Generation of Slco1a4-CreERT2-tdTomato Knock-in Mice for Specific Cerebrovascular Endothelial Cell Targeting.

The cerebrovascular endothelial cells with distinct characteristics line cerebrovascular blood vessels and are the fundamental structure of the blood-brain barrier, which is important for the development and homeostatic maintenance of the central nervous system. Cre-LoxP system-based spatial gene manipulation in mice is critical for investigating the physiological functions of key factors or signaling pathways in cerebrovascular endothelial cells. However, there is a lack of Cre recombinase mouse lines that specifically target cerebrovascular endothelial cells. Here, using a publicly available single-cell RNAseq database, we screened the solute carrier organic anion transporter family member 1a4 (Slco1a4) as a candidate marker of cerebrovascular endothelial cells. Then, we generated an inducible Cre mouse line in which a CreERT2-T2A-tdTomato cassette was placed after the initiation codon ATG of the Slco1a4 locus. We found that tdTomato, which can indicate the endogenous Slco1a4 expression, was expressed in almost all cerebrovascular endothelial cells but not in any other non-endothelial cell types in the brain, including neurons, astrocytes, oligodendrocytes, pericytes, smooth muscle cells, and microglial cells, as well as in other organs. Consistently, when crossing the ROSA26LSL-EYFP Cre reporter mouse, EYFP also specifically labeled almost all cerebrovascular endothelial cells upon tamoxifen induction. Overall, we generated a new inducible Cre line that specifically targets cerebrovascular endothelial cells.

Difference in meal-related esophageal intraluminal impedance change between gastroesophageal reflux disease and functional disorders.

OBJECTIVES: Gastroesophageal reflux disease (GERD) has overlapping symptoms with functional disorders such as functional heart burn. Twenty-four-hour pH with impedance monitoring is useful for differentiation. The intraluminal impedance change with meal in distal esophagus is not investigated. METHODS: We performed a retrospective investigation of clinical files, 24-hour pH with intraluminal impedance monitoring in patients with GERD and functional disorders. The post-reflux swallow induced peristaltic wave (PSPW) index as well as the impedance in distal esophagus before the first meal and 30 min and 60 min after the meal were measured and analyzed. RESULTS: A significant decrease of intraluminal impedance in distal esophagus was noted at 30 min (ΔI30min -301.5 [747.5] Ω, p = 0.018) and recovered at 60 min (ΔI60min -194.6 [766.0] Ω, p = 0.126) after meals in GERD patients. On the other hand, there was no significant change of impedance in patients with functional disorders. There were positive correlations between ΔI30min and PSPW index (correlation = 0.232, p = 0.038). Comparing GERD to functional disorders, the best cut-off value for ΔI30min was -212Ω with 74.4% sensitivity and 60.5% specificity. CONCLUSIONS: The intraluminal impedance in distal esophagus was lowered after meals in GERD patients other than functional disorders. This impedance change was correlated with PSPW index and could help differentiate GERD from functional disorders.

ABA-inducible DEEPER ROOTING 1 improves adaptation of maize to water deficiency.

Root architecture remodelling is critical for forage moisture in water-limited soil. DEEPER ROOTING 1 (DRO1) in Oryza, Arabidopsis, and Prunus has been reported to improve drought avoidance by promoting roots to grow downward and acquire water from deeper soil. In the present study, we found that ZmDRO1 responded more strongly to abscisic acid (ABA)/drought induction in Zea mays ssp. mexicana, an ancestral species of cultivated maize, than in B73. It was proposed that this is one of the reasons why Zea mays ssp. mexicana has a more noticeable change in the downward direction angle of the root and fewer biomass penalties under water-deficient conditions. Thus, a robust, synthetic ABA/drought-inducible promoter was used to control the expression of ZmDRO1B73 in Arabidopsis and cultivated maize for drought-resistant breeding. Interestingly, ABA-inducible ZmDRO1 promoted a larger downward root angle and improved grain yield by more than 40% under water-limited conditions. Collectively, these results revealed that different responses to ABA/drought induction of ZmDRO1 confer different drought avoidance abilities, and we demonstrated the application of ZmDRO1 via an ABA-inducible strategy to alter the root architecture of modern maize to improve drought adaptation in the field.

Generation of an Ihh-mKate2-Dre knock-in mouse line.

Indian hedgehog (Ihh), a member of the Hh family, plays important roles in vertebrate development and homeostasis. To improve our understanding of the function of Ihh-expressing cells and their progeny as well, we generate an Ihh-mKate2tomm20 -Dre knock-in mouse line that can label Ihh-positive cells with a fluorescence protein mKate2 and trace Ihh-positive cells and their progeny via Dre-mediated recombination. Consistent with previous reports, we verified Ihh expression in hypertrophic chondrocytes of growth plate and granulosa cells of ovarian follicles by mKate2 immunostaining, and meanwhile confirmed Dre activity in these cells via a Dre reporter mouse line Rosa26-confetti2. We also found, for the first time, that Ihh can mark some cell types, including retinal ganglion cells, Purkinje cells, and gallbladder epithelial cells. Taken together, the Ihh-mKate2tomm20 -Dre mouse is a genetic tool for examining the precise expression profile of Ihh and tracing Ihh-expressing cells and their progeny.

Genome Editing with AAV-BR1-CRISPR in Postnatal Mouse Brain Endothelial Cells.

Brain endothelial cells (ECs) are an important component of the blood-brain barrier (BBB) and play key roles in restricting entrance of possible toxic components and pathogens into the brain. However, identifying endothelial genes that regulate BBB homeostasis remains a time-consuming process. Although somatic genome editing has emerged as a powerful tool for discovery of essential genes regulating tissue homeostasis, its application in brain ECs is yet to be demonstrated in vivo. Here, we used an adeno-associated virus targeting brain endothelium (AAV-BR1) combined with the CRISPR/Cas9 system (AAV-BR1-CRISPR) to specifically knock out genes of interest in brain ECs of adult mice. We first generated a mouse model expressing Cas9 in ECs (Tie2Cas9). We selected endothelial ß-catenin (Ctnnb1) gene, which is essential for maintaining adult BBB integrity, as the target gene. After intravenous injection of AAV-BR1-sgCtnnb1-tdTomato in 4-week-old Tie2Cas9 transgenic mice resulted in mutation of 36.1% of the Ctnnb1 alleles, thereby leading to a dramatic decrease in the level of CTNNB1 in brain ECs. Consequently, Ctnnb1 gene editing in brain ECs resulted in BBB breakdown. Taken together, these results demonstrate that the AAV-BR1-CRISPR system is a useful tool for rapid identification of endothelial genes that regulate BBB integrity in vivo.

Rapid diagnosis of Talaromyces marneffei infection assisted by metagenomic next-generation sequencing in a HIV-negative patient.

Talaromyces marneffei (T. marneffei), is an opportunistic pathogenic fungus commonly reported in southeast Asia. T. marneffei infection predominantly occurs in patients with immunodeficiency and can be fatal if diagnosis and treatment were delayed. Conventional diagnosis of T. marneffei infection relies heavily on tissue culture and histologic analysis, which is time consuming and has limited positive rate. Rapid and accurate diagnosis of T. marneffei remains urgent for effective therapy and prevention. This case is the first reported T. marneffei infection in non-HIV patients in north China diagnosed by mNGS. The successful diagnosis of T. marneffei infection assistant by mNGS underlies the potential of this technique in rapid etiological diagnosis.

Clinicopathological characteristics and survival in lung signet ring cell carcinoma: a population-based study.

Lung signet-ring cell carcinoma (LSRCC) is a very rare type of lung cancer, the clinical characteristics, and prognosis of which remain to be clarified. In order to explore the clinicopathological and survival-related factors associated with LSRCC, we performed a large population-based cohort analysis of data included in the Surveillance, Epidemiology, and End Results (SEER) registry from 2001 to 2015. A total of 752 LSRCC and 7518 lung mucinous adenocarcinoma (LMAC) patients were incorporated into our analysis, with respective mean ages of 63.8 and 67.5 years at the time of diagnosis. LSRCC patients were significantly more likely than LMAC patients to have distant-stage disease (72.1% vs. 45.8%, p < 0.0001), tumors of a high pathological grade (40.6% vs. 10.8%, p < 0.0001), have undergone chemotherapy (62.1% vs. 39.9%, p<0.0001), be male (52.7% vs. 48.5%, p = 0.03), and be < 40 years old (3.3% vs. 1.3%, p = 0.022), whereas they were less likely to have undergone surgical treatment (52.4% vs. 77.0%, p < 0.0001). LSRCC and LMAC patients exhibited median overall survival (OS) duration of 8 and 18 months (p < 0.0001), respectively, although these differences were not significant after adjusting for confounding variables. Independent factors associated with a favorable patient prognosis included a primary site in the middle or lower lung lobe, underwent surgery, and underwent chemotherapy. However, age ≥80 years, higher grade, distant summary stage disease, and T4 stage disease were linked to poor prognosis. Patient age, tumor grade, primary tumor site, summary stage, T stage, surgery, and chemotherapy were all significantly associated with LSRCC patient prognosis.

Integrative multi-omics analysis of a colon cancer cell line with heterogeneous Wnt activity revealed RUNX2 as an epigenetic regulator of EMT.

Epithelial-mesenchymal transition (EMT) program, which facilitates tumor metastasis, stemness and therapy resistance, is a reversible biological process that is largely orchestrated at the epigenetic level under the regulation of different cell signaling pathways. EMT state is often heterogeneous within individual tumors, though the epigenetic drivers underlying such heterogeneity remain elusive. In colon cancer, hyperactivation of the Wnt/ß-catenin signaling not only drives tumor initiation, but also promotes metastasis in late stage by promoting EMT program. However, it is unknown whether the intratumorally heterogeneous Wnt activity could directly drive EMT heterogeneity, and, if so, what are the underlying epigenetic driver(s). Here, by analyzing a phenotypically and molecularly heterogeneous colon cancer cell line using single-cell RNA sequencing, we identified two distinct cell populations with positively correlated Wnt activity and EMT state. Integrative multi-omics analysis of these two cell populations revealed RUNX2 as a critical transcription factor epigenetically driving the EMT heterogeneity. Both in vitro and in vivo genetic perturbation assays validated the EMT-enhancing effect of RUNX2, which remodeled chromatin landscape and activated a panel of EMT-associated genes through binding to their promoters and/or potential enhancers. Finally, by exploring the clinical data, we showed that RUNX2 expression is positively correlated with metastasis development and poor survival of colon cancer patients, as well as patients afflicted with other types of cancer. Taken together, our work revealed RUNX2 as a new EMT-promoting epigenetic regulator in colon cancer, which may potentially serve as a prognostic marker for tumor metastasis.

IVS2vec: A tool of Inverse Virtual Screening based on word2vec and deep learning techniques.

Inverse Virtual Screening is a powerful technique in the early stage of drug discovery process. This technique can provide important clues for biologically active molecules, which is useful in the following researches of durg discovery. In this work, combining with Word2vec, a natural language processing technique, dense fully connected neural network (DFCNN) algorithm is utilized to build up a prediction model. This model is able to perform a binary classification. Based on the query molecule, the input protein candidates can be classified into two subsets. One set is that potential targets with high possibilities to bind with the query molecule and the other one is that the proteins with low possibilities to bind with the query molecule. This model is named as IVS2vec. IVS2vec also can output a score reflecting binding possibility of the association between a protein and a molecule, which is useful to improve efficiency of research. We applied IVS2vec on several databases related to drug development and shown that our model can detect possible therapeutic targets. In addition, our model can identify targets related to adverse drug reactions which is useful to improve medication safety and repurpose drugs. Moreover, IVS2vec can give a very fast speed to perform prediction jobs. It is suitable for processing a large number of compounds in the chemical databases. We also find that IVS2vec has potential capabilities and outperform other state-of-the-art docking tools such as Autodock vina. In this study, IVS2vec brings many convincing results than Autodock vina in the reverse target searching case of Quercetin.

[Chemical Constituents from the Roots of Rhodomyrtus tomentosa].

Objective: To investigate the chemical constituents of the roots of Rhodomyrtus tomentosa and their antitumor activities. Methods: Various column chromatographic techniques were used to isolate the compounds. The structures were elucidated by their physicochemical properties and spectra data. Moreover, MTT assay was used to evaluate the antitumor activity against melanoma SK-MEI-110 cells. Results: Thirteen compounds were obtained from the roots of Rhodomyrtus tomentosa, and their structures were determined as 2α, 3ß, 23-trihydroxyoleana-11,13( 18)-dien-28-oic acid( 1),3ß,23-dihydroxyolean-18-en-28-oic acid( 2), lupeol( 3), betulin( 4), betulinic acid( 5), friedelin( 6), ß-sitosterol( 7), thero-2, 3-bis-( 4-hydroxy-3-methoxypheyl)-3-methoxy-propanol( 8), evafolin B( 9), ß-hydroxypropiovanillone ( 10), 8, 8'-bis-( dihydroconiferyl)-diferuloylate( 11), gallate acid( 12) and methyl gallate( 13). Conclusion: Compounds 1,2,8 ~ 10 and 13 are isolated from this plant for the first time. Compounds 1 ~ 6 exhibite inhibitory effects on melanoma SK-MEL-110 cells.

A novel treatment regimen for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common, X-linked genetic, skeletal muscle disease, with various regimens of treatment. The objective of this study was to determine the safety and efficacy of a novel treatment regimen for this disease. Thirty boys with DMD were administered prednisone according to the following regimen: in the first year, 1.5 mg/kg/day for the first 3 months, 1.0 mg/kg/day for the next 3 months, 0.75 mg/kg/day for the next 3 months, and 0.5 mg/kg/day for the last 3 months. In the second year, prednisone was administered 0.5 mg/kg on the alternate day for 12 months. The muscle strength (Medical Research Council sum score and Gower's sign), serum enzymes (creatine kinase, creatine kinase isoenzyme-2, and lactate dehydrogenase), pulmonary function (forced vital capacity, maximum voluntary ventilation), body weight, height, and BMI were determined before treatment and 3, 6, 9, 12, and 24 months after treatment. The results showed that the patients' mean Medical Research Council sum score increased from 46.1 at the baseline to 53.6 at 12 months and was maintained at 24 months. Gower's sign disappeared in 22 (73.3%) patients at 12 months and 21 (70.0%) at 24 months. The serum levels of creatine kinase, creatine kinase isoenzyme-2, and lactate dehydrogenase decreased and pulmonary function improved after 24 months of treatment. Significantly increased weight gain, osteoporosis, and cushingoid features were not observed. Our results suggested that this novel prednisone regimen for DMD has similar efficacy and safety as other regimens.