Pathogenicity in Francisella tularensis subspecies

.Sequencing of the non-pathogenic Francisella tularensis sub-species novicida U112, and comparison with two pathogenic sub-species, provides insights into the evolution of pathogenicity in these species.

In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. Differences in HUS frequency among E. coli O157:H7 outbreaks have been noted, but our understanding of bacterial factors that promote HUS is incomplete. In 2006, in an outbreak of E. coli O157:H7 caused by consumption of contaminated spinach, there was a notably high frequency of HUS. We sequenced the genome of the strain responsible (TW14359) with the goal of identifying candidate genetic factors that contribute to an enhanced ability to cause HUS. The TW14359 genome contains 70 kb of DNA segments not present in either of the two reference O157:H7 genomes. We identified seven putative virulence determinants, including two putative type III secretion system effector proteins, candidate genes that could result in increased pathogenicity or, alternatively, adaptation to plants, and an intact anaerobic nitric oxide reductase gene, norV. We surveyed 100 O157:H7 isolates for the presence of these putative virulence determinants. A norV deletion was found in over one-half of the strains surveyed and correlated strikingly with the absence of stx 1 . The other putative virulence factors were found in 8 to 35% of the O157:H7 isolates surveyed, and their presence also correlated with the presence of norV and the absence of stx 1 , indicating that the presence of norV may serve as a marker of a greater propensity for HUS, similar to the correlation between the absence of stx 1 and a propensity for HUS.

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted whole-genome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis.

Single-cell nanosurgery and the ability to manipulate nanometer-sized subcellular structures with optical tweezers has widespread applications in biology, but so far has been limited by difficulties in maintaining the functionality of the transported subcellular organelles. This difficulty arises because of the propensity of optical tweezers to photodamage the trapped object. To address this issue, this paper describes the use of a polarization-shaped optical vortex trap, which exerts less photodamage on the trapped particle than conventional optical tweezers, for carrying out single-cell nanosurgical procedures. This method is also anticipated to find broad use in the trapping of any nanoparticles that are adversely affected by high-intensity laser light.Despite the small size of a mammalian cell, it is an extremely heterogeneous and compartmentalized structure. Proteins and small-molecule metabolites constantly traffic among these intracellular compartments, and it has become increasingly evident that biological specificity (e.g. between proteins) relies heavily on spatial and temporal segregation and compartmentalization of molecules in addition to chemical and molecular specificity 1, 2 . Gaining information with regard to the spatial and temporal distribution and evolution of molecules within cells, therefore, is crucial to the construction of a quantitative model of cellular function. The ability to isolate selectively single subcellular compartments for chemical analysis or transplantation opens new venues for studying the spatial and temporal organization of the cell. For example, the reprogramming of the nucleus of somatic cells may be achieved via nuclear transfer 3, 4 . This paper describes and compares the use of polarizationshaped vortex traps with a Gaussian optical tweezer for performing single-cell nanosurgery.Single-beam optical gradient traps, or optical tweezers, have made significant impact on biophysical and biological research in the past two decades 5-11 . Unfortunately, while optical tweezers offer exquisite sensitivity in its ability to position microparticles and to measure the forces exerted by biological motors 12 , it suffers from one important disadvantage: the trapped particle is localized at the laser focus where light intensity is the highest, often reaching 10 7 -10 8 W/cm 2 . As a result, the laser light used to trap a particle also has a propensity to photobleach and photodamage the particle, especially when the particle is fragile and small (e.g. a subcellular organelle that is fluorescently labeled) for which high laser intensities are often required. To minimize radiation damage to the trapped biological particle, laser wavelengths between ∼800nm to ∼ 1100nm are usually used because of the low absorption cross section of water and biological molecules in this spectral range 6-8, 13-15 . Nevertheless, at the high laser intensities required for trapping and translating subcellular organelles through the dense * Corresponding author. E-mail: chiu@chem.washington.edu. ...

Background-Accurately estimating operative case-time duration is critical for optimizing operating room utilization. Current estimates are inaccurate and prior models include data not available at the time of scheduling. Our objective was to develop statistical models in a large retrospective dataset to improve estimation of case-time duration relative to current standards. Study Design-We developed models to predict case-time duration using linear regression and supervised machine learning (ML). For each of these models, we generated: 1) service-specific models and 2) surgeon-specific models in which surgeons were modeled individually. Our dataset included 46,986 scheduled surgeries performed at our center from January 2014 to December 2017, with 80% used for training and 20% for model testing/validation. Predictions derived from each model were compared to our institutional standard. Models were evaluated based on accuracy, overage (case duration > predicted + 10%), underage (case duration < predicted-10%), and the predictive capability of being within a 10% tolerance threshold. Results-The ML algorithm resulted in the highest predictive capability. The surgeon-specific model was superior to the service-specific model, with higher accuracies, lower percentage of overage and underage, and higher percentage of cases within the 10% threshold. The ability to predict cases within 10% improved from 32% using our institutional standard to 39% with the ML surgeon-specific model. The majority of the information utilized in the models was based on procedure and personnel data rather than patient health status.