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关键词： HHT   Hereditary hemorrhagic telangiectasia   ENG   Endoglin   EC   Endothelial cell   HUVEC   Human umbilical vein endothelial cell   HMVEC-L   Human lung microvascular endothelial cell   AVM   Arteriovenous malformation   ALK   Activin receptor-like kinase   SMAD   Mothers against decapentaplegic homolog   TGF-β   Transforming growth factor-beta   ANGPT   Angiopoietin   siRNA   Small interfering RNA   RT-qPCR   Reverse transcription-quantitative polymerase chain reaction   ERK   Extracellular signal-regulated kinase   VEGF-A   Vascular endothelial growth factor A   VEGFR2   Vascular endothelial growth factor receptor 2   TIE2 and TEK   Tyrosine-protein kinase receptor   FGF2   fibroblast growth factor 2   FGFR   Fibroblast growth factor receptor   NF-kB   Nuclear factor kappa B   Sp1   Specific protein 1   Rac1   Ras-related C3 botulinum toxin substrate 1   GAPDH   Glyceraldehyde 3-phosphate dehydrogenase  

摘要： Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by vascular dysplasia. Mutations of the endoglin (ENG) gene that encodes a co-receptor of the transforming growth factor beta 1 signaling pathway cause type I HHT. ENG is primarily expressed in endothelial cells (ECs), but its interaction with other key angiogenic pathways to control angiogenesis has not been well addressed. The aim of this study is to investigate ENG interplay with VEGFR2, FGFR1 and TIE2 in primary human ECs. ENG was knocked-down with siRNA in human umbilical vein ECs (HUVECs) and human lung microvascular ECs (HMVEC-L). Gene expression was measured by RT-qPCR and Western blotting. Cell signaling pathway activation was analyzed by detecting phosphor-ERK and phosphor-AKT levels. Cell migration and apoptosis were assessed using the Boyden chamber assay and the CCK-8 Kit, respectively. Loss of ENG in HUVECs led to significantly reduced expression of VEGFR2 but not TIE2 or FGFR1, which was also confirmed in HMVEC-L. HUVECs lacking ENG had significantly lower levels of active Rac1 and a substantial reduction of the transcription factor Sp1, an activator of VEGFR2 transcription, in nuclei. Furthermore, VEGF- but not bFGF- or angiopoietin-1-induced phosphor-ERK and phosphorAKT were suppressed in ENG deficient HUVECs. Functional analysis revealed that ENG knockdown inhibited cell migratory but enhanced anti-apoptotic activity induced by VEGF. In contrast, bFGF, angiopoietin-1 and -2 induced HUVEC migration and anti-apoptotic activities were not affected by ENG knockdown. In conclusion, ENG deficiency alters the VEGF/VEGFR2 pathway, which may play a role in HHT pathogenesis. (Translational Research 2021;235:129-143)

摘要： Background and Aims Studies of the identity and pathophysiology of fibrogenic HSCs have been hampered by a lack of genetic tools that permit specific and inducible fate-mapping of these cells in vivo. Here, by single-cell RNA sequencing of nonparenchymal cells from mouse liver, we identified transcription factor 21 (Tcf21) as a unique marker that restricted its expression to quiescent HSCs. Approach and Results Tracing Tcf21(+) cells by Tcf21-CreER (Cre-Estrogen Receptor fusion protein under the control of Tcf21 gene promoter) targeted ~10% of all HSCs, most of which were located at periportal and pericentral zones. These HSCs were quiescent under steady state but became activated on injuries, generating 62%-67% of all myofibroblasts in fibrotic livers and ~85% of all cancer-associated fibroblasts (CAFs) in liver tumors. Conditional deletion of Transforming Growth Factor Beta Receptor 2 (Tgfbr2) by Tcf21-CreER blocked HSC activation, compromised liver fibrosis, and inhibited liver tumor progression. Conclusions In conclusion, Tcf21-CreER-targeted perivenous stellate cells are the main source of myofibroblasts and CAFs in chronically injured livers. TGF-beta signaling links HSC activation to liver fibrosis and tumorigenesis.

关键词： brain microvascular endothelial cells   neural stem cells   neurovascular unit   reconstituting   three-dimensional  

摘要： Neurovascular dysfunction is a primary or secondary cause in the pathogenesis of several cerebrovascular and neurodegenerative disorders, including stroke. Therefore, the overall protection of the neurovascular unit (NVU) is a promising therapeutic strategy for various neurovascular diseases. However, the complexity of the NVU limits the study of the pathological mechanisms of neurovascular dysfunction. Reconstituting the in vitro NVU is important for the pathological study and drug screening of neurovascular diseases. In this study, we generated a spontaneously assembled three-dimensional NVU (3D NVU) by employing the primary neural stem cells and brain microvascular endothelial cells in a Matrigel extracellular matrix platform. This novel model exhibits the fundamental structures and features of the NVU, including neurons, astrocytes, oligodendrocytes, vascular-like structures, and blood-brain barrier-like characteristics. Additionally, under oxygen-glucose deprivation, the 3D NVU exhibits the neurovascular- or oxidative stress-related pathological characteristics of cerebral ischemia and the injuries can be mitigated, respectively, by supplementing with the vascular endothelial growth factor or edaravone, which demonstrated that the availability of 3D NVU in ischemic stroke modeling. Finally, the 3D NVU promoted the angiogenesis and neurogenesis in the brain of cerebral ischemia rats. We expect that the proposed in vitro 3D NVU model will be widely used to investigate the relationships between angiogenesis and neurogenesis and to study the pathology and pharmacology of neurovascular diseases.

关键词： Arsenic   In vitro   Genomics   Mode of action   Carcinogenicity  

摘要： Evidence from both in vivo and in vitro studies suggests that gene expression changes from long-term exposure to arsenite evolve markedly over time, including reversals in the direction of expression change in key regulatory genes. In this study, human uroepithelial cells from the ureter segments of 4 kidney-donors were continuously treated in culture with arsenite at concentrations of 0.1 or 1 mu M for 60 days. Gene expression at 10, 20, 30, 40, and 60 days was determined using Affymetrix human genome microarrays and signal pathway analysis was performed using GeneGo Metacore. Arsenic treated cells continued to proliferate for the full 60-day period, whereas untreated cells ceased proliferating after approximately 30 days. A peak in the number of gene changes in the treated cells compared to untreated controls was observed between 30 and 40 days of exposure, with substantially fewer changes at 10 and 60 days, suggesting remodeling of the cells over time. Consistent with this possibility, the direction of expression change for a number of key genes was reversed between 20 and 30 days, including CFOS and MDM2. While the progression of gene changes was different for each subject, a common pattern was observed in arsenic treated cells over time, with early upregulation of oxidative stress responses (HMOX1, NQ01, TXN, TXNRD1) and down-regulation of immune/inflammatory responses (IKK alpha). At around 30 days, there was a transition to increased inflammatory and proliferative signaling (AKT, CFOS), evidence of epithelial-to-mesenchymal transition (EMT), and alterations in DNA damage responses (MDM2, ATM). A common element in the changing response of cells to arsenite over time appears to involve up-regulation of MDM2 by inflammatory signaling (through AP-1 and NF-kappa B), leading to inhibition of P53 function.

关键词： arborizing vessels   basal cell carcinoma   BCC   blue-white veil   concentric structures   corkscrew vessels   dermatoscopy   dermoscopy   dotted vessels   glomerular vessels   hairpin vessels   infiltrative basal cell carcinoma   large blue-grey ovoid nests   leaf-like areas   morpheaform basal cell carcinoma   multiple blue-grey dots and globules   multiple in-focus dots   multiple small erosions   nodular basal cell carcinoma   nonpigmented basal cell carcinoma   pigmented basal cell carcinoma   polymorphous vessels   rosettes   shiny white structures   shiny white-red structureless background   short-fine telangiectasia   spoke wheel areas   superficial basal cell carcinoma   ulceration  

摘要： Background: Multiple studies have reported on dermoscopic structures in basal cell carcinoma (BCC) and its subtypes, with varying results. Objective: To systematically review the prevalence of dermoscopic structures in BCC and its subtypes. Methods: Databases and reference lists were searched for relevant trials according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Studies were assessed for the relative proportion of BCC dermoscopic features. Random-effects models were used to estimate summary effect sizes. Results: Included were 31 studies consisting of 5950 BCCs. The most common dermoscopic features seen in BCC were arborizing vessels (59%), shiny white structures (49%), and large blue-grey ovoid nests (34%). Arborizing vessels, ulceration, and blue-grey ovoid nests and globules were most common in nodular BCC;short-fine telangiectasia, multiple small erosions, and leaf-like, spoke wheel and concentric structures in superficial BCC;porcelain white areas and arborizing vessels in morpheaform BCC;and arborizing vessels and ulceration in infiltrative BCC. Limitations: Studies had significant heterogeneity. Studies reporting BCC histopathologic subtypes did not provide clinical data on pigmentation of lesions. Conclusion: In addition to arborizing vessels, shiny white structures are a common feature of BCC. A constellation of dermoscopic features may aid in differentiating between BCC histopathologic subtypes.

关键词： non-viral delivery   Solupore   CAR T   CRISPR   CD19 CAR   TRAC  

摘要： Background aims: Next-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches. Here the authors have examined the suitability of the Solupore non-viral delivery system for engineering primary human T cells for cell therapy applications. Methods: The Solupore system was used to deliver messenger RNA (mRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) guide RNA ribonucleoprotein (RNP) cargos to T cells, and efficiency was measured by flow cytometry. Cell perturbation was assessed by immune gene expression profiling, including an electroporation comparator. In vitro and in vivo cytotoxicity of chimeric antigen receptor (CAR) T cells generated using the Solupore system was evaluated using a realtime cellular impedance assay and a Raji-luciferase mouse tumor model, respectively. Results: Efficient transfection was demonstrated through delivery of mRNA and CRISPR CAS9 RNP cargos individually, simultaneously and sequentially using the Solupore system while consistently maintaining high levels of cell viability. Gene expression profiling revealed minimal alteration in immune gene expression, demonstrating the low level of perturbation experienced by the cells during this transfection process. By contrast, electroporation resulted in substantial changes in immune gene expression in T cells. CART cells generated using the Solupore system exhibited efficient cytotoxicity against target cancer cells in vitro and in vivo. Conclusions: The Solupore system is a non-viral means of simply, rapidly and efficiently delivering cargos to primary huma

摘要： Aims Insulin degludec/aspart (IDegAsp) and insulin glargine U300 (IGlarU300) have recently emerged as popular new-generation insulin analogues. The aim of this real-life study was to investigate the patient profiles in which IGlarU300 and IDegAsp were preferred and the insulin combinations after which each of them were mostly used and also to analyse the effect of these two insulin analogues on blood glucose regulation and hypoglycaemia. Materials and Methods The retrospective study included 174 patients that were switched from basal insulin, basal-bolus insulin, or premixed insulin to IGlarU300 or IDegAsp due to uncontrolled blood glucose levels or history of hypoglycaemia. Hypoglycaemia, body weight, body mass index (BMI), fasting plasma glucose (FPG) and HbA1c levels over 3-month periods were evaluated for each patient. Results There were 84 and 90 patients in the IGlarU300 and IDegAsp groups, respectively. Body weight was similar in both groups. Baseline FPG and HbA1c levels in the IGlarU300 and IDegAsp groups were 9.0%, 175.5 mg/dL and 9.4%, 193.5 mg/dL, respectively. A significant decrease was found in FPG and HbA1c levels in both groups (138.5, 7.8 vs 141.5, 8.2;P < .001 for all). Moreover, a significant weight gain was observed in both groups (P < .05 for both). The prevalence of hypoglycaemia in both groups decreased significantly and consistently between months 1 and 9 (P < .001). At month 12, although this decrease continued in the IGlarU300 group (P = .013), no significant decrease was observed in the IDegAsp group (P = .057). Conclusion Both twice-daily IDegAsp +/- bolus insulin and IGlarU300 basal bolus insulin therapies are effective and safe treatment modalities.

关键词： Enterprise architecture   theoretical basis   boundary objects theory   cognitive fit theory   management fashion theory   literature review  

摘要： Enterprise architecture is a collection of artifacts describing various aspects of an organization from an integrated business and IT perspective. Practicing enterprise architecture in organizations implies using these artifacts to facilitate information systems planning and improve business and IT alignment. Despite its long history, the enterprise architecture discipline still remains largely atheoretical and lacks a solid theoretical basis. Based on our previous empirical studies of the practical usage of enterprise architecture artifacts in multiple organizations and broad literature analysis, this conceptual article identifies and discusses in detail 10 theories that can be considered key for understanding how an enterprise architecture practice works: actor-network theory, boundary objects theory, cognitive fit theory, communities of practice theory, decision-making theories, information processing theory, knowledge management theory, management fashion theory, media richness theory, and uncertainty principle. Taken together, these theories offer a comprehensive theoretical view of an enterprise architecture practice explaining the role of enterprise architecture artifacts, their usability, and participation of stakeholders and, therefore, may constitute a theoretical basis of the entire enterprise architecture discipline. Although this article does not elaborate on any of these theories, it brings these theories to light, establishes their critical importance for comprehending an enterprise architecture practice, and positions them as central to the enterprise architecture discourse. Each of these theories can be leveraged by enterprise architecture scholars in their future studies for analyzing enterprise architecture practices through respective theoretical lenses. This article intends to provide fresh theoretical insights on enterprise architecture, spark new waves of theoretical enterprise architecture research, and contribute to the development of a soun

关键词： chemotherapy   human papilloma virus   oropharyngeal carcinoma   radiotherapy   Transoral Robotic Surgery  

摘要： Objectives To analyse the oncological outcomes following primary Transoral Robotic Surgery (TORS) for oropharyngeal squamous cell carcinoma (OPSCC). Design Observational case series. Setting Tertiary centre;first TORS practice to commence in the UK. Participants All consecutive patients undergoing primary TORS with curative intent, with or without adjuvant treatment. Main outcome measures Descriptive analysis of patient and tumour pathology variables. Survival outcomes: Overall, Disease-Specific, Progression-Free and Locoregional control. Results The cohort comprised of 120 patients undergoing TORS with minimum 12-month follow-up data and the following characteristics: mean age 58 years, 91 males (76%), 78 tonsil (65%) and 34 base of tongue primaries (28%), 89% HPV-related OPSCC. The surgical pathology revealed 14 (12%) with positive margins, 19 (16%) had close margins <2mm and 31% with extranodal extension. The treatment was as follows: 39 (33%) treated with TORS alone, 50 (42%) received adjuvant radiotherapy and 31 (26%) received adjuvant radiotherapy with chemotherapy. There were 15 recurrences. Estimated survival for all patients at 3 years (95% CI): overall 85% (78-92), disease-specific 90% (85-96), progression-free 86% (79-92) and locoregional control 90% (84-96). The equivalent survival figures for the HPV-related cases alone were as follows: overall 88% (82-94), disease-specific 93% (87-98), progression-free 88% (81-95) and locoregional control 92% (87-98). Conclusions Whilst TORS has become a common practice in the management of OPSCC in the UK, these are the first reported oncological outcomes. For selected patients, TORS with or without adjuvant therapy is an appropriate treatment modality.

关键词： Human fetal testis   Sertoli cells   Stem cell factor   Spermatogonial stem cells   beta-estradiol  

摘要： Background Mammalian spermatogenesis is responsible for male fertility and is supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs). Sertoli cells provide a supportive microenvironment for SSCs, in part by the production of stem cell factor (SCF), which is a potent regulator of spermatogonia proliferation and survival. Methods We investigated the novel role of beta-estradiol in modulating the proliferation and apoptosis of fetal SSCs via the regulation of SCF secretion in Sertoli cells isolated from human fetal testes. The proliferation of SSCs in the co-culture system was determined by colony formation and BrdU incorporation assays. TUNEL assay was used to measure SSC apoptosis in co-culture in response to treatment with control, beta-estradiol, or the combination of beta-estradiol and the estrogen receptor inhibitor ICI 182780. Results In the system with purified human fetal Sertoli cells (MIS+/c-Kit-/AP-), beta-estradiol upregulated the production of SCF in a dose- and time-dependent manner. In the co-culture system of primary human fetal SSCs (c-Kit+/SSEA-4+/Oct-4+/AP+) and Sertoli cells (MIS+), beta-estradiol markedly increased the proliferation of SSCs. Moreover, SSC apoptosis was significantly inhibited by beta-estradiol and was completely reversed by the combination of beta-estradiol and ICI 182780. Conclusion Here we report, for the first time, that beta-estradiol can induce the increase of SCF expression in human fetal Sertoli cells and regulates the growth and survival of human fetal SSCs. These novel findings provide new perspectives on the current understanding of the role of estrogen in human spermatogenesis.