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Abstract Background Hepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation. Methods Building upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells. Results SphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner. Conclusions This study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.

IntroductionLiver cancers exhibit abnormal (leaky) vasculature, hypoxia and an immunosuppressive microenvironment. Normalization of tumor vasculature is an emerging approach to treat many cancers. Blockmir CD5-2 is a novel oligonucleotide-based inhibitor of the miR-27a interaction with VE-Cadherin, the endothelial-specific cadherin. The combination of a vasoactive medication with inhibition of immune checkpoints such as programmed cell death protein 1 (PD1) has been shown to be effective in treating liver cancer in humans. We aimed to study the effect of CD5-2 combined with checkpoint inhibition (using an antibody against PD1) on liver tumor growth, vasculature and immune infiltrate in the diethylnitrosamine (DEN)-induced liver tumor mouse model.MethodsWe first analyzed human miR-27a and VE-Cadherin expression data from The Cancer Genome Atlas for hepatocellular carcinoma. CD5-2 and/or anti-PD1 antibody were given to the DEN-treated mice from age 7-months until harvest at age 9-months. Tumor and non-tumor liver tissues were analyzed using histology, immunohistochemistry, immunofluorescence and scanning electron microscopy.ResultsHuman data showed high miR-27a and low VE-Cadherin were both significantly associated with poorer prognosis. Mice treated with CD5-2 plus anti-PD1 antibody had significantly smaller liver tumors (50% reduction) compared to mice treated with either agent alone, controls, or untreated mice. There was no difference in tumor number. Histologically, tumors in CD5-2-treated mice had less leaky vessels with higher VE-Cadherin expression and less tumor hypoxia compared to non-CD5-2-treated mice. Only tumors in the combination CD5-2 plus anti-PD1 antibody group exhibited a more favorable immune infiltrate (significantly higher CD3+ and CD8+ T cells and lower Ly6G+ neutrophils) compared to tumors from other groups.DiscussionCD5-2 normalized tumor vasculature and reduced hypoxia in DEN-induced liver tumors. CD5-2 plus anti-PD1 antibody reduced liver tumor size possibly by altering the immune infiltrate to a more immunosupportive one.

Bronchiectasis is the abnormal dilation of the airway which may be caused by various etiologies in children. Beyond the more recognized cause of bacterial and viral infections and primary immunodeficiencies, other genetic conditions such as cystic fibrosis and primary ciliary dyskinesia (PCD) can also contribute to the disease. Currently, there is still debate on whether genome sequencing (GS) or exome sequencing reanalysis (rES) would be beneficial if the initial targeted testing results returned negative. This study aims to provide a back-to-back comparison between rES and GS to explore the best integrated approach for the functional and genetics evaluation for patients referred for assessment of bronchiectasis. In phase 1, an initial 60 patients were analyzed by exome sequencing (ES) with one additional individual recruited later as an affected sibling for ES. Functional evaluation of the nasal nitric oxide test, transmission electron microscopy, and high-speed video microscopy were also conducted when possible. In phase 2, GS was performed on 30 selected cases with trio samples available. To provide a back-to-back comparison, two teams of genome analysts were alternatively allocated to GS or rES and were blinded to each other’s analysis. The time for bioinformatics, analysis, and diagnostic utility was recorded for evaluation. ES revealed five positive diagnoses (5/60, 8.3%) in phase 1, and four additional diagnoses were made by rES and GS (4/30, 13%) during phase 2. Subsequently, one additional positive diagnosis was identified in a sibling by ES and an overall diagnostic yield of 10/61 (16.4%) was reached. Among those patients with a clinical suspicion of PCD (n = 31/61), the diagnostic yield was 26% (n = 8/31). While GS did not increase the diagnostic yield, we showed that a variant of uncertain significance could only be detected by GS due to improved coverage over ES and hence is a potential benefit for GS in the future. We show that genetic testing is an essential component for the diagnosis of early-onset bronchiectasis and is most effective when used in combination with functional tools such as TEM or HSVM. Our comparison of rES vs. GS suggests that rES and GS are comparable in clinical diagnosis.

Abstract Background Gynaecomastia is a fairly common condition in puberty but is rare in prepubertal boys. While it is necessary to exclude possible endocrinopathay in prepubertal gynaecomastia, medication is an important and potentially reversible cause to consider in new onset gynaecomastia. Isoniazid-induced gynaecomastia has been reported in adult males, but none was reported in the paediatric population and general paediatricians may not be aware of this uncommon side effect. Case presentation We hereby report a 11-year-old prepubertal boy who developed gynaecomastia while taking anti-tuberculosis drugs. Investigations excluded endocrinopathies. Gynaecomastia subsided 8 weeks after stopping isoniazid. Conclusion This case is the first paediatric case report describing the association of gynaecomastia with isoniazid use. It is important for general paediatricians to recognize this entity, as prompt diagnosis and cessation of the offending drug can lead to resolution of the problem.

Purpose This study was conducted to evaluate the efficacy of a community-based follow-up program on parenting stress, parenting efficacy, and coping among parents with premature infants. Methods A non-equivalent control group pre-post quasi-experimental design was used. This program consisted of structured home visits and self-help group meetings for 6 months. The experimental group (n=29) received visits by an experienced neonatal intensive care unit (NICU) nurse and the control group (n=27) was visited by a visiting nurse. Data were analyzed using the x2 test, t-test, and analysis of covariance. Results Parents' coping behavior significantly differed in the experimental group compared to the control group (t=3.14, p=.003). In particular, coping subscale I, for maintaining the family situation (t=2.63, p=.011), and subscale III, for understanding the infant's medical situation (t=4.30, p

Abstract Background Vascular endothelial cell alignment in the direction of flow is an adaptive response that protects against aortic diseases such as atherosclerosis. The RhoGTPases are known to regulate this alignment. We have shown previously that ARHGAP18 in endothelial cells is a negative regulator of RhoC and its expression is essential in flow-mediated alignment. Depletion of ARHGAP18 inhibits alignment and results in the induction of a pro-inflammatory phenotype. In embryogenesis, ARHGAP18 was identified as a downstream effector of the Yes-associated protein, YAP, which regulates cell shape and size. Methods We have used siRNA technology to deplete either ARHGAP18 or YAP in human endothelial cells. The in vitro studies were performed under athero-protective, laminar flow conditions. The analysis of YAP activity was also investigated, using high performance confocal imaging, in our ARHGAP18 knockout mutant mice. Results We show here that loss of ARHGAP18, although decreasing the expression of YAP results in its nuclear localisation consistent with activation. We further show that depletion of YAP itself results in its activation as defined by an in increase in its nuclear localisation and an increase in the YAP target gene, CyR61. Depletion of YAP, similar to that observed for ARHGAP18 depletion, results in loss of endothelial cell alignment under high shear stress mediated flow and also in the activation of NFkB, as determined by p65 nuclear localisation. In contrast, ARHGAP18 overexpression results in upregulation of YAP, its phosphorylation, and a decrease in the YAP target gene Cyr61, consistent with YAP inactivation. Finally, in ARHGAP18 deleted mice, in regions where there is a loss of endothelial cell alignment, a situation associated with a priming of the cells to a pro-inflammatory phenotype, YAP shows nuclear localisation. Conclusion Our results show that YAP is downstream of ARHGAP18 in mature endothelial cells and that this pathway is involved in the athero-protective alignment of endothelial cells under laminar shear stress. ARHGAP18 depletion leads to a disruption of the junctions as seen by loss of VE-Cadherin localisation to these regions and a concomitant localisation of YAP to the nucleus.

Cellular senescence is now recognized as one of the hallmarks of aging. Herein, we examine current findings on senescence of the vascular endothelium and its impacts on age-related vascular diseases. Endothelial senescence can result in systemic metabolic changes, implicating senescence in chronic diseases such as diabetes, obesity and atherosclerosis. Senolytics, drugs that eliminate senescent cells, afford new therapeutic strategies for control of these chronic diseases.

Cerebral cavernous malformations (CCMs) are vascular lesions predominantly developing in the central nervous system (CNS), with no effective treatments other than surgery. Loss-of-function mutation in CCM1/krev interaction trapped 1 (KRIT1), CCM2, or CCM3/programmed cell death 10 (PDCD10) causes lesions that are characterized by abnormal vascular integrity. Vascular endothelial cadherin (VE-cadherin), a major regulator of endothelial cell (EC) junctional integrity is strongly disorganized in ECs lining the CCM lesions. We report here that microRNA-27a (miR-27a), a negative regulator of VE-cadherin, is elevated in ECs isolated from mouse brains developing early CCM lesions and in cultured ECs with CCM1 or CCM2 depletion. Furthermore, we show miR-27a acts downstream of kruppel-like factor (KLF)2 and KLF4, two known key transcription factors involved in CCM lesion development. Using CD5-2 (a target site blocker [TSB]) to prevent the miR-27a/VE-cadherin mRNA interaction, we present a potential therapy to increase VE-cadherin expression and thus rescue the abnormal vascular integrity. In CCM1- or CCM2-depleted ECs, CD5-2 reduces monolayer permeability, and in Ccm1 heterozygous mice, it restores dermal vessel barrier function. In a neonatal mouse model of CCM disease, CD5-2 normalizes vasculature and reduces vascular leakage in the lesions, inhibits the development of large lesions, and significantly reduces the size of established lesions in the hindbrain. Furthermore, CD5-2 limits the accumulation of inflammatory cells in the lesion area. Our work has established that VE-cadherin is a potential therapeutic target for normalization of the vasculature and highlights that targeting miR-27a/VE-cadherin interaction by CD5-2 is a potential novel therapy for the devastating disease, CCM.

Background Vascular endothelial cell (EC) alignment in the direction of flow is an adaptive response that protects against aortic diseases, such as atherosclerosis. The Rho GTPases are known to regulate this alignment. Herein, we analyze the effect of ARHGAP18 on the regulation of EC alignment and examine the effect of ARHGAP18 deficiency on the development of atherosclerosis in mice. Methods and Results We used in vitro analysis of ECs under flow conditions together with apolipoprotein E−/−Arhgap18−/− double‐mutant mice to study the function of ARHGAP18 in a high‐fat diet–induced model of atherosclerosis. Depletion of ARHGAP18 inhibited the alignment of ECs in the direction of flow and promoted inflammatory phenotype, as evidenced by disrupted junctions and increased expression of nuclear factor‐κB and intercellular adhesion molecule‐1 and decreased endothelial nitric oxide synthase. Mice with double deletion in ARHGAP18 and apolipoprotein E and fed a high‐fat diet show early onset of atherosclerosis, with lesions developing in atheroprotective regions. Conclusions ARHGAP18 is a protective gene that maintains EC alignments in the direction of flow. Deletion of ARHGAP18 led to loss of EC ability to align and promoted atherosclerosis development.

Chemically extracted acellular nerve allografts loaded with brain-derived neurotrophic factor-transfected or ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells have been shown to repair sciatic nerve injury better than chemically extracted acellular nerve allografts alone, or chemically extracted acellular nerve allografts loaded with bone marrow mesenchymal stem cells. We hypothesized that these allografts compounded with both brain-derived neurotrophic factor- and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells may demonstrate even better effects in the repair of peripheral nerve injury. We cultured bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor and/or ciliary neurotrophic factor and used them to treat sciatic nerve injury in rats. We observed an increase in sciatic functional index, triceps wet weight recovery rate, myelin thickness, number of myelinated nerve fibers, amplitude of motor-evoked potentials and nerve conduction velocity, and a shortened latency of motor-evoked potentials when allografts loaded with both neurotrophic factors were used, compared with allografts loaded with just one factor. Thus, the combination of both brain-derived neurotrophic factor and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells can greatly improve nerve injury.

Disorders of the basal ganglia such as Parkinson's disease (PD) and Huntington's disease are commonly thought of primarily as motor disorders; however, the cognitive symptoms of these diseases such as executive dysfunction, learning, memory and attention deficits are prominent and often more disabling than the hallmark motor symptoms. Cognitive features of PD are often neglected in preclinical studies of PD, likely due to the lack of available animal models to study them. Aphakia mice, which are deficient in the transcription factor Pitx3, model the selective nigrostriatal DA loss in PD. Here we report that aphakia mice are impaired in striatum-dependent cognitive tasks including rotarod learning, T-maze and inhibitory avoidance tasks, but not the striatum-independent social transmission of food preference task. These results suggest that some neuropsychiatric symptoms in PD are related to the pathophysiology of the disease rather than stress associated with disease burden, or medications used to treat PD. Furthermore aphakia mice may be used as a novel model of non-motor symptoms in PD.

Astrocytes have long been perceived only as structural and supporting cells within the central nervous system (CNS). However, the discovery that these glial cells may potentially express receptors capable of responding to endogenous neurotransmitters has resulted in the need to reassess astrocytic physiology. The aim of the current study was to characterise the expression of NMDA receptors (NMDARs) in primary human astrocytes, and investigate their response to physiological and excitotoxic concentrations of the known endogenous NMDAR agonists, glutamate and quinolinic acid (QUIN). Primary cultures of human astrocytes were used to examine expression of these receptors at the mRNA level using RT-PCR and qPCR, and at the protein level using immunocytochemistry. The functionality role of the receptors was assessed using intracellular calcium influx experiments and measuring extracellular lactate dehydrogenase (LDH) activity in primary cultures of human astrocytes treated with glutamate and QUIN. We found that all seven currently known NMDAR subunits (NR1, NR2A, NR2B, NR2C, NR2D, NR3A and NR3B) are expressed in astrocytes, but at different levels. Calcium influx studies revealed that both glutamate and QUIN could activate astrocytic NMDARs, which stimulates Ca2+ influx into the cell and can result in dysfunction and death of astrocytes. Our data also show that the NMDAR ion channel blockers, MK801, and memantine can attenuate glutamate and QUIN mediated cell excitotoxicity. This suggests that the mechanism of glutamate and QUIN gliotoxicity is at least partially mediated by excessive stimulation of NMDARs. The present study is the first to provide definitive evidence for the existence of functional NMDAR expression in human primary astrocytes. This discovery has significant implications for redefining the cellular interaction between glia and neurons in both physiological processes and pathological conditions.

Abstract Background Impaired intrauterine growth, a significant global health problem, contributes to a higher burden of infant morbidity and mortality, mainly in resource-poor settings. Maternal anemia and undernutrition, two important causes of impaired intrauterine growth, are prioritized by global nutrition targets of 2030. We synthesized the evidence on the role of preconception nutrition supplements in reducing maternal anemia and improving intrauterine growth. Methods We undertook a review of the randomized controlled trials (RCTs) assessing the effect of preconception nutrition supplements on maternal hemoglobin, an indicator to estimate maternal anemia, and markers of intrauterine growth including birth weight, length, head circumference, and small for gestational age. Additionally, we examined preterm birth as an important perinatal outcome. We searched PubMed, CINAHL, Web of Science, Cochrane Central, and Embase. We computed summary mean differences and risk ratios (RR) with 95% confidence intervals (CIs) using random-effect models. We employed I 2 and Cochran’s Q test statistics to assess heterogeneity. We used a revised Cochrane risk-of-bias (RoB version 2.0) and GRADE (grading of recommendations, assessment, development, and evaluation) tools to assess the risk of bias and quality of evidence of eligible RCTs, respectively. Results We identified 20 eligible RCTs (n = 27,659 women). Preconception nutrition supplements (iron and folic acid, multiple micronutrients, and a lipid-based nutrient supplement) overall increased maternal hemoglobin by 0.30 g/dL ((0.03, 0.57); I 2 = 79%; n=9). However, we did not find a significant effect of the supplements on birth weight (12.25 gm ((− 22.66, 47.16); I 2 = 55%; n=10)), length (0.15 cm (− 0.26, 0.56); I 2 = 68%; n = 5), head circumference (− 0.23 cm (− 0.88, 0.43); I 2 = 84%; n=4), small for gestational age (RR 0.91 (0.80, 1.04); I 2 = 31%; n=8), or preterm birth (RR 0.93 (0.69,1.25); I 2 = 57%; n=12). In general, the quality of evidence was assessed as very low to moderate. Conclusion Preconception nutrition supplements studied to date appear to reduce maternal anemia. However, it is uncertain whether there are beneficial effects of the supplements on intrauterine growth. Low quality of evidence warrants future well-designed RCTs to produce solid scientific data, particularly of a more comprehensive package of preconception nutrition supplements that include both macro- and micronutrients. Systematic review registration PROSPERO CRD42023464966.

In Chinese society, there are two Confucian teachings that have become guiding values for many teachers and have had profound influence on their educational practices for nearly 2,500 years. These values are "yin cai shi jiao" and "you jiao wu lei." The direct translation of yin cai shi jiao is "to carry out teaching based on the material." The direct translation of "you jiao wu lei" is "to teach without classification." There appears to be a mixture of policy ideologies in the recent education reform in Hong Kong. On one hand, the territory was still implementing the neoliberal reform initiatives that were inherited from the United Kingdom in full strength. On the other hand, part of the new reform proposal was the agenda to improve the equality of educational opportunity within the system. The latter can be seen as trying to fulfill the Confucian ideal of "you jiao wu lei," and the former appears to have an effect of pushing schools toward the ideal of "yin cai shi jiao." However, when these two ideals are combined within the school setting, the result can have a strong impact on the work and psychology of secondary school teachers, and this is the issue that the present article mainly focuses on. In particular, it looks at the consequence of this reform after 2 years of its implementation. Three main questions are addressed in this article. First, how do the teachers perceive such a reform, and how was the reform acted out in the social fabric of everyday school life? Second, if there were difficulties in implementing the reform, what was the nature of the difficulties? (Contains 5 tables.)

Objetivo: Realizar a verificação dos equipamentos de TCA, em atendimento a resolução da ANVISA- RDC n 786, aferindo amostras nos tempos Inicial e Pós Heparina pelos melo método automatizado Hemochron e manual CELITE. Material e métodos: O equipamento hemochron dispõe de lâminas contendo ativador de coagulação, que devem ser preenchidos com 15 μL de sangue total. Os tempos de coagulação foram expressos em segundos. Para a técnica manual, 2 mL de sangue total foram adicionados a um tubo de vidro contendo 12 mg de Celite® 545. Após adição do sangue, o cronômetro foi disparado e a amostra homogeneizada cinco vezes a cada 15 segundos até formação do coágulo. Os testes foram realizados em banho-maria e em duplicata. Os 2 procedimentos foram realizados ao mesmo momento de forma independente, evitando influência nos resultados. Para aprovação dos equipamentos, foi considerado o Erro Total ≤ 20% entre os métodos conforme American Association of Bioanalysts AAB- Table of Grading Limits. Para cálculo do Erro Total Permitido usamos 6,0% de CV obtido dos controle de qualidade do fabricante e consideramos 1,65 para fator multiplicativo de nível de confiança desejado. Resultados: R2 = 0,9892; (r)= 0,9946; Bias = 2,61%; Erro total: 7,29%. Discussão: Na análise estatística observou-se que os resultados dos 2 testes apresentaram excelente correlação e o Erro total foi inferior a especificação esperada. Conclusão: Os 14 equipamentos remotos de TCA foram aprovados.

Diabetes mellitus (DM) affects about 9.3% of the population globally. Hyperhomocysteinemia (HHcy) has been implicated in the pathogenesis of DM, owing to its promotion of oxidative stress, β-cell dysfunction, and insulin resistance. HHcy can result from low status of one-carbon metabolism (OCM) nutrients (e.g., folate, choline, betaine, vitamin B6, B12), which work together to degrade homocysteine by methylation. The etiology of HHcy may also involve genetic variation encoding key enzymes in OCM. This review aimed to provide an overview of the existing literature assessing the link between OCM nutrients status, related genetic factors, and incident DM. We also discussed possible mechanisms underlying the role of OCM in DM development and provided recommendations for future research and practice. Even though the available evidence remains inconsistent, some studies support the potential beneficial effects of intakes or blood levels of OCM nutrients on DM development. Moreover, certain variants in OCM-related genes may influence metabolic handling of methyl-donors and presumably incidental DM. Future studies are warranted to establish the causal inference between OCM and DM and examine the interaction of OCM nutrients and genetic factors with DM development, which will inform the personalized recommendations for OCM nutrients intakes on DM prevention.