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Toll/interleukin-1 receptor (TIR) domain-containing proteins play a critical role in immune responses in diverse organisms, but their function in bacterial systems remains to be fully elucidated. This study, focusing on Escherichia coli, addresses how TIR domain-containing proteins contribute to bacterial immunity against phage attack. Through an exhaustive survey of all E. coli genomes available in the NCBI database and testing of 32 representatives of the 90% of the identified TIR domain-containing proteins, we found that a significant proportion (37.5%) exhibit antiphage activities. These defense systems recognize a variety of phage components, thus providing a sophisticated mechanism for pathogen detection and defense. This study not only highlights the robustness of TIR systems in bacterial immunity, but also draws an intriguing parallel to the diversity seen in mammalian Toll-like receptors (TLRs), enriching our understanding of innate immune mechanisms across life forms and underscoring the evolutionary significance of these defense strategies in prokaryotes.
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Bacteriófagos , Escherichia coli , Domínios Proteicos , Escherichia coli/genética , Escherichia coli/virologia , Escherichia coli/imunologia , Escherichia coli/metabolismo , Bacteriófagos/genética , Bacteriófagos/imunologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Imunidade Inata , Receptores Toll-Like/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-1/genéticaRESUMO
Pancreatic cancer is notoriously lethal due to its late diagnosis and poor patient response to treatments, posing a significant clinical challenge. This study introduced a novel approach that combines a single-cell capturing platform, tumor-targeted silver (Ag) nanoprobes, and precisely docking tapered fiber integrated with Raman spectroscopy. This approach focuses on early detection and progression monitoring of pancreatic cancer. Utilizing tumor-targeted Ag nanoparticles and tapered multimode fibers enhances Raman signals, minimizes light loss, and reduces background noise. This advanced Raman system allows for detailed molecular spectroscopic examination of individual cells, offering more practical information and enabling earlier detection and accurate staging of pancreatic cancer compared to conventional multicellular Raman spectroscopy. Transcriptomic analysis using high-throughput gene screening and transcriptomic databases confirmed the ability and accuracy of this method to identify molecular changes in normal, early, and metastatic pancreatic cancer cells. Key findings revealed that cell adhesion, migration, and the extracellular matrix are closely related to single-cell Raman spectroscopy (SCRS) results, highlighting components such as collagen, phospholipids, and carotene. Therefore, the SCRS approach provides a comprehensive view of the molecular composition, biological function, and material changes in cells, offering a novel, accurate, reliable, rapid, and efficient method for diagnosing and monitoring pancreatic cancer.
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Técnicas Biossensoriais , Nanopartículas Metálicas , Fibras Ópticas , Neoplasias Pancreáticas , Prata , Análise de Célula Única , Análise Espectral Raman , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Análise Espectral Raman/métodos , Análise Espectral Raman/instrumentação , Humanos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Prata/química , Nanopartículas Metálicas/química , Linhagem Celular Tumoral , Desenho de EquipamentoRESUMO
In response to the global threat posed by bacterial pathogens, which are the second leading cause of death worldwide, vaccine development is challenged by the diversity of bacterial serotypes and the lack of immunoprotection across serotypes. To address this, we introduce BacScan, a novel genome-wide technology for the rapid discovery of conserved highly immunogenic proteins (HIPs) across serotypes. Using bacterial-specific serum, BacScan combines phage display, immunoprecipitation, and next-generation sequencing to comprehensively identify all the HIPs in a single assay, thereby paving the way for the development of universally protective vaccines. Our validation of this technique with Streptococcus suis, a major pathogenic threat, led to the identification of 19 HIPs, eight of which conferred 20-100% protection against S. suis challenge in animal models. Remarkably, HIP 8455 induced complete immunity, making it an exemplary vaccine target. BacScan's adaptability to any bacterial pathogen positions it as a revolutionary tool that can expedite the development of vaccines with broad efficacy, thus playing a critical role in curbing bacterial transmission and slowing the march of antimicrobial resistance.
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Proteínas de Bactérias , Animais , Camundongos , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/genética , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/prevenção & controle , Streptococcus suis/imunologia , Streptococcus suis/genética , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Feminino , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/genética , Humanos , Vacinas Bacterianas/imunologiaRESUMO
Recent epidemiological studies have discovered that a lot of cases of porcine epidemic diarrhea virus (PEDV) infection are frequently accompanied by porcine kobuvirus (PKV) infection, suggesting a potential relationship between the two viruses in the development of diarrhea. To investigate the impact of PKV on PEDV pathogenicity and the number of intestinal lymphocytes, piglets were infected with PKV or PEDV or co-infected with both viruses. Our findings demonstrate that co-infected piglets exhibit more severe symptoms, acute gastroenteritis, and higher PEDV replication compared to those infected with PEDV alone. Notably, PKV alone does not cause significant intestinal damage but enhances PEDV's pathogenicity and alters the number of intestinal lymphocytes. These results underscore the complexity of viral interactions in swine diseases and highlight the need for comprehensive diagnostic and treatment strategies addressing co-infections.
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Coinfecção , Infecções por Coronavirus , Intestinos , Kobuvirus , Linfócitos , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Vírus da Diarreia Epidêmica Suína/patogenicidade , Vírus da Diarreia Epidêmica Suína/fisiologia , Suínos , Doenças dos Suínos/virologia , Coinfecção/virologia , Coinfecção/veterinária , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Linfócitos/virologia , Kobuvirus/patogenicidade , Kobuvirus/genética , Intestinos/virologia , Diarreia/virologia , Diarreia/veterinária , Replicação Viral , Gastroenterite/virologia , Gastroenterite/veterinária , Infecções por Picornaviridae/veterinária , Infecções por Picornaviridae/virologiaRESUMO
The COVID-19 pandemic has transformed vaccinology. Rapid deployment of mRNA vaccines has saved countless lives. However, these platforms have inherent limitations including lack of durability of immune responses and mucosal immunity, high cost, and thermal instability. These and uncertainties about the nature of future pandemics underscore the need for exploring next-generation vaccine platforms. Here, we present a novel protein-based, bacteriophage T4 platform for rapid design of efficacious vaccines against bacterial and viral pathogens. Full-length antigens can be displayed at high density on a 120 × 86 nm phage capsid through nonessential capsid binding proteins Soc and Hoc. Such nanoparticles, without any adjuvant, induce robust humoral, cellular, and mucosal responses when administered intranasally and confer sterilizing immunity. Combined with structural stability and ease of manufacture, T4 phage provides an excellent needle-free, mucosal pandemic vaccine platform and allows equitable vaccine access to low- and middle-income communities across the globe.
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Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that has caused significant economic losses in the livestock industry. Peptide vaccines engineered with the protective epitopes of FMDV have provided a safer alternative for disease prevention than the traditional inactivated vaccines. However, the immunogenicity of the peptide is usually poor and therefore an adjuvant is required. Here, we showed that recombinant T4 phages displaying the B-cell epitope of the FMDV VP1 protein (VP1130-158), without additional adjuvants, induced similar levels of antigen-specific IgG1 but higher levels of IgG2a compared to the peptide vaccine. Incorporation of a CD4+ T cell epitope, either 3A21-35 of FMDV 3A protein or P2830-844 of tetanus toxoid, further enhanced the immunogenicity of VP1-T4 phage nanoparticles. Interestingly, the extrinsic adjuvant cannot enhance the immunogenicity of the nanoparticles, indicating the intrinsic adjuvant activities of T4 phage. Furthermore, the recombinant T4 phage can be produced on a large scale within a short period of time at a relatively low-cost using Escherichia coli, heralding its potential in the development of a safe and effective FMDV vaccine.
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Vírus da Febre Aftosa , Febre Aftosa , Vacinas Virais , Animais , Bacteriófago T4 , Febre Aftosa/prevenção & controle , Nanovacinas , Anticorpos Antivirais , Epitopos de Linfócito B , Adjuvantes Imunológicos , Proteínas do CapsídeoRESUMO
Background: Emerging infectious diseases pose a significant threat to both human and animal populations. Rapid de novo identification of protective antigens from a clinical isolate and development of an antigen-matched vaccine is a golden strategy to prevent the spread of emerging novel pathogens. Methods: Here, we focused on Actinobacillus pleuropneumoniae, which poses a serious threat to the pig industry, and developed a general workflow by integrating proteosurfaceomics, secretomics, and BacScan technologies for the rapid de novo identification of bacterial protective proteins from a clinical isolate. Results: As a proof of concept, we identified 3 novel protective proteins of A. pleuropneumoniae. Using the protective protein HBS1_14 and toxin proteins, we have developed a promising multivalent subunit vaccine against A. pleuropneumoniae. Discussion: We believe that our strategy can be applied to any bacterial pathogen and has the potential to significantly accelerate the development of antigen-matched vaccines to prevent the spread of an emerging novel bacterial pathogen.
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Actinobacillus pleuropneumoniae , Pleuropneumonia , Animais , Humanos , Suínos , Antígenos de Bactérias , Vacinas Bacterianas , Proteínas de Bactérias , Pleuropneumonia/microbiologia , Pleuropneumonia/prevenção & controleRESUMO
Vaccines that trigger mucosal immune responses at the entry portals of pathogens are highly desired. Here, we showed that antigen-decorated nanoparticle generated through CRISPR engineering of T4 bacteriophage can serve as a universal platform for the rapid development of mucosal vaccines. Insertion of Flu viral M2e into phage T4 genome through fusion to Soc (Small Outer Capsid protein) generated a recombinant phage, and the Soc-M2e proteins self-assembled onto phage capsids to form 3M2e-T4 nanoparticles during propagation of T4 in E. coli. Intranasal administration of 3M2e-T4 nanoparticles maintains antigen persistence in the lungs, resulting in increased uptake and presentation by antigen-presenting cells. M2e-specific secretory IgA, effector (TEM), central (TCM), and tissue-resident memory CD4+ T cells (TRM) were efficiently induced in the local mucosal sites, which mediated protections against divergent influenza viruses. Our studies demonstrated the mechanisms of immune protection following 3M2e-T4 nanoparticles vaccination and provide a versatile T4 platform that can be customized to rapidly develop mucosal vaccines against future emerging epidemics.
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Vacinas contra Influenza , Nanopartículas , Infecções por Orthomyxoviridae , Animais , Camundongos , Vacinas contra Influenza/genética , Bacteriófago T4/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Escherichia coli/genética , Infecções por Orthomyxoviridae/prevenção & controle , Camundongos Endogâmicos BALB C , Proteínas da Matriz ViralRESUMO
Many phages, such as T4, protect their genomes against the nucleases of bacterial restriction-modification (R-M) and CRISPR-Cas systems through covalent modification of their genomes. Recent studies have revealed many novel nuclease-containing antiphage systems, raising the question of the role of phage genome modifications in countering these systems. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli and demonstrated the roles of T4 genome modifications in countering these systems. Our analysis identified at least 17 nuclease-containing defense systems in E. coli, with type III Druantia being the most abundant system, followed by Zorya, Septu, Gabija, AVAST type 4, and qatABCD. Of these, 8 nuclease-containing systems were found to be active against phage T4 infection. During T4 replication in E. coli, 5-hydroxymethyl dCTP is incorporated into the newly synthesized DNA instead of dCTP. The 5-hydroxymethylcytosines (hmCs) are further modified by glycosylation to form glucosyl-5-hydroxymethylcytosine (ghmC). Our data showed that the ghmC modification of the T4 genome abolished the defense activities of Gabija, Shedu, Restriction-like, type III Druantia, and qatABCD systems. The anti-phage T4 activities of the last two systems can also be counteracted by hmC modification. Interestingly, the Restriction-like system specifically restricts phage T4 containing an hmC-modified genome. The ghmC modification cannot abolish the anti-phage T4 activities of Septu, SspBCDE, and mzaABCDE, although it reduces their efficiency. Our study reveals the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of T4 genomic modification in countering these defense systems. IMPORTANCE Cleavage of foreign DNA is a well-known mechanism used by bacteria to protect themselves from phage infections. Two well-known bacterial defense systems, R-M and CRISPR-Cas, both contain nucleases that cleave the phage genomes through specific mechanisms. However, phages have evolved different strategies to modify their genomes to prevent cleavage. Recent studies have revealed many novel nuclease-containing antiphage systems from various bacteria and archaea. However, no studies have systematically investigated the nuclease-containing antiphage systems of a specific bacterial species. In addition, the role of phage genome modifications in countering these systems remains unknown. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli using all 2,289 genomes available in NCBI. Our studies reveal the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of genomic modification of phage T4 in countering these defense systems.
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Bacteriófago T4 , Enzimas de Restrição-Modificação do DNA , Escherichia coli , Bacteriófago T4/genética , Sistemas CRISPR-Cas , Escherichia coli/enzimologia , Escherichia coli/virologia , Genoma ViralRESUMO
Surface-enhanced Raman scattering (SERS) has great potential in the field of rapid detection of pesticide residues in food. In this paper, a fiber optic SERS sensor excited by evanescent waves was proposed for efficient detection of thiram. Silver nanocubes (Ag NCs) were prepared as SERS active substrates, which had much stronger electromagnetic field intensity than nanospheres under laser excitation due to much more "hot spots". By using the method of electrostatic adsorption and laser induction, Ag NCs were uniformly assembled at the fiber taper waist (FTW) for enhancing the Raman signal. Different from the traditional way of stimulation, evanescent wave excitation greatly increased the interaction area between the excitation and analyte, while reducing the damage of the excited light to the metal nanostructures. The methods proposed in this work have been successfully used to detect the pesticide residues of thiram and showed good detection performance. The detection limits for 4-Mercaptobenzoic acid (4-MBA) and thiram were determined to be 10-9 and 10-8 M, the corresponding enhancement factor could be 1.64 × 105 and 6.38 × 104. Low concentration of thiram was detected in the peels of tomatoes and cucumbers, indicating its feasibility in actual sample detection. The combination of evanescent waves and SERS provides a new direction for the application of SERS sensors, which had great application potential in the field of pesticide residue detection.
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Designing artificial viral vectors (AVVs) programmed with biomolecules that can enter human cells and carry out molecular repairs will have broad applications. Here, we describe an assembly-line approach to build AVVs by engineering the well-characterized structural components of bacteriophage T4. Starting with a 120 × 86 nm capsid shell that can accommodate 171-Kbp DNA and thousands of protein copies, various combinations of biomolecules, including DNAs, proteins, RNAs, and ribonucleoproteins, are externally and internally incorporated. The nanoparticles are then coated with cationic lipid to enable efficient entry into human cells. As proof of concept, we assemble a series of AVVs designed to deliver full-length dystrophin gene or perform various molecular operations to remodel human genome, including genome editing, gene recombination, gene replacement, gene expression, and gene silencing. These large capacity, customizable, multiplex, and all-in-one phage-based AVVs represent an additional category of nanomaterial that could potentially transform gene therapies and personalized medicine.
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Bacteriófago T4 , Genoma Humano , Humanos , Bacteriófago T4/genética , Bacteriófago T4/metabolismo , Vetores Genéticos/genética , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , DNA Viral/genéticaRESUMO
PURPOSE: Deficiency of adenosine deaminase 2 (DADA2), an autosomal recessive autoinflammatory disorder caused by biallelic loss-of-function variants in adenosine deaminase 2 (ADA2), has not been systemically investigated in Chinese population yet. We aim to further characterize DADA2 cases in China. METHODS: A retrospective analysis of patients with DADA2 identified through whole exome sequencing (WES) at seventeen rheumatology centers across China was conducted. Clinical characteristics, laboratory findings, genotype, and treatment response were analyzed. RESULTS: Thirty patients with DADA2 were enrolled between January 2015 and December 2021. Adenosine deaminase 2 enzymatic activity was low in all tested cases to confirm pathogenicity. Median age of disease presentation was 4.3 years and the median age at diagnosis was 7.8 years. All but one patient presented during childhood and two subjects died from complications of their disease. The patients most commonly presented with systemic inflammation (92.9%), vasculitis (86.7%), and hypogammaglobinemia (73.3%) while one patient presented with bone marrow failure (BMF) with variable cytopenia. Twenty-three (76.7%) patients were treated with TNF inhibitors (TNFi), while two (6.7%) underwent hematopoietic stem cell transplantation (HSCT). They all achieved clinical remission. A total of thirty-nine ADA2 causative variants were identified, six of which were novel. CONCLUSION: To establish early diagnosis and improve clinical outcomes, genetic screening and/or testing of ADA2 enzymatic activity should be performed in patients with suspected clinical features. TNFi is considered as first line treatment for those with vascular phenotypes. HSCT may be beneficial for those with hematological disease or in those who are refractory to TNFi.
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Adenosina Desaminase , Peptídeos e Proteínas de Sinalização Intercelular , Humanos , Adenosina Desaminase/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Estudos de Coortes , Estudos Retrospectivos , MutaçãoRESUMO
OBJECTIVES: The genus Streptococcus contains species of important zoonotic pathogens such as those that cause bovine mastitis. Unfortunately, many Streptococcus species have developed antibiotic resistance. Phage lysins are considered promising alternatives to antibiotics because it is difficult for bacteria to develop lysin resistance. However, there remains a lack of phage lysin resources for the treatment of streptococci-induced mastitis. METHODS: We identified the prophage lysin Lys0859 from the genome of the Streptococcus suis SS0859 strain. Lys0859 was subsequently characterized to determine its host range, MIC, bactericidal activity in milk, and ability to clear biofilms in vitro. Finally, to determine the effects of Lys0859 on the treatment of both bovine mastitis and S. suis infection in vivo, we established models of Streptococcus agalactiae ATCC 13813-induced mastitis and S. suis serotype 2 SC19 systemic infection. RESULTS: Our results demonstrate that Lys0859 possesses broad-spectrum lytic activity against Streptococcus and Staphylococcus species isolated from animals with bovine mastitis and 15 serotypes of S. suis isolated from swine. Intramammary and intramuscular injection of Lys0859 reduced the number of bacteria in mammary tissue by 3.75 and 1.45 logs compared with the PBS group, respectively. Furthermore, 100 µg/mouse of Lys0859 administered intraperitoneally at 1 h post-infection protected 83.3% (5/6) of mice from a lethal dose of S. suis infection. CONCLUSIONS: Overall, our results enhance the understanding and development of new strategies to combat both streptococci-induced mastitis and S. suis infection.
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Bacteriófagos , Mastite Bovina , Infecções Estreptocócicas , Fagos de Streptococcus , Streptococcus suis , Feminino , Bovinos , Animais , Suínos , Camundongos , Humanos , Prófagos/genética , Mastite Bovina/tratamento farmacológico , Antibacterianos/farmacologia , Infecções Estreptocócicas/microbiologiaRESUMO
Although the synthesis of α-tertiary amino acids (ATAAs) has been extensively studied, the development of an inexpensive and facile methodology to incorporate multifunctionality on ATAAs remains challenging. In this article, we present a single-step radical approach for the modular synthesis of functionally diverse ATAAs. This synthesis takes place under mild conditions with an absence of metals, photocatalysts, and all other additives. We demonstrate the broad applications of this approach on a variety of aliphatic and aromatic carboxylic acids, alkenes, 1,3-enynes, and oxazolones. The results prove that our method provides excellent functional group tolerance and late-stage applicability, as well as gram-scale synthesis via flow chemistry. Additionally, we include mechanistic studies which reveal that the excited state of oxazolone enolate upon light excitation is a key intermediate that acts as a radical precursor and an efficient reductant.
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Alcenos , Aminoácidos , Alcenos/química , Ácidos Carboxílicos , MetaisRESUMO
Inflammation is an innate immune response of the body against pathogens and other irritants. The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome is a major player in the inflammatory response against pathogenic microorganisms. In this study, we analyzed the relationship between the NLRP3 inflammasome and the influenza virus NS1 protein, which is involved in host immune escape. The canine influenza virus NS1 protein transcriptionally attenuated proinflammatory cytokines by inhibiting the nuclear factor-κB (NF-κB) activator. NS1 also directly interacted with NLRP3 and blocked ASC (Apoptosis-associated speck-like protein containing CARD) oligomerization, which deactivated the NLRP3 inflammasome. In addition, NS1 inhibited pro-caspase 1 cleavage into caspase-1, which prevents maturation of IL-1ß and IL-18 from their respective precursors, eventually reducing the inflammatory response. Taken together, the influenza NS1 protein evades host immunity, and our findings provide a theoretical basis for the prevention and treatment of canine influenza.
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Doenças do Cão , Influenza Humana , Animais , Caspase 1 , Citocinas , Cães , Humanos , Inflamassomos , Vírus da Influenza A Subtipo H3N2/metabolismo , Interleucina-18 , Interleucina-1beta/metabolismo , Irritantes , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas não Estruturais ViraisRESUMO
The rapid emergence of phage-resistant bacterial mutants is a major challenge for phage therapy. Phage cocktails have been considered one approach to mitigate this issue. However, the synergistic effect of randomly selected phages in the cocktails is ambiguous. Here, we rationally designed a phage cocktail consisting of four phages that utilize the lipopolysaccharide (LPS) O antigen, the LPS outer core, the LPS inner core, and the outer membrane proteins BtuB and TolC on the Salmonella enterica serovar Enteritidis cell surface as receptors. We demonstrated that the four-phage cocktail could significantly delay the emergence of phage-resistant bacterial mutants compared to the single phage. To investigate the fitness costs associated with phage resistance, we characterized a total of 80 bacterial mutants resistant to a single phage or the four-phage cocktail. We observed that mutants resistant to the four-phage cocktail were more sensitive to several antibiotics than the single-phage-resistant mutants. In addition, all mutants resistant to the four-phage cocktail had significantly reduced virulence compared to wild-type strains. Our mouse model of Salmonella Enteritidis infection also indicated that the four-phage cocktail exhibited an enhanced therapeutic effect. Together, our work demonstrates an efficient strategy to design phage cocktails by combining phages with different bacterial receptors, which can steer the evolution of phage-resistant strains toward clinically exploitable phenotypes. IMPORTANCE The selection pressure of phage promotes bacterial mutation, which results in a fitness cost. Such fitness trade-offs are related to the host receptor of the phage; therefore, we can utilize knowledge of bacterial receptors used by phages as a criterion for designing phage cocktails. Here, we evaluated the efficacy of a phage cocktail made up of phages that target four different receptors on Salmonella Enteritidis through in vivo and in vitro experiments. Importantly, we found that pressure from phage cocktails with different receptors can drive phage-resistant bacterial mutants to evolve in a direction that entails more severe fitness costs, resulting in reduced virulence and increased susceptibility to antibiotics. These findings suggest that phage cocktail therapy using combinations of phages targeting different important receptors (e.g., LPS or the efflux pump AcrAB-TolC) on the host surface can steer the host bacteria toward more detrimental surface mutations than single-phage therapy, resulting in more favorable therapeutic outcomes.
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Bacteriófagos , Infecções por Salmonella , Camundongos , Animais , Salmonella enteritidis , Bacteriófagos/genética , Lipopolissacarídeos/metabolismo , Virulência , Antígenos O , Antibacterianos/farmacologia , Proteínas de MembranaRESUMO
First discovered in the 1980s, retrons are bacterial genetic elements consisting of a reverse transcriptase and a non-coding RNA (ncRNA). Retrons mediate antiphage defence in bacteria but their structure and defence mechanisms are unknown. Here, we investigate the Escherichia coli Ec86 retron and use cryo-electron microscopy to determine the structures of the Ec86 (3.1 Å) and cognate effector-bound Ec86 (2.5 Å) complexes. The Ec86 reverse transcriptase exhibits a characteristic right-hand-like fold consisting of finger, palm and thumb subdomains. Ec86 reverse transcriptase reverse-transcribes part of the ncRNA into satellite, multicopy single-stranded DNA (msDNA, a DNA-RNA hybrid) that we show wraps around the reverse transcriptase electropositive surface. In msDNA, both inverted repeats are present and the 3' sides of the DNA/RNA chains are close to the reverse transcriptase active site. The Ec86 effector adopts a two-lobe fold and directly binds reverse transcriptase and msDNA. These findings offer insights into the structure-function relationship of the retron-effector unit and provide a structural basis for the optimization of retron-based genome editing systems.
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Escherichia coli , DNA Polimerase Dirigida por RNA , Sequência de Aminoácidos , Sequência de Bases , Microscopia Crioeletrônica , DNA , DNA Bacteriano , Conformação de Ácido NucleicoRESUMO
OBJECTIVE: This study was performed to examine the relationship between the C-reactive protein/albumin ratio (CAR) and the prognosis of patients with lung adenocarcinoma and thus provide a reference for evaluating the prognosis of lung adenocarcinoma. METHODS: The clinical data of 130 patients with lung adenocarcinoma were retrospectively collected and analyzed. The patients' overall survival (OS) time was calculated, and the factors affecting OS were statistically analyzed. RESULTS: The CAR was correlated with sex, clinical stage, brain metastasis, S100 calcium-binding protein B (S100B), interleukin 17, myelin basic protein, squamous cell carcinoma antigen (SCC-Ag), and the lymphocyte count. The median OS was significantly shorter in the high- than low-CAR group (18 vs. 64 months, respectively). The CAR, clinical stage, brain metastasis, S100B, interleukin 17, SCC-Ag, C-reactive protein, albumin, and neutrophil count affected the OS of patients with lung adenocarcinoma. The CAR and clinical stage were independent risk factors for a poor prognosis in patients with lung adenocarcinoma. CONCLUSIONS: The CAR and clinical stage are independent risk factors for OS in patients with lung adenocarcinoma.
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Adenocarcinoma de Pulmão , Neoplasias Encefálicas , Neoplasias Pulmonares , Albuminas/metabolismo , Proteína C-Reativa/metabolismo , Humanos , Interleucina-17 , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Prognóstico , Estudos RetrospectivosRESUMO
Background: Methicillin-resistant Staphylococcus aureus (MRSA) is an important zoonotic pathogen worldwide. Infections due to MRSA are associated with higher mortality rates compared with methicillin-susceptible S. aureus. Meanwhile, bacteriophages have been shown to overcome the emergence of MRSA. Methods: Phage PHB22a, PHB25a, PHB38a, and PHB40a were isolated. Here, we evaluated the ability of a phage cocktail containing phages PHB22a, PHB25a, PHB38a, and PHB40a against MRSA S-18 strain in vivo and in vitro. Phage whole-genome sequencing, host-range determination, lytic activity, and biofilm clearance experiments were performed in vitro. Galleria mellonella larvae and a mouse systemic infection model to evaluate the efficacy of phage therapy in vivo. Results: The phage cocktail exhibited enhanced antibacterial and anti-biofilm effects compared to the single phage. Phage cocktail contained with Ca2+/Zn2+ significantly reduced the number of viable bacteria (24-h or 48-h biofilm) by more than 0.81-log compared to the phage cocktail alone. Furthermore, we demonstrated that the addition of Ca2+ and Zn2+ phage cocktail could increase the survival rate of G. mellonella larvae infected with S. aureus by 10% compared with phage cocktail alone. This was further confirmed in the mouse model, which showed a 2.64-log reduction of host bacteria S-18, when Ca2+ and Zn2+ were included in the cocktail compared with the phage cocktail alone. Conclusion: Our results indicated that phage cocktail supplemented with Ca2+/Zn2+ could effectively remove bacteria in biofilms and mice tissues infected with S. aureus.
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Developing influenza vaccines that protect against a broad range of viruses is a global health priority. Several conserved viral proteins or domains have been identified as promising targets for such vaccine development. However, none of the targets is sufficiently immunogenic to elicit complete protection, and vaccine platforms that can enhance immunogenicity and deliver multiple antigens are desperately needed. Here, we report proof-of-concept studies for the development of next-generation influenza vaccines using the bacteriophage T4 virus-like particle (VLP) platform. Using the extracellular domain of influenza matrix protein 2 (M2e) as a readout, we demonstrate that up to ~1,281 M2e molecules can be assembled on a 120 x 86 nanometer phage capsid to generate M2e-T4 VLPs. These M2e-decorated nanoparticles, without any adjuvant, are highly immunogenic, stimulate robust humoral as well as cellular immune responses, and conferred complete protection against lethal influenza virus challenge. Potentially, additional conserved antigens could be incorporated into the M2e-T4 VLPs and mass-produced in E. coli in a short amount of time to deal with an emerging influenza pandemic.