[Preparation of interleukin-1ß-targeted nanobodies and their effects on apoptosis in hypoxic cardiomyocytes of mice].

Objective: To prepare interleukin-1ß-targeted nanoantibodies and observe their effects on apoptosis in hypoxic cardiomyocyte of mice. Methods: Using DNA recombination technology, the pET-16b and pHEN1 expression vectors were used to construct the prokaryotic expression plasmids of interleukin-1ß-targeted nanobodies (pET-16b-4G6M-VHH, pET-16b-5BVP-VHH, pET-16b-5MVZ-VHH, pHEN1-4G6M-VHH, pHEN1-5BVP-VHH and pHEN1-5MVZ-VHH, where VHH is a variable domain of heavy chain antibody, 4G6M-VHH, 5BVP-VHH, 5MVZ-VHH were three interleukin-1ß-targeted nanoantibodies respectively). The constructed plasmids were transferred into Escherichia coli Rosetta2 (DE3) for induction of expression and nickel column purification, respectively. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were employed to identify the expression product and purified product, and the enzyme-linked immune sorbent assay (ELISA) was performed to determine their affinity. The cardiomyocyte hypoxia model was used with the highest affinity IL-1ß-targeted nanobody (pHEN1-5MVZ-VHH), and cell survival and apoptosis rates were detected (the experiment was divided into normal control group, hypoxia model group, blank plasmid group and 12.5, 25.0, 50.0 µg/ml pHEN1-5MVZ-VHH treatment groups). Results: SDS-PAGE and Western blotting results showed that the anti-interleukin-1ß (IL-1ß) nanobodies with a relative molecular mass of about 15 000 were successfully obtained. Likewise, ELISA results found that the nanobodies expressed in pHEN1 vector group had higher affinity for IL-1ß antigen compared with pET-16b vector group (4G6M-VHH group: 3.20±0.03 vs 1.20±0.03, P<0.001; 5BVP-VHH group: 3.18±0.06 vs 1.21±0.02, P<0.001; 5MVZ-VHH group: 3.38±0.05 vs 1.62±0.04, P<0.001). Additionally, the results of cell survival assay and apoptosis assay detected that compared with the hypoxia model group, HL-1 cell activity was significantly increased in the 25.0 µg/ml and 50.0 µg/ml pHEN1-5MVZ-VHH treatment groups [(75.55±2.23)% vs (46.90±2.51)%, P<0.001; (74.36±1.96)% vs (46.90±2.51)%, P<0.001], and apoptosis rate was significantly reduced [(6.83±0.27)% vs (10.24±0.76)%, P<0.001; (6.68±0.38)% vs (10.24±0.76)%, P<0.001]. Conclusions: 4G6M-VHH, 5BVP-VHH, and 5MVZ-VHH are expressed by both pET-16b and pHEN1 expression vectors and the nanobodies produced by the pHEN1 vector display enhanced antigen affinity. Furthermore, in hypoxic cardiomyocytes, pHEN1-5MVZ-VHH treatment reduces cell apoptosis.

[Efficacy of neoadjuvant therapy on HER2-positive breast cancer: a clinicopathological analysis].

Objective: To investigate the efficacy of neoadjuvant therapy (NAT) on HER2-positive breast cancer and to analyze their clinicopathological features. Methods: A total of 480 cases of HER2-positive breast cancer who received neoadjuvant therapy (NAT), diagnosed at the Department of Pathology of Fudan University Shanghai Cancer Center from 2015 to 2020, were retrospectively identified. Clinicopathological parameters such as age, tumor size, molecular subtype, type of targeted therapy, Ki-67 proliferation index, ER and HER2 immunohistochemical expression, and HER2 amplification status were analyzed to correlate with the efficacy of NAT. Results: Among 480 patients with HER2-positive breast cancer, 209 achieved pathology complete response (pCR) after NAT, with a pCR rate of 43.5%. Of all the cases,457 patients received chemotherapy plus trastuzumab and 23 patients received chemotherapy with trastuzumab and pertuzumab. A total of 198 cases (43.3%) achieved pCR in patients with chemotherapy plus trastuzumab, and 11 cases (47.8%) achieved pCR in patients with chemotherapy plus trastuzumab and pertuzumab. The pCR rate in the latter group was higher, but there was no statistical significance. The results showed that the pCR rate of IHC-HER2 3+patients (49%) was significantly higher than that of IHC-HER2 2+patients (26.1%, P<0.001). The higher the mean HER2 copy number in the FISH assay, the higher the pCR rate was achieved. The expression level of ER was inversely correlated with the efficacy of NAT, and the pCR rate in the ER-positive group (28.2%) was significantly lower than that in the ER-negative group (55.8%, P<0.001). The pCR rate (29.1%) of patients with luminal B type was lower than that of HER2 overexpression type (55.8%, P<0.001). In addition, higher Ki-67 proliferation index was associated with higher pCR rate (P<0.001). The pCR rate was the highest in the tumor ≤2 cm group (57.7%), while the pCR rate in the tumor >5 cm group was the lowest (31.1%). The difference between the groups was significant (P=0.005). Conclusions: HER2 copy numbers, HER2 immunohistochemical expression level, molecular subtype, ER expression level and Ki-67 proliferation index are significantly associated with pCR after NAT. In addition, fluorescence in situ hybridization results, HER2/CEP17 ratio and tumor size could also significantly affect the efficacy of NAT.

[A case report of death from toxic encephalopathy caused by emamectin·chlorfenapyr].

Emamectin·chlorfenapyr is insecticide compounded by emamectin benzoate and chlorfenapyr. There is no special antidote after poisoning, and the mortality rate of patients is very high. We admitted a case of toxic encephalopathy caused by oral administration of emamectin·chlorfenapyr. The clinical manifestations of patient were gastrointestinal symptoms, profuse sweating, high fever, changes in consciousness. After admitted to the hospital, despite active comprehensive treatment, the patient died of ineffective rescue eventually.

[Application and clinical significance of intercellular proximity labeling technique in chronic myelogenous leukemia].

Objective: This study aimed to explore the application of interaction-dependent fucosyl-biotinylation (FucoID), a chemical biology-based proximity labeling technique, in capturing tumor antigen-specific T cells and its clinical value in chronic myelogenous leukemia (CML) . Methods: Flow cytometry and fluorescence microscopy were employed to evaluate the experimental parameters for FucoID in CML. Peripheral blood samples were obtained from 14 newly diagnosed CML patients in the chronic phase. These samples underwent flow cytometry-based sorting and were subsequently labeled with FucoID to facilitate the isolation of tumor cells and T cells, followed by the immunophenotypic identification of tumor antigen-specific T cells. Finally, the diagnostic and therapeutic potential of FucoID in CML was assessed. Results: Initially, the experimental parameters for FucoID in CML were established. The proportion of CD3(+) T cells in patients was (8.96±6.47) %, exhibiting a marked decrease compared with that in healthy individuals at (38.89±22.62) %. The proportion of tumor-specific antigen-reactive T cells was (3.34±4.49) %, which demonstrated interpatient variability. In addition, the proportion of tumor-specific antigen-active T cells in CD4(+) T cells was (3.95±1.72) %, which was generally lower than the proportion in CD8(+) T cells at (5.68±2.18) %. Compared with those in tumor-specific antigen-nonreactive T cells, CCR7(-)CD45RA(-) effector memory T cells and CCR7(-)CD45RA(+) effector T cells were highly enriched in tumor-specific antigen-reactive T cells. Moreover, the intensity of tumor immune reactivity in patients exhibited a significant correlation with white blood cell count (WBC) and hemoglobin (HGB) levels in peripheral blood, while no such correlation was observed with other clinical baseline characteristics. Conclusion: The combination of FucoID and flow cytometry enables the rapid identification and isolation of tumor antigen-specific T cells in CML. The successful application of this method in CML and the implications of our findings suggest its potential clinical value in the field of hematologic malignancies.

[Maresin1 inhibits the NF-κB/caspase-3/GSDME signaling pathway to alleviate hepatic ischemia-reperfusion injury].

Objective: To investigate the role of Maresin1 (MaR1) in hepatic ischemia-reperfusion injury (HIRI). Methods: The HIRI model was established and randomly divided into a sham operation group (Sham group), an ischemia-reperfusion group (IR group), and a MaR1 ischemia-reperfusion group (MaR1+IR group). MaR1 80ng was intravenously injected into each mouse's tail veins 0.5h before anesthesia. The left and middle hepatic lobe arteries and portal veins were opened and clamped. The blood supply was restored after 1h of ischemia. After 6h of reperfusion, the mice were sacrificed to collect blood and liver tissue samples. The Sham's group abdominal wall was only opened and closed. RAW267.4 macrophages were administered with MaR1 50ng/ml 0.5h before hypoxia, followed by hypoxia for 8h and reoxygenation for 2h, and were divided into the control group, the hypoxia-reoxygenation group (HR group), the MaR1 hypoxia-reoxygenation group (MaR1 + HR group), the Z-DEVD-FMK hypoxia-reoxygenation group (HR+Z group), the MaR1 + Z-DEVD-FMK hypoxia-reoxygenation group (MaR1 + HR + Z group), and the Con group without any treatment. Cells and the supernatant above them were collected. One-way analysis of variance was used for inter-group comparisons, and the LSD-t test was used for pairwise comparisons. Results: Compared with the Sham group, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin (IL)-1ß, and IL-18 in the IR group were significantly higher (P < 0.05), with remarkable pathological changes, while the level in the MaR1 + IR group was lower than before (P < 0.05), and the pathological changes were alleviated. Compared with the Con group, the HR group had higher levels of IL-1ß and IL-18 (P < 0.05), while the MaR1 + HR group had lower levels of IL-1ß and IL-18 (P < 0.05). Western blot showed that the expressions of caspase-3, GSDME, and GSDME-N were significantly higher in the HR group and IR group than in the other groups; however, the expression was lower following MaR1 pretreatment. The Z-DEVD-FMK exploration mechanism was inhibited by the expression of caspase-3 in HIRI when using MaR1. Compared with the HR group, the IL-1ß and IL-18 levels and the expressions of caspase-3, GSDME, and GSDME-N in the HR + Z group were decreased (P < 0.05), while the expression of nuclear factor κB was increased, but following MaR1 pretreatment, nuclear factor κB was decreased. There was no significant difference in the results between the MaR1 + H/R group and the MaR1 + H/R + Z group (P > 0.05). Conclusion: MaR1 alleviates HIRI by inhibiting NF-κB activation and caspase-3/GSDME-mediated inflammatory responses.

Does Periodontitis Affect the Association of Biological Aging with Mortality?

The prevalence of periodontitis is increasing with the aging of the global population. Periodontitis has been suggested to accelerate aging and increase mortality. The present nationwide prospective cohort study aimed to determine whether periodontitis could modify the association of biological aging with all-cause and cause-specific mortality in middle-aged and older adults. Participants ≥40 y of age from the Third National Health and Nutrition Examination Survey (NHANES III) were included (n = 6,272). Phenotypic age acceleration (PhenoAgeAccel) was used to evaluate the biological aging process. Moderate/severe periodontitis was defined using a half-reduced Centers for Disease Control and Prevention and American Academy of Periodontology case definition. Multivariable Cox proportional hazard regression was conducted to estimate the association between PhenoAgeAccel and mortality risk, followed by effect modification analysis to test whether periodontitis modified the association. During a median follow-up of 24.5 y, 3,600 (57.4%) deaths occurred. The positive relationships between PhenoAgeAccel and all-cause and cause-specific mortality were nonlinear. After adjusting for potential confounders, the highest quartile of PhenoAgeAccel was associated with increased all-cause mortality in individuals with no/mild periodontitis (hazard ratio for Q4 vs. Q1 [HRQ4vs.Q1] = 1.789; 95% confidence interval [CI], 1.541-2.076). In contrast, the association was enhanced in patients with moderate/severe periodontitis (HRQ4vs.Q1 = 2.446 [2.100-2.850]). Periodontal status significantly modified the association between PhenoAgeAccel and all-cause mortality (P for interaction = 0.012). In subgroup analyses, the modifying effect of periodontitis was observed in middle-aged adults (40-59 y), females, and non-Hispanic Whites. Although cause-specific mortality showed a similar trend, the PhenoAgeAccel × periodontitis interaction did not reach statistical significance. In conclusion, periodontitis might enhance the association of biological aging with all-cause mortality in middle-aged and older adults. Hence, maintaining and enhancing periodontal health is expected to become an intervention to slow aging and extend life span.

PRRX1+MSCs Enhance Mandibular Regeneration during Distraction Osteogenesis.

Bone defect (BD) caused by trauma, infection, congenital defects, or neoplasia is a major cause of physical limitation. Distraction osteogenesis (DO) is a highly effective procedure for bone regeneration, while the concrete mechanism remains unknown. In this study, canine DO and BD models of the mandible were established. The results of micro-computed tomography and histological staining revealed that DO led to an increased mineralized volume fraction and robust new bone formation; in contrast, BD demonstrated incomplete bone union. Mesenchymal stem cells (MSCs) from DO and BD calluses were isolated and identified. Compared with BD-MSCs, DO-MSCs were found to have a stronger osteogenic capability. Single-cell RNA sequencing analysis was further performed to comprehensively define cell differences between mandibular DO and BD calluses. Twenty-six clusters of cells representing 6 major cell populations were identified, including paired related homeobox 1-expressing MSCs (PRRX1+MSCs), endothelial cells (ECs), T cells, B cells, neutrophils, and macrophages. Interestingly, 2 subpopulations in PRRX1+MSCs in the DO group were found to express the marker of neural crest cells (NCCs) and were associated with the process of epithelial-mesenchymal transition. The immunofluorescence assay was performed to further corroborate these results in vivo and in vitro, experimentally validating that continuous distraction maintained the PRRX1+MSCs in an embryonic-like state. Finally, we used CRISPR/Cas9 to knock out (KO) PRRX1 in the context of DO, which significantly blunted the capability of jawbone regeneration, resulting in a diminished NCC-like program and reduction of new bone volume. In addition, the ability of osteogenesis, cell migration, and proliferation in cultured PRRX1KO MSCs was inhibited. Taken together, this study provides a novel, comprehensive atlas of the cell fates in the context of DO regeneration, and PRRX1+MSCs act essential roles.