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Background Rising use of computed tomography (CT) to evaluate patients with trauma has increased both patient costs and risk of cancer from ionizing radiation, without demonstrable improvements in outcome. Patient-centred care mandates disclosure of the potential risks, costs and benefits of diagnostic testing whenever possible. Objective We sought to determine (1) patient preferences regarding emergency department (ED) real-time discussions of risks and costs of CT during their trauma evaluations; and (2) whether varying levels of odds of detection of life-threatening injury (LTI) were associated with changes in patient preferences for CT. Methods Excluding patients already receiving CT and patients with altered mental status, we surveyed adult, English-speaking patients at four Level I verified trauma centres. After informing subjects of cancer risks associated with chest CT, we used hypothetical scenarios with varying LTIs to assess patients' preferences regarding CT. Results Of 941 patients enrolled, 50% were male and their mean age was 42 years. Most patients stated they would prefer to discuss CT radiation risks (73.5%, 95% CI [66.1-80.8]) and costs (53.2%, 95% CI [46.1-60.4]) with physicians. As the odds of detecting LTI decreased, preferences for receiving CT decreased accordingly: LTI 25% (desire 91.2%, 95% CI [89.4-93.1]), LTI 10% (desire 79.3%, 95% CI [76.7-81.9]), LTI 5% (desire 69.1%, 95% CI [66.1-72.1]) and LTI

B cell clonal expansion and cerebrospinal fluid (CSF) oligoclonal IgG bands are established features of the immune response in multiple sclerosis (MS). Clone-specific recombinant monoclonal IgG1 Abs (rAbs) derived from MS patient CSF plasmablasts bound to conformational proteolipid protein 1 (PLP1) membrane complexes and, when injected into mouse brain with human complement, recapitulated histologic features of MS pathology: oligodendrocyte cell loss, complement deposition, and CD68+ phagocyte infiltration. Conformational PLP1 membrane epitopes were complex and governed by the local cholesterol and glycolipid microenvironment. Abs against conformational PLP1 membrane complexes targeted multiple surface epitopes, were enriched within the CSF compartment, and were detected in most MS patients, but not in inflammatory and noninflammatory neurologic controls. CSF PLP1 complex Abs provide a pathogenic autoantibody biomarker specific for MS.

Highly abundant proteins tend to evolve slowly (a trend called E-R anticorrelation), and a number of hypotheses have been proposed to explain this phenomenon. The misfolding avoidance hypothesis attributes the E-R anticorrelation to the abundance-dependent toxic effects of protein misfolding. To avoid these toxic effects, protein sequences (particularly those of highly expressed proteins) would be under selection to fold properly. One prediction of the misfolding avoidance hypothesis is that highly abundant proteins should exhibit high thermostability (i.e., a highly negative free energy of folding, ΔG). Thus far, only a handful of analyses have tested for a relationship between protein abundance and thermostability, producing contradictory results. These analyses have been limited by 1) the scarcity of ΔG data, 2) the fact that these data have been obtained by different laboratories and under different experimental conditions, 3) the problems associated with using proteins' melting energy (Tm) as a proxy for ΔG, and 4) the difficulty of controlling for potentially confounding variables. Here, we use computational methods to compare the free energy of folding of pairs of human-mouse orthologous proteins with different expression levels. Even though the effect size is limited, the most highly expressed ortholog is often the one with a more negative ΔG of folding, indicating that highly expressed proteins are often more thermostable., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Emergency physicians (EPs) frequently deliver care to members of the LGBTQIA+ community in the emergency department. This community suffers from many health disparities important to understand as part of comprehensive care, and these disparities are infrequently discussed in emergency medicine education. Previous data also suggest a need for broader education to increase the comfort of EPs caring for LGBTQIA+ patients. A group of content experts identified key disparities, opportunities for expanded education, and strategies for more inclusive care of LGBTQIA+ patients., Competing Interests: The authors have no potential conflicts to disclose., (© 2022 by the Society for Academic Emergency Medicine.)

Background: Emergency physicians need to recognize the diversity of identities held by sexual and gender minorities, as well as the health implications and inequities experienced by these communities. Identities such as lesbian, gay, bisexual, transgender, queer, questioning, intersex, asexual, aromantic, and many others fall under the LGBTQIA+ acronym. This wide spectrum is seldom discussed in emergency medicine but nonetheless impacts both patient care and patient experience in acute and critical care settings., Aims: This commentary aims to provide a brief but nonexhaustive review of LGBTQIA+ identities and supply a critical framework for applying this understanding to patient encounters in the emergency department, as well as describe the challenges and educational aims at the level of medical school, residency, and postresidency., Materials and Methods: The commonly used and widely accepted definitions of LGBTQIA+ terms are described, as well as implications for patient care and emergency physician education. The authors of this writing group represent the Society for Academic Emergency Medicine, LGBTQ Task Force of the Academy of Diversity Inclusion in Medicine., Results: LGB terms are addressed, with LGBTQIA+ adding "intersex," "asexual," and "+," to include other gender identities and sexual orientations which are not already included. This paper also addresses the terms "transition," "nonbinary," "polyamorous." "two-spirit," "queer," and others. These acronyms and terms continually expand and evolve in the pursuit of inclusivity. Additionally, with some health issues potentially related to medications, hormones, surgery, or to internal or external genitalia, important EM physician tools include gathering an "organ inventory," asking about sexual history, and conducting a physical exam., Discussion: Most persons have congruent biological sex, gender identity, and attraction to the "opposite" gender. However, humans can have every imaginable variation and configuration of chromosomes, genitalia, gender identities, sexual attractions, and sexual behaviors. Terms and definitions are constantly changing and adapting; they may also vary by local culture. Obtaining relevant medical history, conducting an "organ inventory," asking about sexual history in a nonjudgmental way, and conducting a physical exam when warranted can all be important in delivering best possible medical care. Although there has been increased focus on education at the medical school, residency, and faculty level on LGBTQIA+ patient care in the ED, much work remains to be done., Conclusion: Emergency physicians should feel confident in providing a model of care that affirms the sexual and gender identities of all the patient populations we serve. Optimal patient-centric care requires a deeper understanding of the patient's biology, gender identity, and sexual behavior encapsulated into the ever-growing acronym LGBTQIA+., Competing Interests: The authors have no potential conflicts to disclose., (© 2022 by the Society for Academic Emergency Medicine.)

Background: An accurate timescale of evolutionary history is essential to testing hypotheses about the influence of historical events and processes, and the timescale for evolution is increasingly derived from analysis of DNA sequences. But variation in the rate of molecular evolution complicates the inference of time from DNA. Evidence is growing for numerous factors, such as life history and habitat, that are linked both to the molecular processes of mutation and fixation and to rates of macroevolutionary diversification. However, the most widely used methods rely on idealised models of rate variation, such as the uncorrelated and autocorrelated clocks, and molecular dating methods are rarely tested against complex models of rate change. One relationship that is not accounted for in molecular dating is the potential for interaction between molecular substitution rates and speciation, a relationship that has been supported by empirical studies in a growing number of taxa. If these relationships are as widespread as current evidence suggests, they may have a significant influence on molecular dates., Results: We simulate phylogenies and molecular sequences under three different realistic rate variation models-one in which speciation rates and substitution rates both vary but are unlinked, one in which they covary continuously and one punctuated model in which molecular change is concentrated in speciation events, using empirical case studies to parameterise realistic simulations. We test three commonly used "relaxed clock" molecular dating methods against these realistic simulations to explore the degree of error in molecular dates under each model. We find average divergence time inference errors ranging from 12% of node age for the unlinked model when reconstructed under an uncorrelated rate prior using BEAST 2, to up to 91% when sequences evolved under the punctuated model are reconstructed under an autocorrelated prior using PAML., Conclusions: We demonstrate the potential for substantial errors in molecular dates when both speciation rates and substitution rates vary between lineages. This study highlights the need for tests of molecular dating methods against realistic models of rate variation generated from empirical parameters and known relationships., (© 2022. The Author(s).)

Understanding the factors that drive diversification of taxa across the tree of life is a key focus of macroevolutionary research. While the effects of life history, ecology, climate and geography on diversity have been studied for many taxa, the relationship between molecular evolution and diversification has received less attention. However, correlations between rates of molecular evolution and diversification rate have been detected in a range of taxa, including reptiles, plants and birds. A correlation between rates of molecular evolution and diversification rate is a prediction of several evolutionary theories, including the evolutionary speed hypothesis which links variation in mutation rates to differences in speciation rates. If it is widespread, such correlations could also have significant practical impacts, if they are not adequately accounted for in phylogenetic inference of evolutionary rates and timescales. Ray-finned fish (Actinopterygii) offer a prime target to test for this relationship due to their extreme variation in clade size suggesting a wide range of diversification rates. We employ both a sister-pairs approach and a whole-tree approach to test for correlations between substitution rate and net diversification. We also collect life history and ecological trait data and account for potential confounding factors including body size, latitude, max depth and reef association. We find evidence to support a relationship between diversification and synonymous rates of nuclear evolution across two published backbone phylogenies, as well as weak evidence for a relationship between mitochondrial nonsynonymous rates and diversification at the genus level., (© 2022. The Author(s).)

Background: Recovering the historical patterns of selection acting on a protein coding sequence is a major goal of evolutionary biology. Mutation-selection models address this problem by explicitly modelling fixation rates as a function of site-specific amino acid fitness values.However, they are restricted in their utility for investigating directional evolution because they require prior knowledge of the locations of fitness changes in the lineages of a phylogeny., Results: We apply a modified mutation-selection methodology that relaxes assumptions of equlibrium and time-reversibility. Our implementation allows us to identify branches where adaptive or compensatory shifts in the fitness landscape have taken place, signalled by a change in amino acid fitness profiles. Through simulation and analysis of an empirical data set of [Formula: see text]-lactamase genes, we test our ability to recover the position of adaptive events within the tree and successfully reconstruct initial codon frequencies and fitness profile parameters generated under the non-stationary model., Conclusion: We demonstrate successful detection of selective shifts and identification of the affected branch on partitions of 300 codons or more. We successfully reconstruct fitness parameters and initial codon frequencies in simulated data and demonstrate that failing to account for non-equilibrium evolution can increase the error in fitness profile estimation. We also demonstrate reconstruction of plausible shifts in amino acid fitnesses in the bacterial [Formula: see text]-lactamase family and discuss some caveats for interpretation.

Neuromyelitis optica spectrum disorders (NMOSD) constitute rare autoimmune disorders of the CNS that are primarily characterized by severe inflammation of the spinal cord and optic nerve. Approximately 75% of NMOSD patients harbour circulating pathogenic autoantibodies targeting the aquaporin-4 water channel (AQP4). The source of these autoantibodies remains unclear, but parallels between NMOSD and other autoantibody-mediated diseases posit compromised B cell tolerance checkpoints as common underlying and contributing factors. Using a well established assay, we assessed tolerance fidelity by creating recombinant antibodies from B cell populations directly downstream of each checkpoint and testing them for polyreactivity and autoreactivity. We examined a total of 863 recombinant antibodies. Those derived from three anti-AQP4-IgG seropositive NMOSD patients (n = 130) were compared to 733 antibodies from 15 healthy donors. We found significantly higher frequencies of poly- and autoreactive new emigrant/transitional and mature naïve B cells in NMOSD patients compared to healthy donors (P-values < 0.003), thereby identifying defects in both central and peripheral B cell tolerance checkpoints in these patients. We next explored whether pathogenic NMOSD anti-AQP4 autoantibodies can originate from the pool of poly- and autoreactive clones that populate the naïve B cell compartment of NMOSD patients. Six human anti-AQP4 autoantibodies that acquired somatic mutations were reverted back to their unmutated germline precursors, which were tested for both binding to AQP4 and poly- or autoreactivity. While the affinity of mature autoantibodies against AQP4 ranged from modest to strong (Kd 15.2-559 nM), none of the germline revertants displayed any detectable binding to AQP4, revealing that somatic hypermutation is required for the generation of anti-AQP4 autoantibodies. However, two (33.3%) germline autoantibody revertants were polyreactive and four (66.7%) were autoreactive, suggesting that pathogenic anti-AQP4 autoantibodies can originate from the pool of autoreactive naïve B cells, which develops as a consequence of impaired early B cell tolerance checkpoints in NMOSD patients., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Decellularized extracellular matrix (ECM) is an effective tissue repair scaffold. Additionally, ECM has recently been shown to be protective to the lungs. However, current processing is inadequate for effective delivery of ECM to the lungs. Processing methods, such as milling, produce large variability in particle sizes. The size variation produced is ineffective at treating the lung because only a small range of sizes reach the distal regions of the alveoli. The aim of this work is to formulate decellularized ECM to reach the distal lung while retaining the pro-regenerative effects of ECM. We first digested the protein in acid and then electrosprayed the solution into nanoparticles. The average size of the nanoparticles was 225 (±67) nm, within size requirements to reach the alveoli. After characterizing the particles, we measured cytotoxicity of the nanoparticles. Adding 0.125 mg/ml of nanoparticles to the media increased cellular proliferation in A549 alveolar epithelial cells and caused no cytotoxicity in BEAS-2B cells. We added the formed nanoparticles to macrophages derived from murine bone marrow-derived monocytes. The macrophages exposed to the formed nanoparticles expressed cell surface marker CD206 (mannose receptor C type 1), commonly attributed to a pro-regeneration phenotype. Electrosprayed ECM formed nanoparticles may improve bronchoalveolar deposition while maintaining the pro-regenerative benefits shown by other decellularized ECM materials., (© 2018 John Wiley & Sons, Ltd.)

When mutational pressure is weak, the generative process of protein evolution involves explicit probabilities of mutations of different types coupled to their conditional probabilities of fixation dependent on selection. Establishing this mechanistic modeling framework for the detection of selection has been a goal in the field of molecular evolution. Building on a mathematical framework proposed more than a decade ago, numerous methods have been introduced in an attempt to detect and measure selection on protein sequences. In this review, we discuss the structure of the original model, subsequent advances, and the series of assumptions that these models operate under.

Molecular dating analyses allow evolutionary timescales to be estimated from genetic data, offering an unprecedented capacity for investigating the evolutionary past of all species. These methods require us to make assumptions about the relationship between genetic change and evolutionary time, often referred to as a 'molecular clock'. Although initially regarded with scepticism, molecular dating has now been adopted in many areas of biology. This broad uptake has been due partly to the development of Bayesian methods that allow complex aspects of molecular evolution, such as variation in rates of change across lineages, to be taken into account. But in order to do this, Bayesian dating methods rely on a range of assumptions about the evolutionary process, which vary in their degree of biological realism and empirical support. These assumptions can have substantial impacts on the estimates produced by molecular dating analyses. The aim of this review is to open the 'black box' of Bayesian molecular dating and have a look at the machinery inside. We explain the components of these dating methods, the important decisions that researchers must make in their analyses, and the factors that need to be considered when interpreting results. We illustrate the effects that the choices of different models and priors can have on the outcome of the analysis, and suggest ways to explore these impacts. We describe some major research directions that may improve the reliability of Bayesian dating. The goal of our review is to help researchers to make informed choices when using Bayesian phylogenetic methods to estimate evolutionary rates and timescales., (© 2017 Cambridge Philosophical Society.)

Background: The global incidence of melanoma has been increasing faster than any other form of cancer. New therapies offer exciting prospects for improved survival, but the development of resistance is a major problem and there remains a need for additional effective melanoma therapy. Platinum compounds, such as cisplatin, are the most effective chemotherapeutics for a number of major cancers, but are ineffective on metastatic melanoma. They cause monofunctional adducts and intrastrand crosslinks that are repaired by nucleotide excision repair, as well as the more toxic interstrand crosslinks that are repaired by a combination of nuclease activity and homologous recombination., Methods: We investigated the mechanism of melanoma resistance to cisplatin using a panel of melanoma and control cell lines. Cisplatin-induced changes in levels of the key homologous recombination protein RAD51 and compensatory changes in translesion synthesis DNA polymerases were identified by western blotting and qRT-PCR. Flow cytometry, immunofluorescence and western blotting were used to compare the cell cycle and DNA damage response and the induction of apoptosis in cisplatin-treated melanoma and control cells. Ectopic expression of a tagged form of RAD51 and siRNA knockdown of translesion synthesis DNA polymerase zeta were used to investigate the mechanism that allowed cisplatin-treated melanoma cells to continue to replicate., Results: We have identified and characterised a novel DNA damage response mechanism in melanoma. Instead of increasing levels of RAD51 on encountering cisplatin-induced interstrand crosslinks during replication, melanoma cells shut down RAD51 synthesis and instead boost levels of translesion synthesis DNA polymerase zeta to allow replication to proceed. This response also resulted in synthetic lethality to the PARP inhibitor olaparib., Conclusions: This unusual DNA damage response may be a more appropriate strategy for an aggressive and rapidly growing tumour like melanoma that enables it to better survive chemotherapy, but also results in increased sensitivity of cultured melanoma cells to the PARP inhibitor olaparib.

DNA repair pathways present in all cells serve to preserve genome stability, but in cancer cells they also act reduce the efficacy of chemotherapy. The endonuclease ERCC1-XPF has an important role in the repair of DNA damage caused by a variety of chemotherapeutic agents and there has been intense interest in the use of ERCC1 as a predictive marker of therapeutic response in non-small cell lung carcinoma, squamous cell carcinoma and ovarian cancer. We have previously validated ERCC1 as a therapeutic target in melanoma, but all small molecule ERCC1-XPF inhibitors reported to date have lacked sufficient potency and specificity for clinical use. In an alternative approach to prevent the repair activity of ERCC1-XPF, we investigated the mechanism of ERCC1 ubiquitination and found that the key region was the C-terminal (HhH) 2 domain which heterodimerizes with XPF. This ERCC1 region was modified by non-conventional lysine-independent, but proteasome-dependent polyubiquitination, involving Lys33 of ubiquitin and a linear ubiquitin chain. XPF was not polyubiquitinated and its expression was dependent on presence of ERCC1, but not vice versa. To our surprise we found that ERCC1 can also homodimerize through its C-terminal (HhH) 2 domain. We exploited the ability of a peptide containing this C-terminal domain to destabilise both endogenous ERCC1 and XPF in human melanoma cells and fibroblasts, resulting in reductions of up to 85% in nucleotide excision repair and near two-fold increased sensitivity to DNA damaging agents. We suggest that the ERCC1 (HhH) 2 domain could be used in an alternative strategy to treat cancer., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Objectives: Neuromyelitis optica spectrum disorder (NMOSD) is a severe inflammatory disorder of the central nervous system (CNS) targeted against aquaporin-4 (AQP4). The origin and trafficking of AQP4-specific B cells in NMOSD remains unknown., Methods: Peripheral ( n = 7) and splenic B cells ( n = 1) recovered from seven NMOSD patients were sorted into plasmablasts, naïve, memory, and CD27-IgD- double negative (DN) B cells, and variable heavy chain (VH) transcriptome sequences were generated by deep sequencing. Peripheral blood (PB) VH repertoires were compared to the same patient's single-cell cerebrospinal fluid (CSF) plasmablast (PB) VH transcriptome, CSF immunoglobulin (Ig) proteome, and serum Ig proteome. Recombinant antibodies were generated from paired CSF heavy- and light chains and tested for AQP4 reactivity., Results: Approximately 9% of the CSF VH sequences aligned with PB memory B cells, DN B cells, and plasmablast VH sequences. AQP4-specific VH sequences were observed in each peripheral B-cell compartment. Lineage analysis of clonally related VH sequences indicates that CSF AQP4-specific B cells are closely related to an expanded population of DN B cells that may undergo antigen-specific B-cell maturation within the CNS. CSF and serum Ig proteomes overlapped with the VH sequences from each B-cell compartment; the majority of matches occurring between the PB VH sequences and serum Ig proteome., Interpretation: During an acute NMOSD relapse, a dynamic exchange of B cells occurs between the periphery and CNS with AQP4-specific CSF B cells emerging from postgerminal center memory B cells and plasmablasts. Expansion of the PB DN B-cell compartment may be a potential biomarker of NMOSD activity.

In Bayesian phylogenetic analyses of genetic data, prior probability distributions need to be specified for the model parameters, including the tree. When Bayesian methods are used for molecular dating, available tree priors include those designed for species-level data, such as the pure-birth and birth-death priors, and coalescent-based priors designed for population-level data. However, molecular dating methods are frequently applied to data sets that include multiple individuals across multiple species. Such data sets violate the assumptions of both the speciation and coalescent-based tree priors, making it unclear which should be chosen and whether this choice can affect the estimation of node times. To investigate this problem, we used a simulation approach to produce data sets with different proportions of within- and between-species sampling under the multispecies coalescent model. These data sets were then analyzed under pure-birth, birth-death, constant-size coalescent, and skyline coalescent tree priors. We also explored the ability of Bayesian model testing to select the best-performing priors. We confirmed the applicability of our results to empirical data sets from cetaceans, phocids, and coregonid whitefish. Estimates of node times were generally robust to the choice of tree prior, but some combinations of tree priors and sampling schemes led to large differences in the age estimates. In particular, the pure-birth tree prior frequently led to inaccurate estimates for data sets containing a mixture of inter- and intraspecific sampling, whereas the birth-death and skyline coalescent priors produced stable results across all scenarios. Model testing provided an adequate means of rejecting inappropriate tree priors. Our results suggest that tree priors do not strongly affect Bayesian molecular dating results in most cases, even when severely misspecified. However, the choice of tree prior can be significant for the accuracy of dating results in the case of data sets with mixed inter- and intraspecies sampling. [Bayesian phylogenetic methods; model testing; molecular dating; node time; tree prior.]., (© The authors 2016. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Molecular estimates of evolutionary timescales have an important role in a range of biological studies. Such estimates can be made using methods based on molecular clocks, including models that are able to account for rate variation across lineages. All clock models share a dependence on calibrations, which enable estimates to be given in absolute time units. There are many available methods for incorporating fossil calibrations, but geological and climatic data can also provide useful calibrations for molecular clocks. However, a number of strong assumptions need to be made when using these biogeographic calibrations, leading to wide variation in their reliability and precision. In this review, we describe the nature of biogeographic calibrations and the assumptions that they involve. We present an overview of the different geological and climatic events that can provide informative calibrations, and explain how such temporal information can be incorporated into dating analyses., (© 2015 The Author(s).)

ERCC1-XPF is a structure-specific endonuclease that is required for the repair of DNA lesions, generated by the widely used platinum-containing cancer chemotherapeutics such as cisplatin, through the Nucleotide Excision Repair and Interstrand Crosslink Repair pathways. Based on mouse xenograft experiments, where ERCC1-deficient melanomas were cured by cisplatin therapy, we proposed that inhibition of ERCC1-XPF could enhance the effectiveness of platinum-based chemotherapy. Here we report the identification and properties of inhibitors against two key targets on ERCC1-XPF. By targeting the ERCC1-XPF interaction domain we proposed that inhibition would disrupt the ERCC1-XPF heterodimer resulting in destabilisation of both proteins. Using in silico screening, we identified an inhibitor that bound to ERCC1-XPF in a biophysical assay, reduced the level of ERCC1-XPF complexes in ovarian cancer cells, inhibited Nucleotide Excision Repair and sensitised melanoma cells to cisplatin. We also utilised high throughput and in silico screening to identify the first reported inhibitors of the other key target, the XPF endonuclease domain. We demonstrate that two of these compounds display specificity in vitro for ERCC1-XPF over two other endonucleases, bind to ERCC1-XPF, inhibit Nucleotide Excision Repair in two independent assays and specifically sensitise Nucleotide Excision Repair-proficient, but not Nucleotide Excision Repair-deficient human and mouse cells to cisplatin., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Neuromyelitis optica (NMO) is a severe demyelinating disorder of the central nervous system (CNS) associated with the presence of an autoimmune antibody response (AQP4-IgG) against the water channel aquaporin-4 (AQP4). It remains unclear whether pathologic AQP4-IgG in the CNS is produced entirely by peripheral plasma cells or is generated in part by infiltrating B cells. To determine the overlap of AQP4-IgG idiotypes between the CNS and periphery, we compared the immunoglobulin G (IgG) transcriptome of cerebrospinal fluid (CSF) plasmablasts with the CSF and serum IgG proteomes in 7 AQP4-seropositive NMO patients following exacerbation., Methods: CSF variable region Ig heavy- (VH) and light-chain (VL) transcriptome libraries were generated for each patient from CSF plasmablasts by single cell sorting, reverse transcriptase polymerase chain reaction (RT-PCR), and DNA sequencing. Recombinant antibodies were generated from clonally expanded, paired VH and VL sequences and tested for AQP4-reactivity by cell-binding assay. CSF and serum IgG fractions were searched for sequences that matched their respective CSF IgG transcriptome. Matching peptides within the same patient's CSF and serum IgG proteomes were also identified., Results: In each NMO patient, we recovered CSF IgG VH and VL sequences that matched germline-mutated IgG protein sequences from the patient's CSF and serum IgG proteomes. Although a modest variation was observed between patients, the overlap between the transcriptome and proteome sequences was found primarily, but not exclusively, within the CSF. More than 50% of the CSF IgG transcriptome sequences were exclusively found in the CSF IgG proteome, whereas 28% were found in both the CSF and blood IgG proteome, and 18% were found exclusively in the blood proteome. A comparable distribution was noted when only AQP4-specific IgG clones were considered. Similarly, on average, only 50% of the CSF IgG proteome matched corresponding peptide sequences in the serum., Conclusions: During NMO exacerbations, a substantial fraction of the intrathecal Ig proteome is generated by an intrathecal B cell population composed of both novel and peripherally-derived clones. Intrathecal CSF B cell clones may contribute to NMO disease exacerbation and lesion formation and may be an important target for preventative therapies.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system that can cause paralysis and blindness. The pathogenesis of NMO involves binding of immunoglobulin G autoantibodies to aquaporin-4 (AQP4) on astrocytes, which is thought to cause complement-dependent cytotoxicity (CDC) and a secondary inflammatory response leading to oligodendrocyte and neuronal damage. Here, we investigate in vivo the role of antibody-dependent cell-mediated cytotoxicity (ADCC) triggered by AQP4 autoantibodies (AQP4-IgG) in the development of NMO pathology. A high-affinity, human recombinant monoclonal AQP4-IgG was mutated in its Fc region to produce 'NMO superantibodies' with enhanced CDC and/or ADCC effector functions, without altered AQP4 binding. Pathological effects of these antibodies were studied in a mouse model of NMO produced by intracerebral injection of AQP4-IgG and human complement. The original (non-mutated) antibody produced large NMO lesions in this model, with loss of AQP4 and GFAP immunoreactivity, inflammation and demyelination, as did a mutated antibody with enhanced CDC and ADCC effector functions. As anticipated, a mutated AQP4-IgG lacking CDC, but having tenfold enhanced ADCC, produced little pathology. However, unexpectedly, a mutated antibody with ninefold enhanced CDC, but lacking ADCC, produced much less pathology than the original AQP4-IgG. Also, pathology was greatly reduced following administration of AQP4-IgG and complement to mice lacking the FcγIII receptor involved in effector cell activation during ADCC, and to normal mice injected with an Fcγ receptor blocking antibody. Our results provide evidence for the central involvement of ADCC in NMO pathology and suggest ADCC as a new therapeutic target in NMO.

Nitrofurans are commonly used for the treatment of trypanosomal diseases including Chagas disease. More recently, following the fortuitous discovery that nifurtimox was clinically active against neuroblastoma, nitrofuran compounds are being investigated for activity against cancer. Herein, we show that nitrofuran compounds are similarly potent to human malignant melanoma and neuroblastoma cells. Furthermore, a recently discovered nitrofuran compound, NFN1, was 50- to 175-fold more potent than nifurtimox against human melanoma and neuroblastoma cell lines. As nitrofuran compounds are known to act as pro-drugs, producing DNA-damaging reactive intermediates upon activation, we investigated the DNA repair pathways involved. We show that, contrary to research in Escherichia coli, the Nucleotide Excision Repair pathway is not required to repair nitrofuran-induced DNA damage in mammalian cells. Instead, we show that inhibiting repair of single-strand DNA breaks with the poly(ADP-ribose) polymerase (PARP) inhibitor, Olaparib, enhances nitrofuran toxicity in melanoma and neuroblastoma cells. We propose that this is due to mammalian cells utilising Type 2 nitroreductases for nitrofuran activation producing Reactive Oxygen Species which cause DNA damage that is repaired by the Single Strand Break Repair and/or Base Excision Repair pathways, whereas in bacteria and trypanosomes, Type 1 nitroreductases are also utilised resulting in different DNA lesions. In addition we show that, consistent with Reactive Oxygen Species being formed upon nitrofuran activation and the ability of melanin to absorb Reactive Oxygen Species, production of melanin in melanoma cells offers some protection from NFN1- and hydrogen peroxide-induced toxicity. Our data suggest that combinations of Olaparib and nitrofuran compounds may be advantageous for the treatment of melanoma and neuroblastoma, but that the protection offered to melanoma cells by their melanin pigment must be taken into account., (Copyright © 2013 Elsevier B.V. All rights reserved.)

OBJECTIVE To identify linear determinants of human aquaporin 4 (hAQP4) in the context of HLA-DRB1*03:01. DESIGN In this controlled study with humanized experimental animals, HLA-DRB1*03:01 transgenic mice were immunized with whole-protein hAQP4 emulsified in complete Freund adjuvant. To test T-cell responses, lymph node cells and splenocytes were cultured in vitro with synthetic peptides 20 amino acids long that overlap by 10 amino acids across the entirety of hAQP4. The frequency of interferon γ, interleukin (IL) 17, granulocyte-macrophage colony-stimulating factor, and IL-5-secreting CD4+ T cells was determined by the enzyme-linked immunosorbent sport assay. Quantitative immunofluorescence microscopy was performed to determine whether hAQP4281-300 inhibits the binding of anti-hAQP4 recombinant antibody to surface full-length hAQP4. SETTING Academic neuroimmunology laboratories. SUBJECTS Humanized HLA-DRB1*03:01+/+ H-2b-/- transgenic mice on a B10 background. RESULTS Peptide hAQP4281-300 generated a significantly (P &lt;.01) greater TH1 and TH17 immune response than any of the other linear peptides screened. This 20mer peptide contains 2 dominant immunogenic 15mer peptides. hAQP4284-298 induced predominantly an IL-17 and granulocyte-macrophage colony-stimulating factor TH cell phenotype, whereas hAQP4285-299 resulted in a higher frequency of TH1 cells. hAQP4281-300 did not interfere with recombinant AQP4 autoantibody binding. CONCLUSIONS hAQP4281-330 is the dominant linear immunogenic determinant of hAQP4 in the context of HLA-DRB1*03:01. Within hAQP4281-330 are 2 dominant immunogenic determinants that induce differential TH phenotypes. hAQP4 determinants identified in this study can serve as diagnostic biomarkers in patients with neuromyelitis optica and may facilitate the monitoring of treatment responses to pharmacotherapies.

The mitogen-activated protein kinase (MAPK) pathway is important in melanoma. In this pathway, DUSP6 phosphatase negatively controls the activation of extracellular signal-regulated (ERK) kinase. Through comparison of melanoma signalling pathways between immortal mouse melanocytes and their tumourigenic derivatives, retrieved from mouse xenografts, we identified a molecularly distinct subtype of melanoma, characterized by reduced ERK activity and increased DUSP6 expression. Overexpression of DUSP6 enhanced anchorage-independent growth and invasive ability of immortal mouse melanocytes, suggesting that increased DUSP6 expression contributes to melanoma formation in the mouse xenografts. In contrast, reduced tumourigenicity was observed after DUSP6 overexpression in human melanoma cells. A minority of thick human primary melanomas had high DUSP6 expression and the same poor melanoma-specific survival as the majority of thick primaries with low DUSP6 levels. We have demonstrated that DUSP6 is important in melanoma and that it plays a different role in our distinct subtype of mouse melanoma compared with that in classic human melanoma., (© 2011 John Wiley & Sons A/S.)

Increased expression of DNA repair genes contributes to the extreme resistance shown by melanoma to conventional DNA-damaging chemotherapeutics. One such chemotherapeutic effective against a range of other cancers, but not melanoma, is cisplatin. The DNA repair protein, ERCC1, is needed to remove cisplatin-induced DNA damage. We have shown that ERCC1 is essential for melanoma growth and resistance to cisplatin in a mouse xenograft model. Untreated xenografts of our transformed Ercc1-proficient melanocyte cell line grew very rapidly as malignant melanoma. Cisplatin treatment caused initial shrinkage of xenografts, but cisplatin-resistant regrowth soon followed. Cells reisolated into culture had twofold elevated levels of ERCC1 compared to both input cells and cells reisolated from untreated xenografts. An isogenic Ercc1-deficient derivative grew equally well in vitro as the Ercc1-proficient melanocyte cell line. However, in xenografts, the Ercc1-deficient melanomas were much slower to establish and were completely cured by just two cisplatin treatments., (© 2011 John Wiley & Sons A/S.)

Objective: Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory B-cell clonal expansion are well-characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic., Methods: We produced 53 different human IgG1 monoclonal recombinant antibodies (rAbs) by coexpressing paired heavy- and light-chain variable region sequences of 51 plasma cell clones and 2 B-lymphocyte clones from MS cerebrospinal fluid in human tissue culture cells. Chimeric control rAbs were generated from anti-myelin hybridomas in which murine variable region sequences were fused to human constant region sequences. Purified rAbs were exhaustively assayed for reactivity against myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein by immunostaining of transfected cells expressing individual myelin proteins, by protein immunoblotting, and by immunostaining of human brain tissue sections., Results: Whereas humanized control rAbs derived from anti-myelin hybridomas and anti-myelin monoclonal antibodies readily detected myelin antigens in multiple immunoassays, none of the rAbs derived from MS cerebrospinal fluid displayed immunoreactivity to the three myelin antigens tested. Immunocytochemical analysis of tissue sections from MS and control brain demonstrated only weak staining with a few rAbs against nuclei or cytoplasmic granules in neurons, glia, and inflammatory cells., Interpretation: The oligoclonal B-cell response in MS cerebrospinal fluid is not targeted to the well-characterized myelin antigens myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein.

Topical application of thymidine dinucleotides (pTpT) provides some protection against the effects of UV on the skin, however, many details of the protective mechanism have yet to be elucidated. We have used mice with an epidermis-specific knockout for the nucleotide excision repair gene, Ercc1, to investigate the mechanisms of protection. pTpT offered no protection against the pronounced UV-induced short-term erythema and skin thickening responses that are characteristic of DNA repair-deficient skin. It also had no effect on UV-induced apoptosis in Ercc1-deficient cultured keratinocytes. However, in these short-term experiments in both skin and keratinocyte culture pTpT did cause a slight reduction in proliferation. pTpT application during a chronic UV irradiation protocol provided some protection from UVB-induced skin carcinogenesis in epidermis-specific Ercc1 knockout mice. The median tumour free survival time was increased in the pTpT-treated group and treated animals had fewer tumours. In addition, pTpT-treated animals developed fewer large inwardly growing skin lesions than untreated animals. Furthermore, the proliferation response was reduced in chronically irradiated, non-lesional pTpT-treated skin. We conclude that cancer protection by pTpT in our mice is not modulated by an upregulation of DNA repair, as protection appears to be independent of a functional nucleotide excision repair pathway. We hypothesise instead that protection by pTpT is due to a reduction in epidermal proliferation.

Herpesvirions and varicella zoster virus (VZV) DNA were recently reported in all 15 cerebrospinal fluid (CSF) samples from patients with relapsing-remitting multiple sclerosis (MS) obtained within 1 week of exacerbation. Using identical electron microscopic and polymerase chain reaction techniques, including additional primer sets representing different regions of the VZV genome, we found no herpesvirions or VZV DNA in MS CSF or acute MS plaques. Although enzyme-linked immunosorbent assay analysis demonstrated a higher titer of VZV antibody in MS CSF than in inflammatory control samples, recombinant antibodies prepared from clonally expanded MS CSF plasma cells did not bind to VZV. VZV is not a disease-relevant antigen in MS.

Using FACS and single cell reverse transcriptase polymerase chain reaction, we examined the cerebrospinal fluid (CSF) IgG VH repertoires from 10 subjects with a clinically isolated demyelinating syndrome (CIS). B and plasma cell repertoires from individual subjects showed similar VH family germline usage, nearly identical levels of post-germinal center somatic hypermutation, and significant overlap in their clonal populations. Repertoires from 7 of 10 CIS subjects demonstrated a biased usage of VH4 and/or VH2 family gene segments in their plasma or B cell repertoires. V-regionbias, however, was not observed in the corresponding peripheral blood CD19+ B cell repertoires from 2 CIS subjects or in normal healthy adults. Clinically, subjects with VH4 or VH2 CSF IgG repertoire bias rapidly progressed to definite MS, whereas individuals without repertoire bias did not develop MS after a minimum of 2 years of follow-up (p=0.01).

Fluorescence-activated cell sorting (FACS) analysis of B cell subtypes in 17 CSF samples from 15 patients with clinically-definite MS revealed that CD19+ B cells accounted for 2 to 11% (mean 5%) and CD138+ cells constituted 0 to 5% (mean 2%) of total CSF lymphocytes. Further stratification of CD138+ cells based on expression levels of CD19 showed that CD138+19+ plasma blasts constituted 89+/-2% (mean+/-SE) of the CD138+ cell population (P<0.00001), with more mature plasma cells (CD138+19-) constituting the remaining 11+/-2%. Sequence analysis of immunoglobulin variable regions in single CD138+19+ and CD138+19- cells sorted from MS CSF identified many of the same clonal populations in both populations, indicating a continuum of clonally related plasma cell subtypes of which CD138+19+ plasma blasts are most abundant.

A characteristic feature of the CNS inflammatory response in multiple sclerosis (MS) is the intrathecal synthesis of IgG and the presence of oligoclonal bands. A strong correlation between CD138(+) plasma blast numbers in MS cerebrospinal fluid (CeSF) and intrathecal IgG synthesis suggests that these cells are the major Ab-secreting cell type in MS CeSF. Sequencing of V regions from CD138(+) cells in MS CeSF has revealed somatically mutated and expanded IgG clonotypes consistent with an Ag-targeted response. In the present study, single-cell RT-PCR analysis of CD138(+) cells from 11 MS patients representing differing clinical courses and stages of disease identified expansion of CD138(+) cells with functionally rearranged V(H)4 gene segments as an overriding feature of MS CeSF repertoires. V(H)4 dominance was attributed to the preferential selection of specific V(H)4 genes, particularly gene segment V(H)4-39, which displayed a significant enrichment in CeSF compared with MS peripheral blood B cells. A modest increase in V(H)4 prevalence among MS peripheral blood IgG memory cells was also noted, suggesting that factors shaping the CD138 repertoire in CeSF might also influence the peripheral IgG memory cell pool. These results indicate a highly restricted B cell response in MS. Identifying the targets of CeSF plasma cells may yield insights into disease pathogenesis.

Infectious and inflammatory diseases of the CNS are often characterized by a robust B-cell response that manifests as increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands. We previously used laser capture microdissection and single-cell PCR to analyze the IgG variable regions of plasma cells from the brain of a patient with subacute sclerosing panencephalitis (SSPE). Five of eight human IgG1 recombinant antibodies (rAbs) derived from SSPE brain plasma cell clones recognized the measles virus (MV) nucleocapsid protein, confirming that the antibody response in SSPE targets primarily the agent causing disease. In this study, as part of our work on antigen identification, we used four rAbs to probe a random phage-displayed peptide library to determine if epitopes within the MV nucleocapsid protein could be identified with SSPE brain rAbs. All four of the SSPE rAbs enriched phage-displayed peptide sequences that reacted specifically to their panning rAb by enzyme-linked immunosorbent assay. BLASTP searches of the NCBI protein database revealed clear homologies in three peptides and different amino acid stretches within the 65 C-terminal amino acids of the MV nucleocapsid protein. The specificities of SSPE rAbs to these regions of the MV nucleocapsid protein were confirmed by binding to synthetic peptides or to short cDNA expression products. These results indicate the feasibility of using peptide screening for antigen discovery in central nervous system inflammatory diseases of unknown etiology, such as multiple sclerosis, neurosarcoidosis, or Behcet's syndrome.

We generated recombinant antibodies (rAbs) from over-represented IgG sequences expressed by single plasma cells from multiple sclerosis (MS) cerebrospinal fluid (CSF). Panning of a phage-displayed random peptide library with the rAbs revealed several specific peptide sequences. Inhibition assays confirmed specific binding of the peptides to the antigen-binding site of the antibody. The native IgG of MS CSF from which the recombinant antibody was cloned also recognized these peptides. Our data demonstrate that MS rAb reflects the specificity of IgG in the CSF. Thus, the epitopes/mimotopes identified by MS rAb may provide clues to disease-relevant antigens.

Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras(tmDelta4A/tmDelta4A) (exon 4A knock-out) ES cells which express K-ras4B only and K-ras(-/-) (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras(tmDelta4A/tmDelta4A) mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras(tmDelta4A/tmDelta4A) ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras(tmDelta4A/tmDelta4A) and K-ras(-/-) ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras(-/-) cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras(tmDelta4A/tmDelta4A) ES cells were more resistant to etoposide-induced apoptosis than K-ras(-/-) cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice.

Chronic infectious diseases of the central nervous system (CNS) are characterized by intrathecal synthesis of increased amounts of immunoglobulin G (IgG) directed against the agent that causes disease. In other inflammatory CNS diseases such as multiple sclerosis and CNS sarcoid, the targets of the humoral immune response are uncertain. To identify the IgGs expressed by individual CD38(+) plasma cells seen in human brain sections, we merged the techniques of laser capture microdissection (LCM) and single-cell RT-PCR. Frozen brain sections from a patient who died of subacute sclerosing panencephalitis (SSPE), were rapidly immunostained and examined by LCM to dissect individual CD38(+) cells. After cell lysis, we developed two techniques for reverse-transcription (RT) of unpurified total RNA in the cell lysates. The first method performed repeated and rapid freeze-thawing, followed by centrifugation of the cell lysate into tubes for subsequent RT. The second, more successful method performed RT in situ on detergent-solubilized cells directly on the cap surface; subsequent nested PCR identified heavy and light chain sequences expressed by two-thirds of individually isolated plasma cells. These techniques will streamline the identification of gene expression products in single cells from complex tissues and have the potential to identify IgGs expressed in the CNS of inflammatory diseases of unknown etiology.

Increased IgG and oligoclonal bands are found in cerebrospinal fluid of humans with chronic infectious CNS disease. Studies have shown that these oligoclonal bands are antibodies directed against the agent that causes disease. Laser-capture microdissection was used to isolate individual CD38+ plasma cells from the brain of a patient with subacute sclerosing panencephalitis, and single-cell RT-PCR was used to analyze individual IgG heavy and light chains expressed by each cell. Based on overrepresented IgG sequences, we constructed functional recombinant antibodies (recombinant IgGs) and determined their specificities. Five of eight recombinant IgGs recognized measles virus, the cause of subacute sclerosing panencephalitis. These results demonstrate that overrepresented IgG sequences in postmortem brains can be used to produce functional recombinant antibodies that recognize their target antigens. This strategy can be used to identify disease-relevant antigens in CNS inflammatory diseases of unknown etiology.

53BP1 protein is re-localized to the sites of DNA damage after ionizing radiation (IR) and is involved in DNA-damage-checkpoint signal transduction. We examined the dynamics of GFP-53BP1 in living cells. The protein starts to accumulate at the sites of DNA damage 2-3 minutes after damage induction. Fluorescence recovery after photobleaching experiments showed that GFP-53BP1 is highly mobile in non-irradiated cells. Upon binding to the IR-induced nuclear foci, the mobility of 53BP1 reduces greatly. The minimum (M) domain of 53BP1 essential for targeting to IR induced foci consists of residues 1220-1703. GFP-M protein forms foci in mouse embryonic fibroblast cells lacking functional endogenous 53BP1. The M domain contains a tandem repeat of Tudor motifs and an arginine- and glycine-rich domain (RG stretch), which are often found in proteins involved in RNA metabolism, the former being essential for targeting. RNase A treatment dissociates 53BP1 from IR-induced foci. In HeLa cells, dissociation of the M domain without the RG stretch by RNase A treatment can be restored by re-addition of nuclear RNA in the early stages of post-irradiation. 53BP1 immunoprecipitates contain some RNA molecules. Our results suggest a possible involvement of RNA in the binding of 53BP1 to chromatin damaged by IR.

Increased amounts of intrathecally synthesized IgG and oligoclonal bands have long been recognized as a hallmark of multiple sclerosis (MS). B cells and plasma cells are components of the inflammatory infiltrates in both active and chronic MS lesions, and increased numbers of these cells are present in MS cerebrospinal fluid (CSF). Single-cell RT-PCR was used to analyze both the CD19+ B cell and CD138+ plasma cell populations in CSF of two patients with clinically definite MS and of one MS patient whose CSF was obtained after a clinically isolated syndrome, but before the second episode. Sequence analysis of amplified IgG V region sequences identified the rearranged germline segments, extent of somatic mutation, and clonal relationships within and between the two cell populations in the three MS patients. Expanded B cell and plasma cell clones were detected in each MS CSF and in all three patients the CD138+ IgG repertoire was more restricted. However, little if any significant sequence overlap was observed between the CD19+ and CD138+ repertoires of each donor. Detection of plasma cell clones by single-cell PCR will facilitate the in vitro production of recombinant Abs useful in identifying disease-relevant Ags.

The CD19+ B-lymphocyte and CD138+ plasma cell repertoires in cerebrospinal fluid from four patients with monosymptomatic optic neuritis (ON) were analyzed by single-cell reverse transcriptase polymerase chain reaction. Amplified heavy (H)- and light (L)-chain antibody segments were sequenced and used to identify the rearranged germline and J segment of closest homology. Both the B-cell and plasma cell repertoires from ON cerebrospinal fluid demonstrated significant clonal expansion. Up to 75% of the amplified H- and L-chain sequences were contained in overrepresented populations and were somatically mutated, consistent with an antigen-targeted response. The relationship between clonal populations within the CD19+ B lymphocyte and CD138+ plasma cell populations suggests ongoing mutational pressure to refine antigen binding. Our observations demonstrate that an antigen-driven clonal B-lymphocyte and plasma cell response is prominent in the initial stages of central nervous system demyelination and suggest that detection of the disease-relevant antigens in ON may bear on the inciting antigens in chronic inflammatory disorders such as multiple sclerosis.

The prion protein (PrP) and Doppel (Dpl) have many structural and biochemical properties in common, leading to the suggestion that the lack of an obvious phenotype in PrP-deficient mice maybe because of compensation by Dpl. To test this hypothesis and also investigate the function of Dpl we have generated Prnd(-/-) and Prnp(-/-)/Prnd(-/-) mouse lines. Both develop normally and display an identical male sterility phenotype that differs from that reported for another Prnd(-/-) mouse line. Sperm from both our mutant lines were present at normal concentrations, had normal motility, and no morphological abnormalities. Despite only rarely fertilizing oocytes in vivo, because of an inability to perform the acrosome reaction, mutant sperm were capable of fertilization in vitro, albeit at reduced rates compared to wild type. Elevated levels of oxidative DNA damage were found in both types of mutant sperm and resulting embryos failed at an early stage. Therefore we found no evidence that Dpl compensates for the loss of PrP function in mutant mouse lines, but it does have an important anti-oxidant function necessary for sperm integrity and male fertility.

In mammals, the three classical ras genes encode four highly homologous proteins, N-Ras, H-Ras, and the isoforms K-Ras 4A and 4B. Previous studies have shown that K-ras is essential for mouse development and that while K-ras 4A and 4B are expressed during development, K-ras 4A expression is regulated temporally and spatially and occurs in adult kidney, intestine, stomach, and liver. In the present study, the pattern of K-ras 4A expression was examined in a wide range of wild-type adult mouse tissues, and gene targeting was used to generate K-ras 4A-deficient mice to examine its role in development. It was found that K-ras 4A is also expressed in uterus, lung, pancreas, salivary glands, seminal vesicles, bone marrow cells, and cecum, where it was the major K-Ras isoform expressed. Mating between K-ras(tmDelta4A/+) mice produced viable K-ras(tmDelta4A/tmDelta4A) offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras(tmDelta4A/tmDelta4A) mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola x C57BL/6) genetic backgrounds. The results demonstrate that K-Ras 4A, like H- and N-Ras, is dispensable for normal mouse development, at least in the presence of functional K-Ras 4B.

Single-cell RT-PCR was used to sample CD19(+) B cell repertoires in cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) or viral meningitis. Analysis of amplified Ab H and L chain products served to identify the rearranged germline segment and J segment, and to determine the degree of homology for the H and L chain sequence of individual B cells. The B cell repertoire of viral meningitis CSF was predominantly polyclonal, whereas B cell clonal expansion was a prominent feature of the IgG repertoire in three of four MS patients. Two dominant clonal populations in one MS CSF accounted for approximately 70% of the IgG H chain V regions sequenced, while the corresponding IgM repertoires were more heterogeneous. One clonal B cell population revealed multiple L chain rearrangements, raising the possibility of a role for receptor editing in shaping the B cell response in some MS patients. The most immediate implications of identifying rearranged Ig sequences in MS B cells is the potential to accurately recreate recombinant Abs from these overrepresented H and L chains that can be used to discover the relevant Ag(s) in MS.