Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening.

BACKGROUND: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM. METHODS: We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells. RESULTS: We established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy. CONCLUSIONS: Our study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors.

Nitric oxide-induced lipophagic defects contribute to testosterone deficiency in rats with spinal cord injury.

Introduction: Males with acute spinal cord injury (SCI) frequently exhibit testosterone deficiency and reproductive dysfunction. While such incidence rates are high in chronic patients, the underlying mechanisms remain elusive. Methods and results: Herein, we generated a rat SCI model, which recapitulated complications in human males, including low testosterone levels and spermatogenic disorders. Proteomics analyses showed that the differentially expressed proteins were mostly enriched in lipid metabolism and steroid metabolism and biosynthesis. In SCI rats, we observed that testicular nitric oxide (NO) levels were elevated and lipid droplet-autophagosome co-localization in testicular interstitial cells was decreased. We hypothesized that NO impaired lipophagy in Leydig cells (LCs) to disrupt testosterone biosynthesis and spermatogenesis. As postulated, exogenous NO donor (S-nitroso-N-acetylpenicillamine (SNAP)) treatment markedly raised NO levels and disturbed lipophagy via the AMPK/mTOR/ULK1 pathway, and ultimately impaired testosterone production in mouse LCs. However, such alterations were not fully observed when cells were treated with an endogenous NO donor (L-arginine), suggesting that mouse LCs were devoid of an endogenous NO-production system. Alternatively, activated (M1) macrophages were predominant NO sources, as inducible NO synthase inhibition attenuated lipophagic defects and testosterone insufficiency in LCs in a macrophage-LC co-culture system. In scavenging NO (2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO)) we effectively restored lipophagy and testosterone levels both in vitro and in vivo, and importantly, spermatogenesis in vivo. Autophagy activation by LYN-1604 also promoted lipid degradation and testosterone synthesis. Discussion: In summary, we showed that NO-disrupted-lipophagy caused testosterone deficiency following SCI, and NO clearance or autophagy activation could be effective in preventing reproductive dysfunction in males with SCI.

PRPF19 promotes the proliferation, migration, and inhibits autophagy in prostate cancer by suppressing SLC40A1.

Prostate cancer (PCa) is a common cancer and the leading cause of cancer-related death in men. To investigate the role of pre-mRNA processing factor 19 (PRPF19) in proliferation, migration of PCa, and evaluate the potential ability of PRPF19 as a therapeutic target. PRPF19 expression was analyzed from The Cancer Genome Atlas and GEPIA databank. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the transcription of PRPF9 and solute carrier family 40 member 1 (SLC40A1). Immunohistochemistry (IHC) was used to test PRPF9 expression in PCa tissues. The cell viability and 5-ethynyl-2'-deoxyuridine incorporation analysis were performed to assess cell proliferation. Transwell assay was performed to investigate the migration and invasion of cancer cells. Western blot was used to measure the expression level of PRPF9, E-cadherin, Vimentin and α-smooth muscle actin (α-SMA), SLC40A1, LC3, Beclin-1 and ATG7. Immunofluorescence assay was performed to measure LC3 expression in PCa cells. The bioinformatic analysis revealed PRPF19 was highly expressed in PCa which was certified by qRT-PCR, western blot and IHC detection in PCa tissues. The proliferation of PCa cells could be promoted by PRPF19 overexpression and suppressed by PRPF19 knockdown. Moreover, the migration and invasion of PCa cells could be positively regulated by PRPF19 which promoted the expression of E-cadherin, Vimentin, and α-SMA. Furthermore, the expression of LC3, Beclin-1, and ATG7 was negatively regulated by PRPF19, indicating that PRPF19 inhibited autophagy in PCa cells. In the double knockdown of PRPF19 and SLC40A1, PRPF19 repressed the mRNA and reduced protein level of SLC40A1, and SLC40A1 antagonized effects of PRPF19 on proliferation, migration and autophagy of PCa cells. PRPF19 promoted proliferation and migration, and inhibited autophagy in PCa by attenuating SLC40A1 expression, indicating PRPF19 was a potential therapeutic target for PCa treatment.

Loss of the Novel Myelin Protein CMTM5 in Multiple Sclerosis Lesions and Its Involvement in Oligodendroglial Stress Responses.

This study comprehensively addresses the involvement of the protein CKLF-like Marvel transmembrane domain-containing family member 5 (CMTM5) in the context of demyelination and cytodegenerative autoimmune diseases, particularly multiple Sclerosis (MS). An observed reduction in CMTM5 expression in post-mortem MS lesions prompted further investigations in both in vitro and in vivo animal models. In the cuprizone animal model, we detected a decrease in CMTM5 expression in oligodendrocytes that is absent in other members of the CMTM protein family. Our findings also confirm these results in the experimental autoimmune encephalomyelitis (EAE) model with decreased CMTM5 expression in both cerebellum and spinal cord white matter. We also examined the effects of a Cmtm5 knockdown in vitro in the oligodendroglial Oli-neu mouse cell line using the CRISPR interference technique. Interestingly, we found no effects on cell response to thapsigargin-induced endoplasmic reticulum (ER) stress as determined by Atf4 activity, an indicator of cellular stress responses. Overall, these results substantiate previous findings suggesting that CMTM5, rather than contributing to myelin biogenesis, is involved in maintaining axonal integrity. Our study further demonstrates that the knockdown of Cmtm5 in vitro does not modulate oligodendroglial responses to ER stress. These results warrant further investigation into the functional role of CMTM5 during axonal degeneration in the context of demyelinating conditions.

Rational design and performance prediction of organic photosensitizer based on TATA+ dye for hydrogen production by photocatalytic decomposition of water.

In comparison to metal complexes, organic photosensitive dyes employed in photocatalytic hydrogen production exhibit promising developmental prospects. Utilizing the organic dye molecule TA+0 as the foundational structure, a series of innovative organic dyes, denoted as TA1-1 to TA2-6, were systematically designed. Employing first-principles calculations, we methodically explored the modifying effects of diverse electron-donating groups on the R1 and R2 positions to assess their application potential. Our findings reveal that, relative to the experimentally synthesized TATA+03, the TA2-6 molecule boasts a spatial structure conducive to intramolecular electron transfer, showcasing the most negative reduction potential (Ered = -2.11 eV) and the maximum reaction driving force (△G0 2 = -1.26 eV). This configuration enhances its compatibility with the reduction catalyst, thereby facilitating efficient hydrogen evolution. The TA2-6 dye demonstrates outstanding photophysical properties and a robust solar energy capture capacity. Its maximum molar extinction coefficient (ε) stands at 2.616 × 104 M-1·cm-1, representing a remarkable 292.8% improvement over TATA+03. In conclusion, this research underscores the promising potential of the TA2-6 dye as an innovative organic photosensitizer, positioning it as an efficacious component in homogeneous photocatalytic systems.

Novel Dyes Design Based on First Principles and the Prediction of Energy Conversion Efficiencies of Dye-Sensitized Solar Cells.

With the depletion of fossil energy, solar energy has gradually attracted people's attention. Dye-sensitized solar cells have developed rapidly in recent years due to their low cost and high conversion efficiency. In this article, based on the theoretical research on the photovoltaic parameters of DSSCs in the early stages of the research team, we have made an accurate prediction of J sc, V oc, and PCE of C286. (The error in our predicted PCE values was 3.33% relative to the experiment.) Also, we further designed a series of new dyes CH1-CH5 by introducing donors and co-acceptors with C286-C288 as the prototype using the DFT/TDDFT method. The PCE of the designed dyes CH2-CH5 exceed the given dye C286, especially the CH3 and CH4 obtained the PCE of 26.2 and 14.5%. This indicates the proposed dyes offer a dramatic improvement on PCE for DSSC devices. Moreover, the designed dyes such as CH3 and CH4 have great potential to be applied to photovoltaic applications, further enabling the design of novel, highly efficient photoactive materials.

Density functional theory design of double donor dyes and electron transfer on dye/TiO2(101) composite systems for dye-sensitized solar cells.

In this work, we designed a series of double donor organic dyes, named ME101-ME106, based on experimentally synthesized dye WD8, and further investigated their electronic structure, the stability of the dye/TiO2 (101) systems, density of states (DOS) and absorption spectra using density functional theory (DFT) and time-dependent DFT (TDDFT). The molar extinction coefficients of all designed dyes are higher than WD8. It's fascinating that ME106 exhibits a smallest energy gap and 75 nm redshifts compared to WD8. The results of calculations reveal that ME101-ME106/TiO2(101) surfaces are more stable than WD8, double donor dyes have sufficient electron injection driving force and have very strong transfer electron ability. It is expected that the design of double donors can provide a new understanding and guidance for the investigation of high efficiency dye-sensitized devices.

NPD1 inhibits excessive autophagy by targeting RNF146 and wnt/ß-catenin pathway in cerebral ischemia-reperfusion injury.

Objective: Cerebral ischemia-reperfusion (I/R) injury is a common pathological feature in ischemic stroke. Autophagy plays a key role in I/R-induced neuronal death. Neuroprotectin D1 (NPD1) is a docosahexaenoic acid derivative with neuroprotective and anti-inflammatory properties. The purpose of this study was to investigate the mediatory role of NPD1 on I/R-induced injury and to elucidate the underlying mechanisms involved in this process.Methods: An I/R injury model was established in PC12 cells induced by oxygen and glucose deprivation/reoxygenation (OGD/R). NPD1 at increasing doses (5, 10, 20, 50, 100 nM) were added to cells one hour before OGD/R. To investigate the effect of ring finger protein 146 (RFP146) deficiency in I/R injury, PC12 cells were transiently transfected with small interfering RNF146 before further experiment.Results: Compared to the controls, OGD/R-challenged cells exhibited significantly decreased cell viability, induced oxidative stress, and excessive autophagic cell death following OGD/R. Pretreatment with NPD1 protected cells against ischemic injury as evidenced by enhanced cell survival, decreased oxidative stress markers, and a lower level of autophagy compared to drug-free group. OGD/R also increased the level of RFP146 and inhibited the expression of ß-catenin in PC12 cells. NPD1 treatment promoted the production of RNF146 and ß-catenin in cells following OGD/R experiment. Moreover, RNF146 deficiency significantly inhibited ß-catenin expression and reversed the protective effects of NPD1 in OGD/R-induced cells.Conclusion: NPD1 alleviated excessive autophagy via regulating RNF146 and Wnt/ß-catenin signaling, suggesting the potential therapeutic use of NPD1 for the protection against cerebral I/R injury.

[Fine management promotes the development, three-level discipline construction promotes the standardization: the discipline management and three-level discipline construction model of critical care medicine in Affiliated Hospital of Guizhou Medical University].

OBJECTIVE: Critical care medicine is an important part of modern medicine and has become an important comprehensive second-level discipline of clinical medicine. The department of critical care medicine of the Affiliated Hospital of Guizhou Medical University was established in 1994. After 24 years of development, there are currently 90 beds, 6 sub-specialties (including comprehensive ICU A, B, C 3 subspecialties, internal ICU, emergency ICU, pediatric ICU) of the third-level intensive medical discipline development model, involve severe nervous system, severe circulatory system, severe environmental disorders, severe trauma, severe digestion, severe kidney, severe immunity, severe endocrine, severe respiratory disease, severe infection, severe obstetric disease, severe poisoning, and there are corresponding talent echelons. The three-level discipline construction model has been explored and operated for more than three years. The hospital's critical care medicine discipline has established a larger professional discipline in southwestern China. The rapid and standardized development of critical care medicine in an all-round way was promoted, so as to lead the rapid development of critical care medicine in hospitals, cities, provinces and even surrounding provinces, and to achieve mutual learning, complementary advantages, resource sharing, win-win cooperation and coordinated development.

[rotective effect of bone marrow mesenchymal stem cells-derived exosomes against testicular ischemia-reperfusion injury in rats].

OBJECTIVE: To investigate the protective effect of bone marrow mesenchymal stem cells (BMSCs)-derived exosomesagainst testicular ischemia-reperfusion injury (IRI) in rats. METHODS: Rat BMSCs were isolated, cultured and identified in theprimary culture. The exosomes were extracted from the BMSCs and characterized using nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Twenty-four healthy male SD rats were randomly divided into shamoperation group, testicular IRI with saline treatment group and IRI with exosome treatment group. The contralateral testes ofthe rats were collected for pathological observation, aseessment of superoxide dismutase (SOD) and malondialdehyde (MDA), and detection of HMGB1, caspases-3 and cleaved caspase-3 expressions using Western blotting. RESULTS: We successfullyobtained exosomes from rat BMSCs. Testicular IRI significantly impaired testicular spermatogenesis, which was markedlyimproved by treatment with the exosomes (P < 0.05). Testicular IRI also caused significant increase in the protein expression ofHMGB1, caspase-3 and cleaved caspase-3 in the testicular tissue, and treatment with the exosomes obviously amelioratedthese changes (P < 0.05). CONCLUSIONS: BMSCs-derived exosomes protects against testicular IRI due to the anti-oxidant, antiinflammatory and anti-apoptosis activities of the exosomes.

With a new clip technique surgically inducing varicocele in Sprague-Dawley rats.

BACKGROUND: We introduced and recreated a more consistent and effective experimental varicocele rat model by a new clip technique. METHODS: A total of 40 rats were numbered and randomly assigned to 5 groups of 8 each, including sham surgery (Group I), conventional (Group II) and clip groups with 0.7, 0.8, 0.9 mm gap widths, respectively (Group III, IV, V). All of the rats in each group were sacrificed at 8 weeks after initial surgery, and the rats forming out with less than 1 mm diameter of left spermatic vein or no presence of the pampiniform plexus dilation were excluded from the experimental groups. The left spermatic vein (LSV) diameter, testicular weight, left kidney weight to body weight coefficients, kidney and testicular histology were determined. RESULTS: The baseline mean diameter of the LSV in Group I, II and III was 0.22 ± 0.02, 0.23 ± 0.02 and 0.22 ± 0.03 mm, respectively (P = 0.7504). At 8 weeks after initial surgery, varicocele was successfully created in 6/8 (75%), 7/8 (87.5%), 3/8 (37.5%), 3/8 (37.5%) in GroupII-V, no varicocele was observed in Group I. In Group I, II and III, no pathological changes were observed and the left kidney weight to body weight coefficients showed no significant differences. The diameter of LSV was remarkably increased both in Group II and III compared to Group I (1.72 ± 0.13, 1.57 ± 0.19 and 0.25 ± 0.02, respectively), and Group II and III had a smaller testicular weight than the rats in Group I (1.67 ± 0.05, 1.62 ± 0.06, and 1.92 ± 0.12, respectively). CONCLUSIONS: With a new clip technique, surgically inducing varicocele rat model becomes convenient and safe. This appears to improve the effectiveness of the model and this innovation may allow us to further understand the pathophysiology of varicocele.

Effect of auxiliary group for p-type organic dyes in NiO-based dye-sensitized solar cells: The first principal study.

Auxiliary acceptor groups play a crucial role in D-A-π-A structured organic dyes. In this paper, we designed three D-A-π-A structured organic molecules based on the prototype dye QT-1, named ME18-ME20, and further investigated their electronic and optical properties with density functional theory (DFT) and time-dependent DFT (TDDFT). The calculated results indicate that the scope and intensity of dyes' absorption spectra have some outstanding changes by inserting auxiliary groups. ME20 has not only 152nm redshifts to long wave orientation, but also 78% increased oscillator strength compared to QT-1, and its absorption spectrum broadens region even up to 1400nm. Then, we studied the reason that the effect of the introduced different auxiliary acceptor groups in these dyes through their ground states geometries and energy levels, electron transfer and recombination rate.

[Extraction and identification of semen-derived exosomes using PEG6000].

OBJECTIVE: To extract and identify semen-derived exosome using PEG6000. METHODS: Exosomes were extracted from semen specimens from 6 healthy volunteers with step-by-step centrifugations and ultracentrifugation prior to 8% PEG6000 enrichment. The extracted exosomes were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting. RESULTS: The pellets obtained were round or elliptic membrane vesicles 30 to 150 nm in diameter with intact double membranes and contained low electron density material. The pellets expressed CD63, ALIX and TSG101 molecules but not calnexin that was expressed in sperm cells. CONCLUSION: The PEG6000-based method for extraction of exosomes from semen samples facilitates future studies of seminal exosomes.

Collaborative Filtering Recommendation on Users' Interest Sequences.

As an important factor for improving recommendations, time information has been introduced to model users' dynamic preferences in many papers. However, the sequence of users' behaviour is rarely studied in recommender systems. Due to the users' unique behavior evolution patterns and personalized interest transitions among items, users' similarity in sequential dimension should be introduced to further distinguish users' preferences and interests. In this paper, we propose a new collaborative filtering recommendation method based on users' interest sequences (IS) that rank users' ratings or other online behaviors according to the timestamps when they occurred. This method extracts the semantics hidden in the interest sequences by the length of users' longest common sub-IS (LCSIS) and the count of users' total common sub-IS (ACSIS). Then, these semantics are utilized to obtain users' IS-based similarities and, further, to refine the similarities acquired from traditional collaborative filtering approaches. With these updated similarities, transition characteristics and dynamic evolution patterns of users' preferences are considered. Our new proposed method was compared with state-of-the-art time-aware collaborative filtering algorithms on datasets MovieLens, Flixster and Ciao. The experimental results validate that the proposed recommendation method is effective and outperforms several existing algorithms in the accuracy of rating prediction.