Cryo-electron microscopy of the f1 filamentous phage reveals insights into viral infection and assembly.

Phages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ffs have seen an extraordinary range of applications, yet the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. In this work, we use cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods to determine a structure of a filamentous virus including the tips. We show that structure combined with mutagenesis can identify phage domains that are important in bacterial attack and for release of new progeny, allowing new models to be proposed for the phage lifecycle.

Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage.

The central dogma of biology does not allow for the study of glycans using DNA sequencing. We report a liquid glycan array (LiGA) platform comprising a library of DNA 'barcoded' M13 virions that display 30-1,500 copies of glycans per phage. A LiGA is synthesized by acylation of the phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins, such as CD22, on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identify the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo.

Genetically Encoded Fragment-Based Discovery from Phage-Displayed Macrocyclic Libraries with Genetically Encoded Unnatural Pharmacophores.

Genetically encoded macrocyclic peptide libraries with unnatural pharmacophores are valuable sources for the discovery of ligands for many targets of interest. Traditionally, generation of such libraries employs "early stage" incorporation of unnatural building blocks into the chemically or translationally produced macrocycles. Here, we describe a divergent late-stage approach to such libraries starting from readily available starting material: genetically encoded libraries of peptides. A diketone linchpin 1,5-dichloropentane-2,4-dione converts peptide libraries displayed on phage to 1,3-diketone bearing macrocyclic peptides (DKMP): shelf-stable precursors for Knorr pyrazole synthesis. Ligation of diverse hydrazine derivatives onto DKMP libraries displayed on phage that carries silent DNA-barcodes yields macrocyclic libraries in which the amino acid sequence and the pharmacophore are encoded by DNA. Selection of this library against carbonic anhydrase enriched macrocycles with benzenesulfonamide pharmacophore and nanomolar Kd. The methodology described in this manuscript can graft diverse pharmacophores into many existing genetically encoded phage libraries and significantly increase the value of such libraries in molecular discoveries.

Humoral Immunodeficiency and Immune Globulin Replacement Therapy (IGRT) Usage in DiGeorge Syndrome.

PURPOSE: An analysis of patients in the United States Immunodeficiency Network (USIDNET) registry previously described a discordance in the reported prevalence of humoral immune deficiency in patients with DiGeorge syndrome (DGS) and its treatment. The primary purpose of this study is to evaluate the rates of humoral immunodeficiency and immune globulin replacement therapy (IGRT) use in patients with DiGeorge syndrome in the USIDNET registry as of September 2016, and to correlate IGRT use with prior infections and laboratory evidence of immune deficiency. METHODS: Current patients in the USIDNET registry with DGS were identified. Patients who were treated with immune globulin replacement therapy (IGRT) were compared with those who were untreated with respect to their laboratory findings and clinical history. RESULTS: Four hundred seventy-three patients were identified. The use of IGRT in patients with DGS has increased over time from 3 to 6.6%. IGRT was more common in patients with humoral immune deficiency (18.2% of those with hypogammaglobulinemia, 39.1% of those with documented low vaccine titers), but most patients with evidence of humoral immune deficiency remain untreated with IGRT. Patients treated with IGRT were more likely to have experienced episodes of pneumonia, sepsis, and bacterial skin infections (p < 0.01 for all). CONCLUSIONS: Humoral immune deficiencies were more common among patients with DGS than previously reported. IGRT was used most commonly in patients with DGS who demonstrated frequent or severe bacterial infections. There is still a significant deficit between those with DGS who have laboratory evidence of a humoral immune deficiency and those being treated for it.

Hypothermia: A Sign of Sepsis in Young Infants in the Emergency Department?

OBJECTIVES: Diagnosis of sepsis in young infants can be challenging due to the nonspecific signs, which can include hypothermia. Whether the presence of hypothermia in young infants should prompt evaluation for serious infection is unclear. The objectives were to measure the prevalence of serious infection among infants ≤60 days of age with hypothermia in the emergency department (ED) and determine other clinical features of hypothermic infants who have serious infection. METHODS: This is a retrospective analysis of all infants ≤60 days seen in a children's hospital ED from April 2014 to February 2017. Primary outcome was presence of serious infection, defined as urinary tract infection, bacteremia, meningitis, pneumonia, or herpes virus infection. Hypothermia was defined as a rectal temperature of 36.0°C or less. RESULTS: Of 4797 infants ≤60 days of age seen in the ED, 116 had hypothermia. The prevalence of serious infection was 2.6% (3/116) in hypothermic infants compared with 15.2% (61/401) in febrile infants (P < 0.01). Hypothermic infants with serious infections were more likely to have a history of prematurity, apnea, poor feeding, lethargy, ill-appearance, and respiratory signs than hypothermic infants without serious infection. All 3 hypothermic infants with serious infection had other concerning features. CONCLUSIONS: The prevalence of serious infection in hypothermic young infants in the ED is low. Serious infection is unlikely in infants with isolated hypothermia.

Compositional Bias in Naïve and Chemically-modified Phage-Displayed Libraries uncovered by Paired-end Deep Sequencing.

Understanding the composition of a genetically-encoded (GE) library is instrumental to the success of ligand discovery. In this manuscript, we investigate the bias in GE-libraries of linear, macrocyclic and chemically post-translationally modified (cPTM) tetrapeptides displayed on the M13KE platform, which are produced via trinucleotide cassette synthesis (19 codons) and NNK-randomized codon. Differential enrichment of synthetic DNA {S}, ligated vector {L} (extension and ligation of synthetic DNA into the vector), naïve libraries {N} (transformation of the ligated vector into the bacteria followed by expression of the library for 4.5 hours to yield a "naïve" library), and libraries chemically modified by aldehyde ligation and cysteine macrocyclization {M} characterized by paired-end deep sequencing, detected a significant drop in diversity in {L} → {N}, but only a minor compositional difference in {S} → {L} and {N} → {M}. Libraries expressed at the N-terminus of phage protein pIII censored positively charged amino acids Arg and Lys; libraries expressed between pIII domains N1 and N2 overcame Arg/Lys-censorship but introduced new bias towards Gly and Ser. Interrogation of biases arising from cPTM by aldehyde ligation and cysteine macrocyclization unveiled censorship of sequences with Ser/Phe. Analogous analysis can be used to explore library diversity in new display platforms and optimize cPTM of these libraries.

Making a Case for Pediatric Antimicrobial Stewardship Programs.

Although antimicrobials are commonly used in children, it is important to remember that they can have a profound impact on this unique patient population. Inadvertent consequences of antiinfective use in children include antimicrobial resistance, infection caused by Clostridium difficile, increased risk of obesity, and adverse drug events. In addition, compared with adults, children have different dosing requirements, antimicrobial formulation needs, pharmacokinetics, and antimicrobial susceptibility profiles. Therefore, pediatric-specific antimicrobial stewardship efforts are needed to promote appropriate use of antimicrobials in children. The primary purposes of this review article are to provide a rationale behind pediatric-focused antimicrobial stewardship and to describe currently available evidence regarding the initiatives of pediatric antimicrobial stewardship programs (ASPs). A literature search of the Medline database was performed (from inception through March 2015). The studies included in this review focus on antimicrobial stewardship interventions in inpatient pediatric settings. Ten inpatient studies involving pediatric-focused antimicrobial stewardship interventions were identified from the published literature. Four studies used the core strategy of prospective audit with feedback; two used prior approval. The remaining four used supplemental antimicrobial stewardship strategies (guidelines, clinical pathways, and computerized decision support tools). In general, the interventions resulted in decreased antimicrobial use, reduced antimicrobial costs, and fewer prescribing errors. Children have unique medical needs related to antimicrobials and deserve focused ASP efforts. The literature regarding pediatric antimicrobial stewardship interventions is limited, but published interventions may serve as paradigms for developing pediatric ASPs as demonstrated by the general success of these interventions.

Ff-nano, short functionalized nanorods derived from Ff (f1, fd, or M13) filamentous bacteriophage.

F-specific filamentous phage of Escherichia coli (Ff: f1, M13, or fd) are long thin filaments (860 nm × 6 nm). They have been a major workhorse in display technologies and bionanotechnology; however, some applications are limited by the high length-to-diameter ratio of Ff. Furthermore, use of functionalized Ff outside of laboratory containment is in part hampered by the fact that they are genetically modified viruses. We have now developed a system for production and purification of very short functionalized Ff-phage-derived nanorods, named Ff-nano, that are only 50 nm in length. In contrast to standard Ff-derived vectors that replicate in E. coli and contain antibiotic-resistance genes, Ff-nano are protein-DNA complexes that cannot replicate on their own and do not contain any coding sequences. These nanorods show an increased resistance to heating at 70(∘)C in 1% SDS in comparison to the full-length Ff phage of the same coat composition. We demonstrate that functionalized Ff-nano particles are suitable for application as detection particles in sensitive and quantitative "dipstick" lateral flow diagnostic assay for human plasma fibronectin.

Current trends in perioperative antibiotic use: a survey of otolaryngologists.

This study describes the patterns of perioperative antimicrobial use by otolaryngologists during common otolaryngologic surgical procedures. Through the American Academy of Otolaryngology--Head and Neck Surgery Infectious Diseases Committee, a survey was developed to assess the current practice patterns regarding the use of perioperative antibiotics in otolaryngology. A total of 6903 surveys were sent out; 458 were fully or partially completed, and a total of 442 responses were included in the final analysis. Most physicians reported routinely prescribing antibiotics either preoperatively or postoperatively for 12 of the 17 procedures included in the questionnaire despite providers agreeing that there is not enough evidence to support their use. The most common procedure for which antibiotics were prescribed was laryngectomy (91.1%). Antibiotic use is a common practice during the perioperative period for otolaryngologic procedures; however, there is a discrepancy between utilization and evidence of benefit.

Unrecognized viral respiratory tract infections in premature infants during their birth hospitalization: a prospective surveillance study in two neonatal intensive care units.

OBJECTIVE: To determine the frequency and effects of nosocomial respiratory viral infections (RVIs) in premature neonates, including those who may be asymptomatic. STUDY DESIGN: We performed a year-long surveillance for RVIs in infants <33 weeks gestational age admitted to 2 Syracuse neonatal intensive care units. Infants were enrolled within 3 days of neonatal intensive care unit admission and were sampled for RVIs until discharge using a multiplex polymerase chain reaction assay capable of detecting 17 different respiratory viruses or subtypes. RESULTS: Twenty-six of 50 prematurely born infants (52%) tested positive for a respiratory virus at least once during their birth hospitalization. Testing positive for a respiratory virus was significantly associated with longer length of stay (70 days vs 35 days, P = .002) and prolonged ventilatory support (51 vs 13 days, P = .002). Infants who tested positive for a respiratory virus during their birth hospitalization had more than twice the rate of developing bronchopulmonary dysplasia (P < .05). CONCLUSION: Nosocomial RVIs were frequent in our study population, despite the absence of clinical indicators of illness. Length of hospital stay was significantly longer and a diagnosis of bronchopulmonary dysplasia was more common in infants who had respiratory viruses detected.

Diastereoselective alkylation reactions of 1-methylcyclohexa-2,5-diene-1-carboxylic acid.

The deprotonation and alkylation of 1-methylcyclohexa-2,5-diene-1-carboxylic acid has been investigated under a range of conditions. In all cases, the formation of compounds 14 was found to be completely stereoselective, although compound 14c was formed as an impurity when alkyl iodides were used as electrophiles, and doubly-alkylated compounds 17 were formed in some cases when alkyl bromides were used.

Pneumocystis pneumonia.

Pneumocystis (carinii) jiroveci pneumonia can occur in immunocompromised individuals, especially hematopoietic stem and solid organ transplant recipients and those receiving immunosuppressive agents, and is the most common opportunistic infection in persons with advanced human immunodeficiency virus (HIV) infection. The Pneumocystis genus was initially mistaken as a trypanosome and later as a protozoan. Genetic analysis identified the organism as a unicellular fungus. Pneumocystis jiroveci is the species responsible for human infections. A slow indolent time course with symptoms of pneumonia progressing over weeks to months is characteristic in HIV-infected patients. Fulminant respiratory failure associated with fever and dry cough is typical in non-HIV-infected patients. Definitive diagnosis relies on histopathological testing of sputum, induced or sampled by fiberoptic bronchoscopy with bronchoalveolar lavage. The first-line drug for treatment and prevention is trimethoprim-sulfamethoxazole.

Characterization of a dual-function domain that mediates membrane insertion and excision of Ff filamentous bacteriophage.

The filamentous phage Ff (f1, fd, or M13) of Escherichia coli is assembled at the cell membranes by a process that is morphologically similar to that of pilus assembly. The release of the filament virion is mediated by excision from the membrane; conversely, entry into a host cell is mediated by insertion of the virion coat proteins into the membrane. The N-terminal domains of the minor virion protein pIII have the sole role of binding to host receptors during infection. In contrast, the C domain of pIII is required for two opposite functions: insertion of the virion into the membrane during infection and excision at the termination step of assembly/secretion. We identified a 28-residue-long segment in the pIII C domain, which is required for phage entry but dispensable for release from the membrane at the end of assembly. This segment, which we named the infection-competence segment (ICS), works only in cis with the N-terminal receptor-binding domains and does not require the equivalent ICS sequences in other subunits within the virion cap. The ICS contains a predicted amphipathic α-helix and is rich in small amino acids, Gly, Ala, and Ser, which are arranged as a [small]XXX[small]XX[small]XXX[small]XXX[small] motif. Scanning Ala/Gly mutagenesis of ICS showed that small residues are compatible with infection. Overall, organization of the C domain is reminiscent of α-helical pore-forming toxins' membrane insertion domains. The unique ability of pIII to mediate both membrane insertion and excision allowed us to compare these two fundamental membrane transactions and to show that receptor-triggered insertion is a more complex process than excision from membranes.

Filamentous bacteriophage: biology, phage display and nanotechnology applications.

Filamentous bacteriophage, long and thin filaments that are secreted from the host cells without killing them, have been an antithesis to the standard view of head-and-tail bacterial killing machines. Episomally replicating filamentous phage Ff of Escherichia coli provide the majority of information about the principles and mechanisms of filamentous phage infection, episomal replication and assembly. Chromosomally- integrated "temperate" filamentous phage have complex replication and integration, which are currently under active investigation. The latter are directly or indirectly implicated in diseases caused by bacterial pathogens Vibrio cholerae, Pseudomonas aeruginosa and Neisseria meningitidis. In the first half of the review, both the Ff and temperate phage are described and compared. A large section of the review is devoted to an overview of phage display technology and its applications in nanotechnology.

Empyema of preexisting subdural hemorrhage caused by a rare salmonella species after exposure to bearded dragons in a foster home.

An infant had a subdural empyema caused by the rare Salmonella species enterica subspecies houtenae (IV) serotype 44:z4,z23:- after only indirect exposure to exotic reptiles in her foster home. Infants recovering from preexisting subdural hematoma are at risk for development of empyema.

Diminished inflammatory responses to natural pneumovirus infection among older mice.

Immune responses to virus infection undergo significant change as part of the aging process. Here we examine the inflammatory responses of older, but otherwise immunologically naive mice to infection with pneumonia virus of mice (PVM). Although we see no changes in the extent or kinetics of virus replication, we observe diminished local production of inflammatory mediators, including MIP-1alpha, JE/MCP-1, IFN-gamma and IFN-gamma-induced MIG and IP-10, and interleukins (IL)-6 and IL-17. Levels of KC and IL-1alpha remained unchanged. Age-dependent diminished production of proinflammatory mediators was associated with diminished recruitment of granulocytes and reduced severity of clinical responses, including weight loss and respiratory dysfunction. The differences observed when comparing these results to those reported among elderly human subjects may be related to the specific extent of aging and its impact on biochemical and cellular inflammatory responses and/or the role of lifetime virus re-exposure on the clinical outcome from acute pneumovirus disease.

Respiratory dysfunction and proinflammatory chemokines in the pneumonia virus of mice (PVM) model of viral bronchiolitis.

We explore relationships linking clinical symptoms, respiratory dysfunction, and local production of proinflammatory chemokines in the pneumonia virus of mice (PVM) model of viral bronchiolitis. With a reduced inoculum of this natural rodent pathogen, we observe virus clearance by day 9, while clinical symptoms and respiratory dysfunction persist through days 14 and 17 postinoculation, respectively. Via microarray and ELISA, we identify expression profiles of proinflammatory mediators MIP-1alpha, MCP-1, and MIP-2 that correlate with persistent respiratory dysfunction. MIP-1alpha is localized in bronchial epithelium, which is also the major site of PVM replication. Interferon-gamma was detected in lung tissue, but at levels that do not correlate with respiratory dysfunction. Taken together, we present a modification of our pneumovirus infection model that results in improved survival and data that stand in support of a connection between local production of specific mediators and persistent respiratory dysfunction in the setting of acute viral bronchiolitis.

Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII.

Protein III (pIII) of filamentous phage is required for both the beginning and the end of the phage life cycle. The infection starts by binding of the N-terminal N2 and N1 domains to the primary and secondary host receptors, F pilus and TolA protein, respectively, whereas the life cycle terminates by the C-terminal domain-mediated release of the membrane-anchored virion from the cell. It has been assumed that the role of the C-terminal domain of pIII in the infection is that of a tether for the receptor-binding domains N1N2 to the main body of the virion. In a poorly understood process that follows receptor binding, the virion disassembles as its protein(s) become integrated into the host inner membrane, resulting in the phage genome entry into the bacterial cytoplasm. To begin revealing the mechanism of this process, we showed that tethering the functional N1N2 receptor-binding domain to the virion via termination-incompetent C domain abolishes infection. This infection defect cannot be complemented by in trans supply of the functional C domain. Therefore, the C domain of pIII acts in concert with the receptor-binding domains to mediate the post receptor binding events in the infection. Based on these findings, we propose a model in which binding of the N1 domain to the periplasmic portion of TolA, the secondary receptor, triggers in cis a conformational change in the C domain, and that this change opens or unlocks the pIII end of the virion, allowing the entry phase of infection to proceed. To our knowledge, this is the first virus that uses the same protein domain both for the insertion into and release from the host membrane.