We previously demonstrated that the characteristic sequence of matrix attachment regions (MARs) allows transgenes to be maintained episomally in CHO cells. In the present study, six commonly used ...promoters from human cytomegalovirus major immediate-early (CMV), simian vacuolating virus 40 (SV40), Rous sarcoma virus, Homo sapiens ubiquitin C, phosphoglycerate kinase, and β-globin, respectively, were evaluated to determine their effects on transgene expression and stability in CHO cells stably transfected via the episomal vector harbouring characteristic MAR motifs. The CHO cells were transfected with vectors and then screened using G418, after which the stably transfected cells were split into two and further cultured either in the presence or absence of G418. Of the six promoters, the CMV promoter yielded the highest transgene expression levels and the highest transfection efficiency, whereas the SV40 promoter maintained transgene expression more stably during long-term culture than the other promoters did. The CMV and SV40 promoter-containing vectors were furthermore episomally maintained and conferred sustained eGFP expression in the cells even under nonselective conditions. On the basis of these findings, we conclude that the CMV promoter performs best in terms of yielding both high expression levels and high levels of stability using this episomal vector system.
Immunogenic cell death is characterized by damage-associated molecular patterns, which can enhance the maturation and antigen uptake of dendritic cells. Shikonin, an anti-inflammatory and antitumor ...phytochemical, was exploited here as an adjuvant for dendritic cell-based cancer vaccines via induction of immunogenic cell death. Shikonin can effectively activate both receptor- and mitochondria-mediated apoptosis and increase the expression of all five tested damage-associated molecular patterns in the resultant tumor cell lysates. The combination treatment with damage-associated molecular patterns and LPS activates dendritic cells to a high maturation status and enhances the priming of Th1/Th17 effector cells. Shikonin-tumor cell lysate-loaded mature dendritic cells exhibit a high level of CD86 and MHC class II and activate Th1 cells. The shikonin-tumor cell lysate-loaded dendritic cell vaccines result in a strong induction of cytotoxic activity of splenocytes against target tumor cells, a retardation in tumor growth, and an increase in the survival of test mice. The much enhanced immunogenicity and efficacy of the current cancer vaccine formulation, that is, the use of shikonin-treated tumor cells as cell lysates for the pulse of dendritic cells in culture, may suggest a new ex vivo approach for developing individualized, dendritic cells-based anticancer vaccines.
The work is focused on the design of drugs that prevent and treat Alzheimer's disease (AD) and its complications. A series of 3-(4-aminophenyl)-coumarin derivatives designed, synthesised, fully ...characterised and evaluated in vitro/vivo. The biological assay experiments showed that some compounds displayed a clearly selective inhibition for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Among all compounds, compound 4m exhibited the highest AChE inhibition with an IC
50
value of 0.091 ± 0.011 µM and compound 4k exhibited the highest BuChE inhibition with an IC
50
value of 0.559 ± 0.017 µM. A zebrafish behaviour analyser (Zebrobox) was used to determine the behavioural effects of the active compound on the movement distance of the aluminium chloride-induced zebrafish. Compound 4m offered a potential drug design concept for the development of therapeutic or preventive agents for AD and its complications.
With initiation of antiretroviral therapy (ART) containing nucleos(t)ide reverse-transcriptase inhibitors (NRTIs) with anti-hepatitis B virus (HBV) activity, the evolution of HBV serologic markers ...among people living with human immunodeficiency virus (PLWH) who were born in the era of nationwide neonatal HBV vaccination is rarely investigated.
This retrospective cohort study evaluated the changes of HBV serologic markers (hepatitis B surface antigen HBsAg, antibody to hepatitis B surface antigen anti-HBs, and antibody to hepatitis B core antigen anti-HBc) of PLWH who had undergone neonatal HBV vaccination. Clinical characteristics were analyzed and the incidences of evolution of HBV serologic markers were estimated.
Between 2004 and 2020, 608 PLWH (mean age, 24 years) were included and 62.0% initiated tenofovir-containing ART: 13 (2.1%) were HBsAg-positive, 312 (51.3%) tested triple-negative, 209 (34.4%) had vaccine-induced seroprotection against HBV, and 74 (12.2%) tested positive for anti-HBc with or without anti-HBs. Among 492 PLWH who received a median follow-up of 2.8 years, 4 cases of incident HBV infection occurred (0.59 per 100 person-years of follow-up PYFU) in PLWH testing triple-negative at baseline despite ART containing NRTIs with anti-HBV activity. Of PLWH with seroprotection against HBV at baseline, 38 subsequently lost anti-HBs (4.46 per 100 PYFU) and 4 cases of incident HBV infection occurred (0.47 per 100 PYFU). PLWH with an anti-HBs antibody titer ≥100 mIU/mL at baseline (adjusted hazard ratio aHR, 0.10 95% confidence interval {CI}: .02-.42) and CD4 ≥500 cells/µL during follow-up (aHR, 0.51 95% CI: .30-1.00) were less likely to lose HBV seroprotection.
Among young PLWH who had undergone neonatal HBV vaccination, evolution of HBV serologic markers and incident infections occurred despite ART containing NRTIs with anti-HBV activity.
Replacement with nonvaccine serotypes (NVTs) among invasive pneumococcal diseases (IPDs) after the introduction of extended-valency pneumococcal conjugate vaccines varies in predominant serotypes ...across countries. This study analyzed changes in serotype distribution through serotyping, multilocus sequence typing, and antimicrobial susceptibility testing of 168 pediatric IPD isolates obtained from a multihospital-based surveillance system during 2014–2019 in South Korea. Vaccine serotypes (VTs) accounted for 16.1% (19A, 10.1%; 6A, 1.8%; and 19F 1.8%), 82.1% were NVTs (10A, 23.8%; 15A, 8.3%; 12F, 6.5%; 15C, 6.5%; and 15B, 6.0%), and three (1.8%) were nontypeable. Serotype 10A was the most common serotype, with a significant increase from 11.5% in 2014 to 33.3% in 2019 (p < 0.05 for the trend). Other NVTs decreased from 70.4% to 41.7% between 2015 and 2019, most notably in serotype 12F (from 14.8% to 0%). Almost all (95.0%) serotype 10A isolates were ST11189, which were multidrug resistant.
Single-cell RNA sequencing (scRNA-seq) of tissues has revealed remarkable heterogeneity of cell types and states but does not provide information on the spatial organization of cells. To better ...understand how individual cells function within an anatomical space, we developed XYZeq, a workflow that encodes spatial metadata into scRNA-seq libraries. We used XYZeq to profile mouse tumor models to capture spatially barcoded transcriptomes from tens of thousands of cells. Analyses of these data revealed the spatial distribution of distinct cell types and a cell migration-associated transcriptomic program in tumor-associated mesenchymal stem cells (MSCs). Furthermore, we identify localized expression of tumor suppressor genes by MSCs that vary with proximity to the tumor core. We demonstrate that XYZeq can be used to map the transcriptome and spatial localization of individual cells in situ to reveal how cell composition and cell states can be affected by location within complex pathological tissue.
Increasing evidence shows that smoking-obtained nicotine is indicated to improve cognition and mitigate certain symptoms of schizophrenia. In this study, we investigated whether chronic nicotine ...treatment alleviated MK-801-induced schizophrenia-like symptoms and cognitive impairment in mice. Mice were injected with MK-801 (0.2 mg/kg, i.p.), and the behavioral deficits were assessed using prepulse inhibition (PPI) and T-maze tests. We showed that MK-801 caused cognitive impairment accompanied by increased expression of PDZ and LIM domain 5 (Pdlim5), an adaptor protein that is critically associated with schizophrenia, in the prefrontal cortex (PFC). Pretreatment with nicotine (0.2 mg · kg
· d
, s.c., for 2 weeks) significantly ameliorated MK-801-induced schizophrenia-like symptoms and cognitive impairment by reversing the increased Pdlim5 expression levels in the PFC. In addition, pretreatment with nicotine prevented the MK-801-induced decrease in CREB-regulated transcription coactivator 1 (CRTC1), a coactivator of CREB that plays an important role in cognition. Furthermore, MK-801 neither induced schizophrenia-like behaviors nor decreased CRTC1 levels in the PFC of Pdlim5
mice. Overexpression of Pdlim5 in the PFC through intra-PFC infusion of an adreno-associated virus AAV-Pdlim5 induced significant schizophrenia-like symptoms and cognitive impairment. In conclusion, chronic nicotine treatment alleviates schizophrenia-induced memory deficits in mice by regulating Pdlim5 and CRTC1 expression in the PFC.
Introduction
An age‐specific evaluation and management algorithm for reduced bone mineral density (BMD) is suggested for HIV‐positive patients without major risk factors. Whether combination of BMD ...and the Fracture Risk Assessment Tool (FRAX) may detect more individuals for therapeutic interventions remains unclear. We aimed to determine the prevalence of middle‐aged or older HIV‐positive males fitting the criteria of therapeutic interventions with different approaches.
Methods
From July 2016 to February 2018, HIV‐positive male patients aged ≥45 years receiving suppressive antiretroviral therapy were recruited in a cross‐sectional study, at two designated hospitals for HIV care in northern Taiwan. Patients with malignancy, AIDS, pre‐existing bone disease or immobilization were excluded. Information on clinical and demographic characteristics, FRAX questionnaire, activity questionnaire, BMD and serum 25(OH)D was obtained. FRAX scores combined with BMD (FRAX/BMD) and without BMD (FRAX) were calculated. The data were analysed on the basis of major risk factors for fragility fracture and age stratification, FRAX score and BMD results respectively.
Results
We enrolled 330 patients with a mean age of 51.6 years and CD4 610 cells/μL, in whom 98.1% (n = 324) underwent BMD assessment of one site or more. By FRAX, 6.7% (n = 22) reached treatment thresholds (10‐year risk of major osteoporotic fracture ≥20% and/or hip fracture ≥3%). The prevalence of osteopenia (−2.5 <T‐score <−1) and osteoporosis (T‐score ≤−2.5) was 50.3% and 10.8% respectively. Compared with FRAX, FRAX/BMD identified 17.4% (95% CI 12.0% to 22.8%) more individuals who reached treatment thresholds (24.1% vs. 6.7%); even in the low‐risk group (without major risks for fragility fracture, 45 to 49 years, n = 129), FRAX/BMD identified 12.6% (95% CI 7.9% to 19.7%) more candidates (12.6% vs. 0%). Patients with BMI<22 kg/m2 (adjusted OR (aOR) 2.86, 95% CI 1.62 to 5.05) and aged ≥50 years (aOR 3.57, 95% CI 1.92 to 6.66) were more likely not to be identified as requiring treatment by FRAX but would be identified as requiring treatment by FRAX/BMD. The sensitivity and specificity of FRAX to detect candidates for interventions was 18.2% (95% CI 10.3% to 28.6%) and 97.9% (95% CI 95.2% to 99.3%) respectively.
Conclusions
With FRAX as a screening approach among HIV‐positive male patients aged ≥45 years, addition of BMD assessment may detect more candidates for therapeutic management.
Vehicle state and parameter estimation has gradually become an important way to soft-sense some variables that are difficult to measure directly using general sensors. In the traditional Kalman ...filtering algorithm, the selection of the process noise covariance matrix and the measurement noise covariance matrix will directly affect the filtering accuracy of the algorithm. In order to improve the filtering accuracy of the filter algorithm to obtain the optimal solution, based on a 7-DOF nonlinear vehicle dynamics model and the Magic formula tire model, a hybrid algorithm containing an unscented Kalman filter (UKF) and a genetic-particle swarm algorithm (genetic-particle swarm UKF) is used to estimate several vehicle key states. Compared with traditional estimator based on UKF and the unscented particle filter (UPF), the simulation and real vehicle test results show that the proposed estimator based on the genetic-particle swarm UKF algorithm has higher accuracy and less computation requirements than the UKF estimator. And also, the proposed hybrid algorithm has superiority on the convergence speed of the optimization than the UPF algorithm. The results of a real-vehicle experiment demonstrate that the proposed hybrid algorithm can be used effectively for solving the vehicle-state estimation problem.
Hexokinase (HXK) proteins play important roles in catalyzing hexose phosphorylation and sugar sensing and signaling. To investigate the roles of HXKs in cassava tuber root development, seven
genes (
...) were isolated and analyzed. A phylogenetic analysis revealed that the MeHXK family can be divided into five subfamilies of plant HXKs. MeHXKs were clearly divided into type A (MeHXK1) and type B (MeHXK2-7) based on their N-terminal sequences. MeHXK1-5 all had typical conserved regions and similar protein structures to the HXKs of other plants; while MeHXK6-7 lacked some of the conserved regions. An expression analysis of the
genes in cassava organs or tissues demonstrated that
is the dominant HXK in all the examined tissues (leaves, stems, fruits, tuber phloems, and tuber xylems). Notably, the expression of
and the enzymatic activity of HXK were higher at the initial and expanding tuber stages, and lower at the mature tuber stage. Furthermore, the HXK activity of MeHXK2 was identified by functional complementation of the HXK-deficient yeast strain YSH7.4-3C (
,
,
). The gene expression and enzymatic activity of MeHXK2 suggest that it might be the main enzyme for hexose phosphorylation during cassava tuber root development, which is involved in sucrose metabolism to regulate the accumulation of starch.
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