Biochemical analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK–TssFG subcomplex

Gram-negative bacteria use the Type VI secretion system (T6SS) to inject toxic proteins into rival bacteria or eukaryotic cells. However, the mechanism of the T6SS is incompletely understood. In the present study, we investigated a conserved component of the T6SS, TssK, using the antibacterial T6SS of Serratia marcescens as a model system. TssK was confirmed to be essential for effector secretion by the T6SS. The native protein, although not an integral membrane protein, appeared to localize to the inner membrane, consistent with its presence within a membrane-anchored assembly. Recombinant TssK purified from S. marcescens was found to exist in several stable oligomeric forms, namely trimer, hexamer and higher-order species. Native-level purification of TssK identified TssF and TssG as interacting proteins. TssF and TssG, conserved T6SS components of unknown function, were required for T6SS activity, but not for correct localization of TssK. A complex containing TssK, TssF and TssG was subsequently purified in vitro, confirming that these three proteins form a new subcomplex within the T6SS. Our findings provide new insight into the T6SS assembly, allowing us to propose a model whereby TssK recruits TssFG into the membrane-associated T6SS complex and different oligomeric states of TssK may contribute to the dynamic mechanism of the system

Using health worker opinions to assess changes in structural components of quality in a Cluster Randomized Trial.

BACKGROUND: The 'resource readiness' of health facilities to provide effective services is captured in the structure component of the classical Donabedian paradigm often used for assessment of the quality of care in the health sector. Periodic inventories are commonly used to confirm the presence (or absence) of equipment or drugs by physical observation or by asking those in charge to indicate whether an item is present or not. It is then assumed that this point observation is representative of the everyday status. However the availability of an item (consumables) may vary. Arguably therefore a more useful assessment for resources would be one that captures this fluctuation in time. Here we report an approach that may circumvent these difficulties. METHODS: We used self-administered questionnaires (SAQ) to seek health worker views of availability of key resources supporting paediatric care linked to a cluster randomized trial of a multifaceted intervention aimed at improving this care conducted in eight rural Kenyan district hospitals. Four hospitals received a full intervention and four a partial intervention. Data were collected pre-intervention and after 6 and 18 months from health workers in three clinical areas asked to score item availability using an 11-point scale. Mean scores for items common to all 3 areas and mean scores for items allocated to domains identified using exploratory factor analysis (EFA) were used to describe availability and explore changes over time. RESULTS: SAQ were collected from 1,156 health workers. EFA identified 11 item domains across the three departments. Mean availability scores for these domains were often <5/10 at baseline reflecting lack of basic resources such as oxygen, nutrition and second line drugs. An improvement in mean scores occurred in 8 out of 11 domains in both control and intervention groups. A calculation of difference in difference of means for intervention vs. control suggested an intervention effect resulting in greater changes in 5 out of 11 domains. CONCLUSION: Using SAQ data to assess resource availability experienced by health workers provides an alternative to direct observations that provide point prevalence estimates. Further the approach was able to demonstrate poor access to resources, change over time and variability across place

A hybrid method for modelling two dimensional non-breaking and breaking waves

This is the first paper to present a hybrid method coupling a Improved Meshless Local Petrov Galerkin method with Rankine source solution (IMLPG_R) based on the Navier Stokes (NS) equations, with a finite element method (FEM) based on the fully nonlinear potential flow theory (FNPT) in order to efficiently simulate the violent waves and their interaction with marine structures. The two models are strongly coupled in space and time domains using a moving overlapping zone, wherein the information from both the solvers is exchanged. In the time domain, the Runge-Kutta 2nd order method is nested with a predictor-corrector scheme. In the space domain, numerical techniques including ‘Feeding Particles’ and two-layer particle interpolation with relaxation coefficients are introduced to achieve the robust coupling of the two models. The properties and behaviours of the new hybrid model are tested by modelling a regular wave, solitary wave and Cnoidal wave including breaking and overtopping. It is validated by comparing the results of the method with analytical solutions, results from other methods and experimental data. The paper demonstrates that the method can produce satisfactory results but uses much less computational time compared with a method based on the full NS model

Experience developing national evidence-based clinical guidelines for childhood pneumonia in a low-income setting - making the GRADE?

BACKGROUND: The development of evidence-based clinical practice guidelines has gained wide acceptance in high-income countries and reputable international organizations. Whereas this approach may be a desirable standard, challenges remain in low-income settings with limited capacity and resources for evidence synthesis and guideline development. We present our experience using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach for the recent revision of the Kenyan pediatric clinical guidelines focusing on antibiotic treatment of pneumonia. METHODS: A team of health professionals, many with minimal prior experience conducting systematic reviews, carried out evidence synthesis for structured clinical questions. Summaries were compiled and distributed to a panel of clinicians, academicians and policy-makers to generate recommendations based on best available research evidence and locally-relevant contextual factors. RESULTS: We reviewed six eligible articles on non-severe and 13 on severe/very severe pneumonia. Moderate quality evidence suggesting similar clinical outcomes comparing amoxicillin and cotrimoxazole for non-severe pneumonia received a strong recommendation against adopting amoxicillin. The panel voted strongly against amoxicillin for severe pneumonia over benzyl penicillin despite moderate quality evidence suggesting clinical equivalence between the two and additional factors favoring amoxicillin. Very low quality evidence suggesting ceftriaxone was as effective as the standard benzyl penicillin plus gentamicin for very severe pneumonia received a strong recommendation supporting the standard treatment. CONCLUSIONS: Although this exercise may have fallen short of the rigorous requirements recommended by the developers of GRADE, it was arguably an improvement on previous attempts at guideline development in low-income countries and offers valuable lessons for future similar exercises where resources and locally-generated evidence are scarce

Locomotor changes in length and EMG activity of feline medial gastrocnemius muscle following paralysis of two synergists

The mechanism of the compensatory increase in electromyographic activity (EMG) of a cat ankle extensor during walking shortly after paralysis of its synergists is not fully understood. It is possible that due to greater ankle flexion in stance in this situation, muscle spindles are stretched to a greater extent and, thus, contribute to the EMG enhancement. However, also changes in force feedback and central drive may play a role. The aim of the present study was to investigate the short-term (1- to 2-week post-op) effects of lateral gastrocnemius (LG) and soleus (SO) denervation on muscle fascicle and muscle–tendon unit (MTU) length changes, as well as EMG activity of the intact medial gastrocnemius (MG) muscle in stance during overground walking on level (0%), downslope (−50%, presumably enhancing stretch of ankle extensors in stance) and upslope (+50%, enhancing load on ankle extensors) surfaces. Fascicle length was measured directly using sonomicrometry, and MTU length was calculated from joint kinematics. For each slope condition, LG-SO denervation resulted in an increase in MTU stretch and peak stretch velocity of the intact MG in early stance. MG muscle fascicle stretch and peak stretch velocity were also higher than before denervation in downslope walking. Denervation significantly decreased the magnitude of MG fascicle shortening and peak shortening velocity during early stance in level and upslope walking. MG EMG magnitude in the swing and stance phases was substantially greater after denervation, with a relatively greater increase during stance of level and upslope walking. These results suggest that the fascicle length patterns of MG muscle are significantly altered when two of its synergists are in a state of paralysis. Further, the compensatory increase in MG EMG is likely mediated by enhanced MG length feedback during downslope walking, enhanced feedback from load-sensitive receptors during upslope walking and enhanced central drive in all walking conditions

Atomic excitation during recollision-free ultrafast multi-electron tunnel ionization

Modern intense ultrafast pulsed lasers generate an electric field of sufficient strength to permit tunnel ionization of the valence electrons in atoms. This process is usually treated as a rapid succession of isolated events, in which the states of the remaining electrons are neglected. Such electronic interactions are predicted to be weak, the exception being recollision excitation and ionization caused by linearly-polarized radiation. In contrast, it has recently been suggested that intense field ionization may be accompanied by a two-stage `shake-up' reaction. Here we report a unique combination of experimental techniques that enables us to accurately measure the tunnel ionization probability for argon exposed to 50 femtosecond laser pulses. Most significantly for the current study, this measurement is independent of the optical focal geometry, equivalent to a homogenous electric field. Furthermore, circularly-polarized radiation negates recollision. The present measurements indicate that tunnel ionization results in simultaneous excitation of one or more remaining electrons through shake-up. From an atomic physics standpoint, it may be possible to induce ionization from specific states, and will influence the development of coherent attosecond XUV radiation sources. Such pulses have vital scientific and economic potential in areas such as high-resolution imaging of in-vivo cells and nanoscale XUV lithography.Comment: 17 pages, 4 figures, original format as accepted by Nature Physic

Visualization of the <i>Serratia </i>type VI secretion system reveals unprovoked attacks and dynamic assembly

SummaryThe Type VI secretion system (T6SS) is a bacterial nanomachine that fires toxic proteins into target cells. Deployment of the T6SS represents an efficient and widespread means by which bacteria attack competitors or interact with host organisms and may be triggered by contact from an attacking neighbor cell as a defensive strategy. Here, we use the opportunist pathogen Serratia marcescens and functional fluorescent fusions of key components of the T6SS to observe different subassemblies of the machinery simultaneously and on multiple timescales in vivo. We report that the localization and dynamic behavior of each of the components examined is distinct, revealing a multi-stage and dynamic assembly process for the T6SS machinery. We also show that the T6SS can assemble and fire without needing a cell contact trigger, defining an aggressive strategy that broadens target range and suggesting that activation of the T6SS is tailored to survival in specific niches

Genetic diversity of Brazilian isolates of feline immunodeficiency virus

We isolated Feline immunodeficiency virus (FIV) from three adult domestic cats, originating from two open shelters in Brazil. Viruses were isolated from PBMC following co-cultivation with the feline T-lymphoblastoid cell line MYA-1. All amplified env gene products were cloned directly into pGL8MYA. The nucleic acid sequences of seven clones were determined and then compared with those of previously described isolates. The sequences of all of the Brazilian virus clones were distinct and phylogenetic analysis revealed that all belong to subtype B. Three variants isolated from one cat and two variants were isolated from each of the two other cats, indicating that intrahost diversity has the potential to pose problems for the treatment and diagnosis of FIV infection