High-Throughput Screening of Functional Neo-Antigens and Their Specific T-Cell Receptors via the Jurkat Reporter System Combined with Droplet Microfluidics.

T-cell receptor (TCR)-engineered T cells can precisely recognize a broad repertoire of targets derived from both intracellular and surface proteins of tumor cells. TCR-T adoptive cell therapy has shown safety and promising efficacy in solid tumor immunotherapy. However, antigen-specific functional TCR screening is time-consuming and expensive, which limits its application clinically. Here, we developed a novel integrated antigen-TCR screening platform based on droplet microfluidic technology, enabling high-throughput peptide-major histocompatibility complex (pMHC)-to-TCR paired screening with a high sensitivity and low background signal. We introduced DNA barcoding technology to label peptide antigen candidate-loaded antigen-presenting cells and Jurkat reporter cells to check the specificity of pMHC-TCR candidates. Coupled with the next-generation sequencing pipeline, interpretation of the DNA barcodes and the gene expression level of the Jurkat T-cell activation pathway provided a clear peptide-MHC-TCR recognition relationship. Our proof-of-principle study demonstrates that the platform could achieve pMHC-TCR paired high-throughput screening, which is expected to be used in the cross-reactivity and off-target high-throughput paired testing of candidate pMHC-TCRs in clinical applications.

Anisotropic Black Phosphorene Structural Modulation for Thermal Storage and Solar-Thermal Conversion.

Exploiting novel strategies for simultaneously harvesting ubiquitous, renewable, and easily accessible solar energy based on the photothermal effect, and efficiently storing the acquired thermal energy plays a vital role in revolutionizing the current fossil fuel-dominating energy structure. Developing black phosphorene-based phase-change composites with optimized photothermal conversion efficiencyand high latent heat is the most promising way to achieve efficient solar energy harvesting and rapid thermal energy storage. However, exfoliating high-quality black phosphorene nanosheets  remains challenging, Furthermore, an efficient strategy that can construct the aligned black phosphorene frameworks to maximize thermal conductivity enhancement is still lacking. Herein, high-quality black phosphorene nanosheets are prepared by an optimized exfoliating strategy. Meanwhile, by regulating the temperature gradient during freeze-casting, the framework consisting of shipshape aligned black phosphorene at long-range is successfully fabricated, improving the thermal conductivity of the poly(ethylene glycol) matrix up to 1.81 W m-1  K-1 at 20 vol% black phosphorene loading. The framework also endows the composite with excellent phase-change material encapsulation capacity and  high latent heat of 103.91 J g-1 . It is envisioned that the work advances the paradigm of contrasting frameworks with nanosheets toward controllable structure thermal enhancement of the composites.

The Targeting Effect of Cetuximab Combined with PD-L1 Blockade against EGFR-Expressing Tumors in a Tailored CD16-CAR T-Cell Reporter System.

The switchable chimeric antigen receptors (CARs) have shown many advantages in CAR T-cell therapy. However, human primary T-cells are required to evaluate antigen-specific adaptors by IFN-γ assay or FACS analysis, which limits the throughput of adaptor screening. A sensitive and robust CD16-CAR Jurkat NFAT-eGFP reporter system has been developed to assess the therapeutic efficacy of antibody-targeted CAR-T-cell by effectively evaluating the T-cell activation by various tumor cells and the impact of immune checkpoint inhibitor antibodies. This reporter system facilitates the screening of targeted antibodies in a high throughput manner for the development of improved T-cell immunotherapy.

Prenatal diagnosis of polycystic renal diseases: diagnostic yield, novel disease-causing variants, and genotype-phenotype correlations.

BACKGROUND: Polycystic renal disease is a frequent congenital anomaly of the kidneys, but research using chromosomal microarray analysis and exome sequencing in fetuses with polycystic renal disease remains sparse, with most studies focusing on the multisystem or genitourinary system. OBJECTIVE: This study aimed to assess the detection rate of detectable genetic causes of fetal polycystic renal disease at different levels, novel disease-causing variants, and genotype-phenotype correlations. STUDY DESIGN: This study included 220 fetal polycystic renal disease cases from January 2014 to June 2022. Cases were divided into the following 3 groups: isolated multicystic dysplastic kidneys, nonisolated multicystic dysplastic kidneys, and suspected polycystic kidney disease group. We reviewed data on maternal demographics, ultrasonographic results, chromosomal microarray analysis/exome sequencing results, and pregnancy outcomes. RESULTS: In our cohort, chromosomal microarray analysis identified 19 (8.6%) fetuses carrying chromosomal abnormalities, and the most common copy number variation was 17q12 microdeletion (7/220; 3.2%). Furthermore, 94 families chose to perform trio-exome sequencing testing, and 21 fetuses (22.3%) were found to harbor pathogenic/likely pathogenic variants. There was a significant difference in the live birth rate among the 3 groups (91/130 vs 46/80 vs 1/10; P<.001). Among 138 live birth cases, 106 (78.5%) underwent postnatal ultrasound review, of which 95 (89.6%) had a consistent prenatal-postnatal ultrasound diagnosis. CONCLUSION: For both isolated and nonisolated polycystic renal disease, our data showed high detection efficiency with both testing tools. The detection of novel pathogenic variants expands the known disease spectrum of polycystic renal disease-associated genes while enriching our understanding of the genotype-phenotype correlation. Therefore, we consider it feasible to perform chromosomal microarray analysis+exome sequencing testing in fetal polycystic renal disease. Moreover, prenatal-postnatal ultrasound concordance was greater, the live birth rate was higher, and prognosis was better when known genetic disorders were excluded, indicating that genetic testing results significantly influenced pregnancy decisions.

Prenatal diagnosis and outcomes in fetuses with duplex kidney.

OBJECTIVE: Duplex kidney is a relatively frequent form of urinary system abnormality. This study aimed to elucidate the value of chromosomal microarray analysis (CMA) and whole exome sequencing (WES) for duplex kidney and the perinatal outcomes of duplex kidney fetuses. METHODS: This retrospective cohort study included 63 patients with duplex kidney diagnosed using antenatal ultrasound between August 2013 and January 2023. We reviewed the clinical characteristics, genetic test results, and pregnancy outcomes of the patients. RESULTS: Among the 63 cases based on the inclusion criteria, the CMA detected seven (11.1%) clinically significant variants and nine variants of uncertain significance (VUS), and the pathogenic/likely pathogenic (P/LP) copy number variations (CNVs) in the recurrent region that were associated with prenatal duplex kidney included 17q12, 17p13.3, and 22q11.2. No significant disparity was observed in the CMA detection rate between the unilateral and bilateral groups, or between the isolated and non-isolated groups. WES identified three (50%) P/LP single-gene variants in six fetuses with duplex kidney. We detected the following pathogenic genes in the duplex kidney fetuses: KMT2D, SMPD4, and FANCI. Pregnancy termination in cases where clinically significant variants were detected by genetic testing was different in statistical significance from that in cases with negative results (9/10, 90.0% vs 8/48, 16.7%, P < 0.001). CONCLUSION: This study elucidated the value of CMA and WES for fetal duplex kidney, proving that CMA and WES may be useful tools in prenatal diagnosis and genetic counseling.

Dry Friction Properties of Diamond-Coated Silicon Carbide.

Dry friction between seal faces, caused by unstable or extreme operating conditions, significantly affects the running stability and service life of mechanical seals. Therefore, in this work, nanocrystalline diamond (NCD) coatings were prepared on the surface of silicon carbide (SiC) seal rings by hot filament chemical vapor deposition (HFCVD). The friction test results under dry environment reveals that the coefficient of friction (COF) of SiC-NCD seal pairs is about 0.07-0.09, which were reduced by 83-86% compared to SiC-SiC seal pairs. The wear rate of SiC-NCD seal pairs is relatively low, ranging from 1.13 × 10-7 mm3/N·m to 3.26 × 10-7 mm3/N·m under different test conditions, which is due to the fact that the NCD coatings prevent adhesive and abrasive wear between the SiC seal rings. The analysis and observation of the wear tracks illustrate that the excellent tribological performance of the SiC-NCD seal pairs is due to a self-lubricating amorphous layer formed on the worn surface. In conclusion, this work highlights a pathway to enable mechanical seals to satisfy the high application requirements under highly parametric working conditions.

A mucus-inspired solvent-free carbon dot-based nanofluid triggers significant tribological synergy for sulfonated h-BN reinforced epoxy composites.

Nano-filler reinforced polymer-based composites have attracted extensive attention in tribology; however, to date, it is still challenging to construct a favorable lubricating system with excellent compatibility, lubricity and durability using nano-filler reinforced polymer-based composites. Herein, sulfonated boron nitride nano-sheets (h-BN@PSDA) are prepared and used as nano-fillers for epoxy resins (EPs), to improve friction and wear along with thermal conductivity. Furthermore, inspired by the lubricating principle and structure of snail mucus, a solvent-free carbon dot-based nanofluid (F-CDs) is fabricated and used for the first time as the lubricant for h-BN@PSDA/EPs. Both poly (4-styrene sulfonate) and polyether amine grafted on the surface of F-CDs contribute to branched structures and multiple interfacial absorption effects. Extraordinarily low friction and wear are detected after long-term sliding. The average coefficient of friction and wear rate of h-BN@PSDA/EPs composites are reduced by 95.25% and 99.42% respectively, in the presence of the F-CD nanofluid, compared to that of EPs. Besides, the added h-BN nano-sheets increase the thermal conductivity (TC) of EPs from 0.178 to 0.194 W (m-1 K-1). The distinguished lubrication performances are likely due to the formation of a hybrid nanostructure of 0D F-CDs and 2D h-BN@PSDA together with the "rolling-sliding" and "self-mending" effects of added F-CDs.

Spatial transcriptome unveils a discontinuous inflammatory pattern in proficient mismatch repair colorectal adenocarcinoma.

The preexistence of immune cells in the tumor microenvironment substantiates the efficacy of immunotherapy in cancer patients. Although the complex intratumoral immune heterogeneity has been extensively studied in single cell resolution, hi-res spatial investigations are limited. In this study, we performed a spatial transcriptome analysis of 4 colorectal adenocarcinoma specimens and 2 paired distant normal specimens to identify the molecular pattern involved in a discontinuous inflammatory response in pathologically annotated cancer regions. Based on the location of spatially varied gene expression, we unmasked the spatially-varied immune ecosystem and identified the locoregional "warmed-up" immune response in predefined "cold" tumor with substantial infiltration of immune components. This "warmed-up" immune profile was found to be associated with the in-situ copy number variance and the tissue remodeling process. Further, "warmed-up" signature genes indicated improved overall survival in CRC patients obtained from TCGA database.

Chemical Vapor Deposition of <110> Textured Diamond Film through Pre-Seeding by Diamond Nano-Sheets

Diamond films prepared by chemical vapor deposition will exhibit different surface morphologies, which are determined by the texture and the structural perfection of the deposited diamond. In general, its surface morphology can be controlled by adjusting the deposition conditions. In the present work, <110> textured diamond film was deposited on single crystalline silicon through pre-seeding by diamond nanosheets, rather than controlling the deposition conditions. The employed diamond nano-sheets were prepared by cleavage along a plane, exhibiting good crystallinity. Before chemical vapor deposition, the as-prepared diamond nano-sheets were pre-seeded on the surface of single crystalline silicon as nucleation sites for diamond growth. SEM and XRD results show that the prepared diamond films have a <110> texture. FIB observation reveals that diamonds homogeneously grow on the pre-seeded diamond nano-sheets during chemical vapor deposition, achieving the diamond film with <110> texture. Our work provides a new strategy to prepare <110> textured diamond film.

A Novel Proteogenomic Integration Strategy Expands the Breadth of Neo-Epitope Sources.

Tumor-specific antigens can activate T cell-based antitumor immune responses and are ideal targets for cancer immunotherapy. However, their identification is still challenging. Although mass spectrometry can directly identify human leukocyte antigen (HLA) binding peptides in tumor cells, it focuses on tumor-specific antigens derived from annotated protein-coding regions constituting only 1.5% of the genome. We developed a novel proteogenomic integration strategy to expand the breadth of tumor-specific epitopes derived from all genomic regions. Using the colorectal cancer cell line HCT116 as a model, we accurately identified 10,737 HLA-presented peptides, 1293 of which were non-canonical peptides that traditional database searches could not identify. Moreover, we found eight tumor neo-epitopes derived from somatic mutations, four of which were not previously reported. Our findings suggest that this new proteogenomic approach holds great promise for increasing the number of tumor-specific antigen candidates, potentially enlarging the tumor target pool and improving cancer immunotherapy.

Outstanding Bio-Tribological Performance Induced by the Synergistic Effect of 2D Diamond Nanosheet Coating and Silk Fibroin.

Due to their excellent biocompatibility, outstanding mechanical properties, high strength-to-weight ratio, and good corrosion resistance, titanium (Ti) alloys are extensively used as implant materials in artificial joints. However, Ti alloys suffer from poor wear resistance, resulting in a considerably short lifetime. In this study, we demonstrate that the chemical self-assembly of novel two-dimensional (2D) diamond nanosheet coatings on Ti alloys combined with natural silk fibroin used as a novel lubricating fluid synergistically results in excellent friction and wear performance. Linear-reciprocating sliding tests verify that the coefficient of friction and the wear rate of the diamond nanosheet coating under silk fibroin lubrication are reduced by 54 and 98%, respectively, compared to those of the uncoated Ti alloy under water lubrication. The lubricating mechanism of the newly designed system was revealed by a detailed analysis of the involved microstructural and chemical changes. The outstanding tribological behavior was attributed to the establishment of artificial joint lubrication induced by the cross binding between the diamond nanosheets and silk fibroin. Additionally, excellent biocompatibility of the lubricating system was verified by cell viability, which altogether paves the way for the application of diamond coatings in artificial Ti joint implants.

Enhanced thermal transportation across an electrostatic self-assembly of black phosphorene and boron nitride nanosheets in flexible composite films.

For effective heat dissipation in portable electronics, there is a great demand for lightweight and flexible films with superior thermal transport properties. Despite extensive efforts, enhancing the intrinsic low thermal conductivity of polymers while simultaneously maintaining their flexibility is difficult to achieve due to the dilemma of quarrying appropriate filler loading. Herein, a cellulose nanofiber-based film with high in-plane thermal conductivity up to 72.53 W m-1 K-1 was obtained by harnessing the advantage of functionalized boron nitride nanosheets (f-BNNS) and black phosphorene (BP) via the vacuum filtration process. Besides, our unique design based on the electrostatic coupling of black phosphorene and functionalized boron nitride nanosheets significantly reduced the interfacial thermal resistance of the composite films. This work offers new insights into establishing a facile, yet efficient approach to preparing high thermal conductive heat spreaders.

Single-cell transcriptome analysis of the heterogeneous effects of differential expression of tumor PD-L1 on responding TCR-T cells.

Rationale: TCR-T cell therapy plays a critical role in the treatment of malignant cancers. However, it is unclear how TCR-T cells are affected by PD-L1 molecule in the tumor environment. We performed an in-depth evaluation on how differential expressions of tumor PD-L1 can affect the functionality of T cells. Methods: We used MART-1-specific TCR-T cells (TCR-TMART-1), stimulated with MART-127-35 peptide-loaded MEL-526 tumor cells, expressing different proportions of PD-L1, to perform cellular assays and high-throughput single-cell RNA sequencing. Results: Different clusters of activated or cytotoxic TCR-TMART-1 responded divergently when stimulated with tumor cells expressing different percentages of PD-L1 expression. Compared to control T cells, TCR-TMART-1 were more sensitive to exhaustion, and secreted not only pro-inflammatory cytokines but also anti-inflammatory cytokines with increasing proportions of PD-L1+ tumor cells. The gene profiles of chemokines were modified by increased expression of tumor PD-L1, which concurrently downregulated pro-inflammatory and anti-inflammatory transcription factors. Furthermore, increased expression of tumor PD-L1 showed distinct effects on different inhibitory checkpoint molecules (ICMs). In addition, there was a limited correlation between the enrichment of cell death signaling in tumor cells and T cells and increased tumor PD-L1 expression. Conclusion: Overall, though the effector functionality of TCR-T cells was suppressed by increased expression percentages of tumor PD-L1 in vitro, scRNA-seq profiles revealed that both the anti-inflammatory and pro-inflammatory responses were triggered by a higher expression of tumor PD-L1. This suggests that the sole blockade of tumor PD-L1 might inhibit not only the anti-inflammatory response but also the pro-inflammatory response in the complicated tumor microenvironment. Thus, the outcome of PD-L1 intervention may depend on the final balance among the highly dynamic and heterogeneous immune regulatory circuits.