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Currently, no publicly available comprehensive set of specifications exists for the design of ground‐penetrating radar antennas. Specifications are presented and derived for the design of a ground‐penetrating radar antenna based on the antenna's interaction with the rest of the radar system. Typically, ground‐penetrating radar antenna engineers are given basic parameters such as gain and bandwidth to design an antenna. An antenna can be designed to meet those specifications but may not perform well in a ground‐penetrating radar imaging system. By considering the whole system, including the operation parameters, expected targets, and signal processing algorithms, we can constrain the less‐commonly considered performance aspects of an antenna to perform more reliably in a deployment. We propose quantitative specifications for the antenna's coupling, beamwidth, boresight radar cross‐section, pulse integrity, residual ringing, and front‐to‐back ratio. Meeting these antenna specifications will decrease the probability that a deficiency in the antenna design impacts the radar's image output. Finally, a summarised design equation table that will be helpful in the ground‐penetrating radar antenna community at large is offered. [ABSTRACT FROM AUTHOR]

We employ a 77–81 GHz frequency-modulated continuous-wave (FMCW) millimeter-wave radar to sense anomalous vibrations during vehicle transport at highway speeds for the first time. Secure metallic containers can be breached during transport by means of drilling into their sidewalls but detecting a drilling signature is difficult because the large vibrations of transport drown out the small vibrations of drilling. For the first time, we demonstrate that it is possible to use a non-contact millimeter-wave radar sensor to detect this micron-scale intrusive drilling while highway-speed vehicle movement shakes the container. With the millimeter-wave radar monitoring the microdoppler signature of the container's vibrating walls, we create a novel signal-processing pipeline consisting of range–angle tracking, time–frequency analysis, horizontal stripe image convolution, and principal component analysis to create a robust and powerful detection statistic to alarm if drilling is present. To support this pipeline, we develop a statistical model combining the vibrating container and the random vibrations induced by vehicle movement to explore the robustness of the sensor's detection capabilities. The presented results strongly support the inclusion of a millimeter-wave radar vibration sensor into a transport security system. [ABSTRACT FROM AUTHOR]

Introduction: Intubation and mechanical ventilation are common interventions performed in the emergency department (ED). These interventions cause pain and discomfort to patients and necessitate analgesia and sedation. Recent trends in the ED and intensive care unit focus on an analgesia-first model to improve patient outcomes. Initial data from our institution demonstrated an over-emphasis on sedation and an opportunity to improve analgesic administration. As a result of these findings, the ED undertook a quality improvement (QI) project aimed at improving analgesia administration and time to analgesia post-intubation. Methods: We performed a pre-post study between January 2017–February 2019 in the ED. Patients over the age of 18 who were intubated using rapid sequence intubation (RSI) were included in the study. The primary outcome was the rate of analgesia administration; a secondary outcome was time to analgesia administration. Quality improvement interventions occurred in two phases: an initial intervention focused on nursing education only, and a subsequent intervention that included nursing and physician education. Results: During the study period, 460 patients were intubated in the ED and met inclusion/exclusion criteria. Prior to the first intervention, the average rate of analgesia administration was 57.3%; after the second intervention, the rate was 94.9% (P <0.01). Prior to the first intervention, average time to analgesia administration was 36.0 minutes; after the second intervention, the time was 16.6 minutes (P value <0.01). Conclusion: This QI intervention demonstrates the ability of education interventions alone to increase the rate of analgesia administration and reduce the time to analgesia in post-intubation patients. [ABSTRACT FROM AUTHOR]

This article presents a novel investigation into the effect of receiver timing jitter on the quality of imaging of ground-penetrating radar systems. We explicitly show the impact of timing jitter throughout an entire example processing pipeline. The process of coupling removal in systems whose receivers have a random Gaussian-distributed jitter with a high standard deviation leaves randomly distributed residue in the radar image. This residue, called jitter-induced coupling noise, extends even into the expected target domain which may cause false alarms. This residue approximately follows a sum of Gaussian and Gamma distributions based on the higher order derivatives of the coupling signal. Once migrated to create an image, the coupling residue is best described by a Weibull distribution. We show that simple filtering does not remove enough residue and propose two preventative design specifications called the linear slope and migrated probability specifications. Antenna and system designers should use these specifications to assess the true severity of coupling signals in a given jitter environment. [ABSTRACT FROM AUTHOR]

We present the development of the novel elliptical resistively loaded vee dipole (ERLVD). By replacing the constituent subelements with ellipses, the ERLVD has two degrees of design freedom to achieve increased gain, bandwidth, and decreased size compared with the classical resistively loaded vee dipole (RLVD). The prototyped ERLVD with a unity growth rate experimentally achieved a bandwidth ratio of 13:1 (0.49–6.41 GHz) and a smaller arm length of $0.17~\lambda _{\mathrm {low}}$ at 490 MHz. This ERLVD has twice larger the bandwidth ratio, 2–3 dB higher gain, and smaller arm size at 490 MHz compared to similar RLVDs. The second ERLVD with a higher growth rate can achieve much higher relative gain increases of 7–8.5 dB with slightly reduced bandwidth. [ABSTRACT FROM AUTHOR]

The mechanisms underlying discrimination between "self" and "non-self", a central immunological principle, require careful consideration in immune oncology therapeutics where eliciting anti-cancer immunity must be weighed against the risk of autoimmunity due to the self origin of tumors. Whole cell vaccines are one promising immunotherapeutic avenue whereby a myriad of tumor antigens are introduced in an immunogenic context with the aim of eliciting tumor rejection. Despite the possibility collateral damage to healthy tissues, cancer immunotherapy can be designed such that off target autoimmunity remains limited in scope and severity or completely non-existent. Here we provide an immunological basis for reconciling the safety of cancer vaccines, focusing on tumor endothelial cell vaccines, by discussing the following topics: (a) Antigenic differences between neoplastic and healthy tissues that can be leveraged in cancer vaccine design; (b) The layers of tolerance that control T cell responses directed against antigens expressed in healthy tissues and tumors; and, (c) The hierarchy of antigenic epitope selection and display in response to whole cell vaccines, and how antigen processing and presentation can afford a degree of selectivity against tumors. We conclude with an example of early clinical data utilizing ValloVax™, an immunogenic placental endothelial cell vaccine that is being advanced to target the tumor endothelium of diverse cancers, and we report on the safety and efficacy of ValloVax™ for inducing immunity against tumor endothelial antigens. [ABSTRACT FROM AUTHOR]

Angiogenesis is essential for the growth and metastasis of solid tumors. The tumor endothelium exists in a state of chronic activation and proliferation, fueled by the tumor milieu where angiogenic mediators are aberrantly over-expressed. Uncontrolled tumor growth, immune evasion, and therapeutic resistance are all driven by the dysregulated and constitutive angiogenesis occurring in the vasculature. Accordingly, great efforts have been dedicated toward identifying molecular signatures of this pathological angiogenesis in order to devise selective tumor endothelium targeting therapies while minimizing potential autoimmunity against physiologically normal endothelium. Vaccination with angiogenic antigens to generate cellular and/or humoral immunity against the tumor endothelium has proven to be a promising strategy for inhibiting or normalizing tumor angiogenesis and reducing cancer growth. Here we review tumor endothelium vaccines developed to date including active immunization strategies using specific tumor endothelium-associated antigens and whole endothelial cell-based vaccines designed to elicit immune responses against diverse target antigens. Among the novel therapeutic options, we describe a placenta-derived endothelial cell vaccine, ValloVax™, a polyvalent vaccine that is antigenically similar to proliferating tumor endothelium and is supported by pre-clinical studies to be safe and efficacious against several tumor types. [ABSTRACT FROM AUTHOR]

Many bacteria that live in contact with eukaryotic hosts, whether as symbionts or as pathogens, have evolved mechanisms that manipulate host cell behaviour to their benefit. One such mechanism, the type III secretion system, is employed by Gram-negative bacterial species to inject effector proteins into host cells. This function is reflected by the overall shape of the machinery, which resembles a molecular syringe. Despite the simplicity of the concept, the type III secretion system is one of the most complex known bacterial nanomachines, incorporating one to more than hundred copies of up to twenty different proteins into a multi-MDa transmembrane complex. The structural core of the system is the so-called needle complex that spans the bacterial cell envelope as a tripartite ring system and culminates in a needle protruding from the bacterial cell surface. Substrate targeting and translocation are accomplished by an export machinery consisting of various inner membrane embedded and cytoplasmic components. The formation of such a multimembrane-spanning machinery is an intricate task that requires precise orchestration. This review gives an overview of recent findings on the assembly of type III secretion machines, discusses quality control and recycling of the system and proposes an integrated assembly model. [ABSTRACT FROM AUTHOR]

Interleukin-2 (IL-2) therapy has been demonstrated to induce responses in 10-20% of advanced melanoma and renal cell carcinoma patients, which translates into durable remissions in up to half of the responsers. Unfortunately the use of IL-2 has been associated with severe toxicity and death. It has been previously observed and reported that IL-2 therapy causes a major drop in circulating levels of ascorbic acid (AA). The IL-2 induced toxicity shares many features with sepsis such as capillary leakage, systemic complement activation, and a relatively non-specific rise in inflammatory mediators such as TNF-alpha, C-reactive protein, and in advanced cases organ failure. Animal models and clinical studies have shown rapid depletion of AA in conditions of sepsis and amelioration associated with administration of AA [105]. In contrast to other approaches to dealing with IL-2 toxicity, which may also interfere with therapeutic effects, AA possesses the added advantage of having direct antitumor activity through cytotoxic mechanisms and suppression of angiogenesis. Here we present a scientific rationale to support the assessment of intravenous AA as an adjuvant to decrease IL-2 mediated toxicity and possibly increase treatment efficacy. [ABSTRACT FROM AUTHOR]

Abstract: Multidrug-resistant Escherichia coli is an emerging clinical challenge in domestic species. Treatment options in many cases are limited. This study characterized MDR E. coli isolates from urinary tract infections in dogs, collected between 2002 and 2011. Isolates were evaluated in terms of β-lactamase production, phylogenetic group, ST type, replicon type and virulence marker profile. Comparisons were made with antibiotic susceptible isolates also collected from dogs with urinary tract infections. AmpC β-lactamase was produced in 67% of the MDR isolates (12/18). Of these, 8 could be specifically attributed to the CMY-2 gene. None of the isolates tested in either group expressed ESBLs. Phylo-group distribution was as expected in the susceptible isolates, with an over representation of the pathogenic B2 phylo-group (67%). In contrast, the phylogenetic background for the MDR group was mixed, with representation of commensal phylo-groups A and B1. The B2 phylo-group represented the smallest proportion (A, B1, B2 or D was 28%, 22%, 11% and 33%, respectively). Virulence marker profiles, evaluated using Identibac® microarray, discriminated between the two groups. Marker sequences for a core panel of virulence determinants were identified in most of the susceptible isolates, but not in most of the MDR isolates. These findings indicate that for MDR isolates, plasmid-mediated AmpC is an important resistance mechanism, and while still capable of causing clinical disease, there is evidence for a shift towards phylogenetic groups of reduced inferred virulence potential. There was no evidence of zoonotic potential in either the susceptible or MDR urinary tract isolates in this study. [Copyright &y& Elsevier]

Abstract: In the bacterium Escherichia coli, the essential inner membrane protein (IMP) YidC assists in the biogenesis of IMPs and IMP complexes. Our current ideas about the function of YidC are based on targeted approaches using only a handful of model IMPs. Proteome-wide approaches are required to further our understanding of the significance of YidC and to find new YidC substrates. Here, using two-dimensional blue native/SDS-PAGE methodology that is suitable for comparative analysis, we have characterized the consequences of YidC depletion for the steady-state levels and oligomeric state of the constituents of the inner membrane proteome. Our analysis showed that (i) YidC depletion reduces the levels of a variety of complexes without changing their composition, (ii) the levels of IMPs containing only soluble domains smaller than 100 amino acids are likely to be reduced upon YidC depletion, whereas the levels of IMPs with at least one soluble domain larger than 100 amino acids do not, and (iii) the levels of a number of proteins with established or putative chaperone activity (HflC, HflK, PpiD, OppA, GroEL and DnaK) are strongly increased in the inner membrane fraction upon YidC depletion. In the absence of YidC, these proteins may assist the folding of sizeable soluble domains of IMPs, thereby supporting their folding and oligomeric assembly. In conclusion, our analysis identifies many new IMPs/IMP complexes that depend on YidC for their biogenesis, responses that accompany depletion of YidC and an IMP characteristic that is associated with YidC dependence. [Copyright &y& Elsevier]

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 bo3 oxidase and lowered oxygen consumption rates; (iii) increased levels of the chaperones DnaK and GroEL at the cytoplasmic membrane; (iv) a σ32 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. [ABSTRACT FROM AUTHOR]

The article provides an overview of the study which aim to find out the association between asthma control and health outcomes in the U.S. It used a cross-sectional data on asthmatics from the 2006 National Health and Wellness Survey. It determined that there were 2,767 uncontrolled asthmatics and 2,912 controlled asthmatics. It found that the asthma control provide higher physical and mental health-related quality if life, lesser hospital days and medical provider visits on patients.

A simple generic method for optimizing membrane protein over-expression in Escherichia co/i is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the 17 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications. [ABSTRACT FROM AUTHOR]

The polytopic inner membrane protein MaIF is a constituent of the MaEFGK2 maltose transport complex in Escherichia coli. We have studied the biogenesis of MaIF using a combination of in vivo and in vitro approaches. MaIF is targeted via the SRP pathway to the Sec/YidC insertion site. Despite close proximity of nascent MaIF to YidC during insertion, YidC is not required for the insertion of MalF into the membrane. However, YidC is required for the stability of MaIF and the formation of the MaIFGK2 maltose transport complex. Our data indicate that YidC supports the folding of MaIF into a stable conformation before it is incorporated into the maltose transport complex. [ABSTRACT FROM AUTHOR]

Functional and structural studies of membrane proteins usually require overexpression of the proteins in question. Often, however, the ‘trial and error’ approaches that are mainly used to produce membrane proteins are not successful. Our rapidly increasing understanding of membrane protein insertion, folding and degradation means that membrane protein overexpression can be more rationalized, both at the level of the overexpression host and the overexpressed membrane protein. This change of mindset is likely to have a significant impact on membrane protein research. [Copyright &y& Elsevier]

Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to cause abnormalities in renal function. This is an important concern in patients with cardiorenal risk factors, including hypertension, congestive heart failure, edema, renal impairment, and advanced age.Objectives: The goals of this study were to determine the prevalence of cardiorenal risk factors in patients with rheumatoid arthritis (RA) or osteoarthritis and ascertain whether these risk factors are associated with prescribing patterns of cyclooxygenase (COX)-2-selective inhibitors and other NSAIDs.Methods: This was a retrospective, longitudinal claims analysis using data from 19 large independent-practice-model managed care health plans in the United Stated. Three cohorts were identified based on claims for celecoxib, rofecoxib, or other NSAIDs from October 1, 1999, through September 30, 2000. Logistic regression models were used to explore whether baseline cardiorenal risk factors were related to choice of therapy.Results: A total of 77,552 patients received celecoxib (n = 6779 [8.74%]), rofecoxib (n = 7189 [9.27%]), or other NSAIDs (n = 63,584 [81.99%]). Patients prescribed COX-2-selective inhibitors were older than those receiving other NSAIDs and had a diagnosis of RA more often. Overall, 42% of patients had ≥1 cardiorenal risk factor, and approximately one third had hypertension. Cardiorenal risk factors were not related to physicians'' prescribing of celecoxib or rofecoxib, but the presence of any cardiorenal risk factor was associated with an increase in the use of COX-2-selective inhibitors compared with other NSAIDs, from 12% for cerebrovascular disease (point estimate, 1.124; P<0.001) to 74% for chronic renal failure/nephritis (point estimate, 1.738; P=0.025). RA and advanced age were associated with the use of celecoxib rather than rofecoxib.Conclusions: The prevalence of cardiorenal risk factors was found to be similar in patients prescribed celecoxib or rofecoxib for arthritis. Patients with these risk factors were more likely to receive a COX-2-selective inhibitor than other NSAIDs. [Copyright &y& Elsevier]

Accurate human identification using radar has a variety of potential applications, such as surveillance, access control and security checkpoints. Nevertheless, radar-based human identification has been limited to a few motion-based biometrics that are solely reliant on micro-Doppler signatures. This paper proposes for the first time the use of combined radar-based heart sound and gait signals as biometrics for human identification. The proposed methodology starts by converting the extracted biometric signatures collected from 18 subjects to images, and then an image augmentation technique is applied and the deep transfer learning is used to classify each subject. A validation accuracy of 58.7% and 96% is reported for the heart sound and gait biometrics, respectively. Next, the identification results of the two biometrics are combined using the joint probability mass function (PMF) method to report a 98% identification accuracy. To the best of our knowledge, this is the highest reported in the literature to date. Lastly, the trained networks are tested in an actual scenario while being used in an office access control platform to identify different human subjects. We report an accuracy of 76.25%. [ABSTRACT FROM AUTHOR]

Understanding which factors make an individual susceptible to parasitism provides insight on how a parasite infects a host. Numerous factors such as habitat characteristics, body size, and sex have shown differential impacts on parasitism rates. Trends in parasitism rate often exist for similar taxonomic groups, so expanding research to new species helps test the robustness of such trends. A chelonian-specific ectoparasite, Cistudinomyia cistudinis (Diptera: Sarcophagidae), has been documented in case studies dating back a century. Almost all reports of infection by this flesh fly are of a single or few turtles, so there is a lack of information regarding the population dynamics of infection. This lack of information means little is known about which factors make a turtle more or less likely to be parasitized. In this work, we compared the occurrences of parasitism in ornate box turtle (Terrapene ornata) populations across multiple seasons from 2 different habitats in northeastern Kansas (North Topeka and Lawrence). At the population level, we found a significantly higher number of parasitized turtles in the Lawrence population. Within the Lawrence population, we found females were more likely to be parasitized. This study sets baseline infection rates for a long-lived vertebrate host that can help better understand the reasons for parasitism both among and within populations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The expression of virulence factors essential for the invasion of host cells by Salmonella enterica is tightly controlled by a network of transcription regulators. The AraC/XylS transcription factor HilD is the main integration point of environmental signals into this regulatory network, with many factors affecting HilD activity. Long-chain fatty acids, which are highly abundant throughout the host intestine, directly bind to and repress HilD, acting as environmental cues to coordinate virulence gene expression. The regulatory protein HilE also negatively regulates HilD activity, through a protein-protein interaction. Both of these regulators inhibit HilD dimerization, preventing HilD from binding to target DNA. We investigated the structural basis of these mechanisms of HilD repression. Long-chain fatty acids bind to a conserved pocket in HilD, in a comparable manner to that reported for other AraC/XylS regulators, whereas HilE forms a stable heterodimer with HilD by binding to the HilD dimerization interface. Our results highlight two distinct, mutually exclusive mechanisms by which HilD activity is repressed, which could be exploited for the development of new antivirulence leads. [ABSTRACT FROM AUTHOR]

Interleukin-2 (IL-2) therapy has been demonstrated to induce responses in 10-20% of advanced melanoma and renal cell carcinoma patients, which translates into durable remissions in up to half of the responsers. Unfortunately the use of IL-2 has been associated with severe toxicity and death. It has been previously observed and reported that IL-2 therapy causes a major drop in circulating levels of ascorbic acid (AA). The IL-2 induced toxicity shares many features with sepsis such as capillary leakage, systemic complement activation, and a relatively non-specific rise in inflammatory mediators such as TNF-alpha, C-reactive protein, and in advanced cases organ failure. Animal models and clinical studies have shown rapid depletion of AA in conditions of sepsis and amelioration associated with administration of AA (JTM 9:1-7, 2011). In contrast to other approaches to dealing with IL-2 toxicity, which may also interfere with therapeutic effects, AA possesses the added advantage of having direct antitumor activity through cytotoxic mechanisms and suppression of angiogenesis. Here we present a scientific rationale to support the assessment of intravenous AA as an adjuvant to decrease IL-2 mediated toxicity and possibly increase treatment efficacy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 σ32 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. [ABSTRACT FROM AUTHOR]

Abstract: The inner membrane proteome of the model organism Escherichia coli is composed of inner membrane proteins, lipoproteins and peripherally attached soluble proteins. Our knowledge of the biogenesis of inner membrane proteins is rapidly increasing. This is in particular true for the early steps of biogenesis — protein targeting to and insertion into the membrane. However, our knowledge of inner membrane protein folding and quality control is still fragmentary. Furthering our knowledge in these areas will bring us closer to understand the biogenesis of individual inner membrane proteins in the context of the biogenesis of the inner membrane proteome of Escherichia coli as a whole. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes. [Copyright &y& Elsevier]

Bacterial type III protein secretion systems deliver effector proteins into eukaryotic cells in order to modulate cellular processes. Central to the function of these protein-delivery machines is their ability to recognize and secrete substrates in a defined order. Here, we describe a mechanism by which a type III secretion system from the bacterial enteropathogen Salmonella enterica serovar Typhimurium can sort its substrates before secretion. This mechanism involves a cytoplasmic sorting platform that is sequentially loaded with the appropriate secreted proteins. The sequential loading of this platform, facilitated by customized chaperones, ensures the hierarchy in type III protein secretion. Given the presence of these machines in many important pathogens, these findings can serve as the bases for the development of novel antimicrobial strategies. [ABSTRACT FROM AUTHOR]

PURPOSE: To address the need for more accurate risk stratification models for cancer immuno-oncology, this study aimed to develop a machine-learned Bayesian network model (BNM) for predicting outcomes in patients with metastatic renal cell carcinoma (mRCC) being treated with immunotherapy. METHODS: Patient-level data from the randomized, phase III CheckMate 025 clinical trial comparing nivolumab with everolimus for second-line treatment in patients with mRCC were used to develop the BNM. Outcomes of interest were overall survival (OS), all-cause adverse events, and treatment-related adverse events (TRAE) over 36 months after treatment initiation. External validation of the model's predictions for OS was conducted using data from select centers from the International Metastatic Renal Cell Carcinoma Database Consortium (IMDC). RESULTS: Areas under the receiver operating characteristic curve (AUCs) for BNM-based classification of OS using baseline data were 0.74, 0.71, and 0.68 over months 12, 24, and 36, respectively. AUC for OS at 12 months increased to 0.86 when treatment response and progression status in year 1 were included as predictors; progression and response at 12 months were highly prognostic of all outcomes over the 36-month period. AUCs for adverse events and treatment-related adverse events were approximately 0.6 at 12 months but increased to approximately 0.7 by 36 months. Sensitivity analysis comparing the BNM with machine learning classifiers showed comparable performance. Test AUC on IMDC data for 12-month OS was 0.71 despite several variable imbalances. Notably, the BNM outperformed the IMDC risk score alone. CONCLUSION: The validated BNM performed well at prediction using baseline data, particularly with the inclusion of response and progression at 12 months. Additionally, the results suggest that 12 months of follow-up data alone may be sufficient to inform long-term survival projections in patients with mRCC. [ABSTRACT FROM AUTHOR]

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a shortage 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 the function of the type III secretion system encoded by Salmonella pathogenicity island 1. Type III secretion substrate–NanoLuc fusions are readily secreted into the culture supernatant, where they can be quantified by luminometry after removal of bacteria. The NanoLuc‐based secretion assay features a very high signal‐to‐noise ratio and sensitivity down to the nanolitre scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384‐well microplates. We further developed a split NanoLuc‐based assay that enables the real‐time monitoring of type III secretion‐dependent injection of effector–HiBiT fusions into host cells stably expressing the complementing NanoLuc–LgBiT. [ABSTRACT FROM AUTHOR]

Summary: Type III secretion injectisomes are essential virulence factors for many pathogenic bacteria by mediating the transport of effector proteins into eukaryotic host cells. The secretion conduit of injectisomes is formed by a helical assembly of three hydrophobic proteins (SctR, SctS and SctT), an inner rod (SctI) and a needle filament (SctF). SctI is thought to play a role in switching between the secretion of different substrate classes and assembly of the inner rod has been implicated in regulating the length of the needle filament. While high‐resolution structures of the hydrophobic components and of the needle filament have been solved, little is known about the structure and the assembly of the inner rod, which impedes the deeper assessment of its function. Here we show by exhaustive in vivo photocrosslinking that SctI engages in extensive interactions with SctR and SctT throughout its entire length. Our data imply that the inner rod serves as an adapter between the export apparatus and the needle filament by forming one helical turn. We show that assembly of the inner rod does not play a role in needle length control nor in substrate specificity switching. Instead, our findings imply that inner rod assembly must precede assembly of the needle filament. [ABSTRACT FROM AUTHOR]

Summary: Disarming pathogens by targeting virulence factors is a promising alternative to classic antibiotics. Many virulence factors in Gram‐negative bacteria are secreted via the autotransporter (AT) pathway, also known as Type 5 secretion. These factors are secreted with the assistance of two membrane‐based protein complexes: Sec and Bam. To identify inhibitors of the AT pathway, we used transcriptomics analysis to develop a fluorescence‐based high‐throughput assay that reports on the stress induced by the model AT hemoglobin protease (Hbp) when its secretion across the outer membrane is inhibited. Screening a library of 1600 fragments yielded the compound VUF15259 that provokes cell envelope stress and secretion inhibition of the ATs Hbp and Antigen‐43. VUF15259 also impairs β‐barrel folding activity of various outer membrane proteins. Furthermore, we found that mutants that are compromised in outer membrane protein biogenesis are more susceptible to VUF15259. Finally, VUF15259 induces the release of vesicles that appear to assemble in short chains. Taken together, VUF15259 is the first reported compound that inhibits AT secretion and our data are mostly consistent with VUF15259 interfering with the Bam‐complex as potential mode of action. The validation of the presented assay incites its use to screen larger compound libraries with drug‐like compounds. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In Staphylococcus aureus, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, RelSau or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of RelSau is crucial for the adaption of S. aureus to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of RelSau. Growth pattern, transcriptional analyses and in vitro assays yielded the following results: i) in vivo, under relaxed conditions, as well as in vitro, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) RelSau lacking the CTD exhibits net hydrolase activity when expressed in S. aureus but net (p)ppGpp synthetase activity when expressed in E. coli; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, RelSau primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ, and FliR form the gate complex inside the basal body MS ring, although FliO is required for efficient export gate formation in Salmonella enterica. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homohexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150, and Glu-178 residues in the periplasmic domain of FliP (FliPP) inhibited FliP6 ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring. The crystal structure of FliPP derived from Thermotoga maritia, and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP–FliP interactions and that Phe-150, Arg-152, Ser-156, and Pro-158 are responsible for the FliP–FliO interactions. Overexpression of FliP restored motility of a ∆fliO mutant to the wild-type level, suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Copurification assays revealed that FlhA, FlhB, FliQ, and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation. [ABSTRACT FROM AUTHOR]

Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production. [ABSTRACT FROM AUTHOR]

Background Economic evaluations often measure an intervention effect with mean overall survival (OS). Emerging types of cancer treatments offer the possibility of being “cured” in that patients can become long-term survivors whose risk of death is the same as that of a disease-free person. Describing cured and noncured patients with one shared mean value may provide a biased assessment of a therapy with a cured proportion. Objective The purpose of this article is to explain how to incorporate the heterogeneity from cured patients into health economic evaluation. Methods We analyzed clinical trial data from patients with advanced melanoma treated with ipilimumab (Ipi; n = 137) versus glycoprotein 100 (gp100; n = 136) with statistical methodology for mixture cure models. Both cured and noncured patients were subject to background mortality not related to cancer. Results When ignoring cured proportions, we found that patients treated with Ipi had an estimated mean OS that was 8 months longer than that of patients treated with gp100. Cure model analysis showed that the cured proportion drove this difference, with 21% cured on Ipi versus 6% cured on gp100. The mean OS among the noncured cohort patients was 10 and 9 months with Ipi and gp100, respectively. The mean OS among cured patients was 26 years on both arms. When ignoring cured proportions, we found that the incremental cost-effectiveness ratio (ICER) when comparing Ipi with gp100 was $324,000/quality-adjusted life-year (QALY) (95% confidence interval $254,000–$600,000). With a mixture cure model, the ICER when comparing Ipi with gp100 was $113,000/QALY (95% confidence interval $101,000–$154,000). Conclusions This analysis supports using cure modeling in health economic evaluation in advanced melanoma. When a proportion of patients may be long-term survivors, using cure models may reduce bias in OS estimates and provide more accurate estimates of health economic measures, including QALYs and ICERs. [ABSTRACT FROM AUTHOR]

Bacterial type III protein secretion systems inject effector proteins into eukaryotic host cells in order to promote survival and colonization of Gram-negative pathogens and symbionts. Secretion across the bacterial cell envelope and injection into host cells is facilitated by a so-called injectisome. Its small hydrophobic export apparatus components SpaP and SpaR were shown to nucleate assembly of the needle complex and to form the central “cup” substructure of a Salmonella Typhimurium secretion system. However, the in vivo placement of these components in the needle complex and their function during the secretion process remained poorly defined. Here we present evidence that a SpaP pentamer forms a 15 Å wide pore and provide a detailed map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. We further refine the current view of export apparatus assembly, consolidate transmembrane topology models for SpaP and SpaR, and present intimate interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ, indicating how export apparatus and needle filament are connected to create a continuous conduit for substrate translocation. [ABSTRACT FROM AUTHOR]

\nImportance Legionella pneumophila (L.p.) is a bacterial pathogen which is a common causative agent of pneumonia. In humans, it infects alveolar macrophages and transfers hundreds of virulence factors that interfere with cellular signalling pathways and the transcriptomic landscape to sustain its own replication. By this interaction, it has acquired eukaryote-like protein motifs by gene transfer events that partake in the pathogenicity of Legionella. In a computational screening approach for eukaryotic motifs in the transcriptome of Legionella, we identified the L.p. strain Corby protein ABQ55614 as putative histone-deacetylase and named it “suppressing modifier of histones 1” (Smh1). During infection, Smh1 translocated from the Legionella vacuole into the host cytosol. When expressed in human macrophage THP-1 cells, Smh1 was localized predominantly in the nucleus, led to broad histone H3 and H4 deacetylation, blunted expression of a large number of genes (e.g. IL-1β and IL-8), and fostered intracellular bacterial replication. L.p. with a Smh1 knockdown grew normally in media but showed a slight growth defect inside the host cell. Furthermore, Smh1 showed a very potent histone deacetylation activity in vitro, e.g. at H3K14, that could be inhibited by targeted mutation of the putative catalytic centre inferred by analogy with eukaryotic HDAC8, and with the deacetylase inhibitor trichostatin A. In summary, Smh1 displays functional homology with class I/II type HDACs. We identified Smh1 as a new Legionella virulence factor with a eukaryote-like histone-deacetylase activity that moderates host gene expression and might pave the way for further histone modifications. Legionella pneumophila (L.p.) is a prominent bacterial pathogen which is a common causative agent of pneumonia. In order to survive inside the host cell, the human macrophage, it profoundly interacts with host cell processes to advance its own replication. In this study, we identify a bacterial factor, Smh1, with yet unknown function as a host histone deacetylase. The activity of this factor in the host cell leads to attenuated gene expression and increased intracellular bacterial replication. [ABSTRACT FROM AUTHOR]

Background & Aims Hepatocellular 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. Methods Data were collected retrospectively from January 2005 to September 2012 by chart reviews of eligible patients newly diagnosed with HCC at participating institutions. Results Forty-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). Conclusions Initial 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. [ABSTRACT FROM AUTHOR]

Patients with certain cancers are treated with curative intent, but for others the results are less favorable and different therapeutic approaches are needed. Early data suggest that new therapies, which modulate immune responses to cancers, may have potential for long-term survival in a proportion of cases. Therefore, it is timely to consider whether metrics generally used to describe the medical value of therapies for patients with common solid tumors remain appropriate for therapies with curative potential. Literature reviews were conducted to define how various stakeholders describe cure in oncology and to identify the endpoints used in clinical trials for selected solid tumors. The results showed that “cure” is described using various terms that can be divided broadly into lack of disease progression, eradication of cancerous cells, and survival. The review of trial endpoints showed frequent use of median overall survival (OS) and progression- and response-related endpoints. Because these endpoints were mainly described in the context of chemotherapies that are not generally curative, they may not adequately capture outcomes of new therapeutic modalities with potential for long-term survival. More appropriate endpoints may include mean OS, cure fraction, and OS rate at landmark time points. [ABSTRACT FROM AUTHOR]

Background: While the concept of angiogenesis blockade as a therapeutic intervention for cancer has been repeatedly demonstrated, the full promise of this approach has yet to be realized. Specifically, drugs such as VEGF-blocking antibodies or kinase inhibitors suffer from the drawbacks of resistance development, as well as off-target toxicities. Previous studies have demonstrated feasibility of specifically inducing immunity towards tumor endothelium without consequences of systemic autoimmunity in both animal models and clinical settings. Method: Placenta-derived endothelial cells were isolated and pretreated with interferon gamma to enhance immunogenicity. Syngeneic mice received subcutaneous administration of B16 melanoma, 4 T1 mammary carcinoma, and Lewis Lung Carcinoma (LLC), followed by administration of control saline, control placental endothelial cells, and interferon gamma primed endothelial cells (ValloVax™). Tumor volume was quantified. An LLC metastasis model was also established and treated under similar conditions. Furthermore, a safety analysis in non-tumor bearing mice bracketing the proposed clinical dose was conducted. Results: ValloVax™ immunization led to significant reduction of tumor growth and metastasis as compared to administration of non-treated placental endothelial cells. Mitotic inactivation by formalin fixation or irradiation preserved tumor inhibitory activity. Twenty-eight day evaluation of healthy male and female mice immunized with ValloVax™ resulted in no abnormalities or organ toxicities. Conclusion: Given the established rationale behind the potential therapeutic benefit of inhibiting tumor angiogenesis as a treatment for cancer, immunization against a variety of endothelial cell antigens may produce the best clinical response, enhancing efficacy and reducing the likelihood of the development of treatment resistance. These data support the clinical evaluation of irradiated ValloVax™ as an anti-angiogenic cancer vaccine. [ABSTRACT FROM AUTHOR]

One of the most exciting developments in the field of bacterial pathogenesis in recent years is the discovery that many pathogens utilize complex nanomachines to deliver bacterially encoded effector proteins into target eukaryotic cells. These effector proteins modulate a variety of cellular functions for the pathogen's benefit. One of these protein-delivery machines is the type III secretion system (T3SS). T3SSs are widespread in nature and are encoded not only by bacteria pathogenic to vertebrates or plants but also by bacteria that are symbiotic to plants or insects. A central component of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins across the bacterial envelope. Working in conjunction with several cytoplasmic components, the needle complex engages specific substrates in sequential order, moves them across the bacterial envelope, and ultimately delivers them into eukaryotic cells. The central role of T3SSs in pathogenesis makes them great targets for novel antimicrobial strategies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: In an international, randomized Phase III trial ipilimumab demonstrated a significant overall survival benefit in previously treated advanced melanoma patients. This report summarizes health-related quality of life (HRQL) outcomes for ipilimumab with/without gp100 vaccine compared to gp100 alone during the clinical trial's 12 week treatment induction period. Methods: The Phase III clinical trial (MDX010-20) was a double-blind, fixed dose study in 676 previously treated advanced unresectable stage III or IV melanoma patients. Patients were randomized 3:1:1 to receive either ipilimumab (3 mg/kg q3w x 4 doses) + gp100 (peptide vaccine; 1 mg q3w x 4 doses; ipilimumab plus gp100, n = 403); gp100 vaccine + placebo (gp100 alone, n = 136); or ipilimumab + placebo (ipilimumab alone, n = 137). The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) assessed HRQL. Baseline to Week 12 changes in EORTC QLQ-C30 function, global health status, and symptom scores were analyzed for ipilimumab with/without gp100 vaccine compared to gp100 alone. Mean change in scores were categorized "no change" (0-5), "a little" (5-10 points), "moderate" (10-20 points), and "very much" (>20). Results: In the ipilimumab plus gp100 and ipilimumab alone groups, mean changes from baseline to Week 12 generally indicated "no change" or "a little" impairment across EORTC QLQ-C30 global health status, function, and symptom subscales. Significant differences in constipation, favoring ipilimumab, were observed (p<0.05). For ipilimumab alone arm, subscales with no or a little impairment were physical, emotional, cognitive, social function, global health, nausea, pain, dyspnea, constipation, and diarrhea subscales. For the gp100 alone group, the observed changes were moderate to large for global health, role function, fatigue, and for pain. Conclusions: Ipilimumab with/without gp100 vaccine does not have a significant negative HRQL impact during the treatment induction phase relative to gp100 alone in stage III or IV melanoma patients. [ABSTRACT FROM AUTHOR]

Background: The self-sufficient autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a β-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein. Results: Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a β-helical core structure (β-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated β-stem appeared unstable after translocation, demonstrating the importance of an intact β-stem. By interrupting the cleavage site between passenger and β-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development. Conclusions: We developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria. [ABSTRACT FROM AUTHOR]

Objective.— To determine the predictability of future migraine attacks and to describe the effect of migraine on daily life during and between migraine attacks. Background.— Migraine is associated with substantial economic and humanistic burden. There is growing evidence that early intervention with triptans results in better treatment outcomes. However, this is dependent on a patient's preparedness for an attack including having abortive medications readily accessible at headache onset. Methods.— Physician-diagnosed adult migraine sufferers, who treat with prescription or over-the-counter medications, completed 2 self-reported, Internet-based questionnaires, administered at baseline and following the resolution of the next migraine attack. The baseline questionnaire included the Migraine Disability Assessment questionnaire (MIDAS), questions about experiences on days between attacks, predictions of the date, time of day (5 time windows), and sufferer's location (4 places) at the start of their next migraine. At follow-up, information was collected in the similar fashion about the date, time of day, and sufferer's location at the start of their most recent migraine. Results.— A total of 1519 migraine sufferers completed the baseline questionnaire and 877 (57.7%) completed the follow-up. At baseline, 58.7% experienced moderate to severe disability from headache, based on MIDAS. Only 4.0% were able to predict the exact date of their next migraine; 21.24% predicted next migraine within 3 days. Larger proportions (46.6%) were able to accurately predict time of day or location (70.7%) of their next migraine. In the past 3 months, 92.6% reported that they were forced to change daily plans because of migraine. Because of fear of getting a migraine, 20.2% had avoided and 27.0% had changed a work commitment, and 27.3% had avoided and 28.2% had changed social plans. Conclusions.— Migraine sufferers are generally unable to predict onset of the next migraine. Lack of predictability heightens the importance of education and preparedness for a migraine attack which may also reduce fear and anxiety between attacks. ( Headache 2010;50:1296-1305) [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

[Display omitted] • Salt bridges are conserved in hydrophobic core export apparatus of bacterial T3SS. • Salt bridges facilitate incorporation of following subunit into helical complex. • Conserved charged residues may play a role in gating of the export apparatus. • Insight in delicate requirements of assembly and function of unique helical complex. Virulence-associated type III secretion systems (T3SS) are utilized by Gram negative bacterial pathogens for injection of effector proteins into eukaryotic host cells. The transmembrane export apparatus at the core of T3SS is composed of a unique helical complex of the hydrophobic proteins SctR, SctS, SctT, and SctU. These components comprise a number of highly conserved charged residues within their hydrophobic domains. The structure of the closed state of the core complex SctR 5 S 4 T 1 revealed that several of these residues form inter- and intramolecular salt bridges, some of which have to be broken for pore opening. Mutagenesis of individual residues was shown to compromise assembly or secretion of both, the virulence-associated and the related flagellar T3SS. However, the exact role of these conserved charged residues in the assembly and function of T3SS remains elusive. Here we performed an in-depth mutagenesis analysis of these residues in the T3SS of Salmonella Typhimurium, coupled to blue native PAGE, in vivo photocrosslinking and luciferase-based secretion assays. Our data show that these conserved salt bridges are not critical for assembly of the respective protein but rather facilitate the incorporation of the following subunit into the assembling complex. Our data also indicate that these conserved charged residues are critical for type III-dependent secretion and reveal a functional link between SctS E44 and SctT R204 and the cytoplasmic domain of SctU in gating the T3SS injectisome. Overall, our analysis provides an unprecedented insight into the delicate requirements for the assembly and function of the machinery at the core of T3SS. [ABSTRACT FROM AUTHOR]

To examine the role of health-related quality of life (HRQOL) in early treatment discontinuation among women enrolled in a breast cancer clinical trial. A total of 464 women were enrolled in the Eastern Cooperative Oncology Group randomized controlled trial of adjuvant regimens comparing six cycles of cytoxan, adriamycin and 5-flurouricil (5-FU) with a 16-week regimen (weekly therapy with cytoxan, adriamycin, vincristine, methotrexate, and 5-FU) among women with lymph node positive breast cancer. One hundred sixty-four women participated in the HrQL substudy using the Breast Chemotherapy Questionnaire, which was designed to measure HRQOL in women receiving chemotherapy. Changes in global HRQOL score were examined over time as a predictor of early treatment discontinuation using generalized estimation equations (GEE) modeling and Cox proportional hazards regression. We considered early treatment discontinuation as a longitudinal binary variable determined at each time point HRQOL was measured. The results of multivariate GEE model fitting indicated that declines in HRQOL (p = 0.04), older age (p = 0.02), higher degree of nausea (p = 0.02), higher degree of neurosensory toxicity (0.03) and lower degrees of hair loss (p = 0.004) were correlated with early treatment discontinuation. We then fitted a proportional hazard regression model for time to early discontinuation with HRQOL score as a time-dependent covariate. The results were identical. Declines in HRQOL during therapy predicted early treatment discontinuation even after accounting for age and chemotherapy-related side effects. In the age of ever more aggressive treatments for breast cancer, women’s perception of the impact of these treatments on their lives will become more important. [ABSTRACT FROM AUTHOR]