Photoactivated antibiotics to treat intracellular infection of bacteria.

Antibiotic resistance combined with pathogen internalization leads to debilitating infections. Here we test novel superoxide producing, stimuli-activated quantum dots (QDs), to treat an intracellular infection of Salmonella enterica serovar Typhimurium in an osteoblast precursor cell line. These QDs are precisely tuned to reduce dissolved oxygen to superoxide and kill bacteria upon stimulation (e.g., light). We show QDs provide tunable clearance at various multiplicities of infection and limited host cell toxicity by modulating their concentration and stimuli intensity, proving the efficacy of superoxide producing QDs for intracellular infection treatment and establishing a framework for further testing in different infection models.

Potentiating antibiotic efficacy via perturbation of non-essential gene expression.

Proliferation of multidrug-resistant (MDR) bacteria poses a threat to human health, requiring new strategies. Here we propose using fitness neutral gene expression perturbations to potentiate antibiotics. We systematically explored 270 gene knockout-antibiotic combinations in Escherichia coli, identifying 90 synergistic interactions. Identified gene targets were subsequently tested for antibiotic synergy on the transcriptomic level via multiplexed CRISPR-dCas9 and showed successful sensitization of E. coli without a separate fitness cost. These fitness neutral gene perturbations worked as co-therapies in reducing a Salmonella enterica intracellular infection in HeLa. Finally, these results informed the design of four antisense peptide nucleic acid (PNA) co-therapies, csgD, fnr, recA and acrA, against four MDR, clinically isolated bacteria. PNA combined with sub-minimal inhibitory concentrations of trimethoprim against two isolates of Klebsiella pneumoniae and E. coli showed three cases of re-sensitization with minimal fitness impacts. Our results highlight a promising approach for extending the utility of current antibiotics.

Análisis del comportamiento clínico-patológico del cáncer de mama mucinoso: primer estudio multicéntrico en México. Revisión a 10 años / Analysis of the clinical-pathological behaviour of mucinous carcinoma of the breast: First multicentre study in Mexico-review at 10 years

Introducción El carcinoma mucinoso de mama es una histología poco frecuente, al que se le ha atribuido buen pronóstico, sin embargo, hay pocos datos sobre su comportamiento en población mexicana. Material y métodos Estudio retrospectivo, descriptivo y observacional de tres centros oncológicos de referencia en México, se analizaron características clínicas e histopatológicas de pacientes con carcinoma mucinoso de mama en el periodo comprendido de 2007 a 2017. Resultados Se diagnosticaron 152 pacientes con carcinoma mucinoso puro de mama. Mediana de edad de 56 años. Se encontró asociación entre el tamaño tumoral (p = 0,002) y la afección ganglionar (p < 0,001) con la supervivencia global. Para supervivencia libre de enfermedad, se identificó como factor asociado la afección ganglionar y el inmunofenotipo; con una media de supervivencia libre de enfermedad (SLE) de 143,5 meses para luminal A, 115,4 meses para luminal B, 81 meses para triple negativo y 16 meses para Her 2, p < 0,001. Conclusiones La afección ganglionar es un factor de riesgo para recurrencia de cáncer de mama mucinoso. El tamaño tumoral y una mayor afección ganglionar se relacionaron con un pronóstico adverso en la supervivencia global. (AU)

Introduction Mucinous carcinoma of the breast is an infrequent histological type. Prognosis is believed to be good but there are few data on its behaviour in the Mexican population. Material and methods Retrospective, descriptive, observational study performed in 3 oncological referral centres in Mexico. We analysed the clinical and histopathological characteristics of patients with mucinous carcinoma of the breast between 2007 and 2017. Results A total of 152 patients were diagnosed with pure mucinous breast carcinoma. The median age was 56 years. An association was found between tumoural size (p = 0.002) and lymph node involvement (p < 0.001) with overall survival. The factors associated with disease-free survival were lymph node involvement and immunophenotype. Mean disease-free survival was 143.5 months for luminal A, 115.4 months for luminal B, 81 months for triple negative and 16 months for Her 2, p < 0.001. Conclusions Lymph node involvement is a risk factor for recurrence of mucinous carcinoma of the breast. Tumoural size and greater lymph node involvement are related to worse overall survival. (AU)

Facile accelerated specific therapeutic (FAST) platform develops antisense therapies to counter multidrug-resistant bacteria.

Multidrug-resistant (MDR) bacteria pose a grave concern to global health, which is perpetuated by a lack of new treatments and countermeasure platforms to combat outbreaks or antibiotic resistance. To address this, we have developed a Facile Accelerated Specific Therapeutic (FAST) platform that can develop effective peptide nucleic acid (PNA) therapies against MDR bacteria within a week. Our FAST platform uses a bioinformatics toolbox to design sequence-specific PNAs targeting non-traditional pathways/genes of bacteria, then performs in-situ synthesis, validation, and efficacy testing of selected PNAs. As a proof of concept, these PNAs were tested against five MDR clinical isolates: carbapenem-resistant Escherichia coli, extended-spectrum beta-lactamase Klebsiella pneumoniae, New Delhi Metallo-beta-lactamase-1 carrying Klebsiella pneumoniae, and MDR Salmonella enterica. PNAs showed significant growth inhibition for 82% of treatments, with nearly 18% of treatments leading to greater than 97% decrease. Further, these PNAs are capable of potentiating antibiotic activity in the clinical isolates despite presence of cognate resistance genes. Finally, the FAST platform offers a novel delivery approach to overcome limited transport of PNAs into mammalian cells by repurposing the bacterial Type III secretion system in conjunction with a kill switch that is effective at eliminating 99.6% of an intracellular Salmonella infection in human epithelial cells.

Design of a De Novo Aggregating Antimicrobial Peptide and a Bacterial Conjugation-Based Delivery System.

Antibacterial resistance necessitates the development of novel treatment methods for infections. Protein aggregates have recently been applied as antimicrobials to disrupt bacterial homeostasis. Past work on protein aggregates has focused on genome mining for aggregation-prone sequences in bacterial genomes rather than on rational design of aggregating antimicrobial peptides. Here, we use a synthetic biology approach to design an artificial gene encoding a de novo aggregating antimicrobial peptide. This artificial gene, opaL (overexpressed protein aggregator lipophilic), disrupts bacterial homeostasis by expressing extremely hydrophobic peptides. When this hydrophobic sequence is disrupted by acidic residues, consequent aggregation and antimicrobial effect decrease. Further, we developed a probiotic delivery system using the broad-host range conjugative plasmid RK2 to transfer the gene from donor to recipient bacteria. We utilize RK2 to mobilize a shuttle plasmid carrying opaL by adding the RK2 origin of transfer. We show that opaL is nontoxic to the donor, allowing for maintenance and transfer since its expression is under control of a promoter with a recipient-specific T7 RNA polymerase. Upon mating of donor and recipient Escherichia coli, we observe selective growth repression in T7 polymerase-expressing recipients. This technique could be used to target desired pathogens by selecting pathogen-specific promoters to control T7 RNA polymerase expression and provides a basis for the design and delivery of aggregating antimicrobial peptides.

Structure of a Human Astrovirus Capsid-Antibody Complex and Mechanistic Insights into Virus Neutralization.

Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease. IMPORTANCE: Human astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.

Attenuated response of aged mice to respiratory Francisella novicida is characterized by reduced cell death and absence of subsequent hypercytokinemia.

BACKGROUND: Pneumonia and pulmonary infections are major causes of mortality among the growing elderly population. Age associated attenuations of various immune parameters, involved with both innate and adaptive responses are collectively known as immune senescence. These changes are likely to be involved with differences in host susceptibility to disease between young and aged individuals. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this study was to assess potential age related differences in the pulmonary host response in mice to the Gram-negative respiratory pathogen, Francisella novicida. We intranasally infected mice with F. novicida and compared various immune and pathological parameters of the pulmonary host response in both young and aged mice. CONCLUSIONS/SIGNIFICANCE: We observed that 20% of aged mice were able to survive an intranasal challenge with F. novicida while all of their younger cohorts died consistently within 4 to 6 days post infection. Further experiments revealed that all of the aged mice tested were initially able to control bacterial replication in the lungs as well as at distal sites of replication compared with young mice. In addition, the small cohort of aged survivors did not progress to a severe sepsis syndrome with hypercytokinemia, as did all of the young adult mice. Finally, a lack of widespread cell death in potential aged survivors coupled with a difference in cell types recruited to sites of infection within the lung confirmed an altered host response to Francisella in aged mice.