Engineered Bacteriophages Containing Anti-CRISPR Suppress Infection of Antibiotic-Resistant P. aeruginosa.

The therapeutic use of bacteriophages (phages) provides great promise for treating multidrug-resistant (MDR) bacterial infections. However, an incomplete understanding of the interactions between phages and bacteria has negatively impacted the application of phage therapy. Here, we explored engineered anti-CRISPR (Acr) gene-containing phages (EATPs, eat Pseudomonas) by introducing Type I anti-CRISPR (AcrIF1, AcrIF2, and AcrIF3) genes into the P. aeruginosa bacteriophage DMS3/DMS3m to render the potential for blocking P. aeruginosa replication and infection. In order to achieve effective antibacterial activities along with high safety against clinically isolated MDR P. aeruginosa through an anti-CRISPR immunity mechanism in vitro and in vivo, the inhibitory concentration for EATPs was 1 × 108 PFU/mL with a multiplicity of infection value of 0.2. In addition, the EATPs significantly suppressed the antibiotic resistance caused by a highly antibiotic-resistant PA14 infection. Collectively, these findings provide evidence that engineered phages may be an alternative, viable approach by which to treat patients with an intractable bacterial infection, especially an infection by clinically MDR bacteria that are unresponsive to conventional antibiotic therapy. IMPORTANCE Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic Gram-negative bacterium that causes severe infection in immune-weakened individuals, especially patients with cystic fibrosis, burn wounds, cancer, or chronic obstructive pulmonary disease (COPD). Treating P. aeruginosa infection with conventional antibiotics is difficult due to its intrinsic multidrug resistance. Engineered bacteriophage therapeutics, acting as highly viable alternative treatments of multidrug-resistant (MDR) bacterial infections, have great potential to break through the evolutionary constraints of bacteriophages to create next-generation antimicrobials. Here, we found that engineered anti-CRISPR (Acr) gene-containing phages (EATPs, eat Pseudomonas) display effective antibacterial activities along with high safety against clinically isolated MDR P. aeruginosa through an anti-CRISPR immunity mechanism in vitro and in vivo. EATPs also significantly suppressed the antibiotic resistance caused by a highly antibiotic-resistant PA14 infection, which may provide novel insight toward developing bacteriophages to treat patients with intractable bacterial infections, especially infections by clinically MDR bacteria that are unresponsive to conventional antibiotic therapy.

Analysis of Nuclear Encoded Mitochondrial Gene Networks in Cervical Cancer.

BACKGROUND: Cervical cancer (CC) is one of the most common female cancers in many developing and underdeveloped countries. High incidence, late presentation, and mortality suggested the need for molecular markers. Mitochondrial defects due to abnormal expression of nuclear-encoded mitochondrial genes (NEMG) have been reported during cancer progression. Nevertheless, the application of NEMG for the prognosis of CC is still elusive. Herein, we aimed to investigate the associations between NEMG and CC prognosis. MATERIALS AND METHODS: The differentially expressed genes (DEG) in the TCGA-CESC dataset and NEMGs were retrieved from TACCO and Mitocarta2.0 databases, respectively. The impact of methylation on NEMG expression were predicted using DNMIVD and UALCAN tools. HCMDB tool was used to predict genes having metastatic potential. The prognostic models were constructed using DNMIVD, TACCO, GEPIA2, and SurvExpress. The functional enrichment analysis (FEA) was performed using clusterProfiler. The protein-protein interaction network (PPIN) was constructed to identify the hub genes (HG) using String and CytoHubba tools. Independent validation of the HG was performed using Oncomine and Human Protein Atlas databases. The druggable genes were predicted using DGIdb. RESULTS: Among the 52 differentially expressed NEMG, 15 were regulated by DNA methylation. The expression level of 16, 10, and 7 has the potential for CC staging, prediction of metastasis, and prognosis. Moreover, 1 driver gene and 16 druggable genes were also identified. The FEA identified the enrichment of cancer-related pathways, including AMPK and carbon metabolism in cancer. The combined expression of 10 HG has been shown to affect patient survival. CONCLUSION: Our findings suggest that the abnormal expression of NEMGs may play a critical role in CC development and progression. The genes identified in our study may serve as a prognostic indicator and therapeutic target in CC.
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From virus to inflammation, how influenza promotes lung damage.

Despite seasonal vaccines, influenza-related hospitalization and death rates have remained unchanged over the past 5 years. Influenza pathogenesis has 2 crucial clinical components; first, influenza causes acute lung injury that may require hospitalization. Second, acute injury promotes secondary bacterial pneumonia, a leading cause of hospitalization and disease burden in the United States and globally. Therefore, developing an effective therapeutic regimen against influenza requires a comprehensive understanding of the damage-associated immune-mechanisms to identify therapeutic targets for interventions to mitigate inflammation/tissue-damage, improve antiviral immunity, and prevent influenza-associated secondary bacterial diseases. In this review, the pathogenic immune mechanisms implicated in acute lung injury and the possibility of using lung inflammation and barrier crosstalk for developing therapeutics against influenza are highlighted.

Anti-capsular immunity to Streptococcus pneumoniae serotype 22F prevents bacterial transmission in murine colonization and influenza virus co-infection models.

We evaluated the effectiveness of anti-22F serotype immunity in the prevention of Streptococcus pneumoniae (Spn) bacterial transmission during colonization and influenza virus co-infection. Mice were immunized with 22F formulation and later colonized with Spn or co-infected with Spn and influenza virus. The 22F antisera exhibited strong reactivity to 22F bacteria and promoted the opsonic uptake of Spn by the neutrophils. The 22F vaccination led to a significant reduction of bacterial densities in the nasopharynx and prevented bacterial transmission during colonization and co-infection. The transfer of 22F antisera to infant mice resulted in reduced bacterial transmission in colonization and co-infection models.

A PCR-based method for quantifying neutrophils in human nasal secretions.

Neutrophil recruitment to the nasopharynx (NP) is a central event in resolution of NP-initiated microbial infections. A vigorous neutrophil response in infected tissues is also associated with the outcome of adverse tissue pathology. Therefore, differences in infection-induced tissue neutrophil numbers may correlate with pathogenesis events. Existing methods of quantifying neutrophils require evaluation of NP samples within hours of procurement as flow cytometry based cell quantification methods require live neutrophil cells. Therefore, we developed a novel RT-PCR method that could reliably quantify neutrophil counts in frozen NP wash samples. mRNA transcripts of the genes encoding CD16, CD18, and CD62L were identified as neutrophil-specific in NP samples and not significantly variable in response to stimulation by heat killed bacteria, and can be used to derive an accurate assessment of neutrophil content in a sample even in the presence of epithelial cells. Using flow cytometry as a comparator, the method was validated in human NP wash samples. We conclude that this PCR-based method should prove useful for providing a quantitative estimate of neutrophil recruitment to the NP during infection and pathogenesis.

Rate of progression of cognitive decline in Alzheimer's disease: effect of butyrylcholinesterase K gene variation.

OBJECTIVE: To determine whether individuals with Alzheimer's disease (AD) and the K variant allele of butyrylcholinesterase have a slower rate of cognitive decline than those without the K variant allele of butyrylcholinesterase. METHOD: The cognitive status of 339 community based subjects with AD was assessed with the Mini Mental State Examination at baseline and yearly over a three year follow up period. The rates of cognitive decline of subjects with and without the K variant allele were compared. RESULT: Presence of the K allele was associated with a slower average rate of cognitive decline in subjects with severe AD. CONCLUSIONS: This finding is consistent with the suggestion that the K variant of butyrylcholinesterase has an important role in disease progression in AD, and this may have implications for treatment.