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Purpose:Tebentafusp is a first-in-class bispecific fusion protein designed to target gp100 (a melanoma-associated antigen) through a high affinity T-cell receptor (TCR) binding domain and an anti-CD3 T-cell engaging domain, which redirects T cells to kill gp100-expressing tumor cells. Here, we report a multicenter phase I/II trial of tebentafusp in metastatic melanoma (NCT01211262) focusing on the mechanism of action of tebentafusp.Patients and Methods:Eighty-four patients with advanced melanoma received tebentafusp. Treatment efficacy, treatment-related adverse events, and biomarker assessments were performed for blood-derived and tumor biopsy samples obtained at baseline and on-treatment.Results:Tebentafusp was generally well-tolerated and active in both patients with metastatic uveal melanoma and patients with metastatic cutaneous melanoma. A 1-year overall survival rate of 65% was achieved for both patient cohorts. On-treatment cytokine measurements were consistent with the induction of IFNγ pathway–related markers in the periphery and tumor. Notably, tebentafusp induced an increase in serum CXCL10 (a T-cell attractant) and a reduction in circulating CXCR3+ CD8+ T cells together with an increase in cytotoxic T cells in the tumor microenvironment. Furthermore, increased serum CXCL10 or the appearance of rash (likely due to cytotoxic T cells targeting gp100-expressing skin melanocytes) showed a positive association with patient survival.Conclusions:These data suggest that redirecting T cells using a gp100-targeting TCR/anti-CD3 bispecific fusion protein may provide benefit to patients with metastatic melanoma. Furthermore, the activity observed in these two molecularly disparate melanoma classes hints at the broad therapeutic potential of tebentafusp.

Purpose: Tebentafusp is a first-in-class bispecific fusion protein designed to target gp100 (a melanoma-associated antigen) through a high affinity T-cell receptor (TCR) binding domain and an anti-CD3 T-cell engaging domain, which redirects T cells to kill gp100-expressing tumor cells. Here, we report a multicenter phase I/II trial of tebentafusp in metastatic melanoma (NCT01211262) focusing on the mechanism of action of tebentafusp. Patients and Methods: Eighty-four patients with advanced melanoma received tebentafusp. Treatment efficacy, treatment-related adverse events, and biomarker assessments were performed for blood-derived and tumor biopsy samples obtained at baseline and on-treatment. Results: Tebentafusp was generally well-tolerated and active in both patients with metastatic uveal melanoma and patients with metastatic cutaneous melanoma. A 1-year overall survival rate of 65% was achieved for both patient cohorts. On-treatment cytokine measurements were consistent with the induction of IFNγ pathway–related markers in the periphery and tumor. Notably, tebentafusp induced an increase in serum CXCL10 (a T-cell attractant) and a reduction in circulating CXCR3+ CD8+ T cells together with an increase in cytotoxic T cells in the tumor microenvironment. Furthermore, increased serum CXCL10 or the appearance of rash (likely due to cytotoxic T cells targeting gp100-expressing skin melanocytes) showed a positive association with patient survival. Conclusions: These data suggest that redirecting T cells using a gp100-targeting TCR/anti-CD3 bispecific fusion protein may provide benefit to patients with metastatic melanoma. Furthermore, the activity observed in these two molecularly disparate melanoma classes hints at the broad therapeutic potential of tebentafusp.

Efruxifermin (EFX) is a novel fusion biotherapeutic consisting of two modified human fibroblast growth factor (FGF21) variants tethered via short linkers to a hIgG1 Fc domain. EFX is under clinical development by Akero Therapeutics, Inc. for treatment of non-alcoholic steatohepatitis (NASH). Fusion to the IgG Fc moiety extends the circulating half-life of EFX, enabling once-weekly subcutaneous administration. In addition, point mutations in the FGF21 moiety of EFX, relative to native human FGF21, protect against degradation of the C-terminus by the endogenous endopeptidase fibroblast activation protein (FAP), and increase affinity for the FGF21 co-receptor b-Klotho, which together enhance in vivo PD activity of EFX. Accordingly, to support clinical PK a sensitive and specific assay is needed for determination of biologically active (i. e., C-terminal intact) EFX serum concentrations that is minimally cross-reactive with endogenous FGF21 or potential inactive metabolites of EFX. Moreover, the assay needs to have reliability with serum samples from NASH patients, in whom hyperlipidemia and insulin resistance are prevalent. To achieve the analytical aims, we undertook development of a noncompetitive immunosorbent assay design, wherein a biotinylated version of a rat mAb directed against EFX's intact C-terminus serves as the capture reagent, while an affinity purified chicken anti-EFX pAb labeled with Sulfo-TAG functions as the detection reagent. This novel ECLIA design permits measurement of an electrochemiluminescence signal for maximizing assay sensitivity. In brief, serum samples are diluted 1: 10 in buffer and added to the MSD plate coated with rat mAb. After incubation and washing, bound EFX is detected using an affinity purified chicken anti-EFX PAB labeled with SULFO-TAG. After washing and addition of read buffer, the signal responses are measured in a SQ120 reader and concentration in test samples is estimated by dose-interpolation using a weighted 5-PL curve fit with an EFX standard curve ranging from 10–2,000 ng/mL. The new assay allows measurement of serum EFX at concentrations as low at 20 ng/mL. Upon pre-study validation, the assay has met analytical performance criteria outlined in BMV regulatory guidances. We conclude the optimized ECLIA will be useful for reliable assessment of EFX PK in patients with NASH. Presentation: No date and time listed

Initiation of antiretroviral therapy (ART) in early compared with chronic human immunodeficiency virus (HIV) infection is associated with a smaller HIV reservoir. This longitudinal analysis of 60 individuals who began ART during primary HIV infection (PHI) investigates which pre- and posttherapy factors best predict HIV DNA levels (a correlate of reservoir size) after treatment initiation during PHI. The best predictor of HIV DNA at 1 year was pre-ART HIV DNA, which was in turn significantly associated with CD8 memory T-cell differentiation (effector memory, naive, and T-bet−Eomes− subsets), CD8 T-cell activation (CD38 expression) and T-cell immunoglobulin and mucin-domain containing-3 (Tim-3) expression on memory T cells. No associations were found for any immunological variables after 1 year of ART. Levels of HIV DNA are determined around the time of ART initiation in individuals treated during PHI. CD8 T-cell activation and memory expansion are linked to HIV DNA levels, suggesting the importance of the initial host-viral interplay in eventual reservoir size., Human immunodeficiency virus (HIV) DNA persists within the viral reservoir despite therapy. A longitudinal analysis of 60 individuals treated during early HIV infection showed that HIV DNA levels are “set” early in infection and predicted by an activated immune response.

Background Human immunodeficiency virus (HIV)-infected individuals have a higher risk of developing active tuberculosis (TB) than HIV-uninfected individuals, but the mechanisms underpinning this are unclear. We hypothesized that depletion of specific components of Mycobacterium tuberculosis (Mtb)-specific CD4+ and CD8+ T-cell responses contributed to this increased risk. Methods Mtb-specific T-cell responses in 147 HIV-infected and 44 HIV-uninfected control subjects in a TB-endemic setting in Bloemfontein, South Africa, were evaluated. Using a whole-blood flow cytometry assay, we measured expression of interferon gamma, tumor necrosis factor alpha, interleukin 2, and interleukin 17 in CD4+ and CD8+ T cells in response to Mtb antigens (PPD, ESAT-6/CFP-10 [EC], and DosR regulon-encoded α-crystallin [Rv2031c]). Results Fewer HIV-infected individuals had detectable CD4+ and CD8+ T-cell responses to PPD and Rv2031c than HIV-uninfected subjects. Mtb-specific T cells showed distinct patterns of cytokine expression comprising both Th1 (CD4 and CD8) and Th17 (CD4) cytokines, the latter at highest frequency for Rv2031c. Th17 antigen-specific responses to all antigens tested were specifically impaired in HIV-infected individuals. Conclusions HIV-associated impairment of CD4+ and CD8+Mtb-specific T-cell responses is antigen specific, particularly impacting responses to PPD and Rv2031c. Preferential depletion of Th17 cytokine-expressing CD4+ T cells suggests this T-cell subset may be key to TB susceptibility in HIV-infected individuals.

Summary The success of immune system‐based cancer therapies depends on a broad immune response engaging a range of effector cells and mechanisms. Immune mobilizing monoclonal T cell receptors (TCRs) against cancer (ImmTAC™ molecules: fusion proteins consisting of a soluble, affinity enhanced TCR and an anti‐CD3 scFv antibody) were previously shown to redirect CD8+ and CD4+ T cells against tumours. Here we present evidence that IMCgp100 (ImmTAC recognizing a peptide derived from the melanoma‐specific protein, gp100, presented by HLA‐A*0201) efficiently redirects and activates effector and memory cells from both CD8+ and CD4+ repertoires. Using isolated subpopulations of T cells, we find that both terminally differentiated and effector memory CD8+ T cells redirected by IMCgp100 are potent killers of melanoma cells. Furthermore, CD4+ effector memory T cells elicit potent cytotoxic activity leading to melanoma cell killing upon redirection by IMCgp100. The majority of T cell subsets belonging to both the CD8+ and CD4+ repertoires secrete key pro‐inflammatory cytokines (tumour necrosis factor‐α, interferon‐γ, interleukin‐6) and chemokines (macrophage inflammatory protein‐1α‐β, interferon‐γ‐inducible protein‐10, monocyte chemoattractant protein‐1). At an individual cell level, IMCgp100‐redirected T cells display a polyfunctional phenotype, which is a hallmark of a potent anti‐cancer response. This study demonstrates that IMCgp100 induces broad immune responses that extend beyond the induction of CD8+ T cell‐mediated cytotoxicity. These findings are of particular importance because IMCgp100 is currently undergoing clinical trials as a single agent or in combination with check point inhibitors for patients with malignant melanoma.

Initiation of antiretroviral therapy (ART) in early compared with chronic HIV infection is associated with a smaller HIV reservoir. This longitudinal analysis of 63 individuals who commenced ART during primary HIV infection (PHI) investigates which pre-and post-therapy factors associate most closely with reservoir size (HIV DNA) following treatment initiation during PHI. The best predictor of reservoir size at one-year was pre-ART HIV DNA which was in turn significantly associated with CD8 memory differentiation (effector memory, naïve and T-betnegEomesnegsubsets), CD8 T cell activation (CD38 expression) and PD-1 and Tim-3 expression on memory CD4 T cells. No associations were found for any immunological variables following one-year of ART. HIV reservoir size is determined around the time of ART initiation in individuals treated during PHI. CD8 T cell activation and memory expansion are linked to HIV reservoir size, suggesting the importance of the initial host-viral interplay in eventual reservoir size.

INTRODUCTION: There are few data on the frequency of virological remission in African individuals after treatment with antiretroviral therapy (ART) in primary HIV infection (PHI). METHODS: We studied participants (n = 82) from South Africa and Uganda in Short Pulse Antiretroviral Treatment at HIV-1 Seroconversion, the first trial of treatment interruption in African individuals with PHI randomized to deferred ART or 48 weeks of immediate ART. All were female and infected with non-B HIV subtypes, mainly C. We measured HIV DNA in CD4+ T cells, CD4+ cell count, plasma viral load (pVL), cell-associated HIV RNA and T-cell activation and exhaustion. We explored associations with clinical progression and time to pVL rebound after treatment interruption (n = 22). Data were compared with non-African Short Pulse Antiretroviral Treatment at HIV-1 Seroconversion participants. RESULTS: Pretherapy pVL and integrated HIV DNA were lower in Africans compared with non-Africans (median 4.16 vs. 4.72 log10 copies/ml and 3.07 vs. 3.61 log10 copies/million CD4+ T cells, respectively; P

Disease-free infection in HIV-infected adults is associated with human leukocyte antigen-mediated suppression of viremia, whereas in the sooty mangabey and other healthy natural hosts of simian immunodeficiency virus (SIV), viral replication continues unabated. To better understand factors preventing HIV disease, we investigated pediatric infection, where AIDS typically develops more rapidly than in adults. Among 170 nonprogressing antiretroviral therapy-naïve children aged >5 years maintaining normal-for-age CD4 T cell counts, immune activation levels were low despite high viremia (median, 26,000 copies/ml). Potent, broadly neutralizing antibody responses in most of the subjects and strong virus-specific T cell activity were present but did not drive pediatric nonprogression. However, reduced CCR5 expression and low HIV infection in long-lived central memory CD4 T cells were observed in pediatric nonprogressors. These children therefore express two cardinal immunological features of nonpathogenic SIV infection in sooty mangabeys-low immune activation despite high viremia and low CCR5 expression on long-lived central memory CD4 T cells-suggesting closer similarities with nonpathogenetic mechanisms evolved over thousands of years in natural SIV hosts than those operating in HIV-infected adults.

abYsis is a web-based antibody research system that includes an integrated database of antibody sequence and structure data. The system can be interrogated in numerous ways-from simple text and sequence searches to sophisticated queries that apply 3D structural constraints. The publicly available version includes pre-analyzed sequence data from the European Molecular Biology Laboratory European Nucleotide Archive (EMBL-ENA) and Kabat as well as structure data from the Protein Data Bank. A researcher's own sequences can also be analyzed through the web interface. A defining characteristic of abYsis is that the sequences are automatically numbered with a series of popular schemes such as Kabat and Chothia and then annotated with key information such as complementarity-determining regions and potential post-translational modifications. A unique aspect of abYsis is a set of residue frequency tables for each position in an antibody, allowing "unusual residues" (those rarely seen at a particular position) to be highlighted and decisions to be made on which mutations may be acceptable. This is especially useful when comparing antibodies from different species. abYsis is useful for any researcher specializing in antibody engineering, especially those developing antibodies as drugs. abYsis is available at www.abysis.org.

Annually, there are over 2 million incidents of traumatic brain injury (TBI) and treatment options are non-existent. While many TBI studies have focused on the brain, peripheral contributions involving the digestive and immune systems are emerging as factors involved in the various symptomology associated with TBI. We hypothesized that TBI would alter hepatic function, including bile acid system machinery in the liver and brain. The results show activation of the hepatic acute phase response by 2 hours after TBI, hepatic inflammation by 6 hours after TBI and a decrease in hepatic transcription factors, Gli 1, Gli 2, Gli 3 at 2 and 24 hrs after TBI. Bile acid receptors and transporters were decreased as early as 2 hrs after TBI until at least 24 hrs after TBI. Quantification of bile acid transporter, ASBT-expressing neurons in the hypothalamus, revealed a significant decrease following TBI. These results are the first to show such changes following a TBI, and are compatible with previous studies of the bile acid system in stroke models. The data support the emerging idea of a systemic influence to neurological disorders and point to the need for future studies to better define specific mechanisms of action.

A Bacterial Artificial Chromosome (BAC) library was made from wild-caught Simulium squamosum , which is an important vector of human onchocerciasis. The library is composed of 12,288 BACs, with an average insert size of 128 kb, and is expected to contain ~ 1.54 GB of cloned DNA. Random BAC-end sequencing generated over 95 kb of DNA sequence data from which putative S. squamosum gene sequences and novel repetitive DNA families were identified, including DNA transposons, retrotransposons and simple sequence repeats (SSRs). The sequence survey also provided evidence of DNA of microbial origin, and dissection of sample blackflies indicated that some of those used to prepare the library were likely to be parasitized by the mermithid Isomermis lairdi . Hybridisations with a set of three independent blackfly single-copy genes and two Wolbachia genes suggest that the library provides around 13-fold coverage of the S. squamosum genome and about 12-fold coverage of its Wolbachia endosymbiont.

BACKGROUND: Chronic HCV infection is a leading cause of liver-related morbidity globally. The innate and adaptive immune responses are thought to be important in determining viral outcomes. Polymorphisms associated with the IFNL3 (IL28B) gene are strongly associated with spontaneous clearance and treatment outcomes. OBJECTIVE: This study investigates the importance of HLA genes in the context of genetic variation associated with the innate immune genes IFNL3 and KIR2DS3. DESIGN: We assess the collective influence of HLA and innate immune genes on viral outcomes in an Irish cohort of women (n=319) who had been infected from a single source as well as a more heterogeneous cohort (Swiss Cohort, n=461). In the Irish cohort, a number of HLA alleles are associated with different outcomes, and the impact of IFNL3-linked polymorphisms is profound. RESULTS: Logistic regression was performed on data from the Irish cohort, and indicates that the HLA-A*03 (OR 0.36 (0.15 to 0.89), p=0.027) -B*27 (OR 0.12 (0.03 to 0.45), p=

The incidence and severity of infections in childhood is typically greater in males. The basis for these observed sex differences is not well understood, and potentially may facilitate novel approaches to reducing disease from a range of conditions. We here investigated sex differences in HIV-infected children in relation to antiretroviral therapy (ART) initiation and post-treatment outcome. In a South African cohort of 2,101 HIV-infected children, we observed that absolute CD4+ count and CD4% were significantly higher in ART-naïve female, compared to age-matched male, HIV-infected children. Absolute CD4 count and CD4% were also significantly higher in HIV-uninfected female versus male neonates. We next showed that significantly more male than female children were initiated on ART (47% female); and children not meeting criteria to start ART by >5 yrs were more frequently female (59%; p

HLA class I polymorphism has a major influence on adult HIV disease progression. An important mechanism mediating this effect is the impact on viral replicative capacity (VRC) of the escape mutations selected in response to HLA-restricted CD8+ T-cell responses. Factors that contribute to slow progression in pediatric HIV infection are less well understood. We here investigate the relationship between VRC and disease progression in pediatric infection, and the effect of HLA on VRC and on disease outcome in adult and pediatric infection. Studying a South African cohort of >350 ART-naïve, HIV-infected children and their mothers, we first observed that pediatric disease progression is significantly correlated with VRC. As expected, VRCs in mother-child pairs were strongly correlated (p = 0.004). The impact of the protective HLA alleles, HLA-B*57, HLA-B*58:01 and HLA-B*81:01, resulted in significantly lower VRCs in adults (p, Author Summary HLA plays a central role in determining disease outcome in adult HIV infection. A principal mechanism by which this HLA effect is mediated is via viral replicative capacity (VRC), protective HLA alleles such as HLA-B*57 driving the selection of viral escape mutants that reduce VRC. The factors contributing to the diverse disease progression rates observed in infected children, however, remain uncertain. We here address the role of HLA and VRC in pediatric disease progression in a large cohort in Kimberley, South Africa. The findings highlight the consistent and important role of VRC in both adult and pediatric progression. However, the impact of key HLA molecules in shaping disease outcome in adult infection is notably absent in pediatric infection. Further studies of pediatric infection therefore provide the potential to gain critical new insights into HLA-independent mechanisms of HIV disease non-progression that predominate in HIV-infected but healthy, ART-naive children. Understanding these mechanisms remains of direct relevance to the development of future interventions to minimize HIV disease.

IMCgp100, a first in class immunotherapy, is a T cell redirecting bispecific biologic comprising an affinity enhanced T-cell receptor specific for gp100 and an anti-CD3 scFV. Phase I/IIa data has provided evidence of a favourable safety profile, and durable responses in both cutaneous and uveal melanoma have been observed. To complement the clinical studies we have developed a comprehensive biomarker strategy to aid our understanding of pharmacodynamics, patient response and potential mechanisms of resistance which includes analysis of markers in both the tumour and periphery. The data obtained to date provide evidence of the pharmacodynamic effects of the molecule including chemokine/cytokine release, in both the tumor and periphery, and lymphocyte infiltration into tumors. In addition, changes in the levels of certain chemokines following the first dose of IMCgp100 were associated with tumor shrinkage. The biomarker strategy we have developed forms the basis for the support of the on-going Ph II development of IMCgp100 in both cutaneous and uveal melanoma and for other ImmTAC molecules, as single agents and in combination, for the treatment of solid tumours. Citation Format: Cheryl McAlpine, David Krige, Sandra Herrero-Gonzalez, Sarah Franklin, Jacob Hurst, William Shingler, Sanjay Patel, Andy Johnson, Debbie Parker, Christina M. Coughlin, Namir J. Hassan, Bent K. Jakobsen. Biomarker strategy to guide the clinical development of ImmTACTM molecules, a novel class of bispecific T cell engaging biologic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3655. doi:10.1158/1538-7445.AM2017-3655

In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials.Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20

The use and benefits of self-adaptive mutation operators are well known within evolutionary computing. In this paper, we begin by examining the use of self-adaptive mutation in learning classifier systems with the aim of improving their performance as controllers for autonomous mobile robots. We implement the operator in a zeroth level classifier system, and examine its performance in two animat environments. It is shown that although no significant increase in performance is seen over results presented in the literature using a fixed rate of mutation, the operator adapts to an appropriate rateregadless of the initial range. The same concept is then applied to the learning rate parameter, but results show that a modification must be made to produce stable/effective controllers. Finally, results from a fully self-adaptive system are presented, with marked benefits being found in a nonstationary environment.

Highlights • The requirement for protocol adaptation from qPCR to ddPCR is characterised. • A potential loss of sensitivity for ddPCR at low target numbers is reported. • A new bioinformatic tool ‘definetherain’ to improve droplet calling at low input target numbers is devised and applied. • ‘Definetherain’ is made available free of charge and open access at www.definetherain.org.uk., Droplet Digital PCR (ddPCR) represents a new and alternative platform to conventional quantitative-PCR (qPCR) for the quantitation of DNA templates. However, the proposed improvement in sensitivity and reproducibility offered by ddPCR is not yet fully proven, partly because the delineation between positive and negative responses is not always clear. Data are presented demonstrating the sensitivity of the ddPCR system to both reagent concentrations and choice of cut-off for defining positive and negative results. By implementing k-nearest clustering, cut-offs are produced that improve the accuracy of ddPCR where target DNA is present at low copy numbers, a key application of ddPCR. This approach is applied to human albumin and HIV-1 proviral DNA ddPCR quantitative protocols. This tool is coded in JavaScript and has been made available for free in a web browser at http://www.definetherain.org.uk. Optimisation of the analyses of raw ddPCR data using ‘definetherain’ indicates that low target number detection can be improved by its implementation. Further application to patient samples will help define the clinical utility of this approach.

Cellular immune control of HIV is mediated, in part, by induction of single amino acid mutations that reduce viral fitness, but compensatory mutations limit this effect. Here, we sought to determine if higher order constraints on viral evolution exist, because some coordinately linked combinations of mutations may hurt viability. Immune targeting of multiple sites in such a multidimensionally conserved region might render the virus particularly vulnerable, because viable escape pathways would be greatly restricted. We analyzed available HIV sequences using a method from physics to reveal distinct groups of amino acids whose mutations are collectively coordinated (“HIV sectors”). From the standpoint of mutations at individual sites, one such group in Gag is as conserved as other collectively coevolving groups of sites in Gag. However, it exhibits higher order conservation indicating constraints on the viability of viral strains with multiple mutations. Mapping amino acids from this group onto protein structures shows that combined mutations likely destabilize multiprotein structural interactions critical for viral function. Persons who durably control HIV without medications preferentially target the sector in Gag predicted to be most vulnerable. By sequencing circulating viruses from these individuals, we find that individual mutations occur with similar frequency in this sector as in other targeted Gag sectors. However, multiple mutations within this sector are very rare, indicating previously unrecognized multidimensional constraints on HIV evolution. Targeting such regions with higher order evolutionary constraints provides a novel approach to immunogen design for a vaccine against HIV and other rapidly mutating viruses.

Pathogenic deviations (PDs) in humans are disease- causing missense mutations. However, in some cases, these disease-associated residues occur as the wild- type residues in functionally equivalent proteins in other species and these cases are termed ‘compensated pathogenic deviations’ (CPDs). The lack of pathogenicity in a non-human protein is presumed to be explained in most cases by the presence of compensatory mutations, most commonly within the same protein. Identification of structural features of CPDs and detection of specific compensatory events will help us to understand traversal along fitness landscape valleys in protein evolution. We divided mutations listed in the OMIM (Online Mendelian Inheritance in Man) database into PD and CPD data sets and performed two independent analyses: (i) We searched for potential compensatory mutations spatially close to the CPDs and, (ii) using our SAAPdb database, we examined likely structural effects to try to explain why mutations are pathogenic, comparing PDs and CPDs. Our data sets were obtained from a set of 245 human proteins of known structure and contained a total of 2328 mutations of which 453 (from 85 structures) were seen to be compensated in at least one functionally equivalent protein in another (non-human) species. Structural analysis results confirm previous findings that CPDs are, on average, ‘milder’ in their likely structural effects than uncompensated PDs and tend to be on the protein surface. We also showed that the residues surrounding the CPD residue in the folded protein are more often mutated than the residues surrounding an uncompensated mutation, supporting the hypothesis that compensation is largely a result of structurally local mutations.

Background Pseudomonas aeruginosa is a genetically complex bacterium which can adopt and switch between a free-living or biofilm lifestyle, a versatility that enables it to thrive in many different environments and contributes to its success as a human pathogen. Results Transcriptomes derived from growth states relevant to the lifestyle of P. aeruginosa were clustered using three different methods (K-means, K-means spectral and hierarchical clustering). The culture conditions used for this study were; biofilms incubated for 8, 14, 24 and 48 hrs, and planktonic culture (logarithmic and stationary phase). This cluster analysis revealed the existence and provided a clear illustration of distinct expression profiles present in the dataset. Moreover, it gave an insight into which genes are up-regulated in planktonic, developing biofilm and confluent biofilm states. In addition, this analysis confirmed the contribution of quorum sensing (QS) and RpoS regulated genes to the biofilm mode of growth, and enabled the identification of a 60.69 Kbp region of the genome associated with stationary phase growth (stationary phase planktonic culture and confluent biofilms). Conclusion This is the first study to use clustering to separate a large P. aeruginosa microarray dataset consisting of transcriptomes obtained from diverse conditions relevant to its growth, into different expression profiles. These distinct expression profiles not only reveal novel aspects of P. aeruginosa gene expression but also provide a growth specific transcriptomic reference dataset for the research community.