The primordial differentiation of tumor-specific memory CD8+ T cells as bona fide responders to PD-1/PD-L1 blockade in draining lymph nodes.

Blocking PD-1/PD-L1 signaling transforms cancer therapy and is assumed to unleash exhausted tumor-reactive CD8+ T cells in the tumor microenvironment (TME). However, recent studies have also indicated that the systemic tumor-reactive CD8+ T cells may respond to PD-1/PD-L1 immunotherapy. These discrepancies highlight the importance of further defining tumor-specific CD8+ T cell responders to PD-1/PD-L1 blockade. Here, using multiple preclinical tumor models, we revealed that a subset of tumor-specific CD8+ cells in the tumor draining lymph nodes (TdLNs) was not functionally exhausted but exhibited canonical memory characteristics. TdLN-derived tumor-specific memory (TTSM) cells established memory-associated epigenetic program early during tumorigenesis. More importantly, TdLN-TTSM cells exhibited superior anti-tumor therapeutic efficacy after adoptive transfer and were characterized as bona fide responders to PD-1/PD-L1 blockade. These findings highlight that TdLN-TTSM cells could be harnessed to potentiate anti-tumor immunotherapy.

A novel protein FNDC3B-267aa encoded by circ0003692 inhibits gastric cancer metastasis via promoting proteasomal degradation of c-Myc.

BACKGROUND: Gastric cancer (GC) ranks fifth in global cancer incidence and third in mortality rate among all cancer types. Circular RNAs (circRNAs) have been extensively demonstrated to regulate multiple malignant biological behaviors in GC. Emerging evidence suggests that several circRNAs derived from FNDC3B play pivotal roles in cancer. However, the role of circFNDC3B in GC remains elusive. METHODS: We initially screened circFNDC3B with translation potential via bioinformatics algorithm prediction. Subsequently, Sanger sequencing, qRT-PCR, RNase R, RNA-FISH and nuclear-cytoplasmic fractionation assays were explored to assess the identification and localization of circ0003692, a circRNA derived from FNDC3B. qRT-PCR and ISH were performed to quantify expression of circ0003692 in human GC tissues and adjacent normal tissues. The protein-encoding ability of circ0003692 was investigated through dual-luciferase reporter assay and LC/MS. The biological behavior of circ0003692 in GC was confirmed via in vivo and in vitro experiments. Additionally, Co-IP and rescue experiments were performed to elucidate the interaction between the encoded protein and c-Myc. RESULTS: We found that circ0003692 was significantly downregulated in GC tissues. Circ0003692 had the potential to encode a novel protein FNDC3B-267aa, which was downregulated in GC cells. We verified that FNDC3B-267aa, rather than circ0003692, inhibited GC migration in vitro and in vivo. Mechanistically, FNDC3B-267aa directly interacted with c-Myc and promoted proteasomal degradation of c-Myc, resulting in the downregulation of c-Myc-Snail/Slug axis. CONCLUSIONS: Our study revealed that the novel protein FNDC3B-267aa encoded by circ0003692 suppressed GC metastasis through binding to c-Myc and enhancing proteasome-mediated degradation of c-Myc. The study offers the potential applications of circ0003692 or FNDC3B-267aa as therapeutic targets for GC.

Mechanical, Thermal Properties, and Extreme Phase Stability of High-Entropy Diborides (V0.2Nb0.2Ta0.2Cr0.2W0.2)B2.

High-entropy diborides (HEDBs) have gained significant attention in industrial applications due to their vast composition space and tunable properties. We propose a solid solution reaction at high temperatures and pressures that successfully synthesized and sintered a novel, dense, and phase-pure HEDB (V0.2Nb0.2Ta0.2Cr0.2W0.2)B2. A high asymptotic Vickers hardness of 26.3 ± 0.6 GPa and a bulk modulus of 320.5 ± 10.6 GPa were obtained. Additionally, we investigated the thermal oxidation process using TG-DSC from room temperature to 1500 °C and explored the phase stability of HEDBs under high-pressure conditions through in situ high-pressure synchrotron radiation X-ray diffraction. We analyzed the formation of lattice distortion, chemical bonding, and band structure in (V0.2Nb0.2Ta0.2Cr0.2W0.2)B2 using first-principles calculations. Surprisingly, we found that the predominant distortion in diborides occurs in the boron layer, supported by ELF. This may be due to uneven electron transfer rather than a straightforward correlation with the atomic radius. These results provide a novel synthesis process and additional experimental data on the mechanical and thermal properties and high-pressure phase stability of HEDBs. Our study offers further insights into the microscopic structure of lattice distortion in HEDBs, which could prove crucial for the selection and design of engineering advanced HEDBs.

Drawing Inspiration from Nature: Trinitarian Strategies for Designing Polyoxometalates and Metal-Organic Framework-Based Biomimetic Microhoneycomb Electromagnetic Wave-Absorbing Materials.

Trinitarian designs in the morphology, components, and microstructure remain challenging for advanced electromagnetic wave absorption (EMWA) materials with light weight, strong absorption, and well-defined structure-function relationships. Herein, a series of X-doped MoS2/Cu9S5 with multilevel honeycomb structures (X-MoS2/Cu9S5 MHs, X = P, Si, Ge) were designed by space-confined growth and in situ sulfidation of a polyoxometalate-based metal-organic framework. X-MoS2/Cu9S5 MHs possess low density, high surface area, and abundant cation-cuprum and anion-sulfur double vacancies (VCu and VS) simultaneously that are unmatched by conventional EMWA materials. Also, the systematic investigation of the doping effect of various polyoxometalate heteroatoms on VCu and VS in the microhoneycomb has been conducted. Experimental results and density functional theory calculations reveal that the excellent EMWA performance (-56.21 dB) results from the synergistic effect of morphology design, component optimization, and vacancy regulation. This study not only provides an important opportunity to understand a morphology-component-microstructure strategy in electromagnetic wave absorption but also builds a noteworthy bridge between bioinspired engineering and microscale absorbers.

A mouse model of a novel missense mutation (Gly83Arg) in a Chinese kindred manifesting vitreous amyloidosis only.

The purpose of this study is to establish a mouse model of transthyretin (TTR) Gly83Arg gene mutation by the technique of gene targeting for research on hereditary vitreous amyloidosis (HVA) and to confirm whether this point mutation is a genetic feature of HVA. A vector (pBR322-MK-TTR) was constructed to target ES cells. The successfully transfected ES cells were used for blastocyst injection, thus generating F0. F0 and Flp mice were mated to generate F1 (TTR+/-, Flp +/-) mice that lacked the neo gene but carried the Flp gene. F1 mice were mated with C57BL/6N wild type mice to generate F2 (TTR+/-) mice. F3 homozygous and heterozygous mice were generated by mating F2 mice with each other. PCR and sequencing were performed for F3 mice. Amyloid was detected using Congo red stain and polarized light. Immunohistochemistry was used to detect the expression of TTR in the tissues. Quantitative fluorescent PCR and Western blotting were used to detect the expression of TTR mRNA and TTR protein, respectively. Two F0-generation, 2 F1-generation and 15 F3-generation mice were obtained. The gene sequencing of F3 mice showed TTR Gly83Arg mutation. When examined with Congo red and polarized light, the vitreous of TTR Gly83Arg mutant mice tested positive for amyloid. The hearts, livers, brains and kidneys of the experimental group and control group were all negative by Congo red staining. Immunohistochemical staining showed that the vitreous of TTR Gly83Arg mutant mice and the livers of the control mice were positive, but the kidneys, hearts and brains of both groups were negative. Quantitative fluorescent PCR showed that the mRNA expression of mutant mice was significantly lower than that of wild-type mice (F = 0.295, P = 0.023). Western blotting showed that the expression of TTR protein was significantly lower in the model mice than in the wild-type mice (t = 3.224, P = 0.018). TTR gene mutation is indeed a molecular characteristic of HVA and manifest in the eye disease only. A C57BL/6 mouse line carrying the TTR Gly83Arg gene mutation was successfully established. This strain of mice can be used for the study of HVA.

Carbonization of Fe-Based Metal Organic Frameworks with Mesoporous Structure as Electrocatalyst for Catalysis of Oxygen to Hydrogen Peroxide.

H2O2 production from electron reduction of oxygen (O2) is considered to be a potential alternative to the current anthraquinone process. Herein, we reported a mesoporous catalyst with iron-carboxylate metal-organic framework (MOF) as precursors to catalyze O2 to hydrogen peroxide (H2O2). Iron-carboxylate MOF (Fe-MOF) was synthesized by the novel cetyltrimethylammonium bromide (CTAB)-citric acid (CA) double-template method. The SEM and SAXD results revealed octahedral structure of the nanoparticles, as well as the presence of mesopores in the Fe-MOF, while the molar ratio 2.03 of CTAB to CA that resulted in the largest value (0.914 cm3g-1) of the mesopores in the Fe-MOFs. The pyrolysis of Fe-MOF with the largest amount of mesopores resulted in its carbonization and produced γ-Fe2O3@carbon material, significantly reduced the BET surface area from 3036 m2 g-1 to 387 m2 g-1, but increased the average pore diameter up to 5.78 nm and disintegrated their octahedral structures to an irregular morphology of Fe-MOF (550), and modified the carbon matrix with trace oxygen and metal oxides. The γ-Fe2O3@carbon material possessed mesoporous structure, with predominant graphitic carbon in the matrix (graphite to amorphous carbon ratio 0.79), which contributed to increased potential for electron reduction of O2 through a 2e- electron transfer pathway.

Highly efficient CRISPR/Cas9-mediated targeted mutagenesis of multiple genes in Populus.

The typeⅡCRISPR/Cas9 system (Clustered regularly interspaced short palindromic repeats /CRISPR-associated 9) has been widely used in bacteria, yeast, animals and plants as a targeted genome editing technique. In previous work, we have successfully knocked out the endogenous phytoene dehydrogenase (PDS) gene in Populus tomentosa Carr. using this system. To study the effect of target design on the efficiency of CRISPR/Cas9-mediated gene knockout in Populus, we analyzed the efficiency of mutagenesis using different single-guide RNA (sgRNA) that target PDS DNA sequence. We found that mismatches between the sgRNA and the target DNA resulted in decreased efficiency of mutagenesis and even failed mutagenesis. Moreover, complementarity between the 3' end nucleotide of sgRNA and target DNA is especially crucial for efficient mutagenesis. Further sequencing analysis showed that two PDS homologs in Populus, PtPDS1 and PtPDS2, could be knocked out simultaneously using this system with 86.4% and 50% efficiency, respectively. These results indicated the possibility of introducing mutations in two or more endogenous genes efficiently and obtaining multi-mutant strains of Populus using this system. We have indeed generated several knockout mutants of transcription factors and structural genes in Populus, which establishes a foundation for future studies of gene function and genetic improvement of Populus.

Spatiotemporal evolution of agricultural drought and its attribution under different climate zones and vegetation types in the Yellow River Basin of China.

Ecological protection and high-quality development of the Yellow River Basin (YRB) are major national strategies in China. Agricultural drought (AD) is one of the most important stress factors of the ecological security of the YRB. Currently, there is a lack of exploration of the spatiotemporal evolution of AD in the YRB under different climatic zones and vegetation types, and the mechanisms by the driving factors influence AD remain unclear. The Temperature Vegetation Dryness Index (TVDI) for the YRB in China during 2000-2020 was calculated using Land Surface Temperature (LST) and the Normalized Difference Vegetation Index (NDVI). We analyzed the spatiotemporal evolution of AD from the perspective of upstream of the YRB (UYRB), midstream of the YRB (MYRB), and downstream of the YRB (DYRB), as well as different climate zones and vegetation types. The driving factors were selected based on the Pearson correlation analysis, Geographical detector, and Mantel test. Structural equation modeling (SEM) was employed to quantify the direct and indirect effects of the driving factors on AD in the YRB. We found a slowing trend of AD in the YRB, mainly in the Loess Plateau, which is distributed in UYRB and MYRB, but an increasing trend for AD in DYRB. Temperature, which is the most direct influential factor, has exacerbated AD in UYRB and MYRB. However, surface solar radiation (SSR) has the greatest constraining effect on DYRB. AD increased in arid and desert zones, while a decreasing trend is observed for other climatic zones and vegetation types. In arid and semiarid zones, human activities and SSR were the largest indirect factors exacerbating AD. In humid and subhumid zones, the largest indirect factor exacerbating AD was potential evapotranspiration (PET). Temperature is the most direct factor exacerbating AD in cropland and forest, while PET is the largest indirect factor exacerbating AD in grassland. This study elucidates the driving factors and mechanisms of AD in the YRB to provide scientific decision support for mitigating regional drought and promoting regional sustainable development.

Nonlinear effects of agricultural drought on vegetation productivity in the Yellow River Basin, China.

Agricultural drought (AD) is the main environmental factor affecting vegetation productivity (VP) in the Yellow River Basin (YRB). In recent years, the nonlinear effects of AD on VP in the YRB have attracted much attention. However, it is still unclear whether fluctuating AD will have complex nonlinear effects on VP in the YRB, and there are scant previous studies at large scale on whether there is a threshold for nonlinear effects of AD on VP in the YRB. Therefore, this study used a newly developed agricultural drought index to explore nonlinear effects on VP revealing the nonlinear effects of AD on VP in the YRB. First, we developed a kernel temperature vegetation drought index (kTVDI) based on kernel normalized difference vegetation index (kNDVI) and land surface temperature data to study the spatiotemporal variation of AD in the YRB. Second, we used GPP data from solar-induced chlorophyll fluorescence inversion as an indicator to explore the spatiotemporal variation of VP in the YRB. Finally, we used several statistical indicators and a distributed lag nonlinear model (DLNM) to analyze the nonlinear effect of AD on VP in the YRB. The results showed that AD decreased significantly during 2000-2020, mainly in the southeast of the Loess Plateau, while GPP increased significantly in 80.93 % of the YRB. Meanwhile, moderate and severe AD stress limited VP growth, with the negative effects gradually decreasing, while mild AD had an increasingly positive promoting effect on VP. AD stress resulted in a VP decrease of 69.78 %, and severe AD stress resulted in a VP decrease of 65.52 %, mainly distributed in the northern Loess and Ordos Plateau. AD had significant nonlinear effects on VP. The effects of moderate and severe AD on the sustained nonlinear lag of vegetation were more obvious, and those of moderate and severe AD on the nonlinear lag of VP were the largest when the lag was approximately 1 month and 7 months. The effect of AD on the nonlinear hysteresis of VP in YRB was significantly different under different vegetation types, and forests were more able to withstand longer and more severe droughts than grasslands and croplands. The results of the study provide a theoretical basis for evaluating AD and analyzing the nonlinear impact of AD on VP. This will provide scientific basis for studying the mechanism of drought effect on vegetation in other regions.

Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis.

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. However, in many cases, cancer cells form metastatic loci in lymph nodes before further metastasizing to distant organs. To what extent the local and systematic CD8+ T cell responses were influenced by LN metastasis remains obscure. To this end, we set up a murine LN metastasis model combined with a B16F10-GP melanoma cell line expressing the surrogate neoantigen derived from lymphocytic choriomeningitis virus (LCMV), glycoprotein (GP), and P14 transgenic mice harboring T cell receptors (TCRs) specific to GP-derived peptide GP33-41 presented by the class I major histocompatibility complex (MHC) molecule H-2Db. This protocol enables the study of antigen-specific CD8+ T cell responses during LN metastasis. In this protocol, C57BL/6J mice were subcutaneously implanted with B16F10-GP cells, followed by adoptive transfer with naive P14 cells. When the subcutaneous tumor grew to approximately 5 mm in diameter, the primary tumor was excised, and B16F10-GP cells were directly injected into the tumor draining lymph node (TdLN). Then, the dynamics of CD8+ T cells were monitored during the process of LN metastasis. Collectively, this model has provided an approach to precisely investigate the antigen-specific CD8+ T cell immune responses during LN metastasis.

Chromosome Three-Dimensional Structure Reconstruction: An Iterative ShRec3D Algorithm.

The three-dimensional (3D) structure of chromosomes is of great significance to ensure that the genome performs various functions (e.g., gene expression) correctly and replicates and separates correctly in mitosis. Since the emergence of Hi-C in 2009, a new experimental technique in molecular biology, researchers have been paying more and more attention to the reconstruction of chromosome 3D structure. To reconstruct the 3D structure of chromosomes based on Hi-C experimental data, many algorithms have been proposed, among which ShRec3D is one of the most outstanding. In this article, an iterative ShRec3D algorithm is presented to greatly improve the native ShRec3D algorithm. Experimental results show that our algorithm can significantly promote the performance of ShRec3D, and this improvement is applicable to almost all data noise range and signal coverage range, so it is universal.

Strontium-doped hydroxyapatite and its role in osteogenesis and angiogenesis.

For the past 50 years, hydroxyapatite (HA) has been widely used in bone defect repair because it is the main inorganic component of the mineral phase of a human bone. Extensive preclinical and clinical studies have shown that strontium (Sr) can safely and effectively help prevent and treat bone diseases, including osteoporosis. These findings have resulted in the concept of integrating Sr and HA for bone disease management. The doped Sr can improve the physicochemical properties of HA and enhance its angiogenic and bone regeneration ability. Nevertheless, no study has reviewed the design strategy of Sr-doped HA (Sr-HA) to understand its biological roles. Therefore, in this article, we review recent developments in Sr-HA preparation and its effect on osteogenesis and angiogenesis in vitro and in vivo along with key suggestions for future research and development.

Tumor-Specific CD4+ T Cells Restrain Established Metastatic Melanoma by Developing Into Cytotoxic CD4- T Cells.

Cytotoxic CD8+ T cells are the main focus of efforts to understand anti-tumor immunity and immunotherapy. The adoptive transfer of tumor-reactive cytotoxic CD8+ T lymphocytes expanded and differentiated in vitro has long been considered the primary strategy in adaptive anti-tumor immunity, however, the majority of the transferred tumor antigen-specific CD8+ T cells differentiated into CD39+CD69+ exhausted progenies, limiting its effects in repressing tumor growth. Contrarily, less attention has been addressed to the role of CD4+ T cells during tumorigenesis. Using a mouse model of metastatic melanoma, we found that transferring tumor-specific CD4+ T cells into recipients induces substantial regression of the established metastatic tumors. Notably, in vitro activated CD4+ T cells developed into cytotoxic CD4- T cells in vivo and get exhausted gradually. The blockade of PD-L1 signaling resulted in an expansion of tumor specific CD4+ T cells, which could better control the established metastatic melanoma. Moreover, the tumor-specific memory CD4+ T cell can prevent mice from tumor metastasis, and the tumor-specific effector CD4+ T cells can also mitigate the established metastatic tumor. Overall, our findings suggest a novel mechanism of CD4+ T cells in curtailing tumor metastasis and confirm their therapeutic role in combination with PD-L1 blockade in cancer immunotherapy. Hence, a better understanding of cytotoxic CD4- T cell-mediated tumor regression could provide an alternative choice for patients exhibiting suboptimal or no response to CD8+ T cell-based immunotherapies.

A novel strategy to investigate the factors regulating the Treg to Tfr transition during acute viral infection.

Follicular regulatory T cells (Tfrs), a specialized subset of regulatory T cells (Tregs), have a particular role in the control of follicular helper T cell-driven germinal center (GC) responses. Following differentiation signals similar to those received by follicular helper T cells (Tfhs), Tfrs gain expression of characteristic chemokine receptors and transcription factors, such as CXCR5 and Bcl-6, allowing them to migrate into the B-cell follicle and perform in situ suppression. Thus, together with Tfhs, Tfrs help maintaining an optimized GC-reaction. However, the mechanism underlying the Treg-to-Tfr transition remains obscure. Here, we established a highly reproducible protocol for investigating the differentiation of Tregs into Tfrs by constructing spleen-chimeric mice combined with retrovirus transduction. We demonstrated that using this strategy, over 4 folds of Tregs could differentiate into Tfrs in Bcl-6 overexpression group compared to control counterparts (Migr1), and Bcl-6 could efficiently promote Tfr differentiation during acute viral infection. Hence, this method provides us an easy access to investigate the factors that regulate the differentiation program that converts Tregs into Tfrs, which will enhance our understanding of the networks regulating GC-reaction and shed new light on the molecular basis of immune homeostasis.

Screening and identification of HLA-A2-restricted neoepitopes for immunotherapy of non-microsatellite instability-high colorectal cancer.

Colorectal cancer has one of the highest mortality rates among malignant tumors, and most patients with non-microsatellite instability-high (MSI-H) colorectal cancer do not benefit from targeted therapy or immune checkpoint inhibitors. Identification of immunogenic neoantigens is a promising strategy for inducing specific antitumor T cells for cancer immunotherapy. Here, we screened potential high-frequency neoepitopes from non-MSI-H colorectal cancer and tested their abilities to induce tumor-specific cytotoxic T cell responses. Three HLA-A2-restricted neoepitopes (P31, P50, and P52) were immunogenic and could induce cytotoxic T lymphocytes in peripheral blood mononuclear cells from healthy donors and colorectal cancer patients. Cytotoxic T lymphocytes induced in HLA-A2.1/Kb transgenic mice could recognize and lyse mutant neoepitope-transfected HLA-A2+ cancer cells. Adoptive transfer of cytotoxic T lymphocytes induced by the peptide pool of these three neoepitopes effectively inhibited tumor growth and increased the therapeutic effects of anti-PD-1 antibody. These results revealed the potential of high-frequency mutation-specific peptide-based immunotherapy as a personalized treatment approach for patients with non-MSI-H colorectal cancer. The combination of adoptive T cell therapy based on these neoepitopes with immune checkpoint inhibitors, such as anti-PD-1, could provide a promising treatment strategy for non-MSI-H colorectal cancer.

Molecular and cytogenetic identification of new wheat-Dasypyrum breviaristatum additions conferring resistance to stem rust and powdery mildew.

Two cytologically stable wheat-Dasypyrum breviarisatatum addition lines, Y93-1-6-6 and Y93-1-A6-4, were identified by integrated molecular and cytogenetic techniques. C-banding and genomic in situ hybridization (GISH) showed that Y93-1-6-6 and Y93-1-A6-4 were different wheat-D. breviaristatum additions. A total of 51 markers (primer/enzyme combinations), including 6 PCR-based Landmark Unique Gene (PLUG) markers and 45 Sequence-Tagged-Site (STS) markers, were selected from 3,774 primer/enzyme combinations to further characterize these two additions. Marker haploytpes suggested that both D. breviaristatum chromosomes in Y93-1-6-6 and Y93-1-A6-4 were rearranged. Stem rust resistance screening indicated that both additions were highly resistant to race RKQQC, whereas only Y93-1-6-6 was resistant to race TTKSK (Ug99). Powdery mildew resistance screening showed that only Y93-1-6-6 was resistant. Pedigree analysis suggested that the stem rust and powdery mildew resistance of Y93-1-6-6 was derived from D. breviaristatum, indicating that the D. breviaristatum chromosomes in Y93-1-6-6 possess a new powdery mildew resistance gene(s), and new stem rust resistance gene(s). These two additions could be used as stem rust or powdery mildew resistance sources in wheat breeding programs.

The recycling regulation of sodium-hydrogen exchanger isoform 3(NHE3) in epithelial cells.

As the main exchanger of electroneutral NaCl absorption, sodium-hydrogen exchanger isoform 3 (NHE3) circulates in the epithelial brush border (BB) and intracellular compartments in a multi-protein complex. The size of the NHE3 complex changes during rapid regulation events. Recycling regulation of NHE3 in epithelial cells can be roughly divided into three stages. First, when stimulated by Ca2+, cGMP, and cAMP-dependent signaling pathways, NHE3 is converted from an immobile complex found at the apical microvilli (MV) into an easily internalized and mobile form that relocates to a compartment near the base of the MV. Second, NHE3 is internalized by clathrin and albumin-dependent pathways into cytoplasmic endosomal compartments, where the complex is reprocessed and reassembled. Finally, NHE3 is translocated from the recycling endosomes (REs) to the apex of epithelial cells, a process that can be stimulated by an increase in sodium-glucose cotransporter 1 (SGLT1) activity, epidermal growth factor receptor (EGFR) signaling, Ca2+ signaling, and binding to ßPix and SH3 and multiple ankyrin repeat domains 2 (Shank2) proteins. This review describes the molecular steps and protein interactions involved in the recycling movement of NHE3 from the apex of epithelial cells, into vesicles, where it is reprocessed and reassembled, and returned to its original location on the plasma membrane, where it exerts its physiological function.

Decreased NHE3 activity in intestinal epithelial cells in TGEV and PEDV-induced piglet diarrhea.

Transmissible gastroenteritis (TGE) and porcine epidemic diarrhea (PED) are highly transmissible intestinal infections caused by transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV), respectively. They are clinically associated with vomiting, diarrhea, and dehydration in piglets. An imbalance in Na+ uptake by intestinal epithelial cells causes TGEV/PEDV-induced diarrhea. However, the mechanism by which TGEV/PEDV-infection in piglets causes Na+ imbalance diarrhea has not been elucidated. In the present study, we demonstrated that specific inhibition of NHE3 activity caused small intestinal bulging, intestinal wall thinning and severe diarrhea in piglets, consistent with the signs of TGEV/PEDV infection. This study further elucidated the role of NHE3 in TGEV/PEDV-induced diarrhea. In this study, small intestinal epithelial cells (IPEC-J2) were used as a model of infection. The results showed that TGEV/PEDV infection reduced NHE3 activity and Na+ uptake in IPEC-J2 cells. Further studies revealed that the use of NHE3-specific inhibitors could reduce the amount of cell membrane NHE3, thereby decreasing Na+ uptake and ultimately leading to diarrhea. Transcriptomic studies performed on obtained jejunal tissues were also consistent with pre-laboratory results. This study will provide a basis for understanding Na+ imbalance diarrhea caused by TGEV/PEDV, as well as for elucidating the diarrheal pathogenesis of other members of α-animal coronaviruses.

Identification of HLA-A2-Restricted Mutant Epitopes from Neoantigens of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC), one of the deadliest gastrointestinal cancers, has had limited effective therapeutic strategies up to now. Accumulating evidence suggests that effective immunotherapy in cancer patients has been associated with T cells responsive to mutant peptides derived from neoantigens. Here, we selected 35 human leukocyte antigen-A2 (HLA-A2)-restricted mutant (MUT) peptides stemmed from neoantigens of ESCC. Among them, seven mutant peptides had potent binding affinity to HLA-A*0201 molecules and could form a stable peptide/HLA-A*0201 complex. Three mutant peptides (TP53-R267P, NFE2L2-D13N, and PCLO-E4090Q) of those were immunogenic and could induce the cytotoxic T lymphocytes (CTLs) recognizing mutant peptides presented on transfected cells in an HLA-A2-restricted and MUT peptide-specific manner. In addition, the CTL response in immunized HLA-A2.1/Kb transgenic (Tg) mice was enhanced by coupling MUT peptides to peptide WH, a peptide delivery carrier targeting Clec9a+ DCs. Then, the possible binding model conversions between the WT and MUT candidate peptides were analyzed by docking with the pockets of HLA-A*0201 molecule. We therefore propose a novel strategy and epitopes for immunotherapy of ESCC based on neoantigens.

Inhibitory effects of Lactobacillus plantarum metabolites on porcine epidemic diarrhea virus replication.

Porcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus; it causes diarrhea in pigs and is associated with high morbidity and mortality in sucking piglets. In this study, we performed in vitro and in vivo experiments to determine the inhibitory effects of Lactobacillus plantarum metabolites (LPM) on PEDV replication. Gas chromatography-mass spectrometry revealed exopolysaccharides to be one of the main components of LPM. We then determine whether L. plantarum exopolysaccharides (LPE) have an antiviral effect and also detected the expression levels of the apoptosis-related genes Bax and Bcl-2 and of the pro-apoptotic protein caspase-3. Further, we assessed the transcription levels of an immune-related protein (STAT1) and antiviral factors (MX1, MX2, ISG15, ZAP, PKR, and OAS1). Our results showed that the most effective method was to pretreat cells with LPM and that the optimal dose of LPM that could be safely administered to Vero cells was 1/8 times of the stock solution. LPE had a strong inhibitory effect on PEDV; the most effective method of administration was to co-incubate cells with LPE and PEDV, and the optimal concentration of LPE was 1.35 mg/mL. To conclude, LPE prevented PEDV adsorption and also alleviated inflammatory responses and induced early apoptosis of injured cells, but it could not regulate the immune function of cells.