Canonical Wnt signaling directs the generation of functional human PSC-derived atrioventricular canal cardiomyocytes in bioprinted cardiac tissues.

The creation of a functional 3D bioprinted human heart remains challenging, largely due to the lack of some crucial cardiac cell types, including the atrioventricular canal (AVC) cardiomyocytes, which are essential to slow down the electrical impulse between the atrium and ventricle. By utilizing single-cell RNA sequencing analysis and a 3D bioprinting technology, we discover that stage-specific activation of canonical Wnt signaling creates functional AVC cardiomyocytes derived from human pluripotent stem cells. These cardiomyocytes display morphological characteristics and express molecular markers of AVC cardiomyocytes, including transcription factors TBX2 and MSX2. When bioprinted in prefabricated cardiac tissues, these cardiomyocytes successfully delay the electrical impulse, demonstrating their capability of functioning as the AVC cardiomyocytes in vitro. Thus, these findings not only identify canonical Wnt signaling as a key regulator of the AVC cardiomyocyte differentiation in vitro, but, more importantly, provide a critical cellular source for the biofabrication of a functional human heart.

Diagnostic Potential of Serum Glycome Analysis in Lung Cancer: A Glycopattern Study.

Lung cancer is the leading cause of cancer-related death, with high morbidity and mortality rates due to the lack of reliable methods for diagnosing lung cancer at an early stage. Low-dose computed tomography can help detect abnormal areas in the lungs, but only 16% of cases are diagnosed early. Tests for lung cancer markers are often employed to determine genetic expression or mutations in lung carcinogenesis. Serum glycome analysis is a promising new method for early lung cancer diagnosis as glycopatterns exhibit significant differences in lung cancer patients. In this study, we employed a solid-phase chemoenzymatic method to systematically compare glycopatterns in benign cases, adenocarcinoma before and after surgery, and advanced stages of adenocarcinoma. Our findings indicate that serum high-mannose levels are elevated in both benign cases and adenocarcinoma, while complex N-glycans, including fucose and 2,6-linked sialic acid, are downregulated in the serum. Subsequently, we developed an algorithm that utilizes 16 altered N-glycans, 7 upregulated and 9 downregulated, to generate a score based on their intensity. This score can predict the stages of cancer progression in patients through glycan characterization. This methodology offers a potential means of diagnosing lung cancer through serum glycome analysis.

CDC20 promotes radioresistance of prostate cancer by activating Twist1 expression.

Currently, radiotherapy is one of the most attractive treatments for prostate cancer (PCa) patients. However, radioresistance remains a challenging issue and the underlying mechanism is unknown. Growing evidence has demonstrated that CDC20 (Cell division cycle protein 20) plays a pivotal role in a variety of tumors, including PCa. Here, GEPIA database mining and western blot analysis showed that higher expression of CDC20 was observed in PCa tissues and cells. We demonstrated that the expression of CDC20 was increased in PCa cells by irradiation, and knockdown of CDC20 resulted in inhibition of cell proliferation, migration, tumor formation, induced cell apoptosis and increased radiosensitivity in PCa in vitro and in vivo. Furthermore, we observed that CDC20 regulated Twist1 pathway, influencing cell proliferation and migration. These results suggest that targeting CDC20 and Twist1 may be an effective way to improve the radiosensitivity of PCa.

Deciphering Protein O-GalNAcylation: Method Development and Disease Implication.

Mucin-type O-glycosylation is an important protein post-translational modification that is abundantly expressed on cell surface proteins. Protein O-glycosylation plays a variety of roles in cellular biological functions including protein structure and signal transduction to the immune response. Cell surface mucins are highly O-glycosylated and are the main substance of the mucosal barrier that protects the gastrointestinal or respiratory tract from infection by pathogens or microorganisms. Dysregulation of mucin O-glycosylation may impair mucosal protection against pathogens that can invade cells to trigger infection or immune evasion. Truncated O-glycosylation, also known as Tn antigen or O-GalNAcylation, is highly upregulated in diseases such cancer, autoimmune disorders, neurodegenerative diseases, and IgA nephropathy. Characterization of O-GalNAcylation helps decipher the role of Tn antigen in physiopathology and therapy. However, the analysis of O-glycosylation, specifically the Tn antigen, remains challenging due to the lack of reliable enrichment and identification assays compared to N-glycosylation. Here, we summarize recent advances in analytical methods for O-GalNAcylation enrichment and identification and highlight the biological role of the Tn antigen in various diseases and the clinical implications of identifying aberrant O-GalNAcylation.

Lycorine suppresses cell growth and invasion via down-regulation of NEDD4 ligase in bladder cancer.

Recent studies have shown that lycorine, a natural alkaloid compound, plays its anti-cancer role in several human malignancies including bladder cancer. However, the molecular mechanism of lycorine-induced antitumor activity has not been sufficiently investigated. The E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated protein 4 (NEDD4, also known as NEDD4-1) plays a crucial role in tumorigenesis and progression of human cancer. Therefore, depletion of NEDD4 could be a prospective therapeutic strategy for the treatment of cancer. In this study, we investigated whether lycorine restrains tumor by inhibiting the expression of NEDD4 in bladder cancer. We observed that lycorine blocked bladder cancer cell proliferation, colony formation, metastasis and invasion. Moreover, we found that overexpression of NEDD4 in bladder cancer cells significantly promoted cell proliferation and motility, whereas downregulating of the NEDD4 gene expression by lycorine or siRNA suppressed cell growth and movement. Notably, lycorine increased gemcitabine sensitivity in bladder cancer cells. Importantly, lycorine significantly reduced tumor growth, whereas overexpression of NEDD4 accelerated tumor growth and rescued lycorine-triggered tumor inhibition in xenograft mouse model. In conclusion, our study demonstrated that lycorine could exert its antineoplastic activity via suppressing NEDD4 pathway in vitro and in vivo. Therefore, inhibition of NEDD4 expression by lycorine might be a potential efficient strategy for bladder cancer.

The duration and breadth of antibody responses to 3-dose of inactivated COVID-19 vaccinations in healthy blood donors: An observational study.

Objectives: We aimed to evaluate the duration and breadth of antibodies elicited by inactivated COVID-19 vaccinations in healthy blood donors. Methods: We performed serological tests on 1,417 samples from 658 blood donors who received two (n=357), or three (n=301) doses of COVID-19 inactivated vaccine. We also accessed the change in antibody response before and after booster vaccination in 94 participants and their neutralization breadth to the current variants after the booster. Results: Following vaccination, for either the 2- or 3-dose, the neutralizing antibodies (nAbs) peaked with about 97% seropositivity approximately within one month but subsequently decreased over time. Of plasmas collected 6-8 months after the last immunization, the nAb seropositivities were 37% and 85% in populations with 2-dose and 3-dose vaccinations, respectively. The nAbs of plasma samples (collected between 2-6 weeks after the 3rd dose) from triple-vaccinated donors (n=94) showed a geometric mean titer of 145.3 (95% CI: 117.2 to 180.1) against the ancestral B.1, slightly reduced by 1.7-fold against Delta variant, but markedly decreased by 4-6 fold in neutralizing Omicron variants, including the sub-lineages of BA.1 (5.6-fold), BA.1.1 (6.0-fold), BA.2 (4.2-fold), B.2.12.1 (6.2-fold) and BA.4/5 (6.5-fold). Conclusion: These findings suggested that the 3rd dose of inactivated COVID-19 vaccine prolongs the antibody duration in healthy populations, but the elicited-nAbs are less efficient in neutralizing circulating Omicron variants.

Cross-species tropism and antigenic landscapes of circulating SARS-CoV-2 variants.

Mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) may alter viral host tropism and affect the activities of neutralizing antibodies. Here, we investigated 153 RBD mutants and 11 globally circulating variants of concern (VOCs) and variants of interest (VOIs) (including Omicron) for their antigenic changes and cross-species tropism in cells expressing 18 ACE2 orthologs. Several RBD mutations strengthened viral infectivity in cells expressing ACE2 orthologs of non-human animals, particularly those less susceptible to the ancestral strain. The mutations surrounding amino acids (aas) 439-448 and aa 484 are more likely to cause neutralization resistance. Strikingly, enhanced cross-species infection potential in the mouse and ferret, instead of the neutralization-escape scores of the mutations, account for the positive correlation with the cumulative prevalence of mutations in humans. These findings present insights for potential drivers of circulating SARS-CoV-2 variants and provide informative parameters for tracking and forecasting spreading mutations.

Cross-neutralizing antibodies bind a SARS-CoV-2 cryptic site and resist circulating variants.

The emergence of numerous variants of SARS-CoV-2, the causative agent of COVID-19, has presented new challenges to the global efforts to control the COVID-19 pandemic. Here, we obtain two cross-neutralizing antibodies (7D6 and 6D6) that target Sarbecoviruses' receptor-binding domain (RBD) with sub-picomolar affinities and potently neutralize authentic SARS-CoV-2. Crystal structures show that both antibodies bind a cryptic site different from that recognized by existing antibodies and highly conserved across Sarbecovirus isolates. Binding of these two antibodies to the RBD clashes with the adjacent N-terminal domain and disrupts the viral spike. Both antibodies confer good resistance to mutations in the currently circulating SARS-CoV-2 variants. Thus, our results have direct relevance to public health as options for passive antibody therapeutics and even active prophylactics. They can also inform the design of pan-sarbecovirus vaccines.

Virus-Free and Live-Cell Visualizing SARS-CoV-2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors.

The ongoing corona virus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, a genetically engineered sensor of fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process is developed. In ACE2-expressing cells, it is found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.

Cdc20 induces the radioresistance of bladder cancer cells by targeting FoxO1 degradation.

Ionizing radiation is a conventional therapy for cancer patients, but patients often experience distant metastasis and recurrence, which lead to a poor prognosis after the implementation of this treatment. Moreover, the underlying mechanisms by which radioresistance contributes to metastatic potential is still elusive. Here, we explored the molecular mechanisms that contribute to radioresistance in bladder cancer. To achieve this, we established two irradiation-resistant (IR) cell lines, T24R and 5637R, which were derived from parental bladder cancer cell lines. Cell viability was detected by CCK-8 assay, while migration and invasion abilities were examined by wound healing and Transwell chamber assays, respectively. Furthermore, the role of Cdc20 in the regulation of epithelial to mesenchymal transition (EMT) in IR cells was explored by Western blotting, immunoprecipitation and immunofluorescence staining. The IR cells exhibited EMT properties, and our data showed that Cdc20 expression was significantly elevated in IR cells. Remarkably, Cdc20 silencing reversed the EMT phenotype in IR cells. Mechanistically, Cdc20 governed IR-mediated EMT in part by governing forkhead box O1 (FoxO1) degradation. Taken together, our findings showed that the inactivation of Cdc20 or the activation of FoxO1 might be a potential strategy to overcome radioresistance in bladder cancer.

Robust in vitro assay for analyzing the neutralization activity of serum specimens against hepatitis B virus.

Anti-HBs is a well-known marker of protective capability against HBV. However, little is known about the association between the qAnti-HBs determined by immunoassays and the neutralization activity (NAT) derived from functional assays. We developed an in vitro assay for direct measurement of the NAT of human sera. The new assay was highly sensitive, with an analytical sensitivity of 9.6 ± 1.3 mIU/mL for the HBIG standard. For serum detection, the maximum fold dilution required to produce ≥50% inhibition (MDF50) of HBV infection was used as the quantitative index. In vitro NAT evaluations were conducted for a cohort of 164 HBV-free healthy individuals. The results demonstrated that the NAT positively correlated with the qAnti-HBs (R2 = 0.473, p < 0.001). ROC analysis indicated that the optimal cutoff value of the qAnti-HBs to discriminate significant NAT (MDF50 ≥ 8) was 62.9 mIU/mL, with an AUROC of 0.920. Additionally, we found that the qAnti-HBc was another independent parameter positively associated with the NAT (R2 = 0.300, p < 0.001), which suggested that antibodies against other HBV proteins generated by previous HBV exposure possibly also contribute to the NAT. In summary, the new cell-based assay provides a robust tool to analyse the anti-HBV NAT. Abbreviations: HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; Anti-HBs: Hepatitis B surface antibody; HBeAg: Hepatitis B e antigen; Anti-HBc: Hepatitis B core antibody; qAnti-HBs: quantitative hepatitis B surface antibody; qAnti-HBc: quantitative hepatitis B core antibody; qHBeAg: quantitative hepatitis B e antigen; NAT: neutralization activity; HBIG: hepatitis B immune globulin; NTCP: Na+-taurocholate cotransporting polypeptide; IRES: internal ribosome entry site; ccHBV: cell culture derived hepatitis B virus; GE/cell: genome equivalent per cell; MOI: multiplicity of infection; Dpi: day post infection; HepG2-TetOn: a HepG2-derived cell line that expresses the doxycycline-regulated transactivator; ROC: receiver operating characteristic curve; AUROC: area under receiver operating characteristic curve; LLOQ: the lower limits of quantification; MDF50: the maximum fold dilution required to produce ≥50% inhibition; IC50: half maximal inhibitory concentration.

Ultrasound-guided totally implantable venous access device through the right innominate vein in older patients is safe and reliable.

AIM: Ultrasound guidance has become the routine method for catheterization, dramatically reducing failure and complication rates for totally implantable venous access devices (TIVAD) placement. The aim of the present study was to report the safety and efficacy of ultrasound-guided right innominate vein TIVAD placement in older patients. METHODS: Between September 2015 and September 2017, 55 older patients underwent right innominate vein TIVAD placement under ultrasound guidance. Intraoperative fluoroscopy was always carried out. The technical success rate and complications were recorded and retrospectively analyzed. RESULTS: The technical success rate was 100%. The success rate of the first puncture was 96.36% (53/55). The mean operation time was 28 ± 7 min (range 23-39 min), and the mean length of catheter introduction was 19.24 ± 2.65 cm (range 17-21 cm). The overall incidence of complications was 7.27% (4/55), including one arterial puncture with self-limiting hematoma, two cases of catheter-related infection and one case of fibrin sheath. No catheter malposition or catheter fracture was observed. At the time of this study, three TIVAD were pulled out unexpectedly, and 32 TIVAD are still in functional use. CONCLUSIONS: Ultrasound-guided puncture of the right innominate vein is safe and reliable to implant TIVAD, which can provide new options for older patients. Geriatr Gerontol Int 2019; 19: 218-221.