictable dose response that may be due in part to genetically determined differences in metabolic capacity. To better understand the interaction between genotype and response to warfarin therapy, we determined the frequency and functional effects of polymorphisms of the predominant cytochrome P450 subfamily responsible for warfarin metabolism (eg, CYP2C9) in an ethnically defined U.S. patient population. DESIGN: Patients requiring chronic anticoagulation with warfarin sodium (warfarin) were recruited over an 11-month period (June 1999 through May 2000) from the inpatient and outpatient divisions of a tertiary care medical center in this prospective observational study. Clinical and demographic information was collected and CYP2C9 genotype was determined. RESULTS: One hundred fifty-three patients receiving warfarin therapy for at least four weeks and comprising two ethnic groups [33 African Americans (22%) and 120 Caucasians (78%)] were genotyped. The mean weekly warfarin dose (+/-SEM) for all patients [36.9 (+/- 1.5) mg] was not influenced by gender [85 males (56%), 68 females (44%)] or ethnicity (p>0.05 for both), but was significantly affected by age (p = 0.006 for weight adjusted warfarin dose). The frequencies of CYP polymorphisms were as follows: 2C9*2 (24/153) 15.7%, 2C9*3 (23/153) 15.0%. There were no gender differences in polymorphism frequency (CYP2C9*2 frequency = (13/ 85) 15.3% in males, (12/68) 17.6% in females, p=0.74; CYP2C9*3 frequency = (15/85) 17.6% in males and (8/68) 11.8% in females, p = 0.38). CYP polymorphisms were much less common in African Americans than Caucasians [(5/33) 15.2% versus (47/120) 39.2%, respectively p = 0.05)]. Patients with CYP polymorphisms (2C9*2, 2C9*3) had significantly lower warfarin doses compared to patients with wild-type genotypes [30.6 (+/- 2.5) mg versus 40.1 (+/- 1.7) mg, p = 0.0021] . Stepwise backward regression analysis suggested a moderate ability to predict warfarin dose based on CYP genotype (r2 = 0.26), p < 0.01). CONCLUSIONS: CYP2C9 polymorphisms are common, associated with significant reductions in warfarin dose, and partly account for interpatient variability in warfarin sensitivity. As interactions between genetic factors and other variables that influence warfarin effect are more completely understood, CYP analysis may prove a useful adjunct for increasing the safety and efficacy of this agent.

Osteoblastic and vascular endothelial niches, their control on normal hematopoietic stem cells, and their consequences on the development of leukemia.

Stem cell self-renewal is regulated by intrinsic mechanisms and extrinsic signals mediated via specialized microenvironments called "niches." The best-characterized stem cell is the hematopoietic stem cell (HSC). Self-renewal and differentiation ability of HSC are regulated by two major elements: endosteal and vascular regulatory elements. The osteoblastic niche localized at the inner surface of the bone cavity might serve as a reservoir for long-term HSC storage in a quiescent state. Whereas the vascular niche, which consists of sinusoidal endothelial cell lining blood vessel, provides an environment for short-term HSC proliferation and differentiation. Both niches act together to maintain hematopoietic homeostasis. In this paper, we provide some principles applying to the hematopoietic niches, which will be useful in the study and understanding of other stem cell niches. We will discuss altered microenvironment signaling leading to myeloid lineage disease. And finally, we will review some data on the development of acute myeloid leukemia from a subpopulation called leukemia-initiating cells (LIC), and we will discuss on the emerging evidences supporting the influence of the microenvironment on chemotherapy resistance.