The rise in antibiotic resistance has led to an increased research focus on discovery of new antibacterial candidates. While broad-spectrum antibiotics are widely pursued, there is evidence that resistance arises in part from the wide spread use of these antibiotics. Our group has developed a system to produce protein affinity agents, called synbodies, which have high affinity and specificity for their target. In this report, we describe the adaptation of this system to produce new antibacterial candidates towards a target bacterium. The system functions by screening target bacteria against an array of 10,000 random sequence peptides and, using a combination of membrane labeling and intracellular dyes, we identified peptides with target specific binding or killing functions. Binding and lytic peptides were identified in this manner and in vitro tests confirmed the activity of the lead peptides. A peptide with antibacterial activity was linked to a peptide specifically binding Staphylococcus aureus to create a synbody with increased antibacterial activity. Subsequent tests showed that this peptide could block S. aureus induced killing of HEK293 cells in a co-culture experiment. These results demonstrate the feasibility of using the synbody system to discover new antibacterial candidate agents.

Background:
Pandemic influenza is said to 'shift mortality' to younger age groups; but also to spare a subpopulation of the elderly population. Does one of these effects dominate? Might this have important ramifications?

Methods: We estimated age-specific excess mortality rates for all-years for which data were available in the 20th century for Australia, Canada, France, Japan, the UK, and the USA for people older than 44 years of age. We modeled variation with age, and standardized estimates to allow direct comparison across age groups and countries. Attack rate data for four pandemics were assembled.

Results: For nearly all seasons, an exponential model characterized mortality data extremely well. For seasons of emergence and a variable number of seasons following, however, a subpopulation above a threshold age invariably enjoyed reduced mortality. 'Immune escape', a stepwise increase in mortality among the oldest elderly, was observed a number of seasons after both the A(H2N2) and A(H3N2) pandemics. The number of seasons from emergence to escape varied by country. For the latter pandemic, mortality rates in four countries increased for younger age groups but only in the season following that of emergence. Adaptation to both emergent viruses was apparent as a progressive decrease in mortality rates, which, with two exceptions, was seen only in younger age groups. Pandemic attack rate variation with age was estimated to be similar across four pandemics with very different mortality impact.

Conclusions: In all influenza pandemics of the 20th century, emergent viruses resembled those that had circulated previously within the lifespan of then-living people. Such individuals were relatively immune to the emergent strain, but this immunity waned with mutation of the emergent virus. An immune subpopulation complicates and may invalidate vaccine trials. Pandemic influenza does not 'shift' mortality to younger age groups; rather, the mortality level is reset by the virulence of the emerging virus and is moderated by immunity of past experience. In this study, we found that after immune escape, older age groups showed no further mortality reduction, despite their being the principal target of conventional influenza vaccines. Vaccines incorporating variants of pandemic viruses seem to provide little benefit to those previously immune. If attack rates truly are similar across pandemics, it must be the case that immunity to the pandemic virus does not prevent infection, but only mitigates the consequences.

Background:
Theory suggests that individual behavioral responses impact the spread of flu-like illnesses, but this has been difficult to empirically characterize. Social distancing is an important component of behavioral response, though analyses have been limited by a lack of behavioral data. Our objective is to use media data to characterize social distancing behavior in order to empirically inform explanatory and predictive epidemiological models.

Methods:
We use data on variation in home television viewing as a proxy for variation in time spent in the home and, by extension, contact. This behavioral proxy is imperfect but appealing since information on a rich and representative sample is collected using consistent techniques across time and most major cities. We study the April-May 2009 outbreak of A/H1N1 in Central Mexico and examine the dynamic behavioral response in aggregate and contrast the observed patterns of various demographic subgroups. We develop and calibrate a dynamic behavioral model of disease transmission informed by the proxy data on daily variation in contact rates and compare it to a standard (non-adaptive) model and a fixed effects model that crudely captures behavior.

Results:
We find that after a demonstrable initial behavioral response (consistent with social distancing) at the onset of the outbreak, there was attenuation in the response before the conclusion of the public health intervention. We find substantial differences in the behavioral response across age subgroups and socioeconomic levels. We also find that the dynamic behavioral and fixed effects transmission models better account for variation in new confirmed cases, generate more stable estimates of the baseline rate of transmission over time and predict the number of new cases over a short horizon with substantially less error.

Conclusions:
Results suggest that A/H1N1 had an innate transmission potential greater than previously thought but this was masked by behavioral responses. Observed differences in behavioral response across demographic groups indicate a potential benefit from targeting social distancing outreach efforts.

Most current approaches for quantification of RNA species in their natural spatial contexts in single cells are limited by a small number of parallel analyses. Here we report a strategy to dramatically increase the multiplexing capacity for RNA analysis in single cells in situ. In this method, transcripts are detected by fluorescence in situ hybridization (FISH). After imaging and data storage, the fluorescence signal is efficiently removed by photobleaching. This enables the reinitiation of FISH to detect other RNA species in the same cell. Through reiterative cycles of hybridization, imaging and photobleaching, the identities, positions and copy numbers of a large number of varied RNA species can be quantified in individual cells in situ. Using this approach, we analyzed seven different transcripts in single HeLa cells with five reiterative RNA FISH cycles. This approach has the potential to detect over 100 varied RNA species in single cells in situ, which will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies.

The notable increase in biofuel usage by the road transportation sector in Brazil during recent years has significantly altered the vehicular fuel composition. Consequently, many uncertainties are currently found in particulate matter vehicular emission profiles. In an effort to better characterise the emitted particulate matter, measurements of aerosol physical and chemical properties were undertaken inside two tunnels located in the São Paulo Metropolitan Area (SPMA). The tunnels show very distinct fleet profiles: in the Jânio Quadros (JQ) tunnel, the vast majority of the circulating fleet are light duty vehicles (LDVs), fuelled on average with the same amount of ethanol as gasoline. In the Rodoanel (RA) tunnel, the particulate emission is dominated by heavy duty vehicles (HDVs) fuelled with diesel (5% biodiesel). In the JQ tunnel, PM_2.5 concentration was on average 52 μg m^-3, with the largest contribution of organic mass (OM, 42%), followed by elemental carbon (EC, 17%) and crustal elements (13%). Sulphate accounted for 7% of PM_2.5 and the sum of other trace elements was 10%. In the RA tunnel, PM_2.5 was on average 233 μg m^-3, mostly composed of EC (52%) and OM (39%). Sulphate, crustal and the trace elements showed a minor contribution with 5%, 1%, and 1%, respectively. The average OC : EC ratio in the JQ tunnel was 1.59 ± 0.09, indicating an important contribution of EC despite the high ethanol fraction in the fuel composition. In the RA tunnel, the OC : EC ratio was 0.49 ± 0.12, consistent with previous measurements of diesel-fuelled HDVs. Besides bulk carbonaceous aerosol measurement, polycyclic aromatic hydrocarbons (PAHs) were quantified. The sum of the PAHs concentration was 56 ± 5 ng m^-3 and 45 ± 9 ng m^-3 in the RA and JQ tunnel, respectively. In the JQ tunnel, benzo(a)pyrene (BaP) ranged from 0.9 to 6.7 ng m^-3 (0.02–0.1‰ of PM_2.5)] whereas in the RA tunnel BaP ranged from 0.9 to 4.9 ng m^-3 (0.004–0. 02‰ of PM_2.5), indicating an important relative contribution of LDVs emission to atmospheric BaP.

The heat-labile toxins (LT) produced by enterotoxigenic Escherichia coli display adjuvant effects to coadministered antigens, leading to enhanced production of serum antibodies. Despite extensive knowledge of the adjuvant properties of LT derivatives, including in vitro-generated non-toxic mutant forms, little is known about the capacity of these adjuvants to modulate the epitope specificity of antibodies directed against antigens. This study characterizes the role of LT and its non-toxic B subunit (LTB) in the modulation of antibody responses to a coadministered antigen, the dengue virus (DENV) envelope glycoprotein domain III (EDIII), which binds to surface receptors and mediates virus entry into host cells. In contrast to non-adjuvanted or alum-adjuvanted formulations, antibodies induced in mice immunized with LT or LTB showed enhanced virus-neutralization effects that were not ascribed to a subclass shift or antigen affinity. Nonetheless, immunosignature analyses revealed that purified LT-adjuvanted EDIII-specific antibodies display distinct epitope-binding patterns with regard to antibodies raised in mice immunized with EDIII or the alum-adjuvanted vaccine. Notably, the analyses led to the identification of a specific EDIII epitope located in the EF to FG loop, which is involved in the entry of DENV into eukaryotic cells. The present results demonstrate that LT and LTB modulate the epitope specificity of antibodies generated after immunization with coadministered antigens that, in the case of EDIII, was associated with the induction of neutralizing antibody responses. These results open perspectives for the more rational development of vaccines with enhanced protective effects against DENV infections.

About 2.5 × 10⁶ snapshots on microcrystals of photoactive yellow protein (PYP) from a recent serial femtosecond crystallographic (SFX) experiment were reanalyzed to maximum resolution. The resolution is pushed to 1.46 Å, and a PYP structural model is refined at that resolution. The result is compared to other PYP models determined at atomic resolution around 1 Å and better at the synchrotron. By comparing subtleties such as individual isotropic temperature factors and hydrogen bond lengths, we were able to assess the quality of the SFX data at that resolution. We also show that the determination of anisotropic temperature factor ellipsoids starts to become feasible with the SFX data at resolutions better than 1.5 Å.

Methicillin resistant Staphylococcus aureus (MRSA) is currently a major cause of skin and soft tissue infections (SSTI) in the United States. Seasonal variation of MRSA infections in hospital settings has been widely observed. However, systematic time-series analysis of incidence data is desirable to understand the seasonality of community acquired (CA)-MRSA infections at the population level. In this paper, using data on monthly SSTI incidence in children aged 0–19 years and enrolled in Medicaid in Maricopa County, Arizona, from January 2005 to December 2008, we carried out time-series and nonlinear regression analysis to determine the periodicity, trend, and peak timing in SSTI incidence in children at different age: 0-4 years, 5-9 years, 10-14 years, and 15-19 years. We also assessed the temporal correlation between SSTI incidence and meteorological variables including average temperature and humidity. Our analysis revealed a strong annual seasonal pattern of SSTI incidence with peak occurring in early September. This pattern was consistent across age groups. Moreover, SSTIs followed a significantly increasing trend over the 4-year study period with annual incidence increasing from 3.36% to 5.55% in our pediatric population of approximately 290,000. We also found a significant correlation between the temporal variation in SSTI incidence and mean temperature and specific humidity. Our findings could have potential implications on prevention and control efforts against CA-MRSA.

Background: Prior research shows that work in agriculture and construction/extraction occupations increases the risk of environmental heat-associated death.

Purpose: To assess the risk of environmental heat-associated death by occupation.

Methods: This was a case-control study. Cases were heat-caused and heat-related deaths occurring from May-October during the period 2002–2009 in Maricopa County, Arizona. Controls were selected at random from non-heat-associated deaths during the same period in Maricopa County. Information on occupation, age, sex, and race-ethnicity was obtained from death certificates. Logistic regression analysis was used to estimate odds ratios for heat-associated death.

Results: There were 444 cases of heat-associated deaths in adults (18+ years) and 925 adult controls. Of heat-associated deaths, 332 (75%) occurred in men; a construction/extraction or agriculture occupation was described on the death certificate in 115 (35%) of these men. In men, the age-adjusted odds ratios for heat-associated death were 2.32 (95% confidence interval 1.55, 3.48) in association with construction/extraction and 3.50 (95% confidence interval 1.94, 6.32) in association with agriculture occupations. The odds ratio for heat-associated death was 10.17 (95% confidence interval 5.38, 19.23) in men with unknown occupation. In women, the age-adjusted odds ratio for heat-associated death was 6.32 (95% confidence interval 1.48, 27.08) in association with unknown occupation. Men age 65 years and older in agriculture occupations were at especially high risk of heat-associated death.

Conclusion: The occurrence of environmental heat-associated death in men in agriculture and construction/extraction occupations in a setting with predictable periods of high summer temperatures presents opportunities for prevention.

In this study we characterized the relationship between temperature and mortality in central Arizona desert cities that have an extremely hot climate. Relationships between daily maximum apparent temperature (AT_max) and mortality for eight condition-specific causes and all-cause deaths were modeled for all residents and separately for males and females ages <65 and ≥65 during the months May-October for years 2000-2008. The most robust relationship was between ATmax on day of death and mortality from direct exposure to high environmental heat. For this condition-specific cause of death, the heat thresholds in all gender and age groups (AT_max = 90–97 °F; 32.2‒36.1 °C) were below local median seasonal temperatures in the study period (AT_max = 99.5 °F; 37.5 °C). Heat threshold was defined as AT_max at which the mortality ratio begins an exponential upward trend. Thresholds were identified in younger and older females for cardiac disease/stroke mortality (AT_max = 106 and 108 °F; 41.1 and 42.2 °C) with a one-day lag. Thresholds were also identified for mortality from respiratory diseases in older people (AT_max = 109 °F; 42.8 °C) and for all-cause mortality in females (AT_max = 107 °F; 41.7 °C) and males <65 years (AT_max = 102 °F; 38.9 °C). Heat-related mortality in a region that has already made some adaptations to predictable periods of extremely high temperatures suggests that more extensive and targeted heat-adaptation plans for climate change are needed in cities worldwide.