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Estimated to comprise approximately 10% of lung cancer cases, multiple pulmonary lesions pose a diagnostic and therapeutic challenge in thoracic oncology. Distinction between multiple primary lung cancer (MPLC) and intrapulmonary metastasis (IPM) directly affects tumor staging and clinical management. In equivocal cases in which the lesions are histopathologically indistinguishable, targeted sequencing can provide key additional evidence for differential diagnosis. Herein, we describe an unusual patient who presented with seven lung lesions that consisted of primary tumors and metastatic lesions, each showing distinct clonality status based on histomolecular findings. Specifically, the 45-year-old female never-smoker underwent a surgery that removed one invasive lepidic predominant adenocarcinoma and five microinvasive adenocarcinomas. Next-generation sequencing revealed three of the lesions to carry a clonal driver mutation EGFR p.L858R, supporting an IMP diagnosis. EGFR p.L858R was not detected in two other surgical specimens, which instead harbored respective oncogenic BRAF p.G469A and an uncommon EGFR p.G779F. These results led to diagnosis of the two lesions as primary tumors of lineages different from that of the metastases. The patient had achieved a recurrence-free survival of 21 months as of the latest follow-up. In this rare case that presented with evidence of both MPLC and IPM, targeted sequencing proved valuable in facilitating the diagnostic workup.

Objective A colistin-resistant Escherichia coli strain isolated from dog feces was characterized in this study. Methods and results A multiplex PCR assay was used to detect the presence of colistin-resistant mcr genes; it was found that E. coli QDFD216 co-harbored the mcr-1 and mcr-3 genes. Whole-genome sequencing and further bioinformatics analysis revealed that E. coli QDFD216 belonged to serotype O176:H11, fimH1311 type and ST132. The resistance genes bla CTX-M-14, mdfA, dfrA3, acrA, acrB, tolc, and sul3 were present in the chromosome. The mcr-1.1 and mcr-3.7 genes were located in two plasmids of different incompatibility groups. mcr-1.1 was carried by a IncX4-type plasmid within an typical IS26-parA-mcr-1.1-pap2 cassette, while mcr-3.7 was encoded by an IncP1-type plasmid with a genetic structure of TnAs2-mcr-3.7-dgkA-IS26. No additional antibiotic resistance genes were carried by either plasmid. Conclusion This is the first report of an E. coli isolate co-harboring a mcr-1.1-carrying IncX4 plasmid and a mcr-3.7-carrying IncP1 plasmid. The evolution and mechanism of mcr gene co-existence need further study to assess its impact on public health.

Background The emergence and prevalence of plasmid-mediated colistin-resistant bacterial strains in recent years have raised great concerns in clinical medicine. It is urgently needed to develop a cheaper, faster, simpler, sensitive, and specific molecular detection method to identify and monitor the dissemination of the transferable resistant determinants. Methods and results Herein, eight pairs of primers were designed to set up a multiplex PCR method for the rapid and efficient determination of reported mcr genes. This assay can give results within 85 min (35 min for amplification and 50 min for electrophoresis). We validated the feasibility of this assay by testing the presence of mcr genes in 60 colistin-resistant isolates. Conclusion Our multiplex PCR technique exhibits remarkable advantages in the light of clear identification, efficiency of amplification, as well as the time consuming for detection, and thus could be useful for the surveillance and epidemiological research of plasmid-mediated colistin resistance, particularly for the under-resourced laboratories.

Bacteriophages have been recently revisited as an alternative biocontrol tool due to the limitations of antibiotic treatment. In this study, we reported on the biological characteristics and genomic information of vB_KpnS_GH-K3 (abbreviated as GH-K3), a Klebsiella phage of the Siphoviridae family, which was previously isolated from a hospital sewage system. One-step growth curve analysis indicated that the burst size of GH-K3 was 291 PFU/cell. GH-K3 maintained a stable titer in a broad range of pH values (6-10) and temperature (up to 50 °C). Based on bioinformatics analysis, GH-K3 comprises of 49,427 bp containing a total of 77 open reading frames (ORFs), which share high degree of nucleotide similarity and close evolutionary relationships with at least 12 other Klebsiella phages. Of note, GH-K3 gp32 was identified as a unique ORF. The major segment of gp32 sequence at the C-terminus (residues 351-907) was found highly variable as determined by its mismatch with the nucleotide and protein sequences available at NCBI database. Furthermore, HHpred analysis indicated that GH-K3 gp32 contains three domains (PDB ID: 5W6S_A, 3GQ8_A and 1BHE_A) similar to depolymerase (depoKP36) of Klebsiella phage KP36 suggestive of a potential depolymerase activity during host receptor-binding in the processes of phage infection. Altogether, the current data revealed a novel putative depolymerase-like protein which is most likely to play an important role in phage-host interaction.

Periodontitis, a chronic progressive inflammation caused by plaque biofilm, is the main cause of tooth loss in adults. For certain refractory periodontitis cases, it is difficult to achieve a good curative effect using the existing periodontal treatment approaches, which may be due to periodontal pathogenic mechanism in the affected periodontal tissue that the host cannot resist and eliminate. Various pieces of evidence collectively revealed that most studies are focusing on phages in periodontal disease. Several studies have reported periodontitis treatment using phage therapy, highlighting its features including specificity, rapid propagation, and effectiveness on bacteriophage biofilms. In this study, we focus on these reports, aiming to lay the foundation for improved periodontal treatment approaches.

Salmonella enterica subsp. enterica serovar Abortusequi is a frequently reported pathogen causing abortion in mares. In this study, the preventive and therapeutic effects of phage PIZ SAE-01E2 against S. Abortusequi in a mouse model of abortion were investigated. Phage PIZ SAE-01E2 was stable at different temperatures (4 to 70°C) and pH values (pH 4 to 10) and could lyse the majority of the Salmonella serogroup O:4 and O:9 strains tested (25/28). There was no lysogeny-related, toxin, or antibiotic resistance-related gene in the genome of PIZ SAE-01E2. All of these characteristics indicate that PIZ SAE-01E2 has the potential for use in phage therapy. In in vivo experiments, 2 × 103 CFU/mouse of S. Abortusequi ATCC 9842 was sufficient to lead to murine abortion (gestational day 14.5) within 48 h. A single intraperitoneal inoculation of PIZ SAE-01E2 (108 PFU/mouse, multiplicity of infection = 105) 1 h before or after S. Abortusequi challenge provided effective protection to all pregnant mice (10/10). After 24 h of treatment with phage PIZ SAE-01E2, the bacterial loads in both the placenta and the uterus of the infected mice were significantly decreased ( 106 CFU/g). In addition, the levels of inflammatory cytokines in the placenta and blood of the mice in the phage administration groups were significantly reduced (P IMPORTANCES. Abortusequi is an important pathogen that can induce abortions in mares. Although S. Abortusequi has been well controlled in Europe and the United States due to strict breeding and health policies, it is still widespread in African and Asian countries and has proven difficult to control. In China, abortions caused by S. Abortusequi have also been reported in donkeys. So far, there is no commercial vaccine. Thus, exploiting alternative efficient and safe strategies to control S. Abortusequi infection is essential. In this study, a new lytic phage, PIZ SAE-01E2, infecting S. Abortusequi was isolated, and the characteristics of PIZ SAE-01E2 indicated that it has the potential for use in phage therapy. A single intraperitoneal inoculation of PIZ SAE-01E2 before or after S. Abortusequi challenge provided effective protection to all pregnant mice. Thus, PIZ SAE-01E2 showed the potential to block abortions induced by S. Abortusequi in vivo.

Bovine mastitis, an inflammatory disease that occurs frequently in early lactation or the dry period, is primarily caused by bacterial infections. There is growing evidence that Aerococcus viridans (A. viridans) is becoming an important cause of bovine mastitis. The treatment of bovine mastitis is primarily based on antibiotics, which not only leads to a large economic burden but also the development of antibiotic resistance. On the other hand, bacteriophages present a promising alternative treatment strategy. The object of this study was to evaluate the potential of a previously isolated A. viridans phage vB_AviM_AVP (AVP) as an anti-mastitis agent in an experimental A. viridans-induced murine mastitis model. A. viridans N14 was isolated from the milk of clinical bovine mastitis and used to establish a mastitis model in mice. We demonstrated that administration of phage AVP significantly reduced colony formation by A. viridans and alleviated damage to breast tissue. In addition, reduced inflammation was indicated by decreased levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and myeloperoxidase (MPO) activity in the phage-treated group compared to those in the phosphate buffered saline (PBS)-treated group. To the best of our knowledge, this report is the first to show the potential use of phages as a treatment for A. viridans-induced mastitis.

Yersinia enterocolitica is generally considered an important food-borne pathogen worldwide, especially in the European Union. A lytic Yersinia phage X1 (Viruses; dsDNA viruses, no RNA stage; Caudovirales; and Myoviridae) was isolated. Phage X1 showed a broad host range and could effectively lyse 27/51 Y. enterocolitica strains covering various serotypes that cause yersiniosis in humans and animals (such as serotype O3 and serotype O8). The genome of this phage was sequenced and analyzed. No toxin, antibiotic-resistance or lysogeny related modules were found in the genome of phage X1. Studies of phage stability confirmed that X1 had a high tolerance toward a broad range of temperatures (4–60°C) and pH values (4–11) for 1 h. The ability to resist harsh acidic conditions and enzymatic degradation in vitro demonstrated that phage X1 is suitable for oral administration, and in particular, that this phage can pass the stomach barrier and efficiently reach the intestine in vivo without losing infectious ability. The potential of this phage against Y. enterocolitica infection in vitro was studied. In animal experiments, a single oral administration of phage X1 at 6 h post infection was sufficient to eliminate Y. enterocolitica in 33.3% of mice (15/45). In addition, the number of Y. enterocolitica strains in the mice was also dramatically reduced to approximately 103 CFU/g after 18 h compared with 107 CFU/g in the mice without phage treatment. Treatment with phage X1 showed significant improvement by intestinal histopathologic observations. Moreover, proinflammatory cytokine levels (IL-6, TNF-α, and IL-1β) were significantly reduced (P < 0.05). These results indicate that phage X1 is a promising candidate to control infection by Y. enterocolitica in vivo.

Bacteriophage endolysin is one of the most promising antibiotic substitutes, but in Gram-negative bacteria, the outer membrane prevents the lysin from hydrolyzing peptidoglycans and blocks the development of lysin applications. The prime strategy for new antibiotic substitutes is allowing lysin to access the peptidoglycan from outside of the bacteria by reformation of the lysin. In this study, the novel Escherichia coli (E. coli) phage lyase lysep3, which lacks outside-in catalytic ability, was fused with the N-terminal region of the Bacillus amyloliquefaciens lysin including its cell wall binding domain D8 through the best manner of protein fusion based on the predicted tertiary structure of lysep3-D8 to obtain an engineered lysin that can lyse bacteria from the outside. Our results showed that lysep3-D8 could lyse both Gramnegative and Gram-positive bacteria, whereas lysep3 and D8 have no impact on bacterial growth. The MIC of lysep3-D8 on E. coli CVCC1418 is 60 μg/ml; lysep3-D8 can inhibit the growth of bacteria up to 12 h at this concentration. The bactericidal spectrum of lysep3-D8 is broad, as it can lyse of all of 14 E. coli strains, 3 P. aeruginosa strains, 1 Acinetobacter baumannii strain, and 1 Streptococcus strain. Lysep3-D8 has sufficient bactericidal effects on the 14 E. coli strains tested at the concentration of 100 μg/ml. The cell wall binding domain of the engineered lysin can destroy the integrity of the outer membrane of bacteria, thus allowing the catalytic domain to reach its target, peptidoglycan, to lyse the bacteria. Lysep3-D8 can be used as a preservative in fodder to benefit the health of animals. The method we used here proved to be a successful exploration of the reformation of phage lysin.

The emergence and spread of antibiotic-resistant bacteria constitute a critical issue for modern medicine. Patients with antibiotic-resistant bacterial infections consume more healthcare resources and have worse clinical outcomes than patients with antibiotic-sensitive bacterial infections. Phages are natural predators of bacteria and may therefore be a source of useful antibacterial drugs. Phage therapy possess availability for oral administration, penetration through the bacteria cell wall, and eradication bacterial biofilms. All of these advantages give phage therapy the possibility to turn into applications for infectious diseases. In this mini-review, we focus on the brief history of lytic phage therapy, the life cycles of lytic phages and the therapeutic effects of lytic phages.

Infections caused by antibiotic-resistant Escherichia coli are a threat to human and animal health globally. Phage therapy has made great progress for the treatment of drug-resistant infections, but it is still unclear whether E. coli resistance to antibiotics could change the lysis ability of phages. In this study, we demonstrate that over expression of AmpC, an important β-lactamase for ampicillin resistance, promotes lysis of E. coli by phage utilizing OmpA as a receptor. E. coli strains expressing more AmpC showed higher levels of OmpA, an E. coli outer membrane protein known to serve as a receptor for T-even phages, which resulted in increased adsorption and lysis by the phage tested in this study. These data demonstrate that increased ampicillin resistance can increase the sensitivity of E. coli to some lytic phage, which provides evidence for the feasibility of synergistic application of phage and antibiotics.

Klebsiella pneumoniae (K. pneumoniae) spp. are important nosocomial and community-acquired opportunistic pathogens, which cause various infections. We observed that K. pneumoniae strain K7 abruptly mutates to rough-type phage-resistant phenotype upon treatment with phage GH-K3. In the present study, the rough-type phage-resistant mutant named K7RR showed much lower virulence than K7. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis indicated that WcaJ and two undefined glycosyltransferases (GTs)- named GT-1, GT-2- were found to be down-regulated drastically in K7RR as compared to K7 strain. GT-1, GT-2, and wcaJ are all located in the gene cluster of capsular polysaccharide (CPS). Upon deletion, even of single component, of GT-1, GT-2, and wcaJ resulted clearly in significant decline of CPS synthesis with concomitant development of GH-K3 resistance and decline of virulence of K. pneumoniae, indicating that all these three GTs are more likely involved in maintenance of phage sensitivity and bacterial virulence. Additionally, K7RR and GT-deficient strains were found sensitive to endocytosis of macrophages. Mitogen-activated protein kinase (MAPK) signaling pathway of macrophages was significantly activated by K7RR and GT-deficient strains comparing with that of K7. Interestingly, in the presence of macromolecular CPS residues (>250 KD), K7(ΔGT-1) and K7(ΔwcaJ) could still be bounded by GH-K3, though with a modest adsorption efficiency, and showed minor virulence, suggesting that the CPS residues accumulated upon deletion of GT-1 or wcaJ did retain phage binding sites as well maintain mild virulence. In brief, our study defines, for the first time, the potential roles of GT-1, GT-2, and WcaJ in K. pneumoniae in bacterial virulence and generation of rough-type mutation under the pressure of bacteriophage.

Dandan Gao,1 Zhimin Guo,1 Jiabin Wang,2 Gaofeng Hu,1 Yuqiao Su,3 Lijun Chen,1 Qianwen Lv,1 Huimei Yu,2 Jianchun Qin,3 Wei Xu1 1Department of Clinical Laboratory, The First Hospital of Jilin University, Changchun 130021, China; 2Department of Pathology and Pathophysiology, School of Basic Medical Sciences, Jilin University, Changchun 130021, China; 3Department of Biotechnology, College of Plant Sciences, Jilin University, Changchun, Jilin 130062, China Background: Dicerandrol B is a natural antitumor agent that can be isolated from the endophytic fungus, Phomopsis sp. The present study investigated the effects of dicerandrol B on human cervical cancer HeLa cells.Materials and methods: In this study, dicerandrol B was identified by electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. We used MTT to detect the cell viability. Flow cytometry was used to analyze the apoptosis and cell cycle. Western blot was used to examine the expression of related proteins.Results: Dicerandrol B was isolated from the endophytic fungus Phomopsis sp. The MTT assay and flow cytometry showed that dicerandrol B significantly inhibited HeLa cell viability and induced G2/M cell cycle arrest. Western blot analysis demonstrated that dicerandrol B increased the levels of GRP78, ubiquitin, cleaved PARP, and Bax protein, decreased the levels of PARP and Bcl-2 protein, and caused an increase in the Bax/Bcl-2 ratio in HeLa cells. Dicerandrol B increased the production of ROS in HeLa cells, which was attenuated by the antioxidant N-acetyl-l-cysteine.Conclusion: These findings suggest that dicerandrol B induces apoptosis in human HeLa cells, possibly through the endoplasmic reticulum stress and mitochondrial apoptotic pathways. This suggests that dicerandrol B possesses strong anticancer activity in cervical cancer and provides insight into the underlying mechanisms. Keywords: apoptosis, cervical cancer, endoplasmic reticulum stress, mitochondrial damage

Treatment of infections caused by staphylococci has become more difficult because of the emergence of multidrug-resistant strains as well as biofilm formation. In this study, we observed the ability of the phage lysin LysGH15 to eliminate staphylococcal planktonic cells and biofilms formed by Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis All these strains were sensitive to LysGH15, showing reductions in bacterial counts of approximately 4 log units within 30 min after treatment with 20 μg/ml of LysGH15, and the MICs ranged from 8 μg/ml to 32 μg/ml. LysGH15 efficiently prevented biofilm formation by the four staphylococcal species at a dose of 50 μg/ml. At a higher dose (100 μg/ml), LysGH15 also showed notable disrupting activity against 24-h and 72-h biofilms formed by S. aureus and coagulase-negative species. In the in vivo experiments, a single intraperitoneal injection of LysGH15 (20 μg/mouse) administered 1 h after the injection of S. epidermidis at double the minimum lethal dose was sufficient to protect the mice. The S. epidermidis cell counts were 4 log units lower in the blood and 3 log units lower in the organs of mice 24 h after treatment with LysGH15 than in the untreated control mice. LysGH15 reduced cytokine levels in the blood and improved pathological changes in the organs. The broad antistaphylococcal activity exerted by LysGH15 on planktonic cells and biofilms makes LysGH15 a valuable treatment option for biofilm-related or non-biofilm-related staphylococcal infections.IMPORTANCE Most staphylococcal species are major causes of health care- and community-associated infections. In particular, Staphylococcus aureus is a common and dangerous pathogen, and Staphylococcus epidermidis is a ubiquitous skin commensal and opportunistic pathogen. Treatment of infections caused by staphylococci has become more difficult because of the emergence of multidrug-resistant strains as well as biofilm formation. In this study, we found that all tested S. aureus, S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis strains were sensitive to the phage lysin LysGH15 (MICs ranging from 8 to 32 μg/ml). More importantly, LysGH15 not only prevented biofilm formation by these staphylococci but also disrupted 24-h and 72-h biofilms. Furthermore, the in vivo efficacy of LysGH15 was demonstrated in a mouse model of S. epidermidis bacteremia. Thus, LysGH15 exhibits therapeutic potential for treating biofilm-related or non-biofilm-related infections caused by diverse staphylococci.

Bacteriophage can be used as an alternative or complementary therapy to antibiotics for treating multidrug-resistant bacterial infections. However, the rapid emergence of resistant host variants during phage treatment has limited its therapeutic applications. In this study, a potential phage-resistant mechanism of Klebsiella pneumoniae was revealed. After phage GH-K3 treatment, a smooth-type colony, named K7R(B), was obtained from the K. pneumoniae K7 culture. Treatment with IO(4)(−) and/or proteinase K indicated that polysaccharides of K7 played an important role in phage recruitment, and protein receptors on K7 were essential for effective infection by GH-K3. Differences in protein expression between K7 and K7R(B) were quantitatively analyzed by liquid chromatography-tandem mass spectrometry. Among differentially expressed proteins, OmpC, OmpN, KPN_02430, and OmpF were downregulated significantly in K7R(B). trans-Complementation of OmpC in K7R(B) conferred rapid adsorption and sensitivity to GH-K3. In contrast, a single-base deletion mutation of ompC in K7, which resulted in OmpC silencing, led to lower adsorption efficiency and resistance to GH-K3. These assays proved that OmpC is the key receptor-binding protein for GH-K3. In addition, the native K. pneumoniae strains KPP14, KPP27, and KPP36 showed low or no sensitivity to GH-K3. However, these strains became more sensitive to GH-K3 after their native receptors were replaced by OmpC of K7, suggesting that OmpC(K7) was the most suitable receptor for GH-K3. This study revealed that K7R(B) became resistant to GH-K3 due to gene mutation of ompC and that OmpC of K7 is essential for effective infection by GH-K3. IMPORTANCE With increased incidence of multidrug-resistant (MDR) bacterial strains, phages have regained attention as promising potential antibacterial agents. However, the rapid emergence of resistant variants during phage treatment has limited the therapeutic applications of phage. According to our trans-complementation, ompC mutation, and phage adsorption efficiency assays, we identified OmpC as the key receptor-binding protein (RBP) for phage GH-K3, which is essential for effective infection. This study revealed that the phage secondary receptor of K. pneumoniae, OmpC, is the essential RBP not only for phage infecting Gram-negative bacteria, such as Escherichia coli and Salmonella, but also for K. pneumoniae.

Staphylococcus aureus (S. aureus) is a common and dangerous pathogen that causes various infectious diseases. Skin damage, such as burn wounds, are at high risk of Staphylococcus aureus colonization and infection, which increases morbidity and mortality. The phage lysin LysGH15 exhibits highly efficient lytic activity against methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. Apigenin (api) significantly decreases haemolysis of rabbit erythrocytes caused by S. aureus and shows anti-inflammatory function. LysGH15 and api were added to Aquaphor to form an LysGH15-api-Aquaphor (LAA) ointment. The LAA ointment simultaneously exhibited bactericidal activity against S. aureus and inhibited haemolysis. In an LAA-treated mouse model of an MRSA-infected skin wound, the mean bacterial colony count decreased to approximately 102 CFU/mg at 18 h after treatment (and the bacteria became undetectable at 96 h), whereas the mean count in untreated mice was approximately 105 CFU/mg of tissue. The LAA ointment also reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IFN-γ) and accelerated wound healing in the mouse model. These data demonstrate the potential efficacy of a combination of LysGH15 and api for use as a topical antimicrobial agent against S. aureus.

As yet, very few vaccine candidates with activity in animals against Mycobacterium tuberculosis infection have been tested as therapeutic postexposure vaccines. We recently described two pools of mycobacterial proteins with this activity, and here we describe further studies in which four of these proteins (Rv1738, Rv2032, Rv3130, and Rv3841) were generated as a fusion polypeptide and then delivered in a novel yeast-based platform (Tarmogen) which itself has immunostimulatory properties, including activation of Toll-like receptors. This platform can deliver antigens into both the class I and class II antigen presentation pathways and stimulate strong Th1 and Th17 responses. In mice this fusion vaccine, designated GI-19007, was immunogenic and elicited strong gamma interferon (IFN-γ) and interleukin-17 (IL-17) responses; despite this, they displayed minimal prophylactic activity in mice that were subsequently infected with a virulent clinical strain. In contrast, in a therapeutic model in the guinea pig, GI-19007 significantly reduced the lung bacterial load and reduced lung pathology, particularly in terms of secondary lesion development, while significantly improving survival in one-third of these animals. In further studies in which guinea pigs were vaccinated with BCG before challenge, therapeutic vaccination with GI-19007 initially improved survival versus that of animals given BCG alone, although this protective effect was gradually lost at around 400 days after challenge. Given its apparent ability to substantially limit bacterial dissemination within and from the lungs, GI-19007 potentially can be used to limit lung damage as well as facilitating chemotherapeutic regimens in infected individuals.

Receptor interacting protein kinase 3 (RIP3) is a critical regulator of programmed necrotic cell death. Here, we observed that RIP3 was significantly down-regulated in esophageal cancer. And its remaining expression was associated with better response to chemotherapy and prolonged survival. Notably, re-expression of kinase-dead RIP3 also restored cisplatin sensitivity, suggesting that some roles of RIP3 beyond necroptosis may be involved in cisplatin-based chemosensitivity. To investigate the mechanisms, a large-scale quantitative proteomics study was performed after cisplatin treatment in RIP3-knockdown cells. In total, approximately 7000 protein groups were confidently identified, with a false discovery rate of 0.21% at the protein level. Of these proteins, 685 displayed RIP3-dependent changes in abundance. Bioinformatics analyses indicated that DNA repair pathway was stimulated after RIP3 depletion. Functional studies showed that deficient RIP3 upregulated FOSL1 and POLD1 through activation of the HSP90/CDC37 complex and ERK phosphorylation in multiple cell lines. Furthermore, via inhibition of the HSP90/CDC37 complex, ERK and FOSL1 reversed the cisplatin resistance phenotype. These results suggest that RIP3 regulates cisplatin sensitivity through both pronecrotic and non-necrotic functions. RIP3 may be a potential marker for predicting chemosensitivity.

Biofilm formation, one of the most important virulence factors of pathogenic bacteria, protects bacteria against desiccation, antibiotics, phages and host immune responses. However, phage-derived depolymerases show antibiofilm activity and demonstrate great potential to treat infections caused by biofilm-forming bacteria. In this study, the Escherichia coli phage vB_EcoM_ECOO78 was isolated and characterised, and we observed its ability to lyse five out of 34 tested E. coli clinical isolates. The highest phage titre was observed at a multiplicity of infection of 10-5 and a burst size of approximately 74 plaque forming units (PFU)/infection. Electron micrographs indicated that vB_EcoM_ECOO78 belongs to the family Myoviridae. The presence of increasing halos surrounding the lysis plaques formed by vB_EcoM_ECOO78 indicated that this phage may encode a depolymerase. Based on a sequencing analysis, the complete genome of vB_EcoM_ECOO78 was found to be 41,289 bp in size, with a GC content of 53.07%. Additionally, vB_EcoM_ECOO78 has 56 predicted open reading frames, 51 (91.07%) of which are assumed to be functional. A BLAST analysis indicated that ORF42 of vB_EcoM_ECOO78 (Dpo42) has low identity with other reported phage-associated depolymerases. Dpo42 was expressed and purified as a soluble protein using E. coli BL21. The biofilm formation ability of E. coli isolates and the antibiofilm activity of Dpo42 were tested by performing spot assays and using a 96-well micro-titre plate method. Dpo42 degraded the capsular polysaccharides surrounding E. coli and exhibited dose-dependent biofilm-formation prevention activity. Based on these results, Dpo42 appears to be a novel phage-derived depolymerase that represents a new potential strategy for preventing E. coli biofilm formation.

Distal tibiofibular syndesmosis (DTS) has wide anatomic variability in depth of incisura fibularis and shape of tibial tubercles. We designed a 3-year prospective cohort study of 300 young physical training soldiers in an Army Physical Fitness School. Ankle computed tomography (CT) scans showed that 56% of the incisura fibularis were a “C” shape, 25% were a “1” shape, and 19% were a “Г” shape. Furthermore, we invited a randomly selected subcohort of 6 participants in each shape of DTS to undergo a three-dimensional (3D) laser scanning. The “1” shape group showed widest displacement range of the DTS in the y-axis, along with the range of motion (ROM) on the position more than 20° of the ankle dorsiflexion, inversion and eversion. During the 3-year study period, 23 participants experienced recurrent lateral ankle sprains. 7 cases of the incisura fibularis were “C” shape, 13 cases were “1” shape, and 3 cases were “Г” shape. The “1” shape showed highest risk among the three shapes in incident recurrent lateral ankle sprains. We propose that it is possible to classify shapes of DTS according to the shapes of incisura fibularis, and people with “1” shape may have more risk of recurrent lateral ankle sprains.

In recent years, after the emergence of a large number of multidrug-resistant bacteria, phages and phage-associated products for the prevention and control of bacterial disease have revealed prominent advantages as compared with antibiotics. However, bacteria are susceptible to becoming phage-resistant, thus severely limiting the application of phage therapy. In this study, Escherichia coli cells were incubated with lytic bacteriophages to obtain mutants that were resistant to the lytic phages. Then, bacteriophages against the phage-resistant variants were isolated and subsequently mixed with the original lytic phage to prepare a novel phage cocktail for bactericidal use. The data showed that our phage cocktail not only had notable bactericidal effects, including a widened host range and rapid lysis, but also decreased the generation and mutation frequency of phage-resistant strains in vitro. In addition, we tested our cocktail in a murine bacteremia model. The results suggested that compared with the single phage, fewer phage-resistant bacteria appeared during the treatment of phage cocktail, thus prolonging the usable time of the phage cocktail and improving its therapeutic effect in phage applications. Importantly, our preparation method of phage cocktail was proved to be generalizable. Because the bacteriophage against the phage-resistant strain is an ideal guard that promptly attacks potential phage resistance, this guard-killer dual-function phage cocktail provides a novel strategy for phage therapy that allows the natural ecology to be sustained.

Phage-derived lysins can hydrolyse bacterial cell walls and show great potential for combating Gram-positive pathogens. In this study, the potential of LysEF-P10, a new lysin derived from a isolated Enterococcus faecalis phage EF-P10, as an alternative treatment for multidrug-resistant E. faecalis infections, was studied. LysEF-P10 shares only 61% amino acid identity with its closest homologues. Four proteins were expressed: LysEF-P10, the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain (LysEF-P10C), the putative binding domain (LysEF-P10B), and a fusion recombination protein (LysEF-P10B-green fluorescent protein). Only LysEF-P10 showed highly efficient, broad-spectrum bactericidal activity against E. faecalis. Several key functional residues, including the Cys-His-Asn triplet and the calcium-binding site, were confirmed using 3D structure prediction, BLAST and mutation analys. We also found that calcium can switch LysEF-P10 between its active and inactive states and that LysEF-P10B is responsible for binding E. faecalis cells. A single administration of LysEF-P10 (5 μg) was sufficient to protect mice against lethal vancomycin-resistant Enterococcus faecalis (VREF) infection, and LysEF-P10-specific antibody did not affect its bactericidal activity or treatment effect. Moreover, LysEF-P10 reduced the number of Enterococcus colonies and alleviated the gut microbiota imbalance caused by VREF. These results indicate that LysEF-P10 might be an alternative treatment for multidrug-resistant E. faecalis infections.

Multidrug-resistant Escherichia coli has seriously threatened antibiotic resources and international public health. Bacteriophage lysin preparations have been widely considered as valid agents for solving multidrug resistances. Many lysins have been derived to treat diseases caused by Gram-positive bacteria, but only a few lysin preparations have been found that successively treat diseases caused by Gram-negative bacteria. The outer membrane of Gram-negative bacteria effectively blocks the interactions between peptidoglycan in the periplasmic space and bacteriophage lysins, which therefore hampers the antimicrobial effects of bacteriophage lysins. In this study, a new fusion protein (Colicin-Lysep3) was constructed by fusing the translocation and receptor binding domains of colicin A with an E. coli phage lysin, which endows Colicin-Lysep3 bactericidal activity against E. coli from outside of Gram-negative bacteria. These results show that Colicin-Lysep3 could lyse the E. coli broadly in vitro and significantly reduce the number of E. coli in an intestinal infection mouse model. Overall, our findings first demonstrated that a colicin A fragment could enable a bacteriophage lysin to lyse E. coli from the outside, promoting the application of phage lysin preparations in control of Gram-negative bacteria.

Phage lysins are considered promising antimicrobials against resistant bacterial infections. Some lysins have been reported for the prevention and treatment of Gram-positive bacterial infection. Gram-negative bacterial phage lysins, however, can only destroy the bacterial cell wall from inside because of the obstruction of the bacterial outer membrane that prevents direct hydrolysis of the bacterial wall peptidoglycan from the outside, severely restricting the development of lysins against Gram-negative bacteria. In this study, genetic engineering techniques were used to fuse a 5 cationic amino acid polypeptide (KRKRK), a 10 cationic amino acid polypeptide (KRKRKRKRKR), a 15 cationic amino acid polypeptide (KRKRKRKRKRKRKRK), and a polypeptide including both cationic and hydrophobic amino acids (KRKRKFFVAIIP) to the C-terminus of the Escherichia coli phage lysin Lysep3 to obtain four fusion lysins (5aa, 10aa, 15aa, Mix). The bactericidal effects of those four lysins on E. coli were then compared in vitro. Our results showed that the fusion of hydrophobic and positively charged amino acids, Mix, can kill E. coli effectively; the fusion of positively charged amino acids alone at the C-terminus (5aa, 10aa, 15aa) also showed bactericidal activity against E. coli from the outside, with the bactericidal activity gradually increasing with the positive charge at the C-terminus of the lysin. Collectively, improving the positive charge at the C-terminus of E. coli bacteriophage lysin Lysep3 increases its bactericidal ability from outside E. coli, providing a new practical method for the development of anti-Gram-negative bacterial lysins.

N-myc down-regulated gene 1 (NDRG1) has been shown to regulate tumor growth and metastasis in various malignant tumors and also to be dysregulated in esophageal squamous cell carcinoma (ESCC). Here, we show that NDRG1 overexpression (91.9%, 79/86) in ESCC tumor tissues is associated with poor overall survival of esophageal cancer patients. When placed in stable transfectants of the KYSE 30 ESCC cell line generated by lentiviral transduction with the ectopic overexpression of NDRG1, the expression of transducin-like enhancer of Split 2 (TLE2) was decreased sharply, however β−catenin was increased. Mechanistically, NDRG1 physically associates with TLE2 and β−catenin to affect the Wnt pathway. RNA interference and TLE2 overexpression studies demonstrate that NDRG1 fails to active Wnt pathway compared with isogenic wild-type controls. Strikingly, NDRG1 overexpression induces the epithelial mesenchymal transition (EMT) through activating the Wnt signaling pathway in ESCC cells, decreased the expression of E-cadherin and enhanced the expression of Snail. Our study elucidates a mechanism of NDRG1-regulated Wnt pathway activation and EMT via affecting TLE2 and β-catenin expression in esophageal cancer cells. This indicates a pro-oncogenic role for NDRG1 in esophageal cancer cells whereby it modulates tumor progression.

GlobeImmune's Tarmogen(®) immunotherapy platform utilizes recombinant Saccharomyces cerevisiae yeast as a vaccine vector to deliver heterologous antigens for activation of disease-specific, targeted cellular immunity. The vaccines elicit immune-mediated killing of target cells expressing viral and cancer antigens in vivo via a CD8(+) CTL-mediated mechanism. Tarmogens are not neutralized by host immune responses and can be administered repeatedly to boost antigen-specific immunity. Production of the vaccines yields stable off-the-shelf products that avoid the need for patient-specific manufacturing found with other immunotherapeutic approaches. Tarmogens for the treatment of chronic hepatitis B and C and various cancers were well tolerated and immunogenic in phase 1 and 2 clinical trials encompassing >600 subjects. The platform is being widely utilized in basic vaccine research and the most rapid path to success in these endeavors follows from optimal immunoassay selection and execution. This chapter provides detailed methods for the construction and preclinical immunogenicity testing of yeast-based immunotherapeutic products to support the rapid and efficient use of this versatile technology.

Soft tissue defects of the pretibial area and the foot are among the most common complications in patients with lower extremity injuries and remain a challenge for surgeons. This study examined the clinical effects of repairing pretibial and foot soft tissue defects with a medial crural fasciocutaneous flap. Twenty-three injury cases with soft tissue defects in the middle/lower parts of the tibia and ankle/heel were treated. Of these, 8 injuries were in the lower pretibial region, 11 injuries were in the heel, and 4 injuries were in the dorsalis pedis. The dimensions of the soft tissue defects ranged from 7 cm × 5 cm to 18 cm × 10 cm. The crural fasciocutaneous flap was used for wound repair in all cases. The skin harvest dimensions ranged from 12 cm × 6 cm to 23 cm × 12 cm, and the width of the reverse flap pedicle was greater than 4 cm. The patients were followed up for 6 to 27 months postsurgery. All flaps survived and appeared healthy, and leg function was satisfactorily recovered. Five patients suffered from distal flap necrosis and 2 patients suffered from effusion. The area of the crural fasciocutaneous flap harvest appeared to have a good blood supply, and the procedure was easy to perform without injuries to the main vasculature. In conclusion, the medial crural fasciocutaneous flap is a safe and effective clinical intervention for the reconstruction of soft tissue defects of the pretibial area and foot.