bluuebunny/crossref_metadata_2025 huggingface dataset:
Toxigenic clostridia
Authors: Hatheway C L
Published: June 2016

Toxigenic clostridia belonging to 13 recognized species are discussed in this review. Each species or group of organisms is, in general, introduced by presenting the historical aspects of its discovery by early investigators of human and animal diseases. The diseases caused by each species or group are described and usually discussed in relation to the toxins involved in the pathology. Morphological and physiological characteristics of the organisms are described. Finally, the toxins produced by each organism are listed, with a presentation of their biological activities and physical and biochemical characteristics. The complete amino acid sequences for some are known, and some of the genes have been cloned. The term toxin is used loosely to include the various antigenic protein products of these organisms with biological and serological activities which have served as distinguishing characteristics for differentiation and classification. Some of these factors are not truly toxic and have no known role in pathogenicity. Some of the interesting factors common to more than one species or group are the following: neurotoxins, lethal toxins, lecithinases, oxygen-labile hemolysins, binary toxins, and ADP-ribosyltransferases. Problems in bacterial nomenclature and designation of biologically active factors are noted.
Lectins and their application to clinical microbiology
Authors: Slifkin M , Doyle R J
Published: June 2016

Lectins are generally associated with plant or animal components, selectively bind carbohydrates, and interact with procaryotic and eucaryotic cells. Lectins have various specificities that are associated with their ability to interact with acetylaminocarbohydrates, aminocarbohydrates, sialic acids, hexoses, pentoses, and as other carbohydrates. Microbial surfaces generally contain many of the sugar residues that react with lectins. Lectins are presently used in the clinical laboratory to type blood cells and are used in a wide spectrum of applications, including, in part, as carriers of chemotherapeutic agents, as mitogens, for fractionation of animal cells, and for investigations of cellular surfaces. Numerous studies have shown that lectins can be used to identify rapidly certain microorganisms isolated from a clinical specimen or directly in a clinical specimen. Lectins have been demonstrated to be important diagnostic reagents in the major realms of clinical microbiology. Thus, they have been applied in bacteriology, mycology, mycobacteriology, and virology for the identification and/or differentiation of various microorganisms. Lectins have been used successfully as epidemiologic as well as taxonomic markers of specific microorganisms. Lectins provide the clinical microbiologist with cost-effective and potential diagnostic reagents. This review describes the applications of lectins in clinical microbiology.
Resistance to vancomycin and teicoplanin: an emerging clinical problem
Authors: Johnson A P , Uttley A H , Woodford N , George R C
Published: June 2016

Vancomycin and teicoplanin are glycopeptides active against a wide range of gram-positive bacteria. For 30 years following the discovery of vancomycin in 1956, vancomycin resistance was not detected among normally susceptible bacteria recovered from human specimens. Since 1986, however, bacteria resistant to vancomycin or teicoplanin or both have been described. Strains of the genera Leuconostoc, Lactobacillus, Pediococcus, and Erysipelothrix seem inherently resistant to glycopeptides. Species and strains of enterococci and coagulase-negative staphylococci appear to have acquired or developed resistance. There are at least two categories of glycopeptide resistance among enterococci, characterized by either high-level resistance to vancomycin (MIC, greater than or equal to 64 mg/liter) and teicoplanin (MIC, greater than or equal to 8 mg/liter) or lower-level vancomycin resistance (MIC, 32 to 64 mg/liter) and teicoplanin susceptibility (MIC, less than or equal to 1 mg/liter). The two categories appear to have similar resistance mechanisms, although genetic and biochemical studies indicate that they have arisen independently. Among coagulase-negative staphylococci, strains for which vancomycin MICs are up to 20 mg/liter or teicoplanin MICs are 16 to 32 mg/liter have been reported, but cross-resistance between these glycopeptides varies. The selective advantage accorded to glycopeptide-resistant bacteria and the observation that high-level resistance in enterococci is transferable suggest that such resistance may be expected to increase in incidence. Clinicians and microbiologists need to be aware of this emerging problem.
Nutritionally variant streptococci
Authors: Ruoff K L
Published: June 2016

Streptococci requiring either pyridoxal or L-cysteine for growth were first observed 30 years ago as organisms forming satellite colonies adjacent to colonies of "helper" bacteria. Although they were previously considered nutritional mutants of viridans streptococcal species, the nutritionally variant streptococci (NVS) are currently thought to belong to distinct species of the genus Streptococcus. NVS strains may display pleomorphic cellular morphologies, depending on their growth conditions, and are distinguished from most other streptococci by enzymatic and serological characteristics and the presence of a cell wall chromophore. NVS are found as normal inhabitants of the oral cavity, and in addition to their participation in endocarditis, they have been isolated from a wide range of clinical specimens. Endocarditis caused by NVS is often difficult to eradicate; combinations of penicillin and an aminoglycoside are recommended for treatment. The unique physiological features of the NVS contribute to the difficulties encountered in their recovery from clinical specimens and may play a role in the problems associated with successful treatment of NVS endocarditis.
Pathobiology of papillomavirus-related cervical diseases: prospects for immunodiagnosis
Authors: Crum C P , Barber S , Roche J K
Published: June 2016

In recent years, the relationship between human papillomaviruses (HPV) and genital neoplasia has been explored intensively, and a molecular basis for the role of HPV in the genesis of these diseases has been convincingly demonstrated. These findings have provided justification for efforts to apply this molecular information to the early detection and possible prevention of HPV-related neoplasia. The technology of detecting viral nucleic acids in genital fluids brought with it initial hopes that it would serve to identify women at risk for having or developing precancers or cancers of the cervix. Subsequent studies, however, have demonstrated limitations of the technology for predicting future disease. Recently, molecular immunology has complemented these prior efforts, with the intent to identify serological indices of exposure to HPV and perhaps delineate individuals at risk. The molecular basis for this approach, its limitations, and future prospects for immunodiagnosis are the subject of this review.
The tribe Ehrlichieae and ehrlichial diseases
Authors: Rikihisa Y
Published: June 2016

The tribe Ehrlichieae consists of gram-negative minute cocci that are obligate intracellular parasites classified in the family Rickettsiaceae. Although ehrlichial organisms have been observed in leukocytes for many years, only a few species have been cultured in quantities sufficient for biochemical and molecular analyses. Recents studies on 16S-rRNA sequence analysis and energy metabolism showed that the genus Ehrlichia is closely related to the genus Rickettsia. There is, however, no antigenic cross-reactivity between these genera. Ehrlichial organisms cause a disease called "ehrlichiosis," a noncontagious infectious disease known to be transmitted by a tick in several cases and by a fluke in one case. Ehrlichia spp. infect dogs, ruminants, horses, and humans. Recently, two new ehrlichial diseases, Potomac horse fever and human ehrlichiosis, were discovered in the United States. The etiologic agent of Potomac horse fever, Ehrlichia risticii, is closely related to the known human pathogen Ehrlichia sennetsu. The etiologic agent of human ehrlichiosis is related to Ehrlichia canis, a canine pathogen. In contrast to the genus Rickettsia, members of the tribe Ehrlichieae reside primarily in the cytoplasmic vacuoles of monocytes or granulocytes and cause hematologic abnormalities, lymphadenopathy, and other pathologic changes in the host. However, the actual mechanisms whereby Ehrlichia spp. infect leukocytes, multiply in them, and produce various forms of systemic disease have not been defined. Depending on the ehrlichial species involved, serologic or direct microscopic observation of stained blood smears is currently used to diagnose ehrlichial disease.
Serodiagnosis of parasitic diseases
Authors: Maddison S E
Published: June 2016

In this review on serodiagnosis of parasitic diseases, antibody detection, antigen detection, use of monoclonal antibodies in parasitic serodiagnosis, molecular biological technology, and skin tests are discussed. The focus at the Centers for Disease Control on developing improved antigens, a truly quantitative FAST-enzyme-linked immunosorbent assay, and the very specific immunoblot assays for antibody detection is highlighted. The last two assays are suitable for field studies. Identification of patient response in terms of immunoglobulin class or immunoglobulin G subclass isotypes or both is discussed. Immunoglobulin isotypes may asist in defining the stage of some diseases. In other instances, use of a particular anti-isotype conjugate may increase the specificity of the assay. Monoclonal antibodies have played important roles in antigen purification and identification, in competitive antibody assays with increased sensitivity and specificity, and in assays for antigen detection in serum, body fluids, or excreta. Molecular biological technology has allowed significant advances in the production of defined parasitic serodiagnostic antigens.
Antiviral therapy: current concepts and practices
Authors: Bean B
Published: June 2016

Drugs capable of inhibiting viruses in vitro were described in the 1950s, but real progress was not made until the 1970s, when agents capable of inhibiting virus-specific enzymes were first identified. The last decade has seen rapid progress in both our understanding of antiviral therapy and the number of antiviral agents on the market. Amantadine and ribavirin are available for treatment of viral respiratory infections. Vidarabine, acyclovir, ganciclovir, and foscarnet are used for systemic treatment of herpesvirus infections, while ophthalmic preparations of idoxuridine, trifluorothymidine, and vidarabine are available for herpes keratitis. For treatment of human immunodeficiency virus infections, zidovudine and didanosine are used. Immunomodulators, such as interferons and colony-stimulating factors, and immunoglobulins are being used increasingly for viral illnesses. While resistance to antiviral drugs has been seen, especially among AIDS patients, it has not become widespread and is being intensely studied. Increasingly, combinations of agents are being used: to achieve synergistic inhibition of viruses, to delay or prevent resistance, and to decrease dosages of toxic drugs. New approaches, such as liposomes carrying antiviral drugs and computer-aided drug design, are exciting and promising prospects for the future.
Compounds active against cell walls of medically important fungi
Authors: Hector R F
Published: June 2016

A number of substances that directly or indirectly affect the cell walls of fungi have been identified. Those that actively interfere with the synthesis or degradation of polysaccharide components share the property of being produced by soil microbes as secondary metabolites. Compounds specifically interfering with chitin or beta-glucan synthesis have proven effective in studies of preclinical models of mycoses, though they appear to have a restricted spectrum of coverage. Semisynthetic derivatives of some of the natural products have offered improvements in activity, toxicology, or pharmacokinetic behavior. Compounds which act on the cell wall indirectly or by a secondary mechanism of action, such as the azoles, act against diverse fungi but are usually fungistatic in nature. Overall, these compounds are attractive candidates for further development.
Pathogenesis and pathophysiology of bacterial meningitis
Authors: Tunkel A R , Scheld W M
Published: June 2016

Bacterial meningitis remains a disease with associated unacceptable morbidity and mortality rates despite the availability of effective bactericidal antimicrobial therapy. Through the use of experimental animal models of infection, a great deal of information has been gleaned concerning the pathogenic and pathophysiologic mechanisms operable in bacterial meningitis. Most cases of bacterial meningitis begin with host acquisition of a new organism by nasopharyngeal colonization followed by systemic invasion and development of a high-grade bacteremia. Bacterial encapsulation contributes to this bacteremia by inhibiting neutrophil phagocytosis and resisting classic complement-mediated bactericidal activity. Central nervous system invasion then occurs, although the exact site of bacterial traversal into the central nervous system is unknown. By production and/or release of virulence factors into and stimulation of formation of inflammatory cytokines within the central nervous system, meningeal pathogens increase permeability of the blood-brain barrier, thus allowing protein and neutrophils to move into the subarachnoid space. There is then an intense subarachnoid space inflammatory response, which leads to many of the pathophysiologic consequences of bacterial meningitis, including cerebral edema and increased intracranial pressure. Attenuation of this inflammatory response with adjunctive dexamethasone therapy is associated with reduced concentrations of tumor necrosis factor in the cerebrospinal fluid, with diminished cerebrospinal fluid leukocytosis, and perhaps with improvement of morbidity, as demonstrated in recent clinical trials. Further information on the pathogenesis and pathophysiology of bacterial meningitis should lead to the development of more innovative treatment and/or preventive strategies for this disorder.
Outbreaks of food-borne and waterborne viral gastroenteritis
Authors: Hedberg C W , Osterholm M T
Published: June 2016

Norwalk virus infection is the epidemiologic prototype for outbreaks of food-borne and waterborne gastroenteritis. Around the world, Norwalk virus and Norwalk-like viruses appear to be major causes of food-borne and waterborne illness. Assessment of the overall significance of viral agents to the epidemiology of food-borne and waterborne illness is hampered by the lack of surveillance throughout much of the world. In areas where food-borne and waterborne illness surveillance is conducted, outbreaks of viral gastroenteritis are underreported because of the lack of availability of routine laboratory services to confirm the viral etiology. Routine use of epidemiologic criteria as an alternative to laboratory confirmation will allow better assessments of the importance of viral gastroenteritis until effective laboratory methods can be widely implemented. Outbreaks of viral gastroenteritis have been propagated by contamination of water supplies, raw foods, and ill food handlers. Controlling an outbreak depends on identifying and removing the source of contamination. The demonstrated occurrence of person-to-person transmission and the likely occurrence of transmission of Norwalk-like viruses by aerosol make it necessary to evaluate the potential for transmission by food handlers and servers in every outbreak, regardless of primary source.
Nonculture methods for diagnosis of disseminated candidiasis
Authors: Reiss E , Morrison C J
Published: June 2016

Two of the nonculture approaches to the diagnosis of DC, enzymatic-fluorometric determination of serum D-arabinitol and detection of marker antigens in antigenemia (enolase and CWMP), have been commercialized and have shown promise in limited clinical trials. These approaches are not new but are the culmination of efforts made over 10 or more years. Clearly, further fine-tuning of both metabolite and antigen detection is needed to simplify the methods and to improve their sensitivity and specificity so that they will be valuable in guiding clinical treatment decisions. An alternative approach, detection of DC by DNA amplification methods such as PCR, is a special case of a compelling technology and one that is capable of standardization across microbial genera. The availability of simplified PCR diagnostic methods for DC remains a tantalizing prospect. Nevertheless, the development of methods to release DNA from very small numbers of Candida organisms in the blood in a form that is sufficiently free of inhibitors of PCR will require further intensive effort. The maturation of these converging laboratory approaches to nonculture diagnosis of DC leads to more optimism about the eventual use of these methods in clinical laboratories.
Update on clinical significance of coagulase-negative staphylococci
Authors: Kloos W E , Bannerman T L
Published: June 2016

The clinical significance of coagulase-negative Staphylococcus species (CNS) continues to increase as strategies in medical practice lead to more invasive procedures. Hospitalized patients that are immunocompromised and/or suffering from chronic diseases are the most vulnerable to infection. Since CNS are widespread on the human body and are capable of producing very large populations, distinguishing the etiologic agent(s) from contaminating flora is a serious challenge. For this reason, culture identification should proceed to the species and strain levels. A much stronger case can be made for the identification of a CNS etiologic agent if the same strain is repeatedly isolated from a series of specimens as opposed to the isolation of different strains of one or more species. Strain identity initially can be based on colony morphology, and then one or more molecular approaches can be used to gain information on the genotype. Many of the CNS species are commonly resistant to antibiotics that are being indicated for staphylococcal infections, with the exception of vancomycin. The widespread use of antibiotics in hospitals has provided a reservoir of antibiotic-resistant genes. The main focus on mechanisms of pathogenesis has been with foreign body infections and the role of specific adhesins and slime produced by Staphylococcus epidermidis. Slime can reduce the immune response and opsonophagocytosis, thereby interfering with host defense mechanisms. As we become more aware of the various strategies used by CNS, we will be in a better position to compromise their defense mechanisms and improve treatment.
Rationale for cost-effective laboratory medicine
Authors: Robinson A
Published: June 2016

There is virtually universal consensus that the health care system in the United States is too expensive and that costs need to be limited. Similar to health care costs in general, clinical laboratory expenditures have increased rapidly as a result of increased utilization and inflationary trends within the national economy. Economic constraints require that a compromise be reached between individual welfare and limited societal resources. Public pressure and changing health care needs have precipitated both subtle and radical laboratory changes to more effectively use allocated resources. Responsibility for excessive laboratory use can be assigned primarily to the following four groups: practicing physicians, physicians in training, patients, and the clinical laboratory. The strategies to contain escalating health care costs have ranged from individualized physician education programs to government intervention. Laboratories have responded to the fiscal restraints imposed by prospective payment systems by attempting to reduce operational costs without adversely impacting quality. Although cost containment directed at misutilization and overutilization of existing services has conserved resources, to date, an effective cost control mechanism has yet to be identified and successfully implemented on a grand enough scale to significantly impact health care expenditures in the United States.
Identification of veterinary pathogens by use of commercial identification systems and new trends in antimicrobial susceptibility testing of veterinary pathogens
Authors: Watts J L , Yancey R J
Published: June 2016

Veterinary diagnostic microbiology is a unique specialty within microbiology. Although isolation and identification techniques are similar to those used for human pathogens, many veterinary pathogens require unique cultivation or identification procedures. Commercial identification systems provide rapid, accurate identification of human pathogens. However, the accuracy of these systems with veterinary pathogens varies widely depending on the bacterial species and the host animal from which it was isolated. Increased numbers of veterinary strains or species in the data bases of the various systems would improve their accuracy. Current procedures and interpretive criteria used for antimicrobial susceptibility testing of veterinary pathogens are based on guidelines used for human pathogens. The validity of these guidelines for use with veterinary pathogens has not been established. As with fastidious human pathogens, standardized methodologies and quality control isolates are needed for tests of organisms such as Actinobacillus pleuropneumoniae and Haemophilus somnus. Furthermore, interpretive criteria for veterinary antimicrobial agents based on the MIC for veterinary pathogens, the pharmacokinetics of the antimicrobial agent in the host animal, and in vivo efficacy of the antimicrobial agent are needed. This article reviews both the commercial identification systems evaluated with veterinary pathogens and current methods for performing and interpreting antimicrobial susceptibility tests with veterinary pathogens. Recommendations for future improvements in both areas are discussed.
Infectious diseases associated with molluscan shellfish consumption
Authors: Rippey S R
Published: June 2016

A history of shellfish-vectored illnesses (i.e., those associated with consumption of clams, oysters, mussels, and scallops) occurring in the past nine decades is presented. Typhoid fever was a significant public health problem among consumers of raw molluscan shellfish earlier in this century. The development of more effective sewage treatment procedures and the institution of a national program following these outbreaks led to a series of measures which eventually eliminated shellfish-associated typhoid fever. Present-day problems associated with this food source still involve some wastewaterborne bacterial illnesses. However, the principal public health concerns are with wastewater-derived viral pathogens and with bacterial agents of an environmental origin. The nature, occurrence, and magnitude of these public health problems are described.
Virulence of enterococci
Authors: Jett B D , Huycke M M , Gilmore M S
Published: June 2016

Enterococci are commensal organisms well suited to survival in intestinal and vaginal tracts and the oral cavity. However, as for most bacteria described as causing human disease, enterococci also possess properties that can be ascribed roles in pathogenesis. The natural ability of enterococci to readily acquire, accumulate, and share extrachromosomal elements encoding virulence traits or antibiotic resistance genes lends advantages to their survival under unusual environmental stresses and in part explains their increasing importance as nosocomial pathogens. This review discusses the current understanding of enterococcal virulence relating to (i) adherence to host tissues, (ii) invasion and abscess formation, (iii) factors potentially relevant to modulation of host inflammatory responses, and (iv) potentially toxic secreted products. Aggregation substance, surface carbohydrates, or fibronectin-binding moieties may facilitate adherence to host tissues. Enterococcus faecalis appears to have the capacity to translocate across intact intestinal mucosa in models of antibiotic-induced superinfection. Extracellular toxins such as cytolysin can induce tissue damage as shown in an endophthalmitis model, increase mortality in combination with aggregation substance in an endocarditis model, and cause systemic toxicity in a murine peritonitis model. Finally, lipoteichoic acid, superoxide production, or pheromones and corresponding peptide inhibitors each may modulate local inflammatory reactions.
Uses of flow cytometry in virology
Authors: McSharry J J
Published: June 2016

This article reviews some of the published applications of flow cytometry for in vitro and in vivo detection and enumeration of virus-infected cells. Sample preparation, fixation, and permeabilization techniques for a number of virus-cell systems are evaluated. The use of flow cytometry for multiparameter analysis of virus-cell interactions for simian virus 40, herpes simplex viruses, human cytomegalovirus, and human immunodeficiency virus and its use for determining the effect of antiviral compounds on these virus-infected cells are reviewed. This is followed by a brief description of the use of flow cytometry for the analysis of several virus-infected cell systems, including blue tongue virus, hepatitis C virus, avian reticuloendotheliosis virus, African swine fever virus, woodchuck hepatitis virus, bovine viral diarrhea virus, feline leukemia virus, Epstein-Barr virus, Autographa californica nuclear polyhedrosis virus, and Friend murine leukemia virus. Finally, the use of flow cytometry for the rapid diagnosis of human cytomegalovirus and human immunodeficiency virus in peripheral blood cells of acutely infected patients and the use of this technology to monitor patients on antiviral therapy are reviewed. Future prospects for the rapid diagnosis of in vivo viral and bacterial infections by flow cytometry are discussed.
QuantiFERON-TB Gold Assay on Plasma for Confirmation of Presumed Tuberculosis-Related Uveitis
Authors: Zanetti Stefania , Bua Alessandra , Molicotti Paola , Maiore Irene , Pinna Antonio
Published: June 2016

The QuantiFERON-TB Gold assay was used to measure interferon gamma levels in plasma from 4 patients with presumed tuberculosis-related uveitis before, during, and after antitubercular therapy. After treatment, all patients showed clinical improvement. The concentrations showed a reversion to an absence of interferon gamma in one case, decreased in two cases, and remained stable in one case. These results suggest that the QuantiFERON assay may be useful for tuberculosis-related uveitis diagnosis and follow-up.
Identification and Antifungal Susceptibility Profile of Candida guilliermondii and Candida fermentati from a Multicenter Study in China
Authors: Cheng Jing-Wei , Yu Shu-Ying , Xiao Meng , Wang He , Kudinha Timothy , Kong Fanrong , Xu Ying-Chun
Published: June 2016

With molecular sequencing as a gold standard, the Vitek MS, Bruker Biotyper MS, and Vitek-2 Compact systems correctly identified 92.7%, 97.0%, and 15.2% of 164
Candida guillermondii
isolates, respectively, and none of 8
C. fermentati
isolates. All of the isolates showed high susceptibility to echinocandins, but some
C. guilliermondii
isolates showed low azole susceptibility.
Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus
Authors: Piret Jocelyne , Goyette Nathalie , Boivin Guy
Published: June 2016

The plaque reduction assay (PRA) is the gold standard phenotypic method to determine herpes simplex virus (HSV) and human cytomegalovirus (HCMV) susceptibilities to antiviral drugs. However, this assay is subjective and labor intensive. Here, we describe a novel antiviral phenotypic method based on real-time cell analysis (RTCA) that measures electronic impedance over time. The effective drug concentrations that reduced by 50% (EC
50
s) the cytopathic effects induced by HSV-1 and HCMV were evaluated by both methods. The EC
50
s of acyclovir and foscarnet against a reference wild-type (WT) HSV-1 strain in Vero cells were, respectively, 0.5 μM and 32.6 μM by PRA and 0.8 μM and 93.6 μM by RTCA. The EC
50
ratios for acyclovir against several HSV-1 thymidine kinase (TK) mutants were 101.8×, 73.4×, 28.8×, and 35.4× (PRA) and 18.0×, 52.0×, 5.5×, and 87.8× (RTCA) compared to those for the WT. The EC
50
ratios for acyclovir and foscarnet against the HSV-1 TK/DNA polymerase mutant were 182.8× and 9.7× (PRA) and >125.0× and 10.8× (RTCA) compared to the WT. The EC
50
s of ganciclovir and foscarnet against WT HCMV strain AD169 in fibroblasts were, respectively, 1.6 μM and 27.8 μM by PRA and 5.0 μM and 111.4 μM by RTCA. The EC
50
ratios of ganciclovir against the HCMV UL97 mutant were 3.8× (PRA) and 8.2× (RTCA) compared to those for the WT. The EC
50
ratios of ganciclovir and foscarnet against the HCMV UL97/DNA polymerase mutant were 17.1× and 12.1× (PRA) and 14.7× and 4.6× (RTCA) compared to those for the WT. RTCA allows objective drug susceptibility testing of HSV and HCMV and could permit high-throughput screening of new antivirals.
Interaction between Nonstructural Proteins NS4B and NS5A Is Essential for Proper NS5A Localization and Hepatitis C Virus RNA Replication
Authors: Biswas Avik , Treadaway Jason , Tellinghuisen Timothy L.
Published: June 2016

The hepatitis C virus NS5A protein is tethered to cellular membranes via an amphipathic amino-terminal helix that is inserted in-plane into the outer endoplasmic reticulum (ER)-derived membrane leaflet. The charged face of the helix faces the cytoplasm and may contribute to interactions involved in replicase assembly and function. Using an aggressive charge flip mutagenesis strategy, we identified a number of essential residues for replication on the charged face of the NS5A anchor and identified a double charge face mutant that is lethal for RNA replication but generates suppressor mutations in the carboxy-terminal helix of the NS4B protein. This suppressor restores RNA replication of the NS5A helix double flip mutant (D1979K/D1982K) and, interestingly, seems to function by restoring the proper localization of NS5A to the viral replicase. These data add to our understanding of the complex organization and assembly of the viral replicase via NS4B-NS5A interactions.

IMPORTANCE
Information about the functional role of the cytosolic face of the NS5A anchoring helix remains obscure. In this study, we show that while the hydrophobic face of the NS5A anchor helix mediates membrane association, the polar cytosolic face of the helix plays a key role during hepatitis C virus (HCV) replication by mediating the interaction of NS5A with other HCV nonstructural proteins via NS4B. Such an interaction determines the subcellular localization of NS5A by engaging NS5A in the HCV replication process during the formation of a functional HCV replication complex. Thus, collectively, it can be stated that the findings in the present study provide further information about the interactions between the HCV nonstructural proteins during HCV RNA replication and provide a platform to gain more insights about the molecular architecture of HCV replication complexes.
Mutagenesis of
S
-Adenosyl-
l
-Methionine-Binding Residues in Coronavirus nsp14 N7-Methyltransferase Demonstrates Differing Requirements for Genome Translation and Resistance to Innate Immunity
Authors: Case James Brett , Ashbrook Alison W. , Dermody Terence S. , Denison Mark R.
Published: June 2016

Eukaryotic mRNAs possess a methylated 5′-guanosine cap that is required for RNA stability, efficient translation, and protection from cell-intrinsic defenses. Many viruses use 5′ caps or other mechanisms to mimic a cap structure to limit detection of viral RNAs by intracellular innate sensors and to direct efficient translation of viral proteins. The coronavirus (CoV) nonstructural protein 14 (nsp14) is a multifunctional protein with N7-methyltransferase (N7-MTase) activity. The highly conserved
S
-adenosyl-
l
-methionine (SAM)-binding residues of the DxG motif are required for nsp14 N7-MTase activity
in vitro
. However, the requirement for CoV N7-MTase activity and the importance of the SAM-binding residues during viral replication have not been determined. Here, we engineered mutations in murine hepatitis virus (MHV) nsp14 N7-MTase at residues D330 and G332 and determined the effects of these mutations on viral replication, sensitivity to mutagen, inhibition by type I interferon (IFN), and translation efficiency. Virus encoding a G332A substitution in nsp14 displayed delayed replication kinetics and decreased peak titers relative to wild-type (WT) MHV. In addition, replication of nsp14 G332A virus was diminished following treatment of cells with IFN-β, and nsp14 G332A genomes were translated less efficiently both
in vitro
and during viral infection. In contrast, substitution of alanine at MHV nsp14 D330 did not affect viral replication, sensitivity to mutagen, or inhibition by IFN-β compared to WT MHV. Our results demonstrate that the conserved MHV N7-MTase SAM-binding-site residues are not required for MHV viability and suggest that the determinants of CoV N7-MTase activity differ
in vitro
and during virus infection.

IMPORTANCE
Human coronaviruses, most notably severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, cause severe and lethal human disease. Since specific antiviral therapies are not available for the treatment of human coronavirus infections, it is essential to understand the functions of conserved CoV proteins in viral replication. Here, we show that substitution of alanine at G332 in the N7-MTase domain of nsp14 impairs viral replication, enhances sensitivity to the innate immune response, and reduces viral RNA translation efficiency. Our data support the idea that coronavirus RNA capping could be targeted for development of antiviral therapeutics.
Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis
Authors: Cai Hui , Zhang Yu , Lu Mijia , Liang Xueya , Jennings Ryan , Niewiesk Stefan , Li Jianrong
Published: June 2016

Human metapneumovirus (hMPV) is a major causative agent of upper- and lower-respiratory-tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1, which plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated, but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization, whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using a reverse genetics system, recombinant hMPVs (rhMPVs) lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wild-type hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication, and pathogenesis
in vivo
.

IMPORTANCE
The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that phosphorylation of M2-1 is essential for virus replication and pathogenesis
in vivo
. Recombinant hMPVs lacking phosphorylation in M2-1 exhibited limited replication in the upper and lower respiratory tract and triggered strong protective immunity in cotton rats. This work highlights the important role of M2-1 phosphorylation in viral replication and that inhibition of M2-1 phosphorylation may serve as a novel approach to develop live attenuated vaccines as well as antiviral drugs for pneumoviruses.
A shale-hosted Cr isotope record of low atmospheric oxygen during the Proterozoic
Authors: Cole Devon B. , Reinhard Christopher T. , Wang Xiangli , Gueguen Bleuenn , Halverson Galen P. , Gibson Timothy , Hodgskiss Malcolm S.W. , McKenzie N. Ryan , Lyons Timothy W. , Planavsky Noah J.
Published: June 2016

The emergence and expansion of animal life on Earth represents a dramatic shift in the structure and complexity of the biosphere. A lack of firm constraints on surface oxygen levels during the mid-Proterozoic has resulted in heated debate as to whether the rise and earliest diversification of animals was directly linked to a change in environmental oxygen levels or, instead, simply reflects the timing of innovations in gene expression and developmental regulation and was independent of a direct environmental trigger. Here, we present chromium (Cr) isotope data from marine black shales that provide evidence for minimal Cr oxidation throughout the mid-Proterozoic leading up to the diversification of eukaryotes and the rise of animals during the late Neoproterozoic. This observation requires very low background oxygen levels (<1% of present atmospheric levels). Accepting previously proposed estimates of pO2 levels needed to induce Cr isotope fractionation, our data provide support for the persistence of an Earth system in which baseline atmospheric pO2 would have been low enough to inhibit the diversification of animals until ca. 800 Ma. More generally, evidence for a delayed rise of atmospheric oxygen strongly suggests that environmental factors have played a fundamental role in controlling the emergence and expansion of complex life on Earth.
Anoxic oxidation of chromium
Authors: Oze Christopher , Sleep Norman H. , Coleman Robert G. , Fendorf Scott
Published: June 2016

Naturally occurring Cr(VI) has been ascribed to terrestrial Cr(III) oxidation by Mn (di)oxides, generated through reaction of Mn(II) with molecular oxygen (O2). However, hydrogen peroxide (H2O2) is a potential oxidant of Cr(III) that may form in serpentinization (high H2, low O2) systems where chromite [i.e., the main mineralogical source of Cr(III)] is abundant. Accordingly, here we evaluate H2O2 and chromite interactions in serpentinization systems to determine pathways of Cr(III) oxidation that alters the current paradigm of O2-dependent oxidation. Field observations support that metastable H2O2 and Cr(VI) are present in serpentinization-related fluids relatively absent of O2. Further, laboratory experiments demonstrate and support that H2O2 is a kinetically facile oxidant of chromite, especially under alkaline conditions, which provides a variety of alternative means by which Cr(VI) may be generated and supplied to the oceans not directly linked to atmospheric O2. Thus, Cr(III) oxidation pathways, and their influence on the Cr isotopic record, must account for anoxic Cr(III) oxidation in serpentinization systems as well as a variety of H2O2-induced Cr(III) oxidation pathways that may occur in both terrestrial and marine systems.
Critical components of neuromuscular training to reduce ACL injury risk in female athletes: meta-regression analysis
Authors: Sugimoto Dai , Myer Gregory D , Barber Foss Kim D , Pepin Michael J , Micheli Lyle J , Hewett Timothy E
Published: June 2016

Objective

The aim of this study was to determine key components in neuromuscular training that optimise ACL injury reduction in female athletes using meta-regression analyses.

Design

Systematic review and meta-regression.

Data sources

The literature search was performed in PubMed and EBSCO.

Eligibility criteria

Inclusion criteria for the current analysis were: (1) documented the number of ACL injuries, (2) employed a neuromuscular training intervention that aimed to reduce ACL injuries, (3) had a comparison group, (4) used a prospective control study design and (5) recruited female athletes as participants. Two independent reviewers extracted studies which met the inclusion criteria. Methodological quality of included study and strength of recommendation were evaluated. Number of ACL injuries and participants in control and intervention groups, age of participants, dosage of neuromuscular training, exercise variations within neuromuscular training and status of verbal feedback were extracted.

Results

The meta-regression analyses identified age of participants, dosage of neuromuscular training, exercise variations within neuromuscular training and utilisation of verbal feedback as significant predictors of ACL injury reduction (p=0.01 in fixed-effects model, p=0.03 in random-effects model). Inclusion of 1 of the 4 components in neuromuscular training could reduce ACL injury risk by 17.2–17.7% in female athletes. No significant heterogeneity and publication bias effects were detected. Strength of recommendation was rated as A (recommendation based on consistent and good-quality patient-oriented study evidence).

Conclusions

Age of participants, dosage of neuromuscular training, exercise variations within neuromuscular training and utilisation of verbal feedback are predictors that influence the optimisation of prophylactic effects of neuromuscular training and the resultant ACL injury reduction in female athletes.
Community-based participatory research to improve life quality and clinical outcomes of patients with breast cancer (DianaWeb in Umbria pilot study)
Authors: Villarini Milena , Lanari Chiara , Nucci Daniele , Gianfredi Vincenza , Marzulli Tiziana , Berrino Franco , Borgo Alessandra , Bruno Eleonora , Gargano Giuliana , Moretti Massimo , Villarini Anna
Published: June 2016

Introduction

Breast cancer (BC) is the most frequent cancer in Europe and the International Agency for Research on Cancer (IARC) has estimated over 460 000 incident cases per year. Survival among patients with BC has increased in the past decades and EUROCARE-5 has estimated a 5-year relative survival rate of 82% for patients diagnosed in 2000–2007. There is growing evidence that lifestyle (such as a diet based on Mediterranean principles associated with moderate physical activity) may influence prognosis of BC; however, this information is not currently available to patients and is not considered in oncology protocols. Only a few epidemiological studies have investigated the role of diet in BC recurrence and metastasis.

Methods and analysis

DianaWeb is a community-based participatory research dedicated to patients with BC and represents a collaborative effort between participants and research institutions to determine if specified changes in lifestyle would result in improved outcomes in terms of quality of life or survival. The aim of the study is to recruit a large number of participants, to monitor their lifestyle and health status over time, to provide them tips to encourage sustainable lifestyle changes, to analyse clinical outcomes as a function of baseline risk factors and subsequent changes, and to share with patients methodologies and results. DianaWeb uses a specific interactive website (http://www.dianaweb.org/) and, with very few exceptions, all communications will be made through the web. In this paper we describe the pilot study, namely DianaWeb in Umbria.

Ethics and dissemination

DianaWeb does not interfere with prescribed oncological treatments; rather, it recommends that participants should follow the received prescriptions. The results will be used to plan guidelines for nutrition and physical activity for patients with BC. The pilot study was approved by the ethics committee of the University of Perugia (reference number 2015-002), and is supported by Fondazione Cassa di Risparmio di Perugia (2013.0185 021).
Evolution of target organ damage and haemodynamic parameters over 4 years in patients with increased insulin resistance: the LOD-DIABETES prospective observational study
Authors: Gómez-Marcos Manuel Ángel , Recio-Rodríguez José Ignacio , Patino-Alonso María Carmen , Agudo-Conde Cristina , Rodríguez-Sanchez Emiliano , Maderuelo-Fernandez Jose Angel , Gómez-Sánchez Leticia , Gomez-Sanchez Marta , García-Ortiz Luís ,   LOD-DIABETES Group
Published: June 2016

Objectives

We prospectively examined the impact of type 2 diabetes compared with metabolic syndrome (MetS) on the development of vascular disease over 4 years as determined by anatomic and functional markers of vascular disease. By comparing the vascular outcomes of the 2 disorders, we seek to determine the independent effect of elevated glucose levels on vascular disease.

Setting

2 primary care centres in Salamanca, Spain.

Participants

We performed a prospective observational study involving 112 patients (68 with type 2 diabetes and 44 with MetS) who were followed for 4 years.

Primary and secondary outcome measures

Measurements included blood pressure, blood glucose, lipids, smoking, body mass index, waist circumference, Homeostasis Model Assessment Insulin Resistance (HOMA-IR), hs-c-reactive protein and fibrinogen levels. We also evaluated vascular, carotid intima media thickness (IMT), pulse wave velocity (PWV) and ankle/brachial index, heart and renal target organ damage (TOD). The haemodynamic parameters were central (CAIx) and peripheral (PAIx) augmentation indices.

Results

In year 4, participants with type 2 diabetes had increased IMT thickness. These patients had more plaques and an IMT>0.90 mm. In participants with MetS, we only found an increase in the number of plaques. We found no changes in PWV, CAIx and PAIx. The patients with diabetes had a greater frequency of vascular TOD. There were no differences neither in renal nor cardiac percentage of TOD in the patients with MetS or diabetes mellitus type 2.

Conclusions

This prospective study showed that the evolution of vascular TOD is different in participants with type 2 diabetes compared with those with MetS. While IMT and PWV increased in type 2 diabetes, these were not modified in MetS. The renal and cardiac TOD evolution, as well as the PAIx and CAIx, did not change in either group.

Trial registration number

NCT01065155; Results.
Comparative study of Y-shaped and circular floating piles in consolidating clay
Authors: Lv Yaru , Ng Charles W.W. , Lam Sze Yue , Liu Hanlong , Ding Xuanming
Published: June 2016

Although engineers often make use of pile geometry to improve the axial load capacity of piles, geometrical effects on floating piles in consolidating clay are still not fully understood. This paper reports two centrifuge model tests to investigate the responses of a Y-shaped pile and a circular pile subjected to an induced dragload and applied axial loads. Three-dimensional numerical back-analyses were performed considering the elastoplastic slip. The Y-shaped and circular piles developed similar downdrag, but the dragload induced on the Y-shaped pile was larger than that induced on the circular pile. As the axial load increased, the neutral plane shifted upward along a nonlinear path, of which the gradient of the Y-shaped pile was gentler. The ultimate bearing capacity of the Y-shaped pile was 1.73 times that of the circular pile. The dragload on the Y-shaped and circular piles was eventually eliminated at approximately 0.56 and 0.83 times the corresponding ultimate pile capacities, respectively. Three flanges of the Y-shaped pile “hung-up” adjacent soil that settled together with the pile shaft. The lateral extent of vertically nonuniform trapped soil decreased with increasing axial load. Even though the Y-shaped and circular piles encountered a similar serviceability limit state, Y-shaped pile had advantages in bearing capacity.