Cathelicidin AS-12W Derived from the Alligator sinensis and Its Antimicrobial Activity Against Drug-Resistant Gram-Negative Bacteria In Vitro and In Vivo.

Antimicrobial peptides (AMPs) have the potential to treat multidrug-resistant bacterial infections. Cathelicidins are a class of cationic antimicrobial peptides that are found in nearly all vertebrates. Herein, we determined the mature peptide region of Alligator sinensis cathelicidin by comparing its cathelicidin peptide sequence with those of other reptiles and designed nine peptide mutants based on the Alligator sinensis cathelicidin mature peptide. According to the antibacterial activity and cytotoxicity screening, the peptide AS-12W demonstrated broad-spectrum antibacterial activity and exhibited low erythrocyte hemolytic activity. In particular, AS-12W exhibited strong antibacterial activity and rapid bactericidal activity against carbapenem-resistant Pseudomonas aeruginosa in vitro. Additionally, AS-12W effectively removed carbapenem-resistant P. aeruginosa from blood and organs in vivo, leading to improved survival rates in septic mice. Furthermore, AS-12W exhibited good stability and tolerance to harsh conditions such as high heat, high salt, strong acid, and strong alkali, and it also displayed high stability toward trypsin and simulated gastric fluid (SGF). Moreover, AS-12W showed significant anti-inflammatory effects in vitro by inhibiting the production of proinflammatory factors induced by lipopolysaccharide (LPS). Due to its antibacterial mechanism against Escherichia coli, we found that this peptide could neutralize the negative charge on the surface of the bacteria and disrupt the integrity of the bacterial cell membrane. In addition, AS-12W has the ability to bind to the genomic DNA of bacteria and stimulate the production of reactive oxygen species (ROS) within bacteria, which is believed to be the reason for the good antibacterial activity of AS-12W. These results demonstrated that AS-12W exhibits remarkable antibacterial activity, particularly against carbapenem-resistant P. aeruginosa. Therefore, it is a potential candidate for antibacterial drug development.

Antimicrobial polyacrylic acid/tannic acid hydrogel wound dressing facilitating full-thickness skin healing.

Polyphenolic compound-modified hydrogel wound dressings with excellent wet tissue adhesion, antimicrobial properties, stretchability, and full-thickness skin healing properties are still extremely rare so far. Polyphenolic compounds such as tannic acid or dopamine can improve the antibacterial and bioadhesive properties of hydrogels, and are also polymerization inhibitors for free radical polymerization. In this study, polyacrylic acid (PAA) aqueous solution was first synthesized, and then antibacterial PAA-TA hydrogel was prepared by mixing it with tannic acid (TA) and the crosslinker 1,6-hexanediol bis(2-methyl-1-propionic acid azide) (HBMAP). This method avoids the hindrance of the phenolic hydroxyl groups in TA on acrylic acid polymerization, and we were able to obtain a series of TA hydrogels (in the range of 0-15 wt.%. We applied these PAA-TA hydrogels to wound dressings and found that they had excellent adhesion to biological tissues, and the tensile strength and elongation at break of PAA-TA hydrogels with 15 wt.%TA content were as high as 1.72 MPa and 1446.3% in tensile strength evaluation. In addition, microbiological analysis showed that wound dressings had significant antimicrobial activity against Staphylococcus aureus and Escherichia coli. In vitro wound healing experiments confirmed that the wound dressing was biocompatible and could significantly promote the healing of full-thickness skin defects in the guinea pig model. Our work describes an injectable, self-healing, antimicrobial hydrogel that may have promising clinical applications as a wound dressing material.

hsdSA regulated extracellular vesicle-associated PLY to protect Streptococcus pneumoniae from macrophage killing via LAPosomes.

IMPORTANCE: S. pneumoniae is a major human pathogen that undergoes a spontaneous and reversible phase variation that allows it to survive in different host environments. Interestingly, we found hsdSA , a gene that manipulated the phase variation, promoted the survival and replication of S. pneumoniae in macrophages by regulating EV production and EV-associated PLY. More importantly, here we provided the first evidence that higher EV-associated PLY (produced by D39) could form LAPosomes that were single membrane compartments containing S. pneumoniae, which are induced by integrin ß1/NOX2/ROS pathway. At the same time, EV-associated PLY increased the permeability of lysosome membrane and induced an insufficient acidification to escape the host killing, and ultimately prolonged the survival of S. pneumoniae in macrophages. In contrast, lower EV-associated PLY (produced by D39ΔhsdSA ) activated ULK1 recruitment to form double-layered autophagosomes to eliminate bacteria.

Compatibility Study of Peptide and Glycerol Using Chromatographic and Spectroscopic Techniques: Application to a Novel Antimicrobial Peptide Cbf-14 Gel.

The interactions between active pharmaceutical ingredients (APIs) and excipients may lead to API degradation, thereby affecting the safety and efficacy of drug products. Cbf-14 is a synthetic peptide derived from Cathelicidin-BF, showing potential for bacterial and fungal infections. In order to assess impurities in Cbf-14 gel, we developed a two-dimensional liquid chromatography coupled with quadrupole/time-of-flight mass spectrometric method. A total of eleven peptide degradation impurities were identified and characterized. Furthermore, the compatibility tests were conducted to evaluate the interactions of Cbf-14 with glycerol and methylcellulose, respectively. The results revealed that the impurities originated from condensation reactions between Cbf-14 and aldehydes caused by glycerol degradation. Several aldehydes were employed to validate this hypothesis. The formation mechanisms were elucidated as Maillard reactions between primary amino groups of Cbf-14 and aldehydes derived from glycerol degradation. Additionally, the compatibility of Cbf-14 with glycerol from different sources and with varying storage times was investigated. Notably, the interaction products in the gel increased with extended storage time, even when fresh glycerol for injection was added. This study offers unique insights into the compatibility study of peptides and glycerol, contributing to the ongoing quality study of Cbf-14 gel. It also serves as a reference for the design of other peptide preparations and excipients selections.

Peptide MSI-1 inhibited MCR-1 and regulated outer membrane vesicles to combat immune evasion of Escherichia coli.

Polymyxin resistance is conferred by MCR-1 (mobile colistin resistance 1)-induced lipopolysaccharide (LPS) modification of G- bacteria. However, the peptide MSI-1 exerts potent antimicrobial activity against mcr-1-carrying bacteria. To further investigate the potential role of MCR-1 in improving bacterial virulence and facilitating immune evasion, and the immunomodulatory effect of peptide MSI-1, we first explored outer membrane vesicle (OMV) alterations of mcr-1-carrying bacteria in the presence and absence of sub-MIC MSI-1, and host immune activation during bacterial infection and OMV stimulation. Our results demonstrated that LPS remodelling induced by MCR-1 negatively affected OMV formation and protein cargo by E. coli. In addition, MCR-1 diminished LPS-stimulated pyroptosis but facilitated mitochondrial dysfunction, further aggravating apoptosis in macrophages induced by OMVs of E. coli. Similarly, TLR4-mediated NF-κB activation was markedly alleviated once LPS was modified by MCR-1. However, peptide MSI-1 at the sub-MIC level inhibited the expression of MCR-1, further partly rescuing OMV alteration and attenuation of immune responses in the presence of MCR-1 during both infection and OMV stimulation, which can be exploited for anti-infective therapy.

Strongylocentrotus nudus egg polysaccharide (SEP) suppresses HBV replication via activation of TLR4-induced immune pathway.

Chronic hepatitis B virus (HBV) infection is a worldwide public health problem that causes significant liver-related morbidity and mortality. In our previous study, Strongylocentrotus nudus eggs polysaccharide (SEP), extracted from sea urchins, had immunomodulatory and antitumor effects. Whether SEP has anti-HBV activity is still obscure. This study demonstrated that SEP decreased the secretion of hepatitis B surface antigen (HBsAg) and e antigen (HBeAg), as well as the replication and transcription of HBV both in vitro and in vivo. Immunofluorescence and immunohistochemistry results showed that the level of HBV core antigen (HBcAg) was clearly reduced by SEP treatment. Mechanistically, RT-qPCR, western blot, and confocal microscopy analysis showed that SEP significantly increased the expression of toll-like receptor 4 (TLR4) and co-localization with TLR4. The downstream molecules of TLR4, including NF-κb and IRF3, were activated and the expression of IFN-ß, TNF-α, IL-6, OAS, and MxA were also increased, which could suppress HBV replication. Moreover, SEP inhibited other genotypes of HBV and hepatitis C virus (HCV) replication in vitro. In summary, SEP could be investigated as a potential anti-HBV drug capable of modulating the innate immune.

An active domain SA-2 derived from cystatin-SA, and its antifungal activity.

Infections induced by fungi, especially the drug-resistant fungi, are difficult clinical problems. Conventional antifungal treatment is effective but due to resistance, treatment failure, and treatment-related toxicity, there is a need for new antifungal drugs. In this study, SA-2 (YYRRLLRVLRRRW) was derived from Cystatin-SA, a saliva protein with a molecular weight of 14 kDa. Meanwhile, the structure-activity of SA-2 and its mutants was also studied. We detected the antimicrobial activity and cytotoxicity of SA-2 and found that SA-2 had a low cytotoxicity toward mammalian cells but a good inhibitory effect on Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans), with MIC values of 16-64 µg/mL and 8-32 µg/mL, respectively. Interestingly, SA-2 effectively killed fluconazole-resistant C. neoformans and C. albicans within 12 h. This antifungal activity against fluconazole-resistant fungi was comparable to that of amphotericin B. In addition, the C. neoformans-infected mice model was established to evaluate the anti-infective activity of SA-2 in vivo. Results showed that SA-2 significantly reduced the counts of fungi in lung and brain tissues to protect fluconazole-resistant C. neoformans-infected mice from death without changing mice body weights. Moreover, the dramatically increased pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß induced by intranasal infection of C. neoformans could be obviously declined due to the treatment of SA-2, which may be attributed to the elimination of C. neoformans in time in the infected tissue. For the mode of actions underlying SA-2 against C. neoformans, we found that the cationic peptide SA-2 could adhere to the negatively charged fungal cell membrane to increase the surface potential of C. neoformans in a dose-dependent manner, and finally disrupted the integrity of fungal cell membrane, reflecting as a 60% positive rate of propidium iodide uptake of C. neoformans cells after SA-2 (4 × MIC) treatment. Our study indicated that SA-2 has the potential to develop as a new therapeutic agent against infection induced by drug-resistant fungi.

Characterization of structurally related peptide impurities using HPLC-QTOF-MS/MS: application to Cbf-14, a novel antimicrobial peptide.

Cbf-14 (RLLRKFFRKLKKSV), a designed antimicrobial peptide derived from the cathelicidin family, is effective against drug-resistant bacteria. Structurally related peptide impurities in peptide medicines probably have side effects or even toxicity, thus impurity profiling research during the entire production process is indispensable. In this study, a simple liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method using a quadrupole time-of-flight (Q-TOF) mass spectrometer was developed for separation, identification, and characterization of structurally related peptide impurities in Cbf-14. A total of one process-related impurity and thirty-two degradation products were identified, and seven of them have been synthesized and confirmed. These impurities have not been declared in custom synthetic peptides. The degradation products were divided into five categories: fifteen Cbf-14 hydrolysates, five Cbf-14 isomers, four acetyl-Cbf-14 isomers, two aldimine derivatives, and six oxidized impurities. Combined with the peptide synthesis and the stress-testing studies, the origins and the formation mechanisms of these impurities were elucidated, which provides a unique insight for the follow-up quality study of Cbf-14 and other peptide products.

Preparation and properties of water-based acrylic emulsion-assisted flexible building tiles.

The heavy and rigid appearance of conventional burnt building tiles is not suitable for a global sustainable development strategy. Flexible facing tiles with lightweight and environmental materials are highly desirable for the construction industry today. In this work, water-based polymer emulsion-assisted flexible building tiles were prepared. Based on the method of achieving post crosslinking and improving adhesion with inorganic matrix-based materials, WPAs modified with GMA and KH570 display good chemical resistance and low solvent absorption (0.132 in water and 0.289 in ethanol respectively). The optimum mechanical performance of flexible building materials prepared with WPAs can strain 1.406% and stress 1.8658 MPa. The TGA, XRD, SEM and AFM results further indicate the excellent thermal stability and compatibility of flexible building tiles. Hence, flexible building tiles prepared with WPAs can be promising building materials for construction.

Strongylocentrotus nudus Eggs Polysaccharide Enhances Macrophage Phagocytosis Against E.coli Infection by TLR4/STAT3 Axis.

Antibiotics resistance is one of the most significant public health threats globally. Strategies that strengthen host defenses to control pathogen infection has become a hot research field. Macrophages are part of early host defense mechanisms, and are activated via host pattern recognition receptors (PRRs), such as Toll-like receptor 4 (TLR4), which then facilitates phagocytosis and elimination of invading pathogens. However, few activators of PRRs have been approved for clinical use because of their toxic effects. This study aimed to investigate whether Strongylocentrotus nudus eggs polysaccharide (SEP), a non-toxic extract from seafood, contributes to host defense against bacterial infection. Results showed that SEP promoted bacterial clearance by enhancing phagocytosis by macrophages during E. coli infection in vitro, but was inhibited by TLR4 specific inhibitor TAK-242, STAT3 inhibitor Stattic or blockade of CD64. In addition, SEP protected mice from E. coli induced mortality, reduced pulmonary inflammation and inhibited dissemination of bacteria to organs, while TAK-242 retarded the protection of SEP. Overall, SEP strengthened innate host defense and improved the outcome in bacterial infection, suggesting that SEP could be used as a potential immunomodulator in host-directed therapies.

A Polysaccharide From Eupolyphaga sinensis Walker With Anti-HBV Activities In Vitro and In Vivo.

Hepatitis B virus (HBV) infection remains a major global threat to human health worldwide. Recently, the Chinese medicines with antiviral properties and low toxicity have been a concern. In our previous study, Eupolyphaga sinensis Walker polysaccharide (ESPS) has been isolated and characterized, while its antiviral effect on HBV remained unclear. The anti-HBV activity of ESPS and its regulatory pathway were investigated in vitro and in vivo. The results showed that ESPS significantly inhibited the production of HBsAg, HBeAg, and HBV DNA in the supernatants of HepG2.2.15 in a dose-dependent manner; HBV RNA and core protein expression were also decreased by ESPS. The in vivo studies using HBV transgenic mice further revealed that ESPS (20 and 40 mg/kg/2 days) significantly reduced the levels HBsAg, HBeAg, and HBV DNA in the serum, as well as HBV DNA and HBV RNA in mice liver. In addition, ESPS activated the Toll-like receptor 4 (TLR4) pathway; elevated levels of IFN-ß, TNF-α, and IL-6 in the serum were observed, indicating that the anti-HBV effect of ESPS was achieved by potentiating innate immunity function. In conclusion, our study shows that ESPS is a potential anti-HBV ingredient and is of great value in the development of new anti-HBV drugs.

Huangqin decoction ameliorates DSS-induced ulcerative colitis: Role of gut microbiota and amino acid metabolism, mTOR pathway and intestinal epithelial barrier.

BACKGROUND: The clinical treatment of ulcerative colitis (UC) is limited. A traditional Chinese medicinal formula, Huangqin decoction (HQD), is chronicled in Shang Han Lun and is widely used to ameliorate gastrointestinal disorders, such as UC; however, its mechanism is yet to be clarified. PURPOSE: The present study aimed to investigate the effect of HQD on 7-day colitis induced by 3% dextran sulfate sodium (DSS) in mice and further explore the inhibitory effect of metabolites on DSS-damaged FHC cells. METHODS: The therapeutic efficacy of HQD was evaluated in a well-established DSS-induced colitis mice model. The clinical symptoms were analyzed, and biological samples were collected for microscopic examination, metabolomics, metagenomics, and the evaluation of the epithelial barrier function. The mechanism of metabolites regulated by HQD was evaluated in the DSS-induced FHC cell damage model. The samples were collected to detect the physiological functions of the cells. RESULTS: HQD suppressed the inflammation of DSS-induced colitis in vivo, attenuated DSS-induced clinical manifestations, reversed colon length reduction, and reduced histological injury. After HQD treatment, the DSS-induced gut dysbiosis was modulated, and the gut microbiota achieved a new equilibrium state. In addition, HQD activated the mTOR signaling pathway by upregulating amino acid metabolism. Significant phosphorylation of S6 and 4E-BP1 ameliorated intestinal epithelial barrier dysfunction. Moreover, HQD-regulated metabolites protected the epithelial barrier integrity by inhibiting DSS-induced apoptosis of FHC cells and regulating the proteins affecting apoptosis and cell-cell junction. CONCLUSIONS: These findings indicated that the mechanism of HQD was related to regulating the gut microbiota and amino acid metabolism, activating the mTOR signaling pathway, and protecting the intestinal mucosal barrier integrity.

Potent antibacterial and antibiofilm activities of TICbf-14, a peptide with increased stability against trypsin.

The poor stability of peptides against trypsin largely limits their development as potential antibacterial agents. Here, to obtain a peptide with increased trypsin stability and potent antibacterial activity, TICbf-14 derived from the cationic peptide Cbf-14 was designed by the addition of disulfide-bridged hendecapeptide (CWTKSIPPKPC) loop. Subsequently, the trypsin stability and antimicrobial and antibiofilm activities of this peptide were evaluated. The possible mechanisms underlying its mode of action were also clarified. The results showed that TICbf-14 exhibited elevated trypsin inhibitory activity and effectively mitigated lung histopathological damage in bacteria-infected mice by reducing the bacterial counts, further inhibiting the systemic dissemination of bacteria and host inflammation. Additionally, TICbf-14 significantly repressed bacterial swimming motility and notably inhibited biofilm formation. Considering the mode of action, we observed that TICbf-14 exhibited a potent membrane-disruptive mechanism, which was attributable to its destructive effect on ionic bridges between divalent cations and LPS of the bacterial membrane. Overall, TICbf-14, a bifunctional peptide with both antimicrobial and trypsin inhibitory activity, is highly likely to become an ideal candidate for drug development against bacteria.

MSI-1 combats drug-resistant S. aureus by affecting bacterial viability and inhibiting carotenoid pigment production.

Development of novel therapeutic strategies and antibacterial agents against antibiotic-resistant Staphylococcus aureus (S. aureus) is urgent. In this study, antibacterial activities and possible mechanisms of peptide MSI-1 against multiple drug-resistant S. aureus were investigated. Results demonstrated that MSI-1 had potent bacteriostatic activity and bactericidal efficiency against S. aureus, including methicillin-resistant S. aureus (MRSA), vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA), with minimum inhibitory concentrations (MICs) ranging from 4 to 16 µg/mL and bactericidal times from 2-12 h. MSI-1 exhibited a low incidence of developing resistance and additive effects with vancomycin to overcome MRSA and VRSA. Moreover, MSI-1, even at sub-MIC concentrations, inhibited staphyloxanthin (STX) production of S. aureus. This inhibitory effect was unique and effectively sensitized S. aureus to host immune defense. In terms of its modes of action, MSI-1 disrupted the cell membrane of S. aureus by binding to negatively-charged lipoteichoic acid to exert a direct bactericidal effect. Interestingly, MSI-1 interacted with 4,4'-diapophytoene desaturase (CrtN) of S. aureus via ionic bonds, hydrogen bonds, and Pi-Pi or Pi-alkyl interactions, and alanine substitution of the key amino acids contributed to these interactions weakened this STX production inhibition. Thus, in a MRSA-induced skin infection in mice and MRSA/VRSA-induced systemic infection in Galleria mellonella,MSI-1 alleviated staphylococcal scalded skin syndrome to promote mouse skin wound repair and mitigated staphylococcus infection-induced immune melanization to enhance G. mellonella survival. Collectively, MSI-1 has potent antibacterial activity against drug-resistant S. aureus by affecting bacterial viability and exerting its anti-virulence effects. It can be developed as a new antibacterial agent to resist refractory S. aureus infection.

Research and Application of Segmented Acid Fracturing by Temporary Plugging in Ultradeep Carbonate Reservoirs.

The ultradeep carbonate reservoir in Sichuan Basin is characterized by deep burial depth, high temperature, and strong heterogeneity. In the early stage of production, the vertical well acid fracturing is the main reservoir stimulation method, and the horizontal well stimulation technology is not mature enough to release the production capacity of gas wells. Segmented acid fracturing of the ultradeep horizontal wells currently faces the following problems: the strong heterogeneity of reservoir leads to the difficulty of fine segmentation; the high reservoir temperature requires higher performance of working fluid; the reaction rate between acid and rock is fast and the action distance of acid is short, and there is low fracture conductivity under high closure stress. In view of the above problems, the fine segmented design method was studied, and the high-temperature-resistant authigenic acid and gelling acid systems were developed. The viscosity of authigenic acid is greater than 150 mPa s after shearing at 160 °C and 170 s-1 for 50 min, and the highest acid generation concentration is 4.05 mol/L. The gelling acid system has both the properties of high-temperature resistance and low friction resistance; not only canit meet the requirements of the retarding rate and the corrosion inhibition ability when the reservoir temperature is 160 °C but also the resistance reduction rate is up to more than 70%. By alternating injection of authigenic acid and gelling acid, the acid-etched fracture length and conductivity were, respectively, increased by 80% and 45%. The application of this technology in the horizontal well of the ultradeep carbonate reservoir in Sichuan Basin can increase the productivity by 3 times when compared with the vertical well acid fracturing, and a better stimulation effect has been achieved.

PCL-1, a Trypsin-Resistant Peptide, Exerts Potent Activity Against Drug-Resistant Bacteria.

Antimicrobial peptides (AMPs), which hold tremendous promise in overcoming the emergence of drug resistance, are limited in wide clinical applications due to their instability, especially against trypsin. Herein, we designed six peptide mutants based on the cathelicidin CATHPb2, followed by screening. Pb2-1, which showed the best activity against drug-resistant bacteria among these mutants, was selected to be combined with the trypsin inhibitory loop ORB-C to obtain two hybrid peptides: PCL-1 and Pb2-1TI. Notably, both of the hybrid peptides exhibited a remarkable enhancement in trypsin resistance compared with Pb2-1. The tests showed that PCL-1 displayed broad-spectrum antimicrobial activity that was superior to that of Pb2-1TI. In addition, PCL-1 had relatively lower cytotoxicity than Pb2-1TI towards the L02 and HaCaT cell lines and negligible hemolysis, as well as tolerance to high concentrations of salt, extreme pH, and temperature variations. In vivo, PCL-1 effectively improved the survival rate of mice that were systemically infected with drug-resistant Escherichia coli through efficient bacterial clearance from the blood and organs. With regard to mode of action, PCL-1 damaged the integrity of the bacterial cell membrane and attached to the membrane surface while bound to bacterial genomic DNA to eventually kill the bacteria. Altogether, the trypsin-resistant peptide PCL-1 is expected to be a candidate for the clinical treatment of bacterial infections.

Effects of Huangqin Decoction on ulcerative colitis by targeting estrogen receptor alpha and ameliorating endothelial dysfunction based on system pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Huangqin Decoction (HQD), a traditional Chinese medicinal (TCM) formula chronicled in Shang Han Lun, has been used to treat gastrointestinal diseases for nearly 1800 years. OBJECTIVE: To investigate the effects and underlying mechanisms of HQD on ulcerative colitis (UC). METHODS: The bioactive compounds in HQD were obtained from the traditional Chinese medicine systems pharmacology database. Then, the HQD and UC-related targets were analyzed by establishing HQD-Compounds-Targets (H-C-T) and protein-protein interaction (PPI) networks. Enrichment analysis was used for further study. The candidate targets for the effects of HQD on UC were validated using a dextran sulfate sodium-induced UC mouse experiment. RESULTS: The results showed that 51 key targets were gained by matching 284 HQD-related targets and 837 UC-related targets. Combined with H-C-T and PPI network analyses, the key targets were divided into endothelial growth, inflammation and signal transcription-related targets. Further experimental validation showed that HQD targeted estrogen receptor alpha (ESR1) and endothelial growth factor receptors to relieve endothelial dysfunction, thereby improving intestinal barrier function. The expression of inflammatory cytokines and signal transducers was suppressed by HQD treatment and inflammation was inhibited. CONCLUSIONS: HQD may acts on UC via the regulation of targets and pathways related to improving the intestinal mucosal barrier and ameliorating endothelial dysfunction. Additionally, ERS1 may be a new target to explore the mechanisms of UC.

Inhibitory Activity of Honeysuckle Extracts against Influenza A Virus In Vitro and In Vivo.

Honeysuckle has been used in the treatment of influenza virus infection for thousands of years in China. However, its main active components and the functional mechanisms remain to be elucidated. Here, four honeysuckle extracts, including acids extract, flavonoids extract, total extract and acids-flavonoids mixture, were prepared to clarify the main active antiviral components. The cytopathic effect reduction assay showed that all the four extracts inhibited the replication of influenza viruses H1N1, H3N2 and the oseltamivir-resistant mutant strain H1N1-H275Y. The acids-flavonoids mixture had the strongest inhibitory effects in vitro with EC50 values of 3.8, 4.1, and > 20 µg/mL against H1N1, H3N2 and H1N1-H275Y, respectively, showing competitive antiviral activity with oseltamivir and ribavirin. Honeysuckle acids extract also showed the most significant antiviral activity in vivo. Oral administration of the acids extract at a dosage of 600 mg/kg/d effectively alleviated viral pneumonia, maintained body weight and improved the survival rate to 30% of the mice infected with a lethal dose of H1N1. The results of time-of-drug addition experiment and neuraminidase (NA) inhibition assay showed that honeysuckle extracts had a broad-spectrum inhibitory effect against influenza virus NAs. The flavonoid extract showed the strongest inhibitory effect on the NA of influenza virus H7N9 with an IC50 of 24.7 µg/mL. These results suggested that these extracts might exert their antiviral activity by suppressing the release of influenza viruses. Briefly, our findings demonstrate that acids and flavonoids extracts of honeysuckle are the major antiviral active components, and the acids extract has the potential to be developed into an antiviral agent against influenza virus, especially for oseltamivir-resistant viruses.

Technology and Application of Segmented Temporary Plugging Acid Fracturing in Highly Deviated Wells in Ultradeep Carbonate Reservoirs in Southwest China.

The Shuangyu gas reservoirs in China has a reservoir depth of more than 7000 m and a reservoir temperature of 160 °C. It is a fractured porous carbonate gas reservoir, and the gas wells mainly establish production by acid fracturing. In the early stage, the Shuangyu gas reservoirs was mainly developed with vertical wells. After acid fracturing, the natural gas production was low and declined rapidly. To obtain higher natural gas production, a large number of highly deviated wells have been deployed in the Shuangyu gas reservoirs. Because the diameter of a highly deviated well in the Shuangyu gas reservoirs is small, a subsection tool cannot be operated inside it, so it can only adopt temporary plugging acid fracturing. In this paper, a resistance-reducing acid with low friction resistance is optimized and a set of temporary plugging agents with low dissolution rate in the acid solution are selected through laboratory tests. A dynamic temporary plugging instrument is used to evaluate the plugging ability of a temporary plugging agent with different concentrations and combinations for fractures with widths of 2 and 4 mm, developing a temporary plugging agent formulation suitable for temporary plugging segmented acid fracturing of the Shuangyu gas reservoirs. In the YY666 well, the application of the research results of this paper has successfully achieved a three-stage temporary plugging acid fracturing, and the natural gas production after fracturing has been increased by 3-4 times compared with those of surrounding wells.

Inhibitory effects of canagliflozin on pancreatic cancer are mediated via the downregulation of glucose transporter­1 and lactate dehydrogenase A.

Pancreatic cancer is one of the most lethal solid malignancies, with a poor prognosis and a high mortality rate. Pancreatic cancer cells exhibit enhanced glycolysis to maintain their rapid growth. Canagliflozin (CANA) is a sodium­glucose co­transporter 2 inhibitor used for the clinical treatment of diabetes. Recent studies have demonstrated the potential ability of CANA to suppress hepatocellular carcinoma, whereas its therapeutic effects on and mechanisms in pancreatic cancer have rarely been reported. In the present study, the antitumor effects of CANA on pancreatic cancer were investigated. The data obtained indicated that pancreatic cancer growth was effectively suppressed by CANA in a dose­dependent manner, with peak inhibition rates of 54.3 and 57.6% in cultured Capan­1 and PANC­1 cells respectively. The tumor inhibitory rate reached 45.2% in nude mice with PANC­1­derived tumors, suggesting its effective antitumor activity against pancreatic cancer in vitro and/or in vivo. In addition, the combined treatment of Capan­1 and PANC­1 cells with gemcitabine and CANA exhibited a greater efficacy compared with that of treatment with gemcitabine alone. Moreover, glucose uptake and lactate production were decreased, and the mRNA levels of the glycolysis­associated genes, including glucose transporter­1 and lactate dehydrogenase A were decreased, indicating the inhibitory effects caused by the combination treatment on the metabolism of glucose in pancreatic cancer cells. Furthermore, CANA induced apoptosis, notably early apoptosis, and decreased the protein levels of PI3K, p­AKT, p­mTOR and HIF­1α, which indicated that the PI3K/AKT/mTOR signaling pathway was involved in the glycolytic process. These results demonstrated that pancreatic cancer growth was effectively inhibited by CANA via the suppression of glycolysis. This was mediated primarily by the PI3K/AKT/mTOR signaling pathway, revealing the underlying role and potential of this pathway for the clinical treatment of pancreatic cancer. Novel applications for the existing drug CANA can be explored, which could reduce the cost and time required for drug development in the field of drug discovery.