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The independent naming of components of injectisome-type type III secretion systems in different bacterial species has resulted in considerable confusion, impeding accessibility of the literature and hindering communication between scientists of the same field. A unified nomenclature had been proposed by Hueck more than 20 years ago. It found little attention for many years, but usage was sparked again by recent reviews and an international type III secretion meeting in 2016. Here, we propose that the field consistently switches to an extended version of this nomenclature to be no longer lost in translation.

In August 2018, the US FDA granted accelerated approval for nivolumab in small cell lung cancer (SCLC) that has progressed after platinum-based chemotherapy and at least one other line of therapy. The objective of this study was to evaluate the cost-effectiveness of nivolumab vs. usual care as third-line (3 L) therapy for patients with recurrent SCLC (rSCLC) from the health payer perspective. Given the potential for a meaningful fraction of treated patients to achieve long-term response to nivolumab, we also assessed the impact of using mixture cure modeling (MCM) vs. parametric survival modeling on survival estimates and cost-effectiveness from the US Medicare payer perspective. We created a partitioned survival decision model to assess the cost-effectiveness of 3 L nivolumab vs. usual care in rSCLC, based on observed US treatment patterns. Using this approach, we assessed the impact of extrapolating long-term survival from the CheckMate 032 trial, using both MCM and standard parametric curve fits. Nivolumab survival, resource use, and Grade 3/4 adverse event rates were derived from CheckMate 032. Usual care survival, resource use, and costs were derived from an analysis of patients receiving 3 L treatment for rSCLC in the SEER-Medicare registry. We applied 2020 Wholesale Acquisition Cost for drugs and 2020 CMS reimbursement for procedures. Utilities were derived from the literature. We estimated life years (LY), quality-adjusted life years (QALYs), and costs over a lifetime horizon. MCM and parametric survival model extrapolations resulted in 0.43 versus 0.38 more LYs, 0.34 versus 0.30 more QALYs, and $69,308 versus $61,336 more expenditure for nivolumab vs. usual care, respectively. The costs per QALY gained using mixture cure versus parametric survival modeling were $204,386 and $207,431, respectively. Mixture cure modeling was equivalent compared to parametric modeling in estimating the cost-effectiveness of nivolumab-based therapy due to the small fraction of patients achieving a long-term response with nivolumab (12.9%).

Activity of the NLRP3 inflammasome, a critical mediator of inflammation (1), is controlled by accessory proteins (2, 3), post-translational modifications (4, 5), cellular localization (6, 7) and oligomerization (8). How these factors relate, is unclear. We show that the established drug target, Bruton’s Tyrosine Kinase (BTK) (2, 9), integrates several levels of NLRP3 regulation: BTK phosphorylation of four conserved tyrosine residues, by neutralizing the charge of a polybasic linker region, weakens the interaction of NLRP3 with Golgi phospholipids and may thus guide NLRP3 cytosolic localization. BTK activity also promotes NLRP3 oligomerization and subsequent formation of inflammasomes. As NLRP3 tyrosine modification ultimately also impacts on IL-1β release, we propose BTK-mediated, charge-switch-based NLRP3 regulation as a novel and therapeutically tractable step in the control of inflammation.One Sentence SummaryMulti-phosphorylation of NLRP3 by Bruton’s tyrosine kinase modulates NLRP3 cellular localization, inflammasome assembly, and IL-1β release.

MotivationAntibiotic resistance is widely recognized as a severe threat to current medical practice. Each antibiotic therapy drives the emergence and subsequent retention of antibiotics resistance genes within the human gut microbiome. However, the details on how the resistance spreads between bacteria within the human gut remain unknown, as does the role of horizontal gene transfer in this process, too.ResultsWe present a novel approach to the analysis of time-series whole-genome metagenomic sequencing data. This involves partitioning the scaffolds from the metagenomic assembly into groups corresponding to bacterial chromosomes, plasmids and those with prophages and transposons. Using specialized sequencing of the bacteriophages we were able to track the flow of ciprofloxacin resistance genes from bacterial chromosomes, through the plasmids, to prophages and phages.Contactanna.gorska@univr.it.

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a lack of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess secretion and injection through the type III secretion system encoded bySalmonellapathogenicity island 1. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanoliter scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed NanoLuc and split-NanoLuc-based assays that enable the monitoring of type III secretion-dependent injection of effector proteins into host cells.ImportanceThe ability to secrete proteins to the bacterial cell surface, to the extracellular environment, or even into target cells is one of the foundations of interbacterial as well as pathogen-host interaction. While great progress has been made in elucidating assembly and structure of secretion systems, our understanding of their secretion mechanism often lags behind, not last because of the challenge to quantitatively assess secretion function. Here, we developed a luciferase-based assay to enable the simple, quick, quantitative, and high throughput-compatible assessment of secretion and injection through virulence-associated type III secretion systems. The assay allows detection of minute amounts of secreted substrate proteins either in the supernatant of the bacterial culture or within eukaryotic host cells. It thus provides an enabling technology to elucidate the mechanisms of secretion and injection of type III secretion systems and is likely adaptable to assay secretion through other bacterial secretion systems.

Functional injectisomes of the type-3 secretion system assemble into highly defined and stoichiometric bacterial molecular machines essential for infecting human and other eukaryotic cells. However, the mechanism that governs the regulated step-wise assembly process from the nucleation-phase, to ring-assembly, and the filamentous phase into a membrane embedded needle complex is unclear. We here report that the formation of a megadalton-sized needle complexes fromSalmonella entericaserovar Typhimurium (SPI-1,Salmonellapathogenicity island-1) with proper stoichiometries is highly structurally controlled competing against the self-assembly propensity of injectisome components, leading to a highly unusual structurally-pleiotropic phenotype. The structure of the entire needle complex from pathogenic injectisomes was solved by cryo electron microscopy, focused refinements (2.5-4 Å) and co-variation analysis revealing an overall asymmetric arrangement containing cyclic, helical, and asymmetric sub-structures. The centrally located export apparatus assembles into a conical, pseudo-helical structure and provides a structural template that guides the formation of a 24-mer cyclic, surrounding ring, which then serves as a docking interface comprising three different conformations for sixteen N-terminal InvG subunits of the outer secretin ring. Unexpectedly, the secretin ring excludes the 16thprotein chain at the C-terminal outer ring, resulting in a pleiotropic 16/15-mer ring and consequently to an overall 24:16/15 basal body structure. Finally, we report how the transition from the pseudo-helical export apparatus into the helical filament is structurally resolved to generate the protein secretion channel, which provides the structural basis to restrict access of unfolded effector substrates. These results highlight the diverse molecular signatures required for a highly coordinated assembly process and provide the molecular basis for understanding triggering and transport of unfolded proteins through injectisomes.

Export of proteins through type three secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are transported through an export gate complex consisting of three proteins (FliPQR in flagellar systems, SctRST in virulence systems) that were initially annotated as membrane proteins, but which we have recently shown assemble into an extra-membranous helical assembly. A fourth putative membrane protein (FlhB/SctU) is essential to the export process, and also functions to “switch” secretion substrate specificity once the growing hook/needle structures reach their determined length. Here we present the structure of an export gate containing the switch protein from a Vibrio polar flagellar system at 3.2 Å resolution by cryo-electron microscopy. The structure reveals that the FlhB/SctU further extends the helical export gate assembly with its four putative transmembrane helices adopting an out-of-membrane location, wrapped around the other export gate components at the base of the structure. The unusual topology of the switch protein helices creates a loop that wraps around the bottom of the closed export gate complex. Structure-informed mutagenesis suggests that this loop is critical in gating secretion and we propose that a series of conformational changes in the type 3 secretion system trigger opening of the export gate through the interactions between FlhB/SctU and FliPQR/SctRST.

This paper examines the additional clutter generated from the inclusion of a reflective backplate on a multi-static ground-penetrating radar array. Clutter is shown to be introduced via a ringing cavity and multipath propagation. This clutter is target-dependent and difficult to process. Experimental radar images show inclusion of a reflective backplate has a dramatic negative effect on image quality and we present potential clutter mitigation with the inclusion of absorbing material.

Enteropathogenic Yersinia enterocolitica (Ye) enters the host via contaminated food. After colonisation of the small intestine Ye invades the Peyer's patches (PPs) via M cells and disseminates to the mesenteric lymph nodes (MLNs), spleen and liver. Whether Ye uses other invasion routes and which pathogenicity factors are required remains elusive. Oral infection of lymphotoxin-β-receptor deficient mice lacking PPs and MLNs with Ye revealed similar bacterial load in the spleen 1h post infection as wild-type mice, demonstrating a PP-independent dissemination route for Ye. Immunohistological analysis of the small intestine revealed Ye in close contact with mononuclear phagocytes (MPs), specifically CX3CR1(+) monocyte-derived cells (MCs) as well as CD103(+) dendritic cells (DCs). This finding was confirmed by flow cytometry and imaging flow cytometry analysis of lamina propria (LP) leukocytes showing CD103(+) DCs and MCs with intracellular Ye. Uptake of Ye by LP CD103(+) DCs and MCs was dependent on the pathogenicity factor invasin, whereas the adhesin YadA was dispensable as demonstrated by Ye deletion mutants. Furthermore, Ye were found exclusively associated with CD103(+) DCs in the MLNs from wild-type mice, but not from CCR7(-/-) mice, demonstrating a CCR7 dependent transport of Ye by CD103(+) DCs from LP to the MLNs. In contrast, dissemination of Ye to the spleen was dependent on MCs as significantly less Ye could be recovered from the spleen of CX3CR1(GFP/GFP) mice compared to wild-type mice. Altogether, MCs and CD103(+) DCs contribute to immediate invasion and dissemination of Ye. This together with data from other bacteria suggests MPs as general pathogenic entry site in the intestine.

In quantum sensing, precision is typically limited by the maximum time interval over which phase can be accumulated. Memories have been used to enhance this time interval beyond the coherence lifetime and thus gain precision. Here, we demonstrate that by using a quantum memory an increased sensitivity can also be achieved. To this end, we use entanglement in a hybrid spin system comprising a sensing and a memory qubit associated with a single nitrogen-vacancy centre in diamond. With the memory we retain the full quantum state even after coherence decay of the sensor, which enables coherent interaction with distinct weakly coupled nuclear spin qubits. We benchmark the performance of our hybrid quantum system against use of the sensing qubit alone by gradually increasing the entanglement of sensor and memory. We further apply this quantum sensor-memory pair for high-resolution NMR spectroscopy of single 13C nuclear spins., In quantum sensing, memories have been used to enhance measurement precision. Here, the authors demonstrate the use of a memory to increase sensitivity of single 13C nuclear spins spectroscopy by storing the full sensor state and entangling memory and sensor.

Precisely knowing the stoichiometry of their components is critical for investigating structure, assembly, and function of macromolecular machines. This has remained a technical challenge in particular for large, hydrophobic membrane-spanning protein complexes. Here, we determined the stoichiometry of a type III secretion system of Salmonella enterica serovar Typhimurium using two complementary protocols of gentle complex purification combined with peptide concatenated standard and synthetic stable isotope-labeled peptide-based mass spectrometry. Bacterial type III secretion systems are cell envelope-spanning effector protein-delivery machines essential for colonization and survival of many Gram-negative pathogens and symbionts. The membrane-embedded core unit of these secretion systems, termed the needle complex, is composed of a base that anchors the machinery to the inner and outer membranes, a hollow filament formed by inner rod and needle subunits that serves as conduit for substrate proteins, and a membrane-embedded export apparatus facilitating substrate translocation. Structural analyses have revealed the stoichiometry of the components of the base, but the stoichiometry of the essential hydrophobic export apparatus components and of the inner rod protein remain unknown. Here, we provide evidence that the export apparatus of type III secretion systems contains five SpaP, one SpaQ, one SpaR, and one SpaS. We confirmed that the previously suggested stoichiometry of nine InvA is valid for assembled needle complexes and describe a loose association of InvA with other needle complex components that may reflect its function. Furthermore, we present evidence that not more than six PrgJ form the inner rod of the needle complex. Providing this structural information will facilitate efforts to obtain an atomic view of type III secretion systems and foster our understanding of the function of these and related flagellar machines. Given that other virulence-associated bacterial secretion systems are similar in their overall buildup and complexity, the presented approach may also enable their stoichiometry elucidation.

A ciência apresenta muitos traços de correntes filosóficas que existiram na história da humanidade, dentre elas o positivismo. Essa corrente originada no iluminismo tem características marcantes onde a ciência é considerada como uma verdade absoluta, entretanto sabe-se da sua dinamicidade. O objetivo deste trabalho foi evidenciar essas manifestações na educação científica em uma escola de educação básica de Quixelô, Ceará. Para tanto, foram aplicados questionários a alunos do Ensino Fundamental para a coleta de dados. Foi evidenciado que na escola existem correntes positivistas concernentes à educação científica, isto pode ser evidenciado devido os alunos acreditarem que a ciência é uma verdade absoluta, de modo que apresenta respostas concretas e imutáveis. Sendo assim, percebemos que a corrente positivista está presente na educação básica, sendo necessário a explanação por parte dos docentes de como a ciência está em constante ressignificação.Incluir o resumo.

Escherichia coli represents a classical intestinal gram-negative commensal. Despite this commensalism, different E. coli strains can mediate disparate immunogenic properties in a given host. Symbiotic E. coli strains such as E. coli Nissle 1917 (EcN) are attributed beneficial properties, e.g., promotion of intestinal homeostasis. Therefore, we aimed to identify molecular features derived from symbiotic bacteria that might help to develop innovative therapeutic alternatives for the treatment of intestinal immune disorders. This study was performed using the dextran sodium sulphate (DSS)-induced colitis mouse model, which is routinely used to evaluate potential therapeutics for the treatment of Inflammatory Bowel Diseases (IBDs). We focused on the analysis of flagellin structures of different E. coli strains. EcN flagellin was found to harbor a substantially longer hypervariable region (HVR) compared to other commensal E. coli strains, and this longer HVR mediated symbiotic properties through stronger activation of Toll-like receptor (TLR)5, thereby resulting in interleukin (IL)-22–mediated protection of mice against DSS-induced colitis. Furthermore, using bone-marrow–chimeric mice (BMCM), CD11c+ cells of the colonic lamina propria (LP) were identified as the main mediators of these flagellin-induced symbiotic effects. We propose flagellin from symbiotic E. coli strains as a potential therapeutic to restore intestinal immune homeostasis, e.g., for the treatment of IBD patients., A flagellum renders bacteria motile, but this study reveals another property important for symbiosis: the hypervariable region of Escherichia coli flagellin strongly determines activation of TLR5, mediating benefits for the host such as protection against colitis.

We present the design and measurement of a planar ultrawideband $50:200\ \Omega$ Guanella balun operating with a 1 dB differential insertion loss from 0.35 to 3.4 GHz for ultrawideband radar antennas. We excite the Guanella balun with a Ricker wavelet centered around 700 MHz to 1.6 GHz and see little distortion in time domain. The response appears to be the best published low frequency response to date of a compact planar microwave Guanella balun. This Guanella balun importantly enables integrated compact feeding of high-impedance balanced antennas such as Resistivelv-Loaded Vee Dipoles.

A design of a resistively loaded vee dipole (RLVD)fed by an integrated even-mode matched Marchand balun is proposed. The RLVD is designed for use in a pulsed radar application with a bandpass characteristic from 0.5–1.5 GHz. The Marchand balun creates the bandpass response when cascaded with the typical high-pass response of the RLVD. To achieve good matching between the balun and the RLVD, an even-mode matching circuit is developed and incorporated at the balanced ports of the balun. Experimental results show prototype return loss better than −10 dB from 400 MHz to 1.9 GHz and a significant decrease in low-frequency monostatic coupling.

Virulence-associated type III secretion systems (T3SS) serve the injection of bacterial effector proteins into eukaryotic host cells. They are able to secrete a great diversity of substrate proteins in order to modulate host cell function, and have evolved to sense host cell contact and to inject their substrates through a translocon pore in the host cell membrane. T3SS substrates contain an N-terminal signal sequence and often a chaperone-binding domain for cognate T3SS chaperones. These signals guide the substrates to the machine where substrates are unfolded and handed over to the secretion channel formed by the transmembrane domains of the export apparatus components and by the needle filament. Secretion itself is driven by the proton motive force across the bacterial inner membrane. The needle filament measures 20-150 nm in length and is crowned by a needle tip that mediates host-cell sensing. Secretion through T3SS is a highly regulated process with early, intermediate and late substrates. A strict secretion hierarchy is required to build an injectisome capable of reaching, sensing and penetrating the host cell membrane, before host cell-acting effector proteins are deployed. Here, we review the recent progress on elucidating the assembly, structure and function of T3SS injectisomes.

Patients with recurrent or metastatic cancer commonly suffer from debilitating toxicity associated with conventional treatment modalities, as well as disease-related symptoms, often with a concomitant negative impact on health-related quality of life (HRQoL). Patient-reported outcomes (PROs) provide important insights into the patient experience in clinical trials. Nivolumab is a programmed death-1 receptor inhibitor that extends survival in patients with recurrent or metastatic disease in multiple tumor types. In this review, we summarize published PRO analyses from eight phase II-IV clinical trials with nivolumab for the treatment of melanoma, non-small cell lung cancer, renal cell carcinoma (RCC), and squamous cell carcinoma of the head and neck (SCCHN). Symptom burden, physical functioning, and HRQoL were measured using generic, cancer-specific, and tumor type-specific validated PRO instruments. Nivolumab showed sustained stabilization across all tumor types and, in some cases, clinically meaningful improvement in HRQoL, whereas standard of care therapies often led to deteriorations. Exploratory analyses found a positive correlation between baseline HRQoL scores and overall survival in RCC, and between baseline HRQoL scores and healthcare resource utilization in SCCHN, suggesting that patient-reported symptoms at treatment initiation may have clinical value. In the era of value-based oncology care, stakeholders are increasingly interested in PRO findings to guide clinical, regulatory, and reimbursement decisions. However, missing data remain a significant challenge in PRO analyses, including in nivolumab trials. Future clinical trials in immuno-oncology should incorporate PRO data collection, including beyond treatment discontinuation or trial completion to assess the long-term effects of treatment on HRQoL.

SummaryExport of proteins through type three secretion systems is critical for bacterial motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP/FliQ/FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. We here present the structure of this complex at 4.2 Å by cryo-electron microscopy. None of the subunits adopt canonical integral membrane protein topologies and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems localize the export gate as a core component of the periplasmic portion of the machinery, and cross-linking experiments confirm this observation. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.One Sentence SummaryThe core of the T3SS export gate forms a supra-membrane helical assembly

Background & aimsHepatocellular carcinoma (HCC) is the second most common cause of cancer deaths worldwide. The global HCC BRIDGE study was a multiregional, large-scale, longitudinal cohort study undertaken to improve understanding of real-life management of patients with HCC, from diagnosis to death.MethodsData were collected retrospectively from January 2005 to September 2012 by chart reviews of eligible patients newly diagnosed with HCC at participating institutions.ResultsForty-two sites in 14 countries contributed final data for 18 031 patients. Asia accounted for 67% of patients, Europe for 20% and North America for 13%. As expected, the most common risk factor was hepatitis C virus in North America, Europe and Japan, and hepatitis B virus in China, South Korea and Taiwan. The most common Barcelona Clinic Liver Cancer stage at diagnosis was C in North America, Europe, China and South Korea, and A in Taiwan and Japan. Across all stages, first HCC treatment was most frequently transarterial chemoembolization in North America, Europe, China and South Korea, percutaneous ethanol injection or radiofrequency ablation in Japan and resection in Taiwan. Survival from first HCC treatment varied significantly by region, with median overall survival not reached for Taiwan and 60, 33, 31, 24 and 23 months for Japan, North America, South Korea, Europe and China respectively (P < 0.0001).ConclusionsInitial results from the BRIDGE study confirm previously reported regional trends in patient demographic characteristics and HCC risk factors, document the heterogeneity of treatment approaches across regions/countries and underscore the need for earlier HCC diagnosis worldwide.

Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP–FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery., Author summary Many bacteria use the bacterial flagellum for directed movement in various environments. The assembly and function of the bacterial flagellum and the related virulence-associated injectisome relies on protein export via a conserved type III secretion system (T3SS). The multicomponent transmembrane core export apparatus of the flagellar T3SS consists of FlhA/B and FliP/Q/R and must assemble in a highly coordinated manner. In the present study, we determined the role of the transmembrane protein FliO in the maturation of the flagellar core protein export apparatus. We show that FliO functions as a flagellum-specific chaperone during the initial step of export apparatus assembly. FliO facilitates the efficient formation of a stable FliP–FliR core complex and is thus required for quality management and productive assembly of the flagellar export apparatus. Our results suggest a coordinated assembly process of the flagellar core export apparatus that nucleates with the FliO-dependent formation of a FliP–FliR complex. Subsequent incorporation of FliQ, FlhB, and FlhA leads to the assembly of a secretion-competent flagellar T3SS.

Bacterial protein secretion systems serve to translocate substrate proteins across up to three biological membranes, a task accomplished by hydrophobic, membrane-spanning macromolecular complexes. The overexpression, purification, and biochemical characterization of these complexes is often difficult, impeding progress in understanding the structure and function of these systems. Blue native (BN) polyacrylamide gel electrophoresis (PAGE) allows for the investigation of these transmembrane complexes right from their originating membranes, without the need for long preparative steps, and is amenable to the parallel characterization of a number of samples under near-native conditions. Here we present protocols for sample preparation, one-dimensional BN PAGE and two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE, as well as for downstream analysis by staining, immunoblotting, and mass spectrometry on the example of the type III secretion system encoded on Salmonella pathogenicity island 1.

The reliable detection of protein-protein interactions by affinity purification mass spectrometry (AP-MS) is crucial for the understanding of biological processes. Quantitative information can be used to separate truly interacting proteins from false-positives by contrasting counts of proteins binding to specific baits with counts of negative controls. Several approaches have been proposed for computing scores for potential interaction proteins, for example, the commonly used SAINT software. However, it remains a subjective decision where to set the cutoff score for candidate selection; furthermore, no precise control for the expected number of false-positives is provided. In related fields, successful data analysis strongly relies on statistical pre- and post-processing steps, which, so far, have played only a minor role in AP-MS data analysis. We introduce a complete workflow, embedding either the scoring method SAINT or alternatively a two-stage Poisson model into a pre- and post-processing framework. To this end, we investigate different normalization methods and apply a statistical filter adjusted to AP-MS data. Furthermore, we propose permutation and adjustment procedures, which allow the replacement of scores by statistical p values. The performance of the workflow is assessed on simulations as well as on a study focusing on interactions with the T3SS in Salmonella Typhimurium. Preprocessing methods significantly increase the number of detected truly interacting proteins, while a constant false-discovery rate is maintained. The software solution is freely available.

Virulence-associated type III secretion systems serve the injection of bacterial effector proteins into eukaryotic host cells. These effector proteins modulate host cell biology in order to promote colonization and infection, hence type III secretion systems are often essential bacterial pathogenicity factors. The core of type III secretion systems is a cell envelope-spanning macromolecular machine called injectisome. It consists of almost twenty different components in a stoichiometry of one to more than one hundred. Assembly of this 6 MDa complex requires the coordinated integration of components from the cytoplasm, the inner membrane, the periplasm, the outer membrane and even the extracellular space of Gram-negative bacteria. Here, we review injectisome assembly with an emphasis on the techniques that were employed towards its investigation. In particular, we focus on in vivo photocrosslinking, a technique that exploits the encoding of the artificial UV-inducible crosslinking amino acid p-benzoyl-phenylalanine to identify protein-protein interactions and to delineate assembly pathways.

Background: Autotransporter adhesins reach the bacterial cell surface by a complex mechanism. Results: In the case of the autotransporter SadA from Salmonella, a lipoprotein assists in surface display. Conclusion: The similarity to eukaryotic MATH domains suggests that the lipoprotein assists in trimerization of SadA. Significance: Understanding the similarities between autotransport systems might lead to new ways of inhibiting bacterial adhesion., Trimeric autotransporter adhesins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. TAAs form fibrous, adhesive structures on the bacterial cell surface. Their N-terminal extracellular domains are exported through a C-terminal membrane pore; the insertion of the pore domain into the bacterial outer membrane follows the rules of β-barrel transmembrane protein biogenesis and is dependent on the essential Bam complex. We have recently described the full fiber structure of SadA, a TAA of unknown function in Salmonella and other enterobacteria. In this work, we describe the structure and function of SadB, a small inner membrane lipoprotein. The sadB gene is located in an operon with sadA; orthologous operons are only found in enterobacteria, whereas other TAAs are not typically associated with lipoproteins. Strikingly, SadB is also a trimer, and its co-expression with SadA has a direct influence on SadA structural integrity. This is the first report of a specific export factor of a TAA, suggesting that at least in some cases TAA autotransport is assisted by additional periplasmic proteins.

The aim of this study was to investigate the impact of menopausal symptoms and menopausal symptom severity on health-related quality of life (HRQoL), work impairment, healthcare utilization, and costs.Data from the 2005 United States National Health and Wellness Survey were used, with only women 40-64 years without a history of cancer included in the analyses (N=8,811). Women who reported experiencing menopausal symptoms (n=4,116) were compared with women not experiencing menopausal symptoms (n=4,695) on HRQoL, work impairment, and healthcare utilization using regression modeling (and controlling for demographics and health characteristic differences). Additionally, individual menopausal symptoms were used as predictors of outcomes in a separate set of regression models.The mean age of women in the analysis was 49.8 years (standard deviation,±5.9). Women experiencing menopausal symptoms reported significantly lower levels of HRQoL and significantly higher work impairment, and healthcare utilization than women without menopausal symptoms. Depression, anxiety, and joint stiffness were symptoms with the strongest associations with health outcomes.Menopausal symptoms can be a significant humanistic and economic burden on women in middle age.

OBJECTIVE: The current study characterizes health-related quality of life, work productivity, and resource use among postmenopausal women by severity of vasomotor symptoms (VMS). METHODS: Participants were selected from the 2010 US National Health and Wellness Survey. Women aged 40 to 75 years who did not report a history of menstrual bleeding or spotting for 1 year were eligible for analysis (N = 3,267). Cohorts of women with no VMS (n = 1,740), mild VMS (n = 931), moderate VMS (n = 462), and severe VMS (n = 134) were compared after controlling for demographic and health characteristics. Outcome measures were assessed using linear models and included health status, work productivity within the past 7 days, and healthcare resource use within the past 6 months. RESULTS: The mean age of women experiencing severe VMS was 57.92 years. After demographic and health characteristics had been controlled for, women experiencing severe and moderate VMS reported significantly lower mean health status scores compared with women with no symptoms (P < 0.0001). The mean number of menopause symptom-related physician visits was significantly greater among women with severe, moderate, or mild symptoms than among women with no symptoms (P < 0.0001). Among employed women experiencing VMS, women with severe and moderate symptoms had adjusted presenteeism of 24.28% and 14.3%, versus 4.33% in women with mild symptoms (P < 0.001), and activities of daily living impairment of 31.66% and 17.06%, versus 6.16% in women with mild symptoms (P < 0.0001). CONCLUSIONS: In postmenopausal women, a greater severity of VMS is significantly associated with lower levels of health status and work productivity, and greater healthcare resource use.

Bacterial protein secretion systems serve to translocate substrate proteins across up to three biological membranes, a task accomplished by hydrophobic, membrane-spanning macromolecular complexes. The overexpression, purification, and biochemical characterization of these complexes is often difficult, impeding progress in understanding the structure and function of these systems. Blue native (BN) polyacrylamide gel electrophoresis (PAGE) allows for the investigation of these transmembrane complexes right from their originating membranes, without the need for long preparative steps, and is amenable to the parallel characterization of a number of samples under near-native conditions. Here we present protocols for sample preparation, one-dimensional BN PAGE and two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE, as well as for downstream analysis by staining, immunoblotting, and mass spectrometry on the example of the type III secretion system encoded on Salmonella pathogenicity island 1.

Gaining knowledge of the structural makeup of protein complexes is critical to advance our understanding of their formation and functions. This task is particularly challenging for transmembrane protein complexes, and grows ever more imposing with increasing size of these large macromolecular structures. The last 10 years have seen a steep increase in solved high-resolution membrane protein structures due to both new and improved methods in the field, but still most structures of large transmembrane complexes remain elusive. An important first step towards the structure elucidation of these difficult complexes is the determination of their stoichiometry, which we discuss in this review. Knowing the stoichiometry of complex components not only answers unresolved structural questions and is relevant for understanding the molecular mechanisms of macromolecular machines but also supports further attempts to obtain high-resolution structures by providing constraints for structure calculations.

Hepatitis C virus (HCV) affects 170 million patients worldwide and is the leading cause of liver cirrhosis and hepatocellular carcinoma. The aim of the current study is to examine the burden of HCV in the European Union (EU) from a patient perspective.Using data from the 2010 EU National Health and Wellness Survey, patients who reported a diagnosis of HCV (n=332) were compared with a propensity-score-matched non-HCV control group (n=332) on measures of quality of life (using the SF-12v2), work productivity, and healthcare resource utilization in the past 6 months. All analyses applied sampling weights to project to the respective country populations.Projected prevalence estimates of HCV were 0.59% in France, 0.44% in Germany, 1.42% in Italy, 0.82% in Spain, and 0.35% in the UK. HCV patients reported significantly lower levels of emotional role limitations (means=66.4 vs. 70.6, P=0.040), physical functioning (means=63.8 vs. 71.9, P=0.001), general health (means=48.3 vs. 54.4, P=0.004), bodily pain (means=64.3 vs. 70.8, P=0.002), and physical component summary scores (means=42.9 vs. 45.3, P=0.002) than the matched controls. Patients with HCV also reported significantly higher levels of presenteeism (means=27.1 vs. 21.0%, P=0.044) and a greater number of physician visits in the past 6 months (means=9.9 vs. 6.7, P0.001).Using a population-based survey methodology and a propensity-score matching analysis, these results add to the literature by documenting the significant effect that HCV has on a variety of both humanistic and economic outcomes in the EU.

Objective Overall survival is a commonly reported end point in clinical trial publications and a key determinant of therapies’ cost-effectiveness. Patients’ survival times have skewed distributions. Outcomes are typically presented in clinical trials as the difference in median survival times; we compare median survival gain with the measure required for economic evaluation, the mean difference. Study Design We summarize the relationships between median and mean survival in 4 parametric survival distributions and the relationship of the differences in these measures between trial arms and parameterized treatment effects. Parametric estimates of mean survival were compared with median survival in a case study of a recent trial in metastatic melanoma. Results In a trial of alternative therapies in unresectable metastatic melanoma, median overall survival with ipilimumab alone was 10.1 months versus 6.4 months with gp100-alone (hazard ratio 0.66; P = 0.003). A log-normal parametric survivor function fitted the gp100 Kaplan-Meier function and a time ratio of 1.90 applied only after 90 days gave a suitable fit to the Kaplan-Meier function for ipilimumab, with mean survival difference of 7 months, compared with an estimate of 5.7 months employing a Weibull distribution, and with a 3.7-months median difference. Conclusion Parametric assessment of mean survival gain in clinical trials may indicate potential benefits to patients that observed medians may greatly underestimate.

To address the gap in knowledge about the impact of chronic obstructive pulmonary disease (COPD) on older working adults, this study examined quality of life, worker productivity, and healthcare resource utilization among employed adults aged 65 and older with and without COPD. Among 2009 National Health and Wellness Survey (a cross-sectional, internet-based survey representative of the US adult population) respondents, employed adults aged 65 years and older, with COPD (n = 297) and without COPD (n = 3061), were included in analyses. Impact of self-reported COPD diagnosis on mean quality of life (using health utilities and mental, MCS, and physical, PCS, component summary scores from SF-12v2), work productivity and activity impairment (using the WPAI questionnaire), and resource use were examined. Adjusting for demographic and health characteristics such as co-morbidities (weighted to project to the US population) in regression models (linear, negative binomial, or logistic, as appropriate given the outcome measure), older workers with COPD reported significantly lower MCS (52.1 vs. 53.4, p < .05), PCS (40.3 vs. 47.2, p < .05), and health utilities (0.72 vs. 0.79, p < .05) than those without COPD, and significantly greater percentages of impairment while at work (presenteeism) (12.6% vs. 8.7%, p < .0001), overall work impairment (absenteeism and presenteeism combined) (19.3% vs. 10.0%, p < .05), and impairment in daily activities (23.9% vs. 13.7%, p < .05). There were no significant differences in absenteeism or healthcare use. Quality of life and work productivity suffered among employed adults aged 65 years and older with COPD, emphasizing the need for disease management in this population.

To assess factors associated with adherence, particularly pill burden, to combination antiretroviral therapy (cART) using multivariate models.A cross-sectional survey of US adults with a self-reported diagnosis of HIV/AIDS was conducted between April and May 2007. Respondents on a cART regimen of at least 2 nucleoside reverse transcriptase inhibitors plus at least 1 protease inhibitor or non-nucleoside reverse transcriptase inhibitor (n = 461) were included in the analytic sample. Multiple logistic regression models determined independent predictors of complete adherence (defined as never missing or skipping an antiretroviral dose).Fifty-four percent of respondents reported complete adherence to cART. Adherent respondents reported a lower percentage of hospitalizations (11% vs 28%; P.0001) and emergency room visits (26% vs 34%; P.09). Respondents taking the single tablet efavirenz/emtricitabine/tenofovir fixed-dose regimen were significantly more likely to have complete adherence than respondents taking other cART regimens (odds ratio [OR] 2.1, P.05), and higher imputed daily HIV pill count was associated with lower likelihood of complete adherence (OR 0.93, P.05).This study shows the negative impact of higher pill burden on medication adherence, an important factor associated with treatment outcomes in patients with HIV/AIDS.

There has been limited research addressing the effects of constipation on work productivity and healthcare resource use.To assess the effect of chronic constipation on health outcomes and healthcare resource use.Using data from the 2007 National Health and Wellness Survey (NHWS), chronic constipation patients (n = 1,430) were propensity score-matched to controls (n = 1,430) on demographic and clinical characteristics. Differences between groups in health-related quality of life (SF-12v2), work productivity and activity impairment, and resource use in the last 6 months were examined. Mediation analyses were conducted in order to determine whether the relationship between constipation and resource use was caused by a reduction in health status.Chronic constipation patients reported significantly lower levels of health-related quality of life (physical component summary score: 39.57 vs. 43.73; mental component summary score: 43.19 vs. 47.86, all P-values0.01) and significantly higher levels of loss of work productivity and activity impairment (absenteeism: 9.08% vs. 5.20%; presenteeism: 29.52% vs. 19.09%; overall work impairment: 33.65% vs. 21.56%; activity impairment: 46.58% vs. 33.90%, all P-values0.01) compared to the matched controls. Chronic constipation patients also reported significantly more provider (7.73 vs. 5.63) and emergency room visits (0.52 vs. 0.30) in the past 6 months (all P-values0.01). Mediation analyses suggested that increased resource use among chronic constipation patients were partially a result of reduced health status.Compared to matched controls, chronic constipation patients reported greater economic and humanistic burden. Alleviating the humanistic burden associated with constipation may have economic benefits.

To investigate the association between reported oral antidiabetic tolerability issues and work productivity, activity impairment, and indirect costs.Data were collected from the 2006 to 2008 US National Health and Wellness Survey and the Lightspeed Research, using an Internet-based questionnaire (N = 2074).Absenteeism, presenteeism, overall work impairment, and activity impairment increased as the number of tolerability issues increased. Similar results were observed using a diabetes-specific productively measure. Total annual adjusted indirect costs (absenteeism and presenteeism costs summed) were $2759, $5533, $7537, and $8405 for patients with 1, 2, 3, and 4 or more tolerability issues, respectively.The consideration of tolerability profiles of oral antidiabetic agents may lead to improved productivity among treated patients. Furthermore, targeted educational programs regarding risks and management of these issues to employees with type 2 diabetes mellitus may benefit both employers and patients.

Between 2.7 and 3.9 million people are currently infected with the hepatitis C virus (HCV) in the United States. Although many studies have investigated the impact of HCV on direct healthcare costs, few studies have estimated the indirect costs associated with the virus using a nationally-representative dataset.Using data from the 2009 United States (US) National Health and Wellness Survey, patients who reported a hepatitis C diagnosis (n = 695) were compared to controls on labor force participation, productivity loss, and activity impairment after adjusting for demographics, health risk behaviors, and comorbidities. All analyses applied sampling weights to project to the population.Patients with HCV were significantly less likely to be in the labor force than controls and reported significantly higher levels of absenteeism (4.88 vs. 3.03%), presenteeism (16.69 vs. 13.50%), overall work impairment (19.40 vs.15.35%), and activity impairment (25.01 vs. 21.78%). A propensity score matching methodology replicated many of these findings.While much of the work on HCV has focused on direct costs, our results suggest indirect costs should not be ignored when quantifying the societal burden of HCV. To our knowledge, this is the first study which has utilized a large, nationally-representative data source for identifying the impact of HCV on labor force participation and work and activity impairment using both a propensity-score matching and a regression modeling framework.All data were patient-reported (including HCV diagnosis and work productivity), which could have introduced some subjective biases.

Thus far, the role of the Escherichia coli signal recognition particle (SRP) has only been studied using targeted approaches. It has been shown for a handful of cytoplasmic membrane proteins that their insertion into the cytoplasmic membrane is at least partially SRP-dependent. Furthermore, it has been proposed that the SRP plays a role in preventing toxic accumulation of mistargeted cytoplasmic membrane proteins in the cytoplasm. To complement the targeted studies on SRP, we have studied the consequences of the depletion of the SRP component Fifty-four homologue (Ffh) in E. coli using a global approach. The steady-state proteomes and the proteome dynamics were evaluated using one- and two-dimensional gel analysis, followed by mass spectrometry-based protein identification and immunoblotting. Our analysis showed that depletion of Ffh led to the following: (i) impaired kinetics of the biogenesis of the cytoplasmic membrane proteome; (ii) lowered steady-state levels of the respiratory complexes NADH dehydrogenase, succinate dehydrogenase, and cytochrome bo(3) oxidase and lowered oxygen consumption rates; (iii) increased levels of the chaperones DnaK and GroEL at the cytoplasmic membrane; (iv) a σ(32) stress response and protein aggregation in the cytoplasm; and (v) impaired protein synthesis. Our study shows that in E. coli SRP-mediated protein targeting is directly linked to maintaining protein homeostasis and the general fitness of the cell.

Objective The aim of the current study was to examine levels of well-being and productivity and the interrelationships between them in community-dwelling patients with schizophrenia who use atypical antipsychotic medications. Methods A mixed methodology (online and paper surveys) was implemented to recruit patients in the USA over the age of 18 years who were diagnosed with schizophrenia by a healthcare professional and who were able to read and write English. Patients who were currently being treated with an atypical medication and provided well-being data (n = 745) were included in the analyses. Logistic and negative binomial regressions were used to determine the effect of well-being on productivity and resource use. Key findings Almost two-thirds of patients taking atypical antipsychotics reported low levels of well-being (63%). After controlling for demographic and patient characteristics, patients with low levels of well-being were significantly less likely to be employed and productive than patients with moderate or high levels of well-being (P

Type III protein secretion systems are unique bacterial nanomachines with the capacity to deliver bacterial effector proteins into eukaryotic cells. These systems are critical to the biology of many pathogenic or symbiotic bacteria for insects, plants, animals, and humans. Essential components of these systems are multiprotein envelope-associated organelles known as the needle complex and a group of membrane proteins that compose the so-called export apparatus. Here, we show that components of the export apparatus associate intimately with the needle complex, forming a structure that can be visualized by cryo-electron microscopy. We also show that formation of the needle complex base is initiated at the export apparatus and that, in the absence of export apparatus components, there is a significant reduction in the levels of needle complex base assembly. Our results show a substantial coordination in the assembly of the two central elements of type III secretion machines.

In the version of this article initially published, the PDB code associated with the study was given as 6F2E but should have been 6F2D in Table 1 and the data availability statement. The error has been corrected in the HTML and PDF versions of the article.

The purpose of this study was to determine if empirically observed differences in patient perception of, and satisfaction with, onset of effect between an active maintenance treatment and placebo are clinically meaningful to practicing clinicians. A secondary objective was to determine the lowest threshold for a clinically meaningful difference in terms of both between-group differences and maximum acceptable placebo effect.Twelve community-based healthcare professionals participated in a Delphi consensus panel. Panelists were provided with blinded results of two clinical trials showing statistically significant treatment effects for treatment A (budesonide/formoterol [Symbicort*]) over placebo in the proportion of patients who could perceive the medication working right away and the proportion of patients satisfied with this perception. Panelists were then asked to respond to a series of questions to identify a threshold for clinically important differences in patient-perceived onset of effect and satisfaction with speed of onset of effect. All expert panelists participated in two rounds of the Delphi process.Panelists were unanimous in their conclusion that the statistically significant results from the two trials were clinically meaningful. According to these practitioners, the empirical results presented to them, showing that patients could feel a maintenance inhaler therapy work right away, were meaningful to clinical decision-making, and the attribute could potentially improve patient adherence with therapy. A group consensus was reached that a minimum active treatment response for these outcomes should range from 50% to 75% and be 2-3 times larger than the placebo response, with a maximum placebo effect of 26-40%.A Delphi panel study of practitioners was used to establish a meaningful range of response and a minimal important difference for interpreting results of clinical trials in which patient perception of onset of effect and satisfaction with this perception are tested. While the views of this panel may not be generalized to the entire population of practitioners in the United States, results provide insight into how a typical practitioner is likely to view clinical trial results and how the information might be used in clinical practice.

In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS-PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS-PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS-PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS-PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS-PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.

The Sec translocon is a protein-conducting channel that allows polypeptides to be transferred across or integrated into a membrane. Although protein translocation and insertion in Escherichia coli have been studied using only a small set of specific model substrates, it is generally assumed that most secretory proteins and inner membrane proteins use the Sec translocon. Therefore, we have studied the role of the Sec translocon using subproteome analysis of cells depleted of the essential translocon component SecE. The steady-state proteomes and the proteome dynamics were evaluated using one- and two-dimensional gel analysis, followed by mass spectrometry-based protein identification and extensive immunoblotting. The analysis showed that upon SecE depletion (i) secretory proteins aggregated in the cytoplasm and the cytoplasmic σ 32 stress response was induced, (ii) the accumulation of outer membrane proteins was reduced, with the exception of OmpA, Pal, and FadL, and (iii) the accumulation of a surprisingly large number of inner membrane proteins appeared to be unaffected or increased. These proteins lacked large translocated domains and/or consisted of only one or two transmembrane segments. Our study suggests that several secretory and inner membrane proteins can use Sec translocon-independent pathways or have superior access to the remaining Sec translocons present in SecE-depleted cells.