Discovery and functional investigation of BMP4 as a new causative gene for human congenital heart disease.

OBJECTIVE: Aggregating evidence highlights the strong genetic basis underpinning congenital heart disease (CHD). Here BMP4 was chosen as a prime candidate gene causative of human CHD predominantly because BMP4 was amply expressed in the embryonic hearts and knockout of Bmp4 in mice led to embryonic demise mainly from multiple cardiovascular developmental malformations. The aim of this retrospective investigation was to discover a novel BMP4 mutation underlying human CHD and explore its functional impact. METHODS: A sequencing examination of BMP4 was implemented in 212 index patients suffering from CHD and 236 unrelated non-CHD individuals as well as the family members available from the proband carrying a discovered BMP4 mutation. The impacts of the discovered CHD-causing mutation on the expression of NKX2-5 and TBX20 induced by BMP4 were measured by employing a dual-luciferase analysis system. RESULTS: A new heterozygous BMP4 mutation, NM_001202.6:c.318T>G;p.(Tyr106*), was found in a female proband affected with familial CHD. Genetic research of the mutation carrier's relatives unveiled that the truncating mutation was in co-segregation with CHD in the pedigree. The nonsense mutation was absent from 236 unrelated non-CHD control persons. Quantitative biologic measurement revealed that Tyr106*-mutant BMP4 failed to induce the expression of NKX2-5 and TBX20, two genes whose expression is lost in CHD. CONCLUSION: The current findings indicate BMP4 as a new gene predisposing to human CHD, allowing for improved prenatal genetic counseling along with personalized treatment of CHD patients.

NMR Studies of the Interactions between Sialyllactoses and the Polysialytransferase Domain for Polysialylation Inhibition.

It is known that sialyllactose (SL) in mammalians is a major source of sialic acid (Sia), which can further form cytidine monophosphate sialic acid (CMP-Sia), and the final product is polysialic acid (polySia) using polysialyltransferases (polySTs) on the neural cell adhesion molecule (NCAM). This process is called NCAM polysialylation. The overexpression of polysialylation is strongly related to cancer cell migration, invasion, and metastasis. In order to inhibit the overexpression of polysialylation, in this study, SL was selected as an inhibitor to test whether polysialylation could be inhibited. Our results suggest that the interactions between the polysialyltransferase domain (PSTD) in polyST and CMP-Siaand the PSTD and polySia could be inhibited when the 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL) concentration is about 0.5 mM or 6'-SL and 3 mM, respectively. The results also show that SLs (particularly for 3'-SL) are the ideal inhibitors compared with another two inhibitors, low-molecular-weight heparin (LMWH) and cytidine monophosphate (CMP), because 3'-SL can not only be used to inhibit NCAM polysialylation, but is also one of the best supplements for infant formula and the gut health system.

Design, docking optimization, and evaluation of biotin-PEG4-1,8-naphthalimide as a potent and safe antitumor agent with dual targeting of ferroptosis and DNA.

A set of biotin-polyethylene glycol (PEG)-naphthalimide derivatives 4a-4h with dual targeting of ferroptosis and DNA were designed and optimized using docking simulation as antitumor agents. Docking simulation optimization results indicated that biotin-PEG4-piperazine-1,8-naphthalimide 4d should be the best candidate among these designed compounds 4a-4h, and therefore, we synthesized and evaluated it as a novel antitumor agent. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and MGC-803 and U251 xenograft models identified 4d as a good candidate antitumor agent with potent efficacy and safety profiles, compared with amonafide and temozolomide. The findings of the docking simulations, fluorescence intercalator displacement (FID), western blot, comet, 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transmission electron microscopy, and BODIPY-581/591-C11, FerroOrange, and dihydroethidium (DHE) fluorescent probe assays revealed that 4d could induce DNA damage, affect DNA synthesis, and cause cell cycle arrest in the S phase in MGC-803 cells. Also, it could induce lipid peroxidation and thus lead to ferroptosis in MGC-803 cells, indicating that it mainly exerted antitumor effects through dual targeting of ferroptosis and DNA. These results suggested that it was feasible to design, optimize using docking simulation, and evaluate the potency and safety of biotin-PEG-1,8-naphthalimide as a antitumor agent with dual targeting of ferroptosis and DNA, based on a multi-target drug strategy.

Gold nanocluster-based ratiometric fluorescence immunoassay for broad-spectrum screening of five eugenols.

BACKGROUND: Eugenol compounds (EUGs), which share chemical similarities with eugenol, belong to a group of phenolic compounds primarily found in clove oil. They are highly valued by fish dealers due to their exceptional anesthetic properties, playing a crucial role in reducing disease incidence and mortality during the transportation of live fish. Despite their widespread use, the safety of EUGs remains a contentious topic, raising concerns about the safety of aquatic products. This underscores the need for efficient and sensitive analytical methods for detecting EUGs. RESULTS: Nanomaterial-based ratiometric fluorescence immunoassay has gained increasing attention due to its integration of the immunoassay's excellent specificity and compatibility for high-throughput analysis, coupled with the exceptional sensitivity and anti-interference capabilities of ratiometric fluorescence assays. In this study, we developed a sensitive ratiometric fluorescence immunoassay for screening five EUGs. This method employs a broad-specificity monoclonal antibody (mAb) as a recognition reagent, selective for five EUGs. It leverages the horseradish peroxidase (HRP)-triggered formation of fluorescent 2,3-diaminophenazine (DAP) and the quenching of fluorescent gold clusters (Au NCs) for detection. The assay's detection limits for eugenol, isoeugenol, eugenol methyl eugenol, methyl isoeugenol, and acetyl isoeugenol in tilapia fish and shrimp were found to be 9.8/19.5 µg/kg, 0.11/0.22 µg/kg, 19/36 Tilapia ng/kg, 8/16 ng/kg, and 3.0/6.1 µg/kg, respectively. Furthermore, when testing spiked Tilapia fish and shrimp samples, recoveries ranging from 84.1 to 111.9 %, with the coefficients of variation staying below 7.1 % was achieved. SIGNIFICANCE: This work introduces an easy-to-use, broad-specificity, and highly sensitive method for the screening of five EUGs at a pg/mL level, which not only provides a high-throughput strategy for screening eugenol-type fish anesthetics in aquatic products, but also can serve as a benchmark for developing immunoassays for other small molecular pollutants, rendering potent technological support for guarding food safety and human health.

The Bifunctional Effects of Lactoferrin (LFcinB11) in Inhibiting Neural Cell Adhesive Molecule (NCAM) Polysialylation and the Release of Neutrophil Extracellular Traps (NETs).

The expression of polysialic acid (polySia) on the neuronal cell adhesion molecule (NCAM) is called NCAM-polysialylation, which is strongly related to the migration and invasion of tumor cells and aggressive clinical status. Thus, it is important to select a proper drug to block tumor cell migration during clinical treatment. In this study, we proposed that lactoferrin (LFcinB11) may be a better candidate for inhibiting NCAM polysialylation when compared with CMP and low-molecular-weight heparin (LMWH), which were determined based on our NMR studies. Furthermore, neutrophil extracellular traps (NETs) represent the most dramatic stage in the cell death process, and the release of NETs is related to the pathogenesis of autoimmune and inflammatory disorders, with proposed involvement in glomerulonephritis, chronic lung disease, sepsis, and vascular disorders. In this study, the molecular mechanisms involved in the inhibition of NET release using LFcinB11 as an inhibitor were also determined. Based on these results, LFcinB11 is proposed as being a bifunctional inhibitor for inhibiting both NCAM polysialylation and the release of NETs.

V6 vein-preserving superior segmentectomy: A potentially preferable option.

Objective: The increasing identification of pulmonary nodules has led to a growing emphasis on segmentectomy. Nevertheless, the surgical process for segmentectomy is complex and optimizing segmentectomy is a critical clinical concern. This study aimed to evaluate the safety and short- and long-term efficacy of V6-preserving superior segmentectomy. Methods: We performed a retrospective analysis of patients who underwent thoracoscopic superior segmentectomy at our hospital between January 2019 and June 2020. Eligible patients were categorized into an V6 vein-preserving segmentectomy (VVPS) group and a Non V6 vein-preserving segmentectomy (NVVPS) group depending on the preservation of V6. Primary outcome measures encompassed the evaluation of surgical safety (surgical margins, 3-year overall survival, and disease-free survival), whereas secondary measures included postoperative complication rates, operative time, estimated intraoperative blood loss, length of hospital stay, and associated costs. Results: The analysis included a final cohort of 78 patients. In the NVVPS group (n = 43), 95.3 % of patients exceeded the tumor diameter, and no positive surgical margins were observed. The 3-year overall survival (OS) and disease-free survival (DFS) rates for the NVVPS group were 95.3 %, with no significant differences in OS (p = 0.572) and DFS (P = 0.800) compared with the VVPS group. Additionally, the median total hospitalization cost for the NVVPS group was 41,400 RMB (IQR, 38,800-43,400), which was significantly lower than that of the VVPS group, showing statistical significance (P < 0.05). No statistically significant differences were observed in the incidence of postoperative complications and length of stay between the two groups (P > 0.05). Conclusion: V6-preserving superior segmentectomy is a secure and optimized surgical alternative. Its streamlined procedure facilitates easier adoption in primary healthcare facilities, rendering it a superior choice for superior segmentectomy.

Discovery of BMP10 as a new gene underpinning congenital heart defects.

OBJECTIVE: Aggregating evidence convincingly establishes the predominant genetic basis underlying congenital heart defects (CHD), though the heritable determinants contributing to CHD in the majority of cases remain elusive. In the current investigation, BMP10 was selected as a prime candidate gene for human CHD mainly due to cardiovascular developmental abnormalities in Bmp10-knockout animals. The objective of this retrospective study was to identify a new BMP10 mutation responsible for CHD and characterize the functional effect of the identified CHD-causing BMP10 mutation. METHODS: Sequencing assay of BMP10 was fulfilled in a cohort of 276 probands with various CHD and a total of 288 non-CHD volunteers. The available family members from the proband harboring an identified BMP10 mutation were also BMP10-genotyped. The effect of the identified CHD-causative BMP10 mutation on the transactivation of TBX20 and NKX2.5 by BMP10 was quantitatively analyzed in maintained HeLa cells utilizing a dual-luciferase reporter assay system. RESULTS: A novel heterozygous BMP10 mutation, NM_014482.3:c.247G>T;p.(Glu83*), was identified in one proband with patent ductus arteriosus (PDA), which was confirmed to co-segregate with the PDA phenotype in the mutation carrier's family. The nonsense mutation was not observed in 288 non-CHD volunteers. Functional analysis unveiled that Glu83*-mutant BMP10 had no transactivation on its two representative target genes TBX20 and NKX2.5, which were both reported to cause CHD. CONCLUSION: These findings provide strong evidence indicating that genetically compromised BMP10 predisposes human beings to CHD, which sheds light on the new molecular mechanism that underlies CHD and allows for antenatal genetic counseling and individualized precise management of CHD.

Somatic GATA4 mutation contributes to tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most prevalent cyanotic congenital heart pathology and causes infant morbidity and mortality worldwide. GATA-binding protein 4 (GATA4) serves as a pivotal transcriptional factor for embryonic cardiogenesis and germline GATA4 mutations are causally linked to TOF. However, the effects of somatic GATA4 mutations on the pathogenesis of TOF remain to be ascertained. In the present study, sequencing assay of GATA4 was performed utilizing genomic DNA derived from resected heart tissue specimens as well as matched peripheral blood specimens of 62 patients with non-familial TOF who underwent surgical treatment for TOF. Sequencing of GATA4 was also performed using the heart tissue specimens as well as matched peripheral venous blood samples of 68 sporadic cases who underwent heart valve displacement because of rheumatic heart disorder and the peripheral venous whole blood samples of 216 healthy subjects. The function of the mutant was explored by dual-luciferase activity analysis. Consequently, a new GATA4 mutation, NM_002052.5:c.708T>G;p.(Tyr236*), was found in the heart tissue of one patient with TOF. No mutation was detected in the heart tissue of the 68 cases suffering from rheumatic heart disorder or in the venous blood samples of all 346 individuals. GATA4 mutant failed to transactivate its target gene, myosin heavy chain 6. Additionally, this mutation nullified the synergistic transactivation between GATA4 and T-box transcription factor 5 or NK2 homeobox 5, two genes causative for TOF. Somatic GATA4 mutation predisposes TOF, highlighting the significant contribution of somatic variations to the molecular pathogenesis underpinning TOF.

Bioresponsive Chimaeric Polymersomes Mediate Sustained and Liver-Specific siRNA Transfection In Vivo.

The silencing of disease-causing genes with small interfering RNA (siRNA) offers a particularly effective therapeutic strategy for different disorders; however, its clinical efficacy relies on the development of nontoxic and tissue-specific delivery vehicles. Herein, we report that bioresponsive chimaeric polymersomes (BCP) with short poly(ethylenimine) as inner shell mediate highly efficacious, sustained, and liver-specific siRNA transfection in vivo. BCP exhibited remarkable encapsulation efficiencies of siRNA (95-100%) at siRNA-feeding contents of 15-25 wt %, to afford stable, small-sized (55-64 nm), and neutral-charged BCP-siRNA. siApoB-Loaded BCP (BCP-siApoB) outperformed lipofectamine counterparts and silenced 93% of ApoB mRNA in HepG2 cells at 50 nM siApoB without inducing cytotoxicity. Intriguingly, the in vivo studies using wild-type C57BL/6 mice revealed that BCP-siApoB preferentially accumulated in the liver, and a single dose of 4.5 mg/kg achieved over 90% downregulation of ApoB mRNA for at least 10 days. The systemic administration of BCP-siApoB at 4.5 mg/kg every 2 weeks or 1.5 mg/kg weekly in diet-induced obese mice could also achieve up to 80% silencing of ApoB mRNA. The liver specificity and silencing efficacy of BCP-siApoB could further be improved by decorating it with the trivalent N-acetylgalactosamine (TriGalNAc) ligand. These bioresponsive and liver-specific chimaeric polymersomes provide an enabling technology for siRNA therapy of various liver-related diseases.

Synthesis and Biological Evaluation of Novel 2-Amino-1,4-Naphthoquinone Amide-Oxime Derivatives as Potent IDO1/STAT3 Dual Inhibitors with Prospective Antitumor Effects.

Indoleamine-2,3-dioxygenase 1 (IDO1) and signal transducer and activator of transcription 3 (STAT3) have emerged as significant targets in the tumor microenvironment for cancer therapy. In this study, we synthesized three novel 2-amino-1,4-naphthoquinone amide-oxime derivatives and identified them as dual inhibitors of IDO1 and STAT3. The representative compound NK3 demonstrated effective binding to IDO1 and exhibited good inhibitory activity (hIDO1 IC50 = 0.06 µM), leading to its selection for further investigation. The direct interactions between compound NK3 and IDO1 and STAT3 proteins were confirmed through surface plasmon resonance analysis. A molecular docking study of compound NK3 revealed key interactions between NK3 and IDO1, with the naphthoquinone-oxime moiety coordinating with the heme iron. In the in vitro anticancer assay, compound NK3 displayed potent antitumor activity against selected cancer cell lines and effectively suppressed nuclear translocation of STAT3. Moreover, in vivo assays conducted on CT26 tumor-bearing Balb/c mice and an athymic HepG2 xenograft model revealed that compound NK3 exhibited potent antitumor activity with low toxicity relative to 1-methyl-L-tryptophan (1-MT) and doxorubicin (DOX). Overall, these findings provided evidence that the dual inhibitors of IDO1 and STAT3 may offer a promising avenue for the development of highly effective drug candidates for cancer therapy.

The NMR studies of CMP inhibition of polysialylation.

The overexpression of polysialic acid (polySia) on neural cell adhesion molecules (NCAM) promotes hypersialylation, and thus benefits cancer cell migration and invasion. It has been proposed that the binding between the polysialyltransferase domain (PSTD) and CMP-Sia needs to be inhibited in order to block the effects of hypersialylation. In this study, CMP was confirmed to be a competitive inhibitor of polysialyltransferases (polySTs) in the presence of CMP-Sia and triSia (oligosialic acid trimer) based on the interactional features between molecules. The further NMR analysis suggested that polysialylation could be partially inhibited when CMP-Sia and polySia co-exist in solution. In addition, an unexpecting finding is that CMP-Sia plays a role in reducing the gathering extent of polySia chains on the PSTD, and may benefit for the inhibition of polysialylation. The findings in this study may provide new insight into the optimal design of the drug and inhibitor for cancer treatment.

Targeted nanodelivery of siRNA against KRAS G12D inhibits pancreatic cancer.

Pancreatic cancer (PC) stands as a most deadly malignancy due to few effective treatments in the clinics. KRAS G12D mutation is a major driver for most PC cases, and silencing of KRAS G12D is considered as a potential therapeutic strategy for PC, which is nevertheless crippled by lacking a pragmatic delivery system for siRNA against KRAS G12D (siKRAS). Here, we report that cRGD peptide-modified bioresponsive chimaeric polymersomes (cRGD-BCP) mediate highly efficient siKRAS delivery to PANC-1 tumor, potently silencing KRAS G12D mRNA in tumor cells and effectively suppressing PC tumor growth in mice. cRGD-BCP exhibited remarkable encapsulation of siKRAS (loading content > 14 wt.%, loading efficiency > 90%) to form stable and uniform (ca. 68 nm) nanovesicles (cRGD-BCP-siKRAS). Of note, cRGD density greatly impacted the cellular uptake and silencing efficiency of cRGD-BCP-siKRAS in PANC-1 cells, in which an optimal cRGD density of 15.7 mol.% achieved 3.7- and 3.6-fold enhancement of internalization and gene silencing, respectively, compared with non-targeted BCP-siKRAS. cRGD-BCP-siKRAS was practically intact after 3-week storage at 4°C. Intriguingly, cRGD-BCP-siKRAS markedly enhanced the uptake of siKRAS in PANC-1 tumor, and at a siKRAS dose of 3 mg/kg knocked down 90% KRAS G12D gene, resulting in potent tumor inhibition and extraordinary survival benefits (median survival time: 101 days versus 38 (PBS group) and 59 days (BCP-siKRAS)) with 40% mice achieved complete regression. It appears that cRGD-mediated nanodelivery of siKRAS provides a potential cure for pancreatic cancer. STATEMENT OF SIGNIFICANCE: Small interfering RNA (siRNA) emerges as a specific and powerful biopharmaceuticals against cancers; however, inefficient in vivo delivery impedes its clinical translation. In spite of the fact that KRAS G12D mutation has been identified as a major driver for most pancreatic cancer, its notorious non-druggability renders little success on development of molecular targeted drugs. Pancreatic cancer is deemed as current king-of-cancer. Here, we show that cyclic RGD peptide installed bioresponsive polymersomes are able to efficiently deliver siRNA against KRAS G12D to pancreatic tumor, resulting in 90% gene knock-down and effective tumor inhibition. Strikingly, two out of five mice have been cured. This targeted nanodelivery of siRNA provides a high-efficacy treatment strategy for pancreatic cancer.

Sub-axillary cosmetic incision versus single-incision thoracoscopic surgery for primary spontaneous pneumothorax.

BACKGROUND: In recent years, single-incision thoracoscopic surgery (SITS) has been increasingly applied as an optimal treatment option for primary spontaneous pneumothorax (PSP). However, most SITS techniques are used in the fourth to sixth intercostal space between the anterior axillary and mid axillary lines. To find out more concealed incisions, this study performed PSP surgery via the sub-axillary cosmetic incision (SACI) technique. METHODS: A total of 128 PSP patients were subjected to video-assisted thoracoscopic surgery (VATS) between January 2017 and January 2019 at our institution. These patients were evaluated and assigned into SACI (n = 21) and SITS (n = 57) groups. Propensity score matching (PSM) was performed based on patients' backgrounds, and the enrolled cohort was divided into 21 pairs. The incision satisfaction was assessed at 2 weeks and 6 months post-surgery. RESULTS: The 21 pairs with matching baseline characteristics in the two groups did not exhibit significant differences in their backgrounds and surgical results. However, compared with the SITS group, the operation time was longer in the SACI group (p = 0.013). There were no post-operative complications in both groups. At 2 weeks and 6 months, incision satisfaction scores in the SACI group were significantly lower than those in the SITS group (p = 0.022 and p = 0.039, respectively). There were no recurrences of ipsilateral pneumothorax in both groups. CONCLUSIONS: SACI is a safe and feasible surgical method for PSP treatment. In addition, incision concealment can be used for patients with incision needs.

SARS-CoV-2 Structural Proteins Modulated Blood-Testis Barrier-Related Proteins through Autophagy in the Primary Sertoli Cells.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) disrupts the blood-testis barrier (BTB), resulting in alterations in spermatogenesis. However, whether BTB-related proteins (such as ZO-1, claudin11, N-cadherin, and CX43) are targeted by SARS-CoV-2 remains to be clarified. BTB is a physical barrier between the blood vessels and the seminiferous tubules of the animal testis, and it is one of the tightest blood-tissue barriers in the mammalian body. In this study, we investigated the effects of viral proteins, via ectopic expression of individual viral proteins, on BTB-related proteins, the secretion of immune factors, and the formation and degradation of autophagosomes in human primary Sertoli cells. Our study demonstrated that ectopic expression of viral E (envelope protein) and M (membrane protein) induced the expressions of ZO-1 and claudin11, promoted the formation of autophagosomes, and inhibited autophagy flux. S (spike protein) reduced the expression of ZO-1, N-cadherin, and CX43, induced the expression of claudin11, and inhibited the formation and degradation of autophagosomes. N (nucleocapsid protein) reduced the expression of ZO-1, claudin11, and N-cadherin. All the structural proteins (SPs) E, M, N, and S increased the expression of the FasL gene, and the E protein promoted the expression and secretion of FasL and TGF-ß proteins and the expression of IL-1. Blockage of autophagy by specific inhibitors resulted in the suppression of BTB-related proteins by the SPs. Our results indicated that SARS-CoV-2 SPs (E, M, and S) regulate BTB-related proteins through autophagy.

GP73 enhances the ox-LDL-induced inflammatory response in THP-1 derived macrophages via affecting NLRP3 inflammasome signaling.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease with its molecular basis incompletely understood. Here, we determined whether the Golgi phosphoprotein 73 (GP73), a novel protein highly related to inflammation and disrupted lipid metabolism, was involved in the development of atherosclerosis. METHODS: Public microarray databases of human vascular samples were analyzed for expression patterns. Apolipoprotein-E-gene-deficient (ApoE-/-) mice (8-week-old) were randomly assigned to either a chow diet group or a high-fat diet group. The levels of serum GP73, lipid profiles and key inflammatory cytokines were determined by ELISA. The aortic root plaque was isolated and used for by Oil Red O staining. PMA-differentiated THP-1 macrophages were transfected with GP73 small interfering RNA (siRNA) or infected with adenovirus expressing GP73, and then stimulated with oxidized low density lipoprotein (ox-LDL). The expressions of pro-inflammatory cytokines and signal pathway key targets were determined by ELISA kit and Western blot respectively. In addition, ichloro-dihydro-fluorescein diacetate (DCFH-DA) was used to measure the intracellular ROS levels. RESULTS: The expressions of GP73 and NLRP3 were substantially upregulated in human atherosclerotic lesions. There were significant linear correlations between GP73 and inflammatory cytokines expressions. High-fat diet-induced atherosclerosis and increased levels of plasma inflammatory mediators (IL-1ß, IL-18, and TNF-α) were observed in ApoE-/- mice. Besides, the expressions of GP73 in the aorta and serum were significantly upregulated and positively correlated with the NLRP3 expression. In the THP-1 derived macrophages, ox-LDL treatment upregulated the expressions of GP73 and NLRP3 proteins and activated the inflammatory responses in a concentration-dependent and time-dependent manner. Silencing of GP73 attenuated the inflammatory response and rescued the decreased migration induced by ox-LDL, inhibiting the NLRP3 inflammasome signaling and the ROS and p-NF-κB activation. CONCLUSIONS: We demonstrated that GP73 promoted the ox-LDL-induced inflammation in macrophages by affecting the NF-κB/NLRP3 inflammasome signaling, and may play a role in atherosclerosis.

Identification of preoperative risk factors associated with prolonged length of stay after lobectomy.

Objective: To examine the association between length of stay (LOS) after lobectomy and operative adverse events and define the best predictors and risk factors associated with prolonged LOS after lobectomy. Methods: Data from patients undergoing thoracoscopic lobectomy in the Thoracic Surgery Department of our center between January 2015 and December 2021 were retrospectively analyzed. The association between operative adverse events and LOS after lobectomy was explored using receiver operating characteristic (ROC) curves, and multivariate logistic regression analyses were used to identify preoperative risk factors associated with prolonged LOS after lobectomy. Results: Prolonged LOS after lobectomy was defined as a LOS after lobectomy that is > 3.5 days based on an optimal diagnostic value for operative adverse events (AUC = 0.882). Of the included patients, 20.9% (91/435) exceeded this threshold, of whom 52.7% (48/91) exhibited operative adverse events. The preoperative risk factors associated with prolonged LOS after lobectomy were age≥60 years old (OR = 9.632, 95%CI 1.126-75.66, p = 0.03), being a current smoker (OR = 2.702, 95%CI 1.547-4.72, P < 0.001), an American Society of Anesthesiology (ASA) classification of 2 or higher (OR = 1.845, 95%CI 1.06-3.211, P = 0.03), ASA = 3 (OR = 9.133, 95%CI 3.281-25.425, P < 0.001), and Stage IIIA disease (OR = 6.565, 95%CI 2.823-15.271, P < 0.001). Prolonged LOS after lobectomy was significantly associated with the incidence of different operative adverse events, including conversion to thoracotomy, an operative duration of ≥300 min, blood transfusion events, chest tube drainage time, postoperative complications, and postoperative interventions (P < 0.001). Conclusion: The risk of prolonged LOS after lobectomy is higher in patients that are ≥60 years old, current smokers, exhibit an ASA classification of 2 or higher, and have a stage IIIA disease. Early identification of these risk factors can enhance the treatment offered to high-risk patients, thereby reducing the rates of operative adverse events and optimizing resource utilization.

Highly efficient bioconversion of icariin to icaritin by whole-cell catalysis.

BACKGROUND: Icaritin is an aglycone of flavonoid glycosides from Herba Epimedii. It has good performance in the treatment of hepatocellular carcinoma in clinical trials. However, the natural icaritin content of Herba Epimedii is very low. At present, the icaritin is mainly prepared from flavonoid glycosides by α-L-rhamnosidases and ß-glucosidases in two-step catalysis process. However, one-pot icaritin production required reported enzymes to be immobilized or bifunctional enzymes to hydrolyze substrate with long reaction time, which caused complicated operations and high costs. To improve the production efficiency and reduce costs, we explored α-L-rhamnosidase SPRHA2 and ß-glucosidase PBGL to directly hydrolyze icariin to icaritin in one-pot, and developed the whole-cell catalytic method for efficient icaritin production. RESULTS: The SPRHA2 and PBGL were expressed in Escherichia coli, respectively. One-pot production of icaritin was achieved by co-catalysis of SPRHA2 and PBGL. Moreover, whole-cell catalysis was developed for icariin hydrolysis. The mixture of SPRHA2 cells and PBGL cells transformed 200 g/L icariin into 103.69 g/L icaritin (yield 95.23%) in 4 h in whole-cell catalysis under the optimized reaction conditions. In order to further increase the production efficiency and simplify operations, we also constructed recombinant E. coli strains that co-expressed SPRHA2 and PBGL. Crude icariin extracts were also efficiently hydrolyzed by the whole-cell catalytic system. CONCLUSIONS: Compared to previous reports on icaritin production, in this study, whole-cell catalysis showed higher production efficiency of icaritin. This study provides promising approach for industrial production of icaritin in the future.

Discovery of novel sulfonamide chromone-oxime derivatives as potent indoleamine 2,3-dioxygenase 1 inhibitors.

A series of chromone-oxime derivatives containing piperazine sulfonamide moieties were designed, synthesized and evaluated for their inhibitory activities against IDO1. These compounds displayed moderate to good inhibitory activity against IDO1 with IC50 values in low micromolar range. Among them, compound 10m bound effectively to IDO1 with good inhibitory activities (hIDO1 IC50 = 0.64 µM, HeLa IDO1 IC50 = 1.04 µM) and were selected for further investigation. Surface plasmon resonance analysis confirmed the direct interaction between compound 10m and IDO1 protein. Molecular docking study of the most active compound 10m revealed key interactions between 10m and IDO1 in which the chromone-oxime moiety coordinated to the heme iron and formed several hydrogen bonds with the porphyrin ring of heme and ALA264, consistent with the observation by UV-visible spectra that 10m induced a Soret peak shift from 403 to 421 nm. Moreover, compound 10m exhibited no cytotoxicity at its effective concentration in MTT assay. Consistently, in vivo assays results demonstrated that 10m displayed potent antitumor activity with low toxicity in CT26 tumor-bearing Balb/c mice, in comparison with 1-methyl-l-tryptophan (1-MT) and 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide (IDO5L). In brief, the results suggested that chromone-oxime derivatives containing sulfonamide moieties might serve as IDO1 inhibitors for the development of new antitumor agents.

Discovery of GJC1 (Cx45) as a New Gene Underlying Congenital Heart Disease and Arrhythmias.

As the most prevalent type of birth malformation, congenital heart disease (CHD) gives rise to substantial mortality and morbidity as well as a socioeconomic burden. Although aggregating investigations highlight the genetic basis for CHD, the genetic determinants underpinning CHD remain largely obscure. In this research, a Chinese family suffering from autosomal dominant CHD (atrial septal defect) and arrhythmias was enrolled. A genome-wide genotyping with microsatellite markers followed by linkage assay as well as sequencing analysis was conducted. The functional effects of the discovered genetic mutation were characterized by dual patch-clamp electrophysiological recordings in N2A cells and propidium iodide uptake assays in HeLa cells. As a result, a novel genetic locus for CHD and arrhythmias was located on chromosome 17q21.31-q21.33, a 4.82-cM (5.12 Mb) region between two markers of D17S1861 and D17S1795. Sequencing assays of the genes at the mapped locus unveiled a novel heterozygous mutation in the GJC1 gene coding for connexin 45 (Cx45), NM_005497.4:c.550A>G;p.R184G, which was in co-segregation with the disease in the whole family and was not observed in 516 unrelated healthy individuals or gnomAD. Electrophysiological analyses revealed that the mutation significantly diminished the coupling conductance in homomeric cell pairs (R184G/R184G) and in cell pairs expressing either R184G/Cx45 or R184G/Cx43. Propidium iodide uptake experiments demonstrated that the Cx45 R184G mutation did not increase the Cx45 hemichannel function. This investigation locates a new genetic locus linked to CHD and arrhythmias on chromosome 17q21.31-q21.33 and indicates GJC1 as a novel gene predisposing to CHD and arrhythmias, implying clinical implications for prognostic risk assessment and personalized management of patients affected with CHD and arrhythmias.

Discovery of two biotin-PEG4­diarylidenyl piperidone prodrugs as potent antitumor agents with good efficacy, limited toxicity, and low resistance.

Two biotin-polyethylene glycol (PEG)4­diarylidenyl piperidone (DAP) prodrugs, compounds 3a and 3b, were designed as antineoplastic agents and synthesized by coupling biotin to bifluoro- and binitro-substituted DAP derivatives (DAP-F and DAP-NO2) through a PEG4 linker, respectively. The results of the MTT (3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide) assay and a SW480 xenograft model identified compounds 3a and 3b as candidate antitumor agents with good efficacy, limited toxicity, and low resistance, as compared to the original drugs (DAP-F and DAP-NO2), cisplatin, and doxorubicin (dox). The results of a preliminary pharmacokinetic study showed that compounds 3a and 3b slowly released their original drug DAP-F and DAP-NO2 within 12 h after intraperitoneal injection, respectively. Western blot analysis and computer docking simulations indicated that DAP-F, DAP-NO2, and compounds 3a and 3b were indeed inhibitors of signal transducer and activator of transcription 3 (STAT3) and the antitumor effects of compounds 3a and 3b were exerted by sequentially interacting with the SH2-binding domain followed by the DNA-binding domain after releasing the original drugs DAP-F and DAP-NO2, respectively. These results suggest that the targeted prodrug model led to good antitumor efficacy with reduced toxicity, while a dual STAT3-binding model may promote antitumor efficacy and resistance.