Using a modified piggyBac transposon-combined Cre/loxP system to produce selectable reporter-free transgenic bovine mammary epithelial cells for somatic cell nuclear transfer.

Transposon systems are widely used for genetic engineering in various model organisms. PiggyBac (PB) has recently been confirmed to have highly efficient transposition in the mouse germ line and mammalian cell lines. In this study, we used a modified PB transposon system mediated by PB transposase (PBase) mRNA carrying the human lactoferrin gene driven by bovine ß-casein promoter to transfect bovine mammary epithelial cells (BMECs), and the selectable reporter in two stable transgenic BMEC clones was removed using cell-permeant Cre recombinase. These reporter-free transgenic BMECs were used as donor cells for somatic cell nuclear transfer (SCNT) and exhibited a competence of SCNT embryos similar to stable transgenic BMECs and nontransgenic BMECs. The comprehensive information from this study provided a modified approach using an altered PB transposon system mediated by PBase mRNA in vitro and combined with the Cre/loxP system to produce transgenic and selectable reporter-free donor nuclei for SCNT. Consequently, the production of safe bovine mammary bioreactors can be promoted.

FABP4 mediates endoplasmic reticulum stress and autophagy to regulate endometrial epithelial cell function during early sheep gestation.

Dynamic changes in the endometrium are crucial for establishing early pregnancy in ruminants. Blastocyst elongation and implantation require hormones and nutrients to be secreted from the maternal endometrium. The fatty acid-binding protein FABP4 is a widely expressed fatty acid transport protein that promotes cell proliferation, migration, and invasion and is involved in conceptus implantation. However, the mechanism underlying the functional regulation of endometrial epithelial cells (EECs) by FABP4 during ovine peri-implantation remains unclear. We simulated hormonal changes in vitro in sheep EECs (SEECs) during the peri-implantation period and found that it elevated FABP4 expression. FABP4 inhibition significantly reduced cell migration, endoplasmic reticulum stress, and autophagy, suggesting that FABP4 regulates endometrial function in sheep. Moreover, the FABP4 inhibitor BMS309403 counteracted hormone-mediated functional changes in SEECs, and an endoplasmic reticulum stress activator and autophagy inhibitor reversed the abnormal secretion of prostaglandins induced by FABP4 inhibition. These results suggest that FABP4 affects ovine endometrial function during early gestation by regulating endoplasmic reticulum stress and autophagy in SEECs.

Establishment and characterization of a sheep endometrial epithelial cell line.

Endometrial epithelial cells play a significant role in the "dialogue" between the embryo and the mother, but in vitro studies to clarify this are hampered by the limited lifespan of primary cells. As such, it is necessary to develop an in vitro model to study endometrial function. Morphological analysis showed that the pEECs were homogeneous, formed characteristic cobblestone monolayers, and expressed the epithelial cell-specific marker, cytokeratin 18. The isolated and purified cells were transfected with a plasmid encoding human telomerase reverse transcriptase (TERT) gene, pCI-neo-TERT, to establish an immortal endometrial epithelial cell line (iEECs). The transfected cells were cultured with G418 and monoclonal cells were selected for expanded culture. Expression of TERT mRNA was detected by RT-qPCR and protein was quantitated by Western blot. TERT expression was stable and continued to be active with no signs of aging. Assays for cell proliferation and apoptosis indicated higher proliferation and cellular activity in iEECs than pEECs. After stimulated by interferon tau (IFN-τ), both iEECs and pEECs showed similar upregulation levels in all the underlying genes. Taken together, these findings demonstrate that iEECs retained the basic morphology and function of pEECs, providing a robust in vitro model for study of the function of ovine endometrial epithelial cells.

Multiple Cross Displacement Amplification Coupled with Lateral Flow Biosensor (MCDA-LFB) for rapid detection of Legionella pneumophila.

BACKGROUND: Legionella pneumophila is an opportunistic waterborne pathogen of significant public health problems, which can cause serious human respiratory diseases (Legionnaires' disease). Multiple cross displacement amplification (MCDA), a isothermal nucleic acid amplification technique, has been applied in the rapid detection of several bacterial agents. In this report, we developed a MCDA coupled with Nanoparticles-based Lateral Flow Biosensor (MCDA-LFB) for the rapid detection of L. pneumophila. RESULTS: A set of 10 primers based on the L. pneumophila specific mip gene to specifically identify 10 different target sequence regions of L. pneumophila was designed. The optimal time and temperature for amplification are 57 min and 65 °C. The limit of detection (LoD) is 10 fg in pure cultures of L. pneumophila. No cross-reaction was obtained and the specificity of MCDA-LFB assay was 100%. The whole process of the assay, including 20 min of DNA preparation, 35 min of L. pneumophila-MCDA reaction, and 2 min of sensor strip reaction, took a total of 57 min (less than 1 h). Among 88 specimens for clinical evaluation, 5 (5.68%) samples were L. pneumophila-positive by MCDA-LFB and traditional culture method, while 4(4.55%) samples were L. pneumophila-positive by PCR method targeting mip gene. Compared with culture method, the diagnostic accuracy of MCDA-LFB method was higher. CONCLUSIONS: In summary, the L. pneumophila-MCDA-LFB method we successfully developed is a simple, fast, reliable and sensitive diagnostic tool, which can be widely used in basic and clinical laboratories.

Isolation of a New Polysaccharide from Dandelion Leaves and Evaluation of Its Antioxidant, Antibacterial, and Anticancer Activities.

Dandelion, in China, has a long history as a medicinal and edible plant, and possesses high nutritional and medical value. The present study aimed to isolate a new polysaccharide (DLP-3) from dandelion leaves and to evaluate its antioxidant, antibacterial, and anticancer activities. The structure of DLP-3 was analyzed using HPLC, FT-IR, SEM, GC-MS, and NMR spectroscopy. DLP-3 mainly consisted of Man, Rha, GlcA, Glc, Gal, and Ara with molar ratios of 2.32, 0.87, 1.21, 3.84, 1.00, and 1.05, respectively, with a molecular weight of 43.2 kDa. The main linkages of DLP-3 contained (1→4)-α-d-Glc, (1→4,6)-α-d-Glc, (1→6)-α-d-Gal, (1→2)-α-d-Man, (1→4)-α-d-Man, ß-l-Ara-(1→, and α-l-Rha-(1→. DLP-3 exhibited a smooth surface, purely flake-like structure, and a triple helix conformation. Moreover, DLP-3 presented obvious antioxidant and antibacterial activities in a concentration-dependent manner. DLP-3 showed significant anticancer activities by inhibiting tumor cell proliferation. These findings provide a theoretical basis for the application of DLP-3 as a natural functional active substance in functional foods.

Ribavirin augments doxorubicin's efficacy in human hepatocellular carcinoma through inhibiting doxorubicin-induced eIF4E activation.

Activation of eukaryotic translation initiation factor 4E (eIF4E) is a cellular survival mechanism in response to chemotherapy in cancers. In this work, we demonstrate that targeting eIF4E by ribavirin sensitizes hepatocellular carcinoma (HCC) cell response to doxorubicin. Ribavirin inhibits growth and survival of HCC cells, and to a greater extent than in normal liver cells. Its combination with doxorubicin achieves greater efficacy than single drug in vitro and in vivo. Ribavirin suppresses phosphorylation of molecules involved in Akt/mTOR/eIF4E pathway. Overexpression of the phosphomimetic form (S209D) but not the nonphosphorylatable form (S209A) eIF4E significantly reverses the inhibitory effects of ribavirin. Interestingly, doxorubicin significantly increases p-eIF4E(S209) level in a dose- and time-dependent manner, suggesting that doxorubicin induces eIF4E activation in HCC cells. In addition, eIF4E activation induced by doxorubicin in HCC cells is inhibited by ribavirin. Our work demonstrates the greater efficacy of ribavirin and doxorubicin combination and its underlying mechanisms.

Antibiotic tigecycline enhances cisplatin activity against human hepatocellular carcinoma through inducing mitochondrial dysfunction and oxidative damage.

Targeting mitochondrial metabolism has been recently demonstrated to be a promising therapeutic strategy for the treatment of various cancer. In this work, we demonstrate that antibiotic tigecycline is selectively against hepatocellular carcinoma (HCC) through inducing mitochondrial dysfunction and oxidative damage. Tigecycline is more effective in inhibiting proliferation and inducing apoptosis of HCC than normal liver cells. Importantly, tigecycline significantly enhances the inhibitory effects of chemotherapeutic drug cisplatin in HCC in vitro and in vivo. Mechanistically, tigecycline specifically inhibits mitochondrial translation as shown by the decreased protein levels of Cox-1 and -2 but not Cox-4 or Grp78, and increased mRNA levels of Cox-1 and -2 but not Cox-4 in HCC cells exposed to tigecycline. In addition, tigecycline significantly induces mitochondrial dysfunction in HCC cells via decreasing mitochondrial membrane potential, complex I and IV activities, mitochondrial respiration and ATP levels. Tigecycline also increases levels of mitochondrial superoxide, hydrogen peroxide and ROS levels. Consistent with oxidative stress, oxidative damage on DNA, protein and lipid are also observed in tigecycline-treated cells. Importantly, antioxidant N-acetyl-l-cysteine (NAC) reverses the effects of tigecycline, suggesting that oxidative stress is required for the action of tigecycline in HCC cells. We further show that HCC cells have higher level of mitochondrial biogenesis than normal liver cells which might explain the different sensitivity to tigecycline between HCC and normal liver cells. Our work is the first to demonstrate that tigecycline is a promising candidate for HCC treatment and highlight the therapeutic value of targeting mitochondrial metabolism in HCC.

Alkoxysulfonyl radical species: acquisition and transformation towards sulfonate esters through electrochemistry.

Sulfonyl radical mediated processes have been considered as a powerful strategy for the construction of sulfonyl compounds. However, an efficient and high atom-economical radical approach to the synthesis of sulfonate esters is still rare, owing to the limited tactics to achieve alkoxysulfonyl radicals. Herein, an electrochemical anodic oxidation of inorganic sulfites with alcohols is developed to afford alkoxysulfonyl radical species, which are utilized in subsequent alkene difunctionalization to provide various sulfonate esters. This transformation features excellent chemoselectivity and broad functional group tolerance. This new discovery presents the potential prospect for the construction of sulfonate esters, and enriches the electrochemical reaction type.

Anti-cancer Potential of Polysaccharide Extracted From Polygonatum sibiricum on HepG2 Cells via Cell Cycle Arrest and Apoptosis.

Polygonatum sibiricum is one of the most widely used traditional Chinese medicine in China. Polygonatum sibiricum polysaccharide (PSP) is the main functional component of Polygonatum sibiricum. In this study, a water-soluble polysaccharide (PSP-1) was first isolated from Polygonatum sibiricum with a molecular weight of 38.65 kDa. Structural analysis was performed via methylation and FT-IR spectroscopy analyses, which in combination with NMR spectroscopy, revealed that PSP-1 has a → 4-α-D-Glcp-1 → backbone with the substitution at O-6 with the ß-D-Glcp-1 → residues. Furthermore, PSP-1 exhibited potent and concentration-dependent anticancer effects, inducing HepG2 cell apoptosis and arresting the cell cycle at the G1 phase. Moreover, PSP-1 also decreased the mitochondrial membrane potential, damaged the nucleus of HepG2 cells, and increased the activity of caspase-9 and-3 in the intrinsic apoptotic pathways to induce HepG2 cell apoptosis. To conclude, PSP-1 might be a good candidate for the treatment of liver cancer, and this work provides important information for understanding the relationship between structure and antitumor activity of PSP-1, which is relevant for the treatment of hepatocellular carcinoma in clinic.

Extraction, purification, structural characterization, and antioxidant activity of a novel polysaccharide from Lonicera japonica Thunb.

A novel water-soluble polysaccharide (HEP-4) with a molecular weight of 1.98 × 105Da was extracted from honeysuckle. Structural characterization was performed using high-performance liquid chromatography (HPLC), gas chromatography, Fourier transform-infrared (FT-IR) spectrum, nucleus magnetic resonance (NMR) spectra, and scanning electron microscopy. The results showed that HEP-4 is primarily composed of mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose with a mole ratio of 6.74:1.56:1.04:14.21:4.31:5.4, and the major types of the glycosidic bond types of HEP-4 were 1-α-D-Glcp, 1,4-ß-D-Glcp, 1-ß-D-Arap, 1,3,4-ß-D-Arap, and 1,3,6-ß-D-Manp. The results of bioactivity experiments revealed that HEP-4 had antioxidant in vitro. In addition, HEP-4 inhibited H2O2-induced oxidative damage and increased the activity of HepG2 cells by reducing MDA levels and inhibiting ROS production. Meanwhile, HEP-4 significantly enhanced the activities of GSH-Px and CAT, indicating that HEP-4 exerts a protective effect on H2O2-induced oxidative stress. These results indicate that HEP-4 could be a potential natural antioxidant.

Corrigendum: Anti-cancer potential of polysaccharide extracted from Polygonatum sibiricum on HepG2 cells via cell cycle arrest and apoptosis.

[This corrects the article DOI: 10.3389/fnut.2022.938290.].

Immune checkpoint inhibitor-related adverse events in lung cancer: Real-world incidence and management practices of 1905 patients in China.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are the standard treatment for advanced lung cancer, but immune-related adverse events (irAEs) remain poorly understood, especially in a real-world setting. METHODS: A multicenter observational study was conducted. Medical records of lung cancer patients treated with ICIs at 26 hospitals from January 1, 2015, to February 28, 2021, were retrieved. Types of ICIs included antiprogrammed cell death 1 or antiprogrammed cell death ligand 1 (PD-L1) monotherapy, anticytotoxic T-lymphocyte antigen-4 monotherapy, or combination therapy. RESULTS: In total, 1905 patients with advanced lung cancer were evaluated. The median age was 63 (range 28-87) years, and the male/female ratio was 3.1:1 (1442/463). The primary histological subtype was adenocarcinoma (915). A total of 26.9% (512/1905) of the patients developed 671 irAEs, and 5.8% (110/1905) developed 120 grade 3-5 irAEs. Median duration from ICI initiation to irAEs onset was 56 (range 0-1160) days. The most common irAEs were thyroid dysfunction (7.2%, 138/1905), pneumonitis (6.5%, 124/1905), and dermatological toxicities (6.0%, 115/1905). A total of 162 irAEs were treated with steroids and 11 irAEs led to death. Patients with positive PD-L1 expression (≥1%) and who received first-line ICI treatment developed more irAEs. Patients who developed irAEs had a better disease control rate (DCR, 71.3% [365/512] vs. 56.0% [780/1145]; p < 0.001). CONCLUSIONS: The incidence rate of irAEs was 26.9% in a real-world setting. IrAEs might be related to a better DCR, but clinicians should be more aware of irAE recognition and management in clinical practice.

Feasibility and Safety of Neoadjuvant Alectinib in Pulmonary Invasive Mucinous Adenocarcinoma with ALK Rearrangement: Case Report and Literature Review.

BACKGROUND: Pulmonary invasive mucinous adenocarcinoma (IMA) is a rare variant of lung adenocarcinoma that rarely shows anaplastic lymphoma kinase (ALK) rearrangement. Alectinib (tyrosine kinase inhibitors) has been listed as category 1 recommendations for advanced ALK + NSCLC first-line therapy due to low toxicity and excellent efficacy, and its median progression-free survival is 34.8 months. Here, we report a case of a patient with ALK-rearranged lung IMA who showed favorable results to neoadjuvant alectinib. CASE: A 67-year-old man with no history of smoking was diagnosed with clinical stage as IIIB invasive mucinous adenocarcinoma based on clinical symptoms, chest CT and pathological findings. The anaplastic lymphoma kinase (ALK) fusion status was assessed by real-time PCR. After acquiring informed consent from the patient, we offered neoadjuvant alectinib at a dosage of 150 mg twice per day for three cycles (84 days), all lesions were undetectable on chest CT. Later, a thoracoscopic left lobectomy was performed. The postoperative pathological showed that a small amount of tumor cells remained, and the TNM stage was downstaged as T1aN0M0 IA. CONCLUSION: To our knowledge, this is the first case discussing the treatment of ALK-rearranged IMA of the lung with neoadjuvant alectinib. Alectinib is an effective ALK inhibitor, and in cases of lung adenocarcinoma with ALK rearrangement, alectinib treatment is a reasonable and priority option. Neoadjuvant alectinib may be clinically feasible and well tolerated in locally advanced NSCLC.

Strategically prolonged release of multiple antibacterial components from a thin film coating for synergistic effects against Staphylococcus aureus infections.

Biomaterial-associated infections (BAIs) remain a major challenge in clinical surgery because they can potentially cause serious disabilities in patients. This study investigated the use of a multilayer coating technology that can co-deliver two therapeutic components, gentamicin and OP-145 peptide, to treat Staphylococcus aureus effectively. A biocompatible and biodegradable thin film was produced via layer-by-layer assembly using an antibacterial peptide and gentamicin. The thin film was systematically characterized, showing controllable features such as thickness, transparency, cargo loading, and cargo release. In vitro tests showed that the thin film has fewer toxicity problems than soluble cargos; compared to cargos in a soluble form, the thin film has minor impacts on mammalian cells' metabolism. Additionally, the antibacterial cargos assembled on the thin film can effectively inhibit the formation of biofilms and show effective in vitro antibacterial potency. In vivo studies illustrated that the thin film can inhibit the progression of S. aureus in a mouse model, indicating the effectiveness of the thin film structure in the clinic. This study demonstrates that a thin film composed of gentamicin and OP-145 could be employed to prevent BAIs in clinical surgery.

Suppression of cyclooxygenase 2 increases chemosensitivity to sesamin through the Akt­PI3K signaling pathway in lung cancer cells.

Safe, affordable and efficacious agents are urgently required for cancer prevention. Sesamin, a lipid­soluble lignan from sesame (Sesamum indicum) displays anticancer activities through an unknown mechanism. In the present study, the anticancer activity of sesamin via cyclooxygenase 2 (COX2) was investigated in lung cancer. Quantitative polymerase chain reaction was performed to determine the mRNA expression levels of COX2 in cells, while western blot analysis was used to determine its protein expression levels. Cell proliferation was evaluated by Cell Counting Kit­8 assay, while apoptosis and cell cycle analyses were conducted by flow cytometry. The results indicated that COX2 expression was upregulated in lung cancer cell lines compared with human normal lung epithelial cell line BEAS­2B and sesamin was demonstrated to decrease the levels of COX2, inhibit the proliferation of lung cancer cells and promote their apoptosis in a concentration­dependent manner. Furthermore, decreased COX2 expression potentiated sesamin­induced apoptosis and G1­phase arrest, which was correlated with the suppression of gene products associated with cell apoptosis (Bcl­2 and Bax) and the cell cycle (cyclin E1). In addition, cotreatment with the COX2 inhibitor CAY10404 and sesamin downregulated the expression of downstream molecules of COX2 [including interleukin (IL)1ß, IL6 and tumor necrosis factor α] compared with CAY10404 or sesamin alone. Furthermore, cotreatment with sesamin and CAY10404 markedly reduced the levels of phosphorylated protein kinase B (pAkt) and phosoinositide 3 kinase (PI3K) in three lung cancer cell lines. PI3K expression was observed to be under the control of COX2, possibly forming a negative feedback loop. In addition, PI3K depletion induced apoptosis and G1­phase arrest in A549 cells. These results suggested that sesamin blocked the pAkt­PI3K signaling pathway by downregulating the expression of COX2, therefore resulting in cell cycle arrest and increased apoptosis in vitro. In conclusion, inhibition of COX2 increased the sensitivity of lung cancer cells to sesamin by modulating pAkt­PI3K signaling. These results may aid the development of more selective agents to overcome cancer.

Urinary circulating DNA profiling in non-small cell lung cancer patients following treatment shows prognostic potential.

BACKGROUND: Disease relapse in non-small cell lung cancer (NSCLC) requires close monitoring for early detection. The aim of the current study examines the use of urinary circulating DNA for patients after first line therapies. METHODS: EGFR positive NSCLC patients in stages I-III were profiled using digital droplet PCR (ddPCR). Urinary circulating DNA was collected prior to treatment and all monitored patients had detectable EGFR mutations. Post treatment urinary DNA measurements were taken at multiple time intervals. Results were matched to disease-free survival. RESULTS: Among the 213 patients recruited, 130 had matched EGFR profiles to corresponding tumor tissues. Concentrations of mutant DNA varied with different patients and mean concentration was 220±237 copies/mL. Measurements taken post-treatment showed a significant number of patients with undetectable EGFR mutations in their urine samples. Other patients registered a significant decline in urinary DNA concentrations. For measurements taken post treatment (6-month), we observed a significant increase of positively identified EGFR mutations in urine samples. In the patient group with higher urinary DNA concentration, 91% of the cohort experienced recurrence. CONCLUSIONS: Our results indicated that urinary DNA measurements can potentially be useful for disease monitoring of minimal residual disease (MRD) in NSCLC. This can complement current serial radiographic imaging to provide early detection for lung cancer relapse.

Antibiotic drug levofloxacin inhibits proliferation and induces apoptosis of lung cancer cells through inducing mitochondrial dysfunction and oxidative damage.

Lung cancer is the leading cause of cancer death worldwide and its clinical management remains challenge. Here, we repurposed antibiotic levofloxacin for lung cancer treatment. We show that levofloxacin is effectively against a panel of lung cancer cell lines via inhibiting proliferation and inducing apoptosis, regardless of cellular origin and genetic pattern, in in vitro cell culture system and in vivo xenograft lung tumor model. Mechanistically, levofloxacin inhibits activities of mitochondrial electron transport chain complex I and III, leading to inhibition of mitochondrial respiration and reduction of ATP production. In addition, levofloxacin significantly increases levels of ROS, mitochondrial superoxide and hydrogen peroxide in vitro and oxidative stress markers (HEL and 4-HNE) in vivo. Antioxidants, such as NAC and vitamin C, prevent the inhibitory effects of levofloxacin, confirming the induction of oxidative damage as the mechanism of its action in lung cancer cells. Our work demonstrates that levofloxacin is a useful addition to the treatment of lung cancer. Our work also suggests that targeting mitochondria may be an alternative therapeutic strategy for lung cancer treatment.