Chemogenetic inhibition of subicular seizure-activated neurons alleviates cognitive deficit in male mouse epilepsy model.

Cognitive deficit is a common comorbidity in temporal lobe epilepsy (TLE) and is not well controlled by current therapeutics. How epileptic seizure affects cognitive performance remains largely unclear. In this study we investigated the role of subicular seizure-activated neurons in cognitive impairment in TLE. A bipolar electrode was implanted into hippocampal CA3 in male mice for kindling stimulation and EEG recording; a special promoter with enhanced synaptic activity-responsive element (E-SARE) was used to label seizure-activated neurons in the subiculum; the activity of subicular seizure-activated neurons was manipulated using chemogenetic approach; cognitive function was assessed in object location memory (OLM) and novel object recognition (NOR) tasks. We showed that chemogenetic inhibition of subicular seizure-activated neurons (mainly CaMKIIα+ glutamatergic neurons) alleviated seizure generalization and improved cognitive performance, but inhibition of seizure-activated GABAergic interneurons had no effect on seizure and cognition. For comparison, inhibition of the whole subicular CaMKIIα+ neuron impaired cognitive function in naïve mice in basal condition. Notably, chemogenetic inhibition of subicular seizure-activated neurons enhanced the recruitment of cognition-responsive c-fos+ neurons via increasing neural excitability during cognition tasks. Our results demonstrate that subicular seizure-activated neurons contribute to cognitive impairment in TLE, suggesting seizure-activated neurons as the potential therapeutic target to alleviate cognitive impairment in TLE.

Senescent cells re-engineered to express soluble programmed death receptor-1 for inhibiting programmed death receptor-1/programmed death ligand-1 as a vaccination approach against breast cancer.

Various types of vaccines have been proposed as approaches for prevention or delay of the onset of cancer by boosting the endogenous immune system. We previously developed a senescent-cell-based vaccine, induced by radiation and veliparib, as a preventive and therapeutic tool against triple-negative breast cancer. However, the programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) pathway was found to play an important role in vaccine failure. Hence, we further developed soluble programmed death receptor-1 (sPD1)-expressing senescent cells to overcome PD-L1/PD-1-mediated immune suppression while vaccinating to promote dendritic cell (DC) maturity, thereby amplifying T-cell activation. In the present study, sPD1-expressing senescent cells showed a particularly active status characterized by growth arrest and modified immunostimulatory cytokine secretion in vitro. As expected, sPD1-expressing senescent tumor cell vaccine (STCV/sPD-1) treatment attracted more mature DC and fewer exhausted-PD1+ T cells in vivo. During the course of the vaccine studies, we observed greater safety and efficacy for STCV/sPD-1 than for control treatments. STCV/sPD-1 pre-injections provided complete protection from 4T1 tumor challenge in mice. Additionally, the in vivo therapeutic study of mice with s.c. 4T1 tumor showed that STCV/sPD-1 vaccination delayed tumorigenesis and suppressed tumor progression at early stages. These results showed that STCV/sPD-1 effectively induced a strong antitumor immune response against cancer and suggested that it might be a potential strategy for TNBC prevention.

P-side-up thin-film AlGaInP-based light emitting diodes with direct ohmic contact of an ITO layer with a GaP window layer.

A twice wafer-transfer technique can be used to fabricate high-brightness p-side-up thin-film AlGaInP-based light-emitting diodes (LEDs) with an indium-tin oxide (ITO) transparent conductive layer directly deposited on a GaP window layer, without using postannealing. The ITO layer can be used to improve light extraction, which enhances light output power. The p-side-up thin-film AlGaInP LED with an ITO layer exhibited excellent performance stability (e.g., emission wavelength and output power) as the injection current increased. This stability can be attributed to the following factors: 1) Refractive index matching, performed by introducing ITO between the epoxy and the GaP window layer enhances light extraction; and 2) The ITO layer is used as the current spreading layer to reduce the thermal accumulation in the epilayers.

Single-cell mapping reveals several immune subsets associated with liver metastasis of pancreatic ductal adenocarcinoma.

BACKGROUND: Identifying a metastasis-correlated immune cell composition within the tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) will help to develop promising and innovative therapeutic strategies. However, the dynamics of immune cell lineages in the TME of advanced PDAC remains elusive. METHODS: Twenty-six samples from 11 patients (including 11 primary tumor tissues, 10 blood, and 5 lymph nodes) with different stages were used to develop a multiscale immune profile. High-dimensional single-cell analysis with mass cytometry was performed to search for metastasis-correlated immune changes in the microenvironment. The findings were further validated by published single-cell RNA sequencing (scRNA-seq) data and multiplex fluorescent immunohistochemistry. FINDINGS: High-dimensional single-cell profiling revealed that the three immune-relevant sites formed a distinct immune atlas. Interestingly, the PDAC microenvironment with the potential for metastatic spread to the liver was characterized by a decreased proportion of CD103+PD-1+CD39+ T cells with cytotoxic and exhausted functional status and an increased proportion of CD73+ macrophages. Analysis of scRNA-seq data of PDAC further confirmed the identified subsets and revealed strong potential interactions via various ligand-receptor pairs between the identified T subsets and the macrophages. Moreover, stratified patients with different immune compositions correlated with clinical outcomes of PDAC. CONCLUSIONS: Our study uncovered metastasis-correlated immune changes, suggesting that ecosystem-based patient classification in PDAC will facilitate the identification of candidates likely to benefit from immunotherapy. FUNDING: This work was supported by the National Key Research and Development Program of China, the Shanghai International Science and Technology Collaboration Program, the Shanghai Sailing Program, and the Key Laboratory of diagnosis and treatment of severe hepato-pancreatic diseases of Zhejiang Province.

Learning to Learn Adaptive Classifier-Predictor for Few-Shot Learning.

Few-shot learning aims to learn a well-performing model from a few labeled examples. Recently, quite a few works propose to learn a predictor to directly generate model parameter weights with episodic training strategy of meta-learning and achieve fairly promising performance. However, the predictor in these works is task-agnostic, which means that the predictor cannot adjust to novel tasks in the testing phase. In this article, we propose a novel meta-learning method to learn how to learn task-adaptive classifier-predictor to generate classifier weights for few-shot classification. Specifically, a meta classifier-predictor module, (MPM) is introduced to learn how to adaptively update a task-agnostic classifier-predictor to a task-specialized one on a novel task with a newly proposed center-uniqueness loss function. Compared with previous works, our task-adaptive classifier-predictor can better capture characteristics of each category in a novel task and thus generate a more accurate and effective classifier. Our method is evaluated on two commonly used benchmarks for few-shot classification, i.e., miniImageNet and tieredImageNet. Ablation study verifies the necessity of learning task-adaptive classifier-predictor and the effectiveness of our newly proposed center-uniqueness loss. Moreover, our method achieves the state-of-the-art performance on both benchmarks, thus demonstrating its superiority.

Corrections to "Learning to Learn Adaptive Classifier-Predictor for Few-Shot Learning".

In the above article [1], the results of "Fully-supervised (Upper bound)" in Tables III and IV were inadvertently set to intermediate records that were used as placeholders. This error has no effect on any of the interpretations and conclusions. Tables I and II of this amendment show the corrected results (highlighted in italics) of the original Tables III and IV.

[Interleukin-12 over-expression in malignant melanoma B16 cells reduces programmed death-1 expression on T cells in mice with immune reconstitution].

OBJECTIVE: To investigate whether interleukin-12 (IL-12) over-expression in malignant melanoma B16 cells affects the expression level of programmed death-1 (PD-1) on T cells in mice during immune microenvironment reconstruction. METHODS: B16 cells were transfected with an IL-12 expression lentiviral vector, and IL-12 over-expression in the cells was verified qPCR and ELISA. Plate cloning assay was used to compare the cell proliferation activity between B16 cells and B16/IL-12 cells. The expression of IL-12 protein in B16/IL-12 cells-derived tumor tissue were detected by ELISA. C57BL/6 mice were inoculated with B16 cells or B16/IL-12 cells, and 14 days later the proportion of T cells with high expression of PD-1 in the tumor-draining lymph nodes was detected by flow cytometry. Mouse models of immune reconstitution established by 650 cGy X-ray radiation were inoculated with B16 (B16+RT group) or B16/IL-12 (B16/IL-12+RT group) cells, with the mice without X-ray radiation prior to B16 cell inoculation as controls. Tumor growth in the mice was recorded at different time points, and on day 14, flow cytometry was performed to detect the proportion of T cells with high PD-1 expression in the tumor-draining lymph nodes and in the tumor tissue. RESULTS: B16 cells infected with the IL-12-overexpressing lentiviral vector showed significantly increased mRNA and protein levels of IL-12 (P < 0.001) without obvious changes in cell viability (P>0.05). B16/IL-12 cells expressed higher levels of IL-12 than B16 cells in vivo (P < 0.01). In the tumor-bearing mouse models, the proportion of CD4 + PD-1+ T cells was significantly lower in B16/IL-12 group than in B16 group (P < 0.01). In the mice with X-ray radiation-induced immune reconstitution, PD-1 expressions on CD4+ T cells (P < 0.05) and CD8+ T cells (P < 0.01) were significantly higher in B16+ RT group than in the control mice and in B16/IL-12+RT group (P < 0.01 or 0.001); the tumors grew more slowly in B16/IL-12+RT group than in B16 + RT group (P < 0.001). CONCLUSIONS: During immune microenvironment reconstruction, overexpression IL-12 in the tumor microenvironment can reduce the percentage of PD-1 + T cells and suppress the growth of malignant melanoma in mice.

Evaluation the diagnostic accuracy of albuminuria detection in semi-quantitative urinalysis.

BACKGROUND: Taiwan has the highest end-stage renal disease prevalence in the world, and the costs on the maintenance of dialysis imposes a great financial burden on National Health Insurance. Routine urinalysis provides an opportunity for the early detection of microalbuminuria. We evaluated the accuracy of semi-quantitative chemical methods from Siemens Novus Pro12 dipstick for albumin-creatinine ratio (ACR). METHODS: We collected 1029 random urine samples and performed urinary analytic tests by Siemens Novus with Pro12 dipsticks and also calculated the urinary ACR. The reference method was performed by Hitachi LST008, a quantitative assay. The percentage of exact agreement in ACR was 81.9% between Siemens Novus and Hitachi LST008. The percentage of agreement within 1 level between the 2 methods was 98.5%. When ACR > 30 mg/g was defined as the threshold for positive results, the sensitivity, specificity, positive, and negative predictive values for microalbuminuria were 87.2%, 91.6%, 91.5%, and 87.3%, respectively. There were 778 cases with negative results of urinary protein, analyzed by conventional dipsticks. 149 of 778 (19.2%) cases were positive, measured by Pro12 dipsticks, and 111 of 149 (74.5%) cases were confirmed positive ACR by Hitachi LST008. CONCLUSIONS: Urinary ACR measured by Siemens Novus with Pro12 dipsticks was shown to be a reliable test for detection of microalbuminuria.

Bismuth shield affecting CT image quality and radiation dose in adjacent and distant zones relative to shielding surface: A phantom study.

BACKGROUND: To quantify image quality and radiation doses in regions adjacent to and distant from bismuth shields in computed tomography (CT). METHODS: An American College of Radiology accreditation phantom with four solid rods embedded in a water-like background was scanned to verify CT number (CTN) accuracy when using bismuth shields. CTNs, image noise, and contrast-to-noise ratios (CNRs) were determined in the phantom at 80-140 kVp. Image quality was investigated on image portions in the zones adjacent (A zone) to and distant (D zone) from a bismuth shield. Surface radiation doses were measured using thermoluminescent dosimeters. Streak artefacts were graded on a 3-point-scale. RESULTS: Changes in CTN caused by a bismuth shield resulted in changes in X-ray spectra. CTN changes were more apparent in the A zone than in the D zone, particularly for a low tube voltage. The degrees of CTN changes and image noise were proportional to the thickness of the bismuth shields. A 1-ply bismuth shield reduced surface radiation doses by 7.2%-15.5%. The overall CNRs were slightly degraded, and streak artefacts were acceptable. CONCLUSIONS: Using a bismuth shield could result in significant CTN changes and perceivable artefacts, particularly for a superficial organ close to the shield, and is not recommended for quantification CT examinations or follow-up CT examinations.

Increase Trichomonas vaginalis detection based on urine routine analysis through a machine learning approach.

Trichomonas vaginalis (T. vaginalis) detection remains an unsolved problem in using of automated instruments for urinalysis. The study proposes a machine learning (ML)-based strategy to increase the detection rate of T. vaginalis in urine. On the basis of urinalysis data from a teaching hospital during 2009-2013, individuals underwent at least one urinalysis test were included. Logistic regression, support vector machine, and random forest, were used to select specimens with a high risk of T. vaginalis infection for confirmation through microscopic examinations. A total of 410,952 and 428,203 specimens from men and women were tested, of which 91 (0.02%) and 517 (0.12%) T. vaginalis-positive specimens were reported, respectively. The prediction models of T. vaginalis infection attained an area under the receiver operating characteristic curve of more than 0.87 for women and 0.83 for men. The Lift values of the top 5% risky specimens were above eight. While the most risky vigintile was picked out by the models and confirmed by microscopic examination, the incremental cost-effectiveness ratios for T. vaginalis detection in men and women were USD$170.1 and USD$29.7, respectively. On the basis of urinalysis, the proposed strategy can significantly increase the detection rate of T. vaginalis in a cost-effective manner.

[Soluble PD-1 over-expression enhances the anti-tumor effect of senescence tumor cell vaccine against breast cancer cell growth in tumor-bearing mice].

OBJECTIVE: To investigate whether soluble PD-1 overexpression in 4T1 senescence tumor cells enhances the antitumor effect of senescence tumor cell vaccine (STCV) against breast tumor cells in a tumor-bearing mouse model. METHODS: 4T1 cells were treated with interferon-γ (IFN-γ) and the expression of PD-L1 was detected by flow cytometry. CCK8 assay was used to compare the cell proliferation activity between 4T1 cells and 4T1 cells infected by a lentiviral vector of sPD-1 (4T1/sPD-1 cells), and the expressions of sPD-1 mRNA and protein in 4T1/sPD-1 cells were detected using qPCR and Western blotting. The culture supernatant of 4T1/sPD-1 cells was added in 4T1 cells pre-treated with IFN-γ, and PD-1-positive 4T1 cells were detected with flow cytometry. Senescence ß-galactosidase staining kit was used to detect the senescent 4T1 and 4T1/sPD-1 cells following exposure to X-ray radiation and Veliparib. Balb/c mice bearing subcutaneous 4T1 tumor xenografts were treated with injections of PBS, 4T1 STCV, or 4T1/sPD-1 STCV, and tumor growth was observed. RESULTS: Stimulation with IFN-γ concentration-dependently up-regulated PD-L1 expression by as much as (84.80 ± 1.03)% in 4T1 cells (P < 0.001). sPD-1 overexpression in 4T1 cells did not significantly affect the cell proliferation. Treatment of 4T1 cells with 4T1/sPD-1 cell culture supernatant significantly increased the proportion of PD-1 + cells from (6.893 ± 0.271)% to (55.450 ± 0.555)% (P < 0.001). X-ray irradiation combined with Veliparib caused obvious senescence in 4T1 and 4T1/sPD-1 cells. In the tumor-preventing experiment, tumor formation occurred in all the mice in PBS group; 28.787% of the mice in 4T1 STCV group and 55.556% in 4T1/sPD-1 STCV group showed no tumor formation. In the tumor treatment experiment, tumor formation occurred in all the mice in PBS groups while in 4T1 STCV and 4T1/sPD-1 STCV groups, 11.111% and 38.89% of the mice were tumor-free during the observation period, respectively. CONCLUSIONS: Senescence tumor cells vaccine has antitumor effect against breast cancer in mice, and sPD-1 overexpression can enhance this effect of the vaccine.

Notch1 signaling in melanoma cells promoted tumor-induced immunosuppression via upregulation of TGF-ß1.

BACKGROUND: The receptors of Notch family play an important role in controlling the development, differentiation, and function of multiple cell types. The aim of this study is to investigate the role of Notch1 signaling upon immune suppression induced by melanoma cells. METHODS: Melanoma cell line B16 cells were transfected by lentivirus containing mouse Notch1 gene or Notch1 shRNA to generate B16 cell line that highly or lowly expressed Notch1. Notch1 in anti-tumor immune response was comprehensively appraised in murine B16 melanoma tumor model in immunocompetent and immunodeficient mice. The ratios of CD3+CD8+ cytotoxic T cells, CD49b+NK cells, CD4+CD25+FoxP3+ Tregs and Gr1+CD11b+ MDSCs in tumor-DLN or spleen were examined by flow cytometry. After the co-culture of B16 cells and CD8+ T cells, the effects of Notch1 on the proliferation and activation of T cells were assessed by CCK8 assay, CFSE dilution and Chromium-release test. The mRNA expression and supernatant secretion of immunosuppressive cytokines, TGF-ß1, VEGF, IL-10 and IFN-γ were measured by RT-PCR and ELISA, respectively. RESULTS: Downregulation or overexpression of Notch1 in B16 melanoma cells inhibited or promoted tumor growth in immunocompetent mice, respectively. Notch1 expression in B16 melanoma cells inhibited the infiltration of CD8+ cytotoxic T lymphocytes and NK cells and reduced IFN-γ release in tumor tissue. It could also enhance B16 cell-mediated inhibition of T cell proliferation and activation, and upregulate PD-1 expression on CD4+ and CD8+ T cells. The percentage of CD4+CD25+FoxP3+ Tregs and Gr1+CD11b+MDSCs were significantly increased in tumor microenvironment, and all these were attributed to the upregulation of TGF-ß1. CONCLUSION: These findings suggested that Notch1 signaling in B16 melanoma cells might inhibit antitumor immunity by upregulation of TGF-ß1.

[Two HLA-loci mismatched sibling cord blood transplantation in a severe beta-thalassemia patient].

Allogeneic hematopoietic stem cell transplantation is the only curative therapy for severe beta-thalassemia. This time, the experience of utilizing HLA 2-loci mismatched sibling cord blood transplantation (CBT) in a child with severe beta-thalassemia was firstly reported in our country. A 3-year-male patient had been diagnosed with severe beta-thalassemia at 6 months of age (HbF 86.6%, HbA1 1.7%, HbA2 1.7%, beta globin gene mutation CD17, A-->T/IVS-II-654, C-->T). The patient's HLA typing was A 24,11, B 58,35 and DRB1 03,15. During a subsequent maternal pregnancy. The prenatal diagnosis for thalassemia and prenatal HLA typing analysis were performed on 18 weeks of pregnancy. The results indicated that the male fetus was a heterozygote (beta globin gene mutation N/CD17, A-->T), HLA typing was A 24,11, B 58,51 and DRB1 03,12. 120 ml cord blood was collected at time of delivery, the total numbers of nucleated cells, CFU-GM and CD34(+) cells were 1.830 x 10(9), 16.653 x 10(5) and 3.11 x 10(6), respectively. A new conditioning regimen including: hypertransfusion, continuous i.v. desferrioxamine, busulfan, cyclophosphamide, antithymocyte globulin plus hydroxyurea and fludarabine. GVHD prophylaxis comprised cyclosporin A and mycophenolate mofetil. The viability of cord blood at the time infusion was 92%, The total numbers of nucleated cells, CFU-GM and CD34(+) cells in the transfused cord blood were 12.06 x 10(7)/kg, 1.098 x 10(5)/kg, and 2.04 x 10(6)/kg, respectively. Results showed that the patient's clinical course after cord blood transplantation was unremarkable. Acute GVHD grade I developed on day 15, methylprednisolone 2 mg/kg was given to cure. Neutrophil engraftment (ANC > 0.5 x 10(9)/L) on day 17, platelet engraftment (> 50 x 10(9)/L) on day 50. The patients became independent from red blood cell transfusion since day 80 (when his hemoglobin level kept > 12.5 g/L). His beta globin gene mutation and HLA typing were all the same as the donor's analyzed on day 60 and 200. There was also a switch in blood group from A pre-transplant to O post-transplant. It is concluded that the new conditioning and GVHD prophylaxis regimens allow a successful engraftment in this case. This observation may contribute in developing UCBT as an alternative when matched sibling donors are not available.