Vaccine Protection against Bacillus cereus-Mediated Respiratory Anthrax-Like Disease in Mice

Bacillus cereus strains harboring a pXO1-like virulence plasmid cause respiratory anthrax-like disease in humans, particularly in welders. We developed mouse models for intraperitoneal as well as aerosol challenge with spores of B. cereus G9241, harboring pBCXO1 and pBC218 virulence plasmids. Compared to wild-type B. cereus G9241, spores with a deletion of the pBCXO1-carried protective antigen gene (pagA1) were severely attenuated, whereas spores with a deletion of the pBC218-carried protective antigen homologue (pagA2) were not. Anthrax vaccine adsorbed (AVA) immunization raised antibodies that bound and neutralized the pagA1-encoded protective antigen (PA1) but not the PA2 orthologue encoded by pagA2. AVA immunization protected mice against a lethal challenge with spores from B. cereus G9241 or B. cereus Elc4, a strain that had been isolated from a fatal case of anthrax-like disease. As the pathogenesis of B. cereus anthrax-like disease in mice is dependent on pagA1 and PA-neutralizing antibodies provide protection, AVA immunization may also protect humans from respiratory anthrax-like death.     

The variability of MR axon radii estimates in the human white matter

The noninvasive quantification of axonal morphology is an exciting avenue for gaining understanding of the function and structure of the central nervous system. Accurate non‐invasive mapping of micron‐sized axon radii using commonly applied neuroimaging techniques, that is, diffusion‐weighted MRI, has been bolstered by recent hardware developments, specifically MR gradient design. Here the whole brain characterization of the effective MR axon radius is presented and the inter‐ and intra‐scanner test–retest repeatability and reproducibility are evaluated to promote the further development of the effective MR axon radius as a neuroimaging biomarker. A coefficient‐of‐variability of approximately 10% in the voxelwise estimation of the effective MR radius is observed in the test–retest analysis, but it is shown that the performance can be improved fourfold using a customized along‐tract analysis.     

Fifty years of research on the physiology and pharmacology of the autonomic nervous system

Clinical Autonomic Research -     

Allergen immunotherapy for respiratory allergy: Quality appraisal of observational comparative effectiveness studies using the REal Life Evidence AssessmeNt Tool. An EAACI methodology committee analysis

BackgroundObservational comparative effectiveness studies in allergen immunotherapy (AIT) represent an important evidence source answering research questions that can be challenging to obtain from randomized controlled trials (RCTs), such as long‐term benefits of AIT, the effects on asthma prevention and the onset of new allergen sensitizations. However, observational studies are prone to several sources of bias, which limit their reliability.The REal Life Evidence AssessmeNt Tool (RELEVANT) was recently developed to assist in quality appraisal of observational comparative research to enable identification of useful nonrandomized studies to be considered within guideline development.ObjectiveTo systematically appraise the quality of published observational comparative AIT studies using RELEVANT.MethodsObservational studies comparing AIT to pharmacotherapy for respiratory allergies, assessing as outcome measures reduction of symptoms and/or medication use reduction, were retrieved by computerized bibliographic searches. According to RELEVANT, a failure to meet any one of primary items (background, design, measures, analysis, results, discussion/interpretation, and conflict of interest) represents a critical flaw, significantly undermining the validity of the study results.ResultsThe 14 studies identified supported the benefit of AIT in real‐life, which persists after treatment discontinuation. However, none of them met all the 7 primary RELEVANT criteria. The main defects were reported in the design (28.6% of studies), measures and analysis (64.3% of studies), and results (78.6% of studies) items, due to selection bias and lack of methods for adjusting controls. Half of the studies did not report on conflict of interest.ConclusionThere is a need for more robust observational research in AIT. RELEVANT appears as an easy‐to‐use and sensitive tool for quality appraisal in AIT studies.     

Kaplan-Meier Survival Analysis Overestimates the Risk of Revision Arthroplasty: A Meta-analysis

Background

Although Kaplan-Meier survival analysis is commonly used to estimate the cumulative incidence of revision after joint arthroplasty, it theoretically overestimates the risk of revision in the presence of competing risks (such as death). Because the magnitude of overestimation is not well documented, the potential associated impact on clinical and policy decision-making remains unknown.

Questions/purposes

We performed a meta-analysis to answer the following questions: (1) To what extent does the Kaplan-Meier method overestimate the cumulative incidence of revision after joint replacement compared with alternative competing-risks methods? (2) Is the extent of overestimation influenced by followup time or rate of competing risks?

Methods

We searched Ovid MEDLINE, EMBASE, BIOSIS Previews, and Web of Science (1946, 1980, 1980, and 1899, respectively, to October 26, 2013) and included article bibliographies for studies comparing estimated cumulative incidence of revision after hip or knee arthroplasty obtained using both Kaplan-Meier and competing-risks methods. We excluded conference abstracts, unpublished studies, or studies using simulated data sets. Two reviewers independently extracted data and evaluated the quality of reporting of the included studies. Among 1160 abstracts identified, six studies were included in our meta-analysis. The principal reason for the steep attrition (1160 to six) was that the initial search was for studies in any clinical area that compared the cumulative incidence estimated using the Kaplan-Meier versus competing-risks methods for any event (not just the cumulative incidence of hip or knee revision); we did this to minimize the likelihood of missing any relevant studies. We calculated risk ratios (RRs) comparing the cumulative incidence estimated using the Kaplan-Meier method with the competing-risks method for each study and used DerSimonian and Laird random effects models to pool these RRs. Heterogeneity was explored using stratified meta-analyses and metaregression.

Results

The pooled cumulative incidence of revision after hip or knee arthroplasty obtained using the Kaplan-Meier method was 1.55 times higher (95% confidence interval, 1.43–1.68; p < 0.001) than that obtained using the competing-risks method. Longer followup times and higher proportions of competing risks were not associated with increases in the amount of overestimation of revision risk by the Kaplan-Meier method (all p > 0.10). This may be due to the small number of studies that met the inclusion criteria and conservative variance approximation.

Conclusions

The Kaplan-Meier method overestimates risk of revision after hip or knee arthroplasty in populations where competing risks (such as death) might preclude the occurrence of the event of interest (revision). Competing-risks methods should be used to more accurately estimate the cumulative incidence of revision when the goal is to plan healthcare services and resource allocation for revisions.     

Prevalence and Incidence of Epilepsy in an Elderly and Low-Income Population in the United States

Background and Purpose

The purpose of this study was to estimate the incidence and prevalence of epilepsy among an elderly and poor population in the United States.

Methods

Arizona Medicaid claims data from January 1, 2008 to December 31, 2010 were used for this analysis. Subjects who were aged ≥65 years and were continuously enrolled in any Arizona Medicaid health plans (eligible to patients with low income) for ≥12 months between January 1, 2008 and December 31, 2009 were considered eligible for inclusion in the study cohort. In addition to meeting the aforementioned criteria, incident and prevalent cases must have had epilepsy-related healthcare claims. Furthermore, incident cases were required to have a 1-year "clean" period immediately preceding the index date. Negative binomial and logistic regression models were used to assess the factors associated with epilepsy incidence and prevalence.

Results

The estimated epilepsy incidence and prevalence for this population in 2009 were 7.9 and 19.3 per 1,000 person-years, respectively. The incidence and prevalence rates were significantly higher for patients with comorbid conditions that were potential risk factors for epilepsy and were of younger age than for their non-comorbid and older counterparts (p<0.05). The prevalence rates were significantly higher for non-Hispanic Blacks and male beneficiaries than for non-Hispanic Whites and female beneficiaries, respectively (p<0.05).

Conclusions

This patient population had higher epilepsy incidence and prevalence compared with the general US population. These differences may be at least in part attributable to their low socioeconomic status.     

Oncogenic KRAS engages an RSK1/NF1 pathway to inhibit wild-type RAS signaling in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4B^G12D), and nontransforming cytosolic double mutant (BirA-KRAS4B^G12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRAS^G12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.

A total of 60,430 new cases of pancreatic cancer were estimated for 2021, and the 5-y relative survival rate has consistently remained below 11% (1). About 85% of these pancreatic cancer tumors are pancreatic ductal adenocarcinoma (PDAC) (2). Poor outcomes of PDAC cases result from late diagnoses leading to unresectable and heterogeneous tumors as well as ineffective therapies, which only prolong survival on the order of months (3–5). Mutations in the KRAS proto-oncogene are present in over 90% of PDAC cases and are associated with a poor prognosis (6). Furthermore, mice expressing mutant KRAS in the pancreas develop precursor lesions, which sporadically progress into frank PDAC. This progression is accelerated when combined with other mutations or deletion of tumor suppressor genes (7–11). Additionally, independent studies have shown that the maintenance of murine PDAC cells require KRAS (12–14).As a RAS GTPase, KRAS acts as a molecular switch at the plasma membrane that relays growth factor signaling from receptor tyrosine kinases to downstream pathways such as RAF/MEK and PI3K/AKT (15). GTP binding alters the conformation of the KRAS G domain, thereby creating binding sites for downstream effectors to trigger enzymatic cascades that promote cell transformation (16–19). Intrinsically, KRAS slowly hydrolyzes GTP into GDP to halt signaling; however, GTPase activating proteins (GAPs) such as neurofibromin 1(NF1) catalyze this process (20). In contrast, guanine nucleotide exchange factors, such as son of sevenless homolog 1 (SOS1), catalyze the exchange of GTP for bound GDP. In most PDAC cases, KRAS is mutated at the 12th residue located in the G domain from glycine to either a valine (G12V), or more commonly, aspartate (G12D). These mutations sterically prevent the “arginine finger domain” of GAPs from entering the GTPase site, thereby blocking extrinsic allosteric GTPase activation and stabilizing RAS-GTP (21, 22). Activating mutations in KRAS constitutively trigger RAF/MEK and PI3K/AKT pathways leading to increased cell proliferation as well as other prooncogenic behaviors (15). KRAS signaling not only relies on the G domain but also the C-terminal hypervariable domain (HVR), which is required to stabilize KRAS on membranes where signaling is most efficient (23–26). Independent studies suggest that specific biochemical and cellular consequences of KRAS activation are attributed to the unique properties of the HVR of the predominant splice form KRAS4B, namely the polybasic domain and the lipid anchor (27–30). Localization of RAS proteins to the plasma membrane requires the prenylation of the CAAX motif (23). Additionally, for KRAS4B, the hypervariable region contains a highly polybasic domain consisting of several consecutive lysines, which can interact with the negative charges on the polar heads of phospholipids and stabilize protein interactions (31). Structural and biochemical characterization of the HVR and G domain has contributed to a better understanding of the signaling outputs of KRAS and led to KRAS-targeting strategies.Various approaches to inhibit KRAS include direct inhibition, expression interference, mislocalization, and targeting of downstream effectors (32). Thus far, direct inhibitors against KRAS have only successfully targeted the G12C mutant, which comprises 2.9% of KRAS mutant PDAC (21, 33). For other KRAS mutants, targeting downstream effectors of KRAS in pancreatic cancer remains an alternative approach. Unfortunately, dual inhibition of MEK and AKT pathways was ineffective in PDAC patients (34). Difficulty in targeting KRAS due to adaptive resistance and feedback regulation motivates a better understanding of KRAS biology (35). For example, although PDAC typically features a mutant KRAS, there may be a role for its wild-type (WT) counterpart as well as WT RAS paralogs (HRAS and NRAS), which are GAP sensitive and subject to signaling feedback. While oncogenic KRAS has been shown to activate WT HRAS and NRAS via allosteric stimulation of SOS1 (36), WT KRAS has been proposed to be a tumor suppressor in some KRAS mutant cancers based on the commonly observed mutant-specific allele imbalance that occurs throughout tumor progression (37). Additionally, the reintroduction of WT KRAS abolished tumor T cell acute lymphoblastic leukemia development and impaired tumor growth in KRAS mutant lung cancer cells in vivo (37–39). The discovery of novel KRAS protein interactors involved in downstream signaling or feedback and compensatory pathways may elucidate why inhibition of downstream pathways have had limited clinical impact in PDAC. Here, we perform proximity labeling experiments by expressing a fusion of BirA^R118G biotin ligase and KRAS in PDAC cells, which, in the presence of high concentrations of biotin, generates reactive biotinoyl-AMP that labels lysines of nearby proteins, such as interactors of its fusion partner KRAS (40–42). The biotinylated interactor proteins can be isolated by streptavidin pulldown and analyzed by proteomics to identify novel protein interactors (43–45). Because covalent labeling occurs in living cells, enzymatic labeling may potentially identify transient interactors and protein complexes.Two recent studies used proximity-dependent biotin identification (BioID) labeling methods to identify KRAS interactors in 293T and colon cancer cells (46, 47). These studies uncovered and validated the functional relevance of PIP5KA1 and mTORC2 in PDAC cells. However, BirA-KRAS screens in PDAC models have not yet been performed. Since the tumor context may determine protein expression and relevant interactions, we sought to perform a BirA-KRAS screen in PDAC cells. We hypothesize that proximity labeling with BioID presents a means for identifying new mutant KRAS-specific interactions in PDAC, which may unveil new insights into therapeutic design for this malignancy.