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The efficacy of PD-1 monoclonals such as pembrolizumab can be modulated by the signals delivered via their Fc region. Tumour/inflammation associated proteases can generate F(ab')2 fragments of therapeutic monoclonals, and subsequent recognition of F(ab')2 epitopes by circulating anti-hinge antibodies (AHA) can then, potentially, link F(ab')2 binding to the target antigen with novel Fc signalling. Although elevated in inflammatory diseases, AHA levels in cancer patients have not been investigated and functional studies utilising the full repertoire of AHA present in sera have been limited. AHA levels in pembrolizumab treated melanoma patients (n = 23) were therefore compared to those of normal donors and adalimumab treated patients. A subset of melanoma patients and the majority of adalimumab patients had elevated levels of AHA reactive with F(ab')2 fragments of IgG4 anti-PD-1 monoclonals (nivolumab, pembrolizumab) and IgG1 therapeutic monoclonals (rituximab, adalimumab). Survival analysis was restricted by the small patient numbers but those melanoma patients with the highest levels (>75% percentile, n = 5) of pembrolizumab-F(ab')2 reactive AHA had significantly better overall survival post pembrolizumab treatment (p = 0.039). In vitro functional studies demonstrated that the presence of AHA+ sera restored the neutrophil activating capacity of pembrolizumab to its F(ab')2 fragment. Neither pembrolizumab nor its F(ab')2 fragments can induce NK cell or complement dependent cytotoxicity (CDC). However, AHA+ sera in combination with pembrolizumab-F(ab')2 provided Fc regions that could activate NK cells. The ability of AHA+ sera to restore CDC activity was more restricted and observed using only one pembrolizumab and one adalimumab patient serum in combination with rituximab- F(ab')2. This study reports the presence of elevated AHA levels in pembrolizumab treated melanoma patients and highlight the potential for AHA to provide additional Fc signaling. The issue of whether tumour associated proteolysis of PD-1 mAbs and subsequent AHA recognition impacts on treatment efficacy requires further study., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2023 Hock et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Between 2013 and 2019, 63 presumed Chinook salmon Oncorhynchus tshawytscha sampled primarily in the Strait of Georgia (0.63% of total sample) were identified as potential Chinook-Coho ( Oncorhynchus kisutch ) hybrids by the presence of anomalous microsatellite genotypes. Their hybrid origin was confirmed by single nucleotide polymorphism amplification of two species-specific amplicons. Mitochondrial DNA indicated that most of these fish resulted from the hybridization of Coho salmon females and Chinook salmon males. Although no diagnostic external features were identified, several individuals displayed an abnormal scale arrangement on the caudal peduncle. One hybrid juvenile examined for meristics exhibited a pyloric caeca count intermediate between published values for Chinook and Coho salmon. Most hybrids originated in the Cowichan River during the 2014 brood year. Their prevalence in the watershed is a naturally occurring event, likely exacerbated by prolonged low water levels which limit habitat and delay Chinook salmon spawning, in addition to the differential abundance of the parental species. This research is the first to document ongoing natural hybridization (Chinook-Coho salmon crosses) and link it to habitat and climatic changes, and includes the identification of eight F1 adults and two juvenile backcross or F2 hybrids. The potential negative impacts of hybridization, particularly in Coho salmon through potential introgression, warrant hybrid identification as an ecosystem monitoring tool within a survey program., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Objectives: We set out to create Consensus Guidelines, based on current evidence and relative risks of adverse effects and the costs of different treatments, which reflect the views of the British Rhinological Society (BRS) Council on where the use of biologics should be positioned within treatment pathways for CRSwNP, specifically in the setting of the National Health Service (NHS)., Design: An expert panel of 16 members was assembled. A review of the literature and evidence synthesis was undertaken and circulated to the panel. We used the RAND/UCLA methodology with a multi-step process to make recommendations on the use of biologics., Setting: N/A., Participants: N/A., Results: Recommendations were made, based on underlying disease severity, prior treatments and co-morbidities. A group of patients for whom biologics were considered an appropriate treatment option for CRSwNP was defined., Conclusions: Although biologics are not currently available for the treatment of CRSwNP, the BRS Council have defined a group of patients who have higher rates of "failure" with current treatment pathways, higher resource use and are more likely to suffer with uncontrolled symptoms. We would urge NICE to consider approval of biologics for such indications without applying further restrictions on use., (© 2021 John Wiley & Sons Ltd.)

Loss-of-function mutations in the DNA demethylase TET2 are associated with the dysregulation of hematopoietic stem cell differentiation and arise in approximately 10% of de novo acute myeloid leukemia (AML). TET2 mutations coexist with other mutations in AML, including TP53 mutations, which can indicate a particularly poor prognosis. Ascorbate can function as an epigenetic therapeutic in pathological contexts involving heterozygous TET2 mutations by restoring TET2 activity. How this response is affected when myeloid leukemia cells harbor mutations in both TET2 and TP53 is unknown. Therefore, we examined the effects of ascorbate on the SKM-1 AML cell line that has mutated TET2 and TP53 . Sustained treatment with ascorbate inhibited proliferation and promoted the differentiation of these cells. Furthermore, ascorbate treatment significantly increased 5-hydroxymethylcytosine, suggesting increased TET activity as the likely mechanism. We also investigated whether ascorbate affected the cytotoxicity of Prima-1 Met , a drug that reactivates some p53 mutants and is currently in clinical trials for AML. We found that the addition of ascorbate had a minimal effect on Prima-1 Met -induced cytotoxicity, with small increases or decreases in cytotoxicity being observed depending on the timing of treatment. Collectively, these data suggest that ascorbate could exert a beneficial anti-proliferative effect on AML cells harboring both TET2 and TP53 mutations whilst not interfering with targeted cytotoxic therapies such as Prima-1 Met ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Smith-Díaz, Magon, McKenzie, Hampton, Vissers and Das.)

Global change is altering ecosystems at an unprecedented rate. The resulting shifts in species ranges and reproductive timing are opening the potential for hybridization between closely related species which could dramatically alter the genetic diversity, adaptive capacity and evolutionary trajectory of interbreeding taxa. Here, we used behavioural breeding experiments, in vitro fertilization experiments, and whole-transcriptome gene expression data to assess the potential for and consequences of hybridization between Chinook and Coho salmon. We show that behavioural and gametic prezygotic barriers between socio-economically valuable Chinook and Coho salmon are incomplete. Postzygotically, we demonstrate a clear transcriptomic response to hybridization among F 1 Chinook-Coho offspring. Genes transgressively expressed within hybrids were significantly enriched with genes encoded in the nucleus but localized to the mitochondrion, suggesting a potential role for mito-nuclear incompatibilities as a postzygotic mechanism of hybrid breakdown. Chinook and Coho salmon are expected to continue to respond to climate change with shifts in migration timing and habitat use, potentiating hybridization between these species. The downstream consequences of hybridization on the future of these threatened salmon, and the ecosystems they inhabit, is unknown.

The PD-1-targeting IgG 4 antibody pembrolizumab has significant anti-tumor activity in a proportion of stage IV melanoma patients. A subset of patients develop anti-drug antibodies (ADA) which can form immune complexes (IC) with pembrolizumab. Although IC can induce powerful, Fc-mediated, immune-regulatory effects, their functional impact during pembrolizumab treatment is unclear. The functional effects of IC generated in vitro using pembrolizumab and patient-derived ADA was, therefore, investigated. Screening identified a patient whose trough serum samples from three treatment cycles contained both ADA with neutralizing activity and low levels of pembrolizumab. This patient responded well to therapy over 2 years and had ongoing, infusion-related, hypersensitivity reactions despite the later absence of detectable ADA. The components of IC were mimicked by forming a complex of pembrolizumab by absorption onto a solid phase with or without subsequent exposure to the ADA + patient sera. Complexes comprised of pembrolizumab alone significantly inhibited TLR4 (LPS)-driven IL-10 production by PBMC and stimulated the generation of reactive oxygen species by granulocytes. In contrast, soluble and solid-phase F(ab´)2 fragments of pembrolizumab had no effect demonstrating the requirement for cross-linked Fc regions. IC containing pembrolizumab and ADA could additionally induce complement and NK activation. The results of this study demonstrate that, when oligomerized, the Fc region of pembrolizumab alone can provide immuno-regulatory signals. Furthermore, IC containing both pembrolizumab and patient-generated ADA can induce additional signals. These Fc-mediated signals may modulate both hypersensitivity reactions and anti-tumor responses associated with pembrolizumab therapy.

Adalimumab is a TNF specific monoclonal widely used therapeutically. Monitoring adalimumab levels is important for guiding treatment strategies and is predominantly performed using an ELISA. The homogeneous mobility shift assay (HMSA) has many advantages over an ELISA for adalimumab monitoring but current HMSA methodologies do not discriminate between adalimumab and other TNF specific monoclonals such as infliximab. The development and validation of a competitive binding HMSA (cHMSA) specific for adalimumab is reported here. The cHMSA had a lower limit of quantitation of 1.25 μg/ml and the intra-assay and inter-assay coefficents of variation (CV) were <20%. No signal was detected in adalimumab naïve control serum including those containing rheumatoid factor or infliximab. The majority (14/20) of adalimumab patient samples containing anti-adalimumab antibodies gave a cHMSA signal >3 standard deviations lower than the controls. The performance of the cHMSA and an ELISA was compared using adalimumab patient samples (n = 82). There was a strong correlation between the assays (r = 0.91) and the intra-class correlation coefficient (0.88) was indicative of good-excellent inter-assay reliability. Bland-Altman plots showed little overall bias and comparison of the sub-groups defined using cut-points (1.25 or 7.3 μg/ml) gave percent agreement (>90%) and Cohens kappa (95% CI: 0.61-0.93) values indicative of substantial-almost perfect agreement. These results demonstrate that cHMSA provides an accurate and specific method for monitoring adalimumab levels and can additionally provide an initial screen for the presence of anti-adalimumab antibodies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The mitonuclear species concept hypothesizes that incompatibilities between interacting gene products of the nuclear and mitochondrial genomes are a major factor establishing and maintaining species boundaries. However, most of the data available to test this concept come from studies of genetic variation in mitochondrial DNA, and clines in the mitochondrial genome across contact zones can be produced by a variety of forces. Here, we show that using a combination of population genomic analyses of the nuclear and mitochondrial genomes and studies of mitochondrial function can provide insight into the relative roles of neutral processes, adaptive evolution, and mitonuclear incompatibility in establishing and maintaining mitochondrial clines, using Atlantic killifish (Fundulus heteroclitus) as a case study. There is strong evidence for a role of secondary contact following the last glaciation in shaping a steep mitochondrial cline across a contact zone between northern and southern subspecies of killifish, but there is also evidence for a role of adaptive evolution in driving differentiation between the subspecies in a variety of traits from the level of the whole organism to the level of mitochondrial function. In addition, studies are beginning to address the potential for mitonuclear incompatibilities in admixed populations. However, population genomic studies have failed to detect evidence for a strong and pervasive influence of mitonuclear incompatibilities, and we suggest that polygenic selection may be responsible for the complex patterns observed. This case study demonstrates that multiple forces can act together in shaping mitochondrial clines, and illustrates the challenge of disentangling their relative roles., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Scope: Blueberry polyphenols are thought to confer cardiovascular health benefits, but have limited bioavailability. They undergo extensive metabolism and their phenolic acid metabolites are likely to be the mediators of bioactivity. The effect of blueberry-derived phenolic acids on one aspect of inflammation, monocyte adhesion to vascular endothelial cells, is investigated., Methods and Results: The major blueberry-derived phenolic acids in human plasma are identified and quantified. Three test mixtures representing compounds present at 0-4 h (Early), 4-24 h (Late), or 0-24 h (Whole) are used to investigate the effect on adhesion of monocytes to tumor necrosis factor alpha (TNFα)-activated endothelial cells. The Late mixture reduces monocyte adhesion, but there is no effect of the Early or Whole mixtures. Exclusion of syringic acid from each mixture results in inhibition of monocyte adhesion. Exposure to the phenolic acid mixtures has no effect on the endothelial surface expression of adhesion molecules intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or E-selectin, suggesting that other molecular mechanisms are responsible for the observed effect., Conclusion: This study shows that physiological concentrations of blueberry polyphenol metabolites can help maintain cardiovascular health by regulating monocyte adhesion to the vascular endothelium., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The impact of changes in novel agent (NA) usage on the survival of multiple myeloma (MM) patients in real-world hospital settings is unclear. In New Zealand (NZ) in 2011, frontline bortezomib became available and thalidomide availability was expanded., Aim: This retrospective study analyses the impact these change had on the survival of MM patients treated at a NZ hospital., Methods: Clinical and overall survival (OS) data were collected on MM patients who were treated at Christchurch Hospital during 2000-2009 (pre-cohort, n = 337) and 2011-2017 (post-cohort, n = 343). Outcomes were compared using pre-cohort data truncated at 2011., Results: Patients in the post-cohort had significant increases (P < 0.001) in not only NA usage (85 vs 55%) and OS (median = 56 vs 44 months) but also the proportion (74 vs 49%) of young patients (age < 70) who received an autologous stem cell transplant (ASCT). Separate analysis of older patients demonstrated that those in the post-cohort had significantly longer OS (median OS 28 vs 17, P < 0.001) although 5-year relative survival remained less than 50%. Separate analysis of young patients demonstrated that those in the post-cohort had significantly increased initial OS with the survival curves converging at 5 years. Although ASCT-treated patients had similar OS in each cohort, their progression-free survival (PFS) was significantly increased in the post-cohort (median 40 vs 20 months, P < 0.0001)., Conclusion: In the setting of a NZ hospital the increased availability of NA was associated with a significant improvement in both the OS of older patients and the PFS of ASCT patients., (© 2018 Royal Australasian College of Physicians.)

Diabetic bladder dysfunction (DBD) is a well-recognized and common symptom affecting up to 50% of all diabetic patients. DBD has a broad range of clinical presentations ranging from overactive to underactive bladder symptoms that develops in middle-aged to elderly patients with long standing and poorly controlled diabetes. Low efficacy of current therapeutics and lifestyle interventions combined with high national healthcare costs highlight the need for more research into bladder dysfunction pathophysiology and novel treatment options. Cellular senescence is an age-related physiologic process in which cells undergo irreversible growth arrest induced by replicative exhaustion and damaging insults. While controlled senescence negatively regulates cell proliferation and promotes tissue regeneration, uncontrolled senescence is known to result in tissue dysfunction through enhanced secretion of inflammatory factors. This review presents previous scientific findings and current hypotheses that characterize diabetic bladder dysfunction. Further, we propose the novel hypothesis that cellular senescence within the urothelial layer of the bladder contributes to the pro-inflammatory/pro-oxidant environment and symptoms of diabetic bladder dysfunction. Our results show increased cellular senescence in the urothelial layer of the bladder; however, whether this phenomenon is the cause or effect of DBD is unknown. The urothelial layer of the bladder is made up of transitional epithelia specialized to contract and expand with demand and plays an active role in transmission by modulating afferent activity. Transition from normal functioning urothelial cells to secretory senescence cells would not only disrupt the barrier function of this layer but may result in altered signaling and sensation of bladder fullness; dysfunction of this layer is known to result in symptoms of frequency and urgency. Future DBD therapeutics may benefit from targeting and preventing early transition of urothelial cells to senescent cells.

Background: The measurement of anti-drug antibody (ADA) levels in adalimumab (ADAL)-treated and infliximab (IFX)-treated patients is critical for guiding therapeutic strategies. The homogeneous mobility shift assay (HMSA) and affinity capture elution (ACE) assay provide effective, drug-tolerant formats for measuring total ADA levels. However, their ability to discriminate between ADA from samples with or without neutralizing capacity is unclear and therefore was analyzed in this study., Methods: Sera from ADAL and IFX patients with low drug levels (<1 mcg/mL) were analyzed by ACE, HMSA, and bridging assay. Neutralizing capacity was determined by competitive ligand-binding assay., Results: HMSA and ACE detected high ADA levels in all ADAL (19/42) and IFX (27/64) samples with neutralizing capacity. ADA was also detected in most of the samples without neutralizing capacity, but levels were significantly lower (P < 0.0001). Receiver operator characteristic curve analysis demonstrated that for both assays, ADA levels were a strong discriminatory marker of neutralizing ADA (area under the curve > 0.9, P < 0.0001). Using a signal >8× background as a cut-point, neutralizing ADA could be identified with high specificity (HMSA > 95%, ACE > 85%) and sensitivity (HMSA > 70%, ACE > 80%). The detection of multimeric drug-ADA complexes after HMSA was also a highly specific marker (specificity > 95%) of neutralizing ADA in both ADAL and IFX patients. Results using ACE and HMSA were highly correlated., Conclusions: Results obtained after HMSA and ACE analysis are strongly correlated, and in both assays, high ADA levels are a specific marker of neutralizing capacity. The detection of multimeric complexes by HMSA also selectively identifies sera with neutralizing capacity. These data support the use of these assays as quantitative rather than simple qualitative measures of ADA.

The fossa navicularis is an anatomical variant of the skull base thought to be a rare finding. It represents a bony depression in the skull base. The authors here report the case of a fossa navicularis magna in a 9-year-old female who had been treated for recurrent episodes of meningitis.A literature review was also done to highlight the unique features and clinical importance of this distinctive radiological skull base finding. The literature search covered papers from the 19th century up to 2018. Earlier authors described "fossa navicularis" as a very rare skull base finding. So far, only three cases of fossa navicularis with associated clival or intracranial infection have been reported in the literature. This is the fourth reported case, and the defect was closed endoscopically via a transnasal route. This morphological skull base anomaly should be considered in the differential diagnoses for an unexplained skull base infective pathology.Skull base surgeons should be aware of the existence of the fossa navicularis because of its clinical importance in rendering a prompt diagnosis and appropriate treatment.

Life history strategies, physiological traits, and behavior are thought to covary along a "pace of life" axis, with organisms at the fast end of this continuum having higher fecundity, shorter lifespan, and more rapid development, growth, and metabolic rates. Countergradient variation represents a special case of pace of life variation, in which high-latitude organisms occupy the fast end of the continuum relative to low-latitude conspecifics when compared at a common temperature. Here, we use Atlantic killifish (Fundulus heteroclitus) to explore the role of mitochondrial properties as a mechanism underlying countergradient variation, and thus variation in the pace of life. This species is found along the Atlantic coast of North America, through a steep latitudinal thermal gradient. The northern subspecies has faster development, more rapid growth, higher routine metabolic rate, and higher activity than the southern subspecies when compared at a common temperature. The northern subspecies also has greater mitochondrial respiratory capacity in the liver, although these differences are not evident in other tissues. The increased respiratory capacity of liver mitochondria in northern fish is associated with increases in the activity of multiple electron transport complexes, which largely reflects an increase in the amount of inner mitochondrial membrane per mitochondrion in the northern fish. There are also differences in the lipid composition of liver mitochondrial membranes, including differences in cardiolipin species, which could also influence respiratory capacity. These data suggest that variation in mitochondrial properties could, at least in part, underlie variation in the pace of life in Atlantic killifish.

Immunological and performance characteristics were explored in Romney sheep from lines selected for either resistance or resilience to parasite infection. At a mean 78 days-of-age, twin lambs from a line selected for resistance (RT) and lambs from a line selected for resilience (RL) were infected with the intestinal nematode Trichostrongylus colubriformis for 100 days (I) while their twin remained as an uninfected control (C). Compared with RL, RT animals had lower levels of circulating CD4 + T-cells (P = 0.003) but a greater proportion of these were activated (CD4 + CD25 + ) in response to infection (P = 0.007). Differences between the lines in humoral immune responses to nematode infection varied with higher levels of T. colubriformis specific immunoglobulin (Ig) E in RT-I than RL-I (P = 0.002) but similar levels of both IgG (P = 0.926) and IgA (P = 0.321) responses. Temporal differences in the immune response also existed between the lines with RT-I animals displaying an earlier peak and more rapid reduction in FEC and an earlier peak in T. colubriformis specific IgA. In addition, compared with their RT-C and RL-C counterparts, infection caused a 22% reduction in feed intake from day 56 (P = 0.001) with total feed intake reduced by 15% and 9% for RT-I and RL-I, respectively. Cumulative liveweight gain was greatest for RL animals (P = 0.026) and relative to RT-C and RL-C was reduced by 5.8 kg and 4.9 kg for RT-I and RL-I, respectively. Overall, the selection lines appear to have differences in immunological characteristics that are both dependent on, and independent of parasite infection. Further, the difference in growth in the uninfected animals coupled with the similar cost of infection suggests the lower liveweight gain of RT-I compared with RL-I may be due to inherent differences between the lines in their growth potential, rather than a greater cost of infection in animals selected for resistance., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

The modulation of T cell responses by Helicobacter pylori is thought to potentiate both H. pylori persistence and development of gastric pathologies including cancer. Release of outer membrane vesicles (OMV) by H. pylori provides a potential vehicle for modulation of the immune system. Although OMV are thought to have T cell suppressive activity, this has not yet been demonstrated. Their suppressive activity was investigated in this study using the responses of peripheral blood mononuclear cells (PBMC) to T cell stimuli as a readout. We demonstrate that addition of OMV to PBMC significantly inhibits subsequent T cell proliferation in a cyclo-oxygenase-2 (COX-2)-dependent manner. Addition of OMV did not significantly modulate PBMC apoptosis, but induced strong expression of COX-2 by the monocytes present and significantly increased levels of PGE2 and IL-10. These effects were independent of vacuolating cytotoxin expression. Together, these findings demonstrate that OMV can suppress human T cell responses and that the predominant mechanism is not through a direct effect on the T cells but results from the induction of COX-2 expression in monocytes. This increased COX-2 activity may modulate not only H. pylori-directed immune responses but also wider immune responses., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Understanding factors involved in maintaining stable hybrid zones is important for predicting the ultimate fate of the interacting taxa, but the relative importance of mechanisms such as ecological selection and intrinsic reproductive isolation remains unclear. Most studies of reproductive isolation in hybrid zones have focused either on zones with strongly bimodal patterns in genotype or phenotype frequencies, with relatively strong isolation, or unimodal zones with relatively weak isolation, whereas less is known about more intermediate classes of hybrid zone. Here, we utilize a hybrid zone of this intermediate type occurring between northern and southern subspecies of Atlantic killifish, Fundulus heteroclitus, to identify isolating mechanisms playing a role in maintaining this type of zone. The two subspecies differ in environmental tolerance, and we found some evidence of microhabitat preference between subspecies within a small tidal creek at the centre of the hybrid zone. There was also an association between sex, mitochondrial genotype and habitat within this creek. Fertilization success did not differ between consubspecific and heterosubspecific crosses, but hatching success was significantly lower for crosses involving southern males and northern females, and crosses between southern females and northern males had altered developmental rates. Southern females and northern males showed patterns consistent with positive assortative mating. Together, these results indicate a role for a combination of factors including assortative mating and/or early hybrid inviability in the maintenance of this hybrid zone and suggest that hybrid zones with intermediate levels of reproductive isolation are likely to be maintained by multiple interacting isolating mechanisms., (© 2017 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2017 European Society For Evolutionary Biology.)

Chronic lymphocytic leukemia (CLL) is associated with T cell dysfunction. Activated CLL cells are found within the lymphoid tumor micro-environment and overcoming immuno-suppression induced by these cells may improve anti-CLL immune responses. However, the mechanisms by which activated CLL cells inhibit T cell responses, and reagents targeting such mechanisms have not been identified. Here we demonstrate that the ability of in vitro activated CLL cells to suppress T cell proliferation is not reversed by the presence of ecto-nuclease inhibitors or blockade of IL-10, PD-1 and CTLA-4 pathways. Caffeine is both an adenosine receptor antagonist and a phosphatidylinositol-3-kinase, p110δ (PI3Kδ) inhibitor and, at physiologically relevant levels, significantly reversed suppression. Significant reversal of suppression was also observed with the PI3Kδ specific inhibitor Idelalisib but not with adenosine receptor specific antagonists. Furthermore, addition of caffeine or Idelalisib to activated CLL cells significantly inhibited phosphorylation of AKT, a downstream kinase of PI3K, but did not affect CLL viability. These results suggest that caffeine, in common with Idelalisib, reduces the immuno-suppressive activity of activated CLL cells by inhibiting PI3Kδ. These findings raise the possibility that these compounds may provide a useful therapeutic adjunct by reducing immuno-suppression within the tumor micro-environment of CLL.

Assigning relative importance of spawning and nursery habitats for threatened and endangered teleosts, such as those seen in the Gulf of Mexico (GoM), relies on the proper identification of the early life-history stages of the species of concern. Here, sequencing a portion of the mitochondrial DNA (mtDNA) control region (CR) I as barcodes is recommended to identify istiophorid (billfish) larvae in the Atlantic Ocean because of its high resolution and the intrinsic value of the levels of genetic variation that can be extracted from these data. The universality of the primers employed here demonstrates their utility for not only the positive identification of istiophorids in the GoM, but for any larval teleost occurring in areas recognized as larval hotspots worldwide., (© 2016 The Fisheries Society of the British Isles.)

Background: Chronic lymphocytic leukaemia (CLL) is associated with an increased incidence and aggressiveness of skin cancers, particularly cutaneous squamous cell carcinoma (cSCC), but little is known about cSCC incidence in Australasian CLL patients., Aim: In this retrospective study, we analysed the incidence of cSCC in patients seen at a tertiary hospital in New Zealand (NZ)., Methods: We retrospectively assessed the clinical history and histology data of CLL patients (n = 371) who presented to the Haematology Department, Christchurch Hospital, NZ during the period 1996-2015. Baseline characteristics, incidence of second cancers, treatment details and overall survival were analysed., Results: During follow-up (median = 11.8 years), 221 second cancers were recorded in 88 patients. Of these cancers, 185 were cSCC, removed from 61 patients. In 56% of these patients, >1 cSCC was removed, and the majority of cSCC occurred following the treatment for CLL. The cumulative incidence of a first cSCC was 11% at 5 years, whereas the cumulative incidence of a subsequent cSCC was 88% at 5 years. The incidence of cSCC in male patients was threefold higher than that reported for the general NZ population., Conclusion: NZ CLL patients have a high incidence of cSCC relative to the levels observed in the general population, which are themselves among the highest in the world. The careful monitoring of CLL patients is warranted, particularly those who have a progressive disease or have had a first cSCC removed., (© 2016 Royal Australasian College of Physicians.)

Many members of the Apicomplexa contain a remnant chloroplast, known as an apicoplast. The apicoplast encodes numerous genes, and loss of the organelle is lethal. Here, we present a summary of what is known about apicoplast transcription. Unlike plant chloroplasts, there is a single RNA polymerase, and initial transcription is polycistronic. RNA is then cleaved into tRNA, mRNA and rRNA molecules. Significant levels of antisense transcription have been reported, together with a single case of RNA editing. Polycistronic transcription is also observed in the related algae Chromera and Vitrella, which retain a photosynthetic chloroplast. Surprisingly, a polyU tail is added to Chromera and Vitrella transcripts which encode proteins involved in photosynthesis. No such tail is added to Plasmodium transcripts. Transcription in the Apicomplexa is remarkably similar to that seen in the chloroplast of the related peridinin dinoflagellate algae, reflecting the common evolutionary origins of the organelle., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Soil-dwelling bacteria of the phylum actinomycetes generally harbor either GlnR or AmtR as a global regulator of nitrogen metabolism. Mycobacterium smegmatis harbors both of these canonical regulators; GlnR regulates the expression of key genes involved in nitrogen metabolism, while the function and signal transduction pathway of AmtR in M. smegmatis remains largely unknown. Here, we report the structure and function of the M. smegmatis AmtR and describe the role of AmtR in the regulation of nitrogen metabolism in response to nitrogen availability. To determine the function of AmtR in M. smegmatis, we performed genome-wide expression profiling comparing the wild-type versus an ∆amtR mutant and identified significant changes in the expression of 11 genes, including an operon involved in urea degradation. An AmtR consensus-binding motif (CTGTC-N 4 -GACAG) was identified in the promoter region of this operon, and ligand-independent, high-affinity AmtR binding was validated by both electrophoretic mobility shift assays and surface plasmon resonance measurements. We confirmed the transcription of a cis-encoded small RNA complementary to the gene encoding AmtR under nitrogen excess, and we propose a post-transcriptional regulatory mechanism for AmtR. The three-dimensional X-ray structure of AmtR at 2.0Å revealed an overall TetR-like dimeric structure, and the alignment of the M. smegmatis AmtR and Corynebacterium glutamicum AmtR regulatory domains showed poor structural conservation, providing a potential explanation for the lack of M. smegmatis AmtR interaction with the adenylylated P II protein. Taken together, our data suggest an AmtR (repressor)/GlnR (activator) competitive binding mechanism for transcriptional regulation of urea metabolism that is controlled by a cis-encoded small antisense RNA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Steep genetic clines resulting from recent secondary contact between previously isolated taxa can either gradually erode over time or be stabilized by factors such as ecological selection or selection against hybrids. We used patterns of variation in 30 nuclear and two mitochondrial SNPs to examine the factors that could be involved in stabilizing clines across a hybrid zone between two subspecies of the Atlantic killifish, Fundulus heteroclitus. Increased heterozygote deficit and cytonuclear disequilibrium in populations near the center of the mtDNA cline suggest that some form of reproductive isolation such as assortative mating or selection against hybrids may be acting in this hybrid zone. However, only a small number of loci exhibited these signatures, suggesting locus-specific, rather than genomewide, factors. Fourteen of the 32 loci surveyed had cline widths inconsistent with neutral expectations, with two SNPs in the mitochondrial genome exhibiting the steepest clines. Seven of the 12 putatively non-neutral nuclear clines were for SNPs in genes related to oxidative metabolism. Among these putatively non-neutral nuclear clines, SNPs in two nuclear-encoded mitochondrial genes (SLC25A3 and HDDC2), as well as SNPs in the myoglobin, 40S ribosomal protein S17, and actin-binding LIM protein genes, had clines that were coincident and concordant with the mitochondrial clines. When hybrid index was calculated using this subset of loci, the frequency distribution of hybrid indices for a population located at the mtDNA cline center was non-unimodal, suggesting selection against advanced-generation hybrids, possibly due to effects on processes involved in oxidative metabolism.

Background: Hearing loss is the most common sensory deficit in elderly patients, and is often under-recognised and poorly managed. It is essential for all clinicians to have awareness and knowledge in this field to enable the institution of early and appropriate care., Objective: The goal of this article is to review the causes, diagnosis and management of hearing loss as it applies to elderly patients. The review describes a useful approach that clinicians can apply to daily practice., Discussion: For elderly patients presenting with hearing loss, the basic assessment should include history, physical examination and pure tone audiometry. Management depends on the cause and type of hearing loss, and options include medical therapy, surgery and amplification. In the absence of a simple and correctable cause, consider referring patients to an otolaryngologist for further assessment.

The genetic structure of a hybrid zone can provide insights into the relative roles of the various factors that maintain the zone. Here, we use a multilocus approach to characterize a hybrid zone between two subspecies of killifish (Fundulus heteroclitus, Walbaum 1792) found along the Atlantic coast of North America. We first analysed clinal variation along the Atlantic coast using a single-nucleotide polymorphism in the mitochondrial DNA (mtDNA) displacement loop (D-loop) and a panel of nine nuclear microsatellite markers. A model constraining all clines to the same width and centre was not significantly different from a model in which the clines were allowed to vary independently. Locus-by-locus analysis indicated that the majority of nuclear clines shared the same centre as the mtDNA cline, and the widths of these clines were also narrower than that predicted by a neutral model, suggesting that selection is operating to maintain the hybrid zone. However, two of the nuclear clines had widths greater than the neutral prediction and had centres that were displaced relative to the mtDNA cline centre. We also found that a marsh located near the centre of the mtDNA cline demonstrated a bimodal distribution of nuclear hybrid index values, suggesting a deficit of first-generation hybrids and backcrossed genotypes. Thus, selection against hybrid genotypes may be playing a role in maintaining this hybrid zone and the associated steep nuclear and mtDNA clines.

Background: Myeloid-derived suppressor cells (MDSC) are powerful suppressors of immune responses. MDSC numbers are increased in some renal transplant recipients (RTR) and there is increasing interest in their potential role in promoting both transplant tolerance and transplant associated malignancy. However the factors influencing MDSC mobilisation are unknown, and the relative temporal changes in the granulocytic (G-MDSC) and monocytic (Mo-MDSC) subsets of MDSC have not been defined., Methods: The circulating frequencies of MDSC and dendritic cells (DC) were analysed by multicolour flow cytometry in RTR (n = 8) prior to transplant and at regular intervals out to 1 year post-transplant., Results: In RTRs, numbers of both G-MDSC and Mo-MDSC increased rapidly following transplant and peaked within 8 days, whilst DC numbers decreased. A second peak in G-MDSC numbers was observed in 7/8 patients within 3 months and 2 patients had a further 3rd peak in G-MDSC numbers which, in one patient, corresponded to a rejection event. Mo-MDSC numbers underwent less fluctuation and a subsequent peak in numbers was observed in only 2/8 patients. Respective kidney donors (n = 5) underwent only small transient increases in MDSC following surgery. Overall, there was little correlation between increases in MDSC and the occurrence of detectable clinical events or treatment changes., Conclusions: In RTRs, MDSC numbers increase rapidly and peak following commencement of immunosuppression, but then fluctuate in response to as yet undefined stimuli. Further studies are required to identify the factors modulating MDSC mobilisation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Chronic lymphocytic leukaemia (CLL) is associated with immunosuppression. The activation of CLL cells induced by interaction with other cell types, particularly activated T-cells, within the tumour micro-environment is thought to be important for CLL progression. However it is unclear whether activated CLL cells (CLL(Act)) have immunosuppressive capacity. We report that co-culture of CLL cells with normal PBMC in the context of CD3/CD28 T-cell activation generates CLL(Act) with increased CD38 expression that are capable of suppressing the proliferative responses of both CD4+ and CD8+ T-cells. The suppression required cell contact but did not involve induction of T-cell apoptosis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Patients undergoing surgery for colorectal cancer are at high risk of post-operative venous thromboembolism (VTE). Thromboprophylaxis has been shown to have significant risk reduction, although there remains some controversy surrounding the optimal duration of pharmacological prophylaxis. Our institution does not routinely practise extended prophylaxis. The aim of this study was to retrospectively review the rate of post-operative thromboprophylaxis in colorectal cancer patients, and incidence of symptomatic VTE., Methods: We conducted a retrospective audit of 200 consecutive patients who underwent colorectal surgery for cancer. Data to 90 days post-operatively were collected from medical records and imaging and phone calls to patients and family practitioners., Results: Of the patients, 98% received pharmacological prophylaxis, with a median duration of eight days. Eight (4%) symptomatic VTEs were diagnosed within the 90-day follow-up period: two deep vein thrombosis (DVTs), five pulmonary emboli (PE) and one patient with both PE and DVT. A higher proportion of patients developed DVT/PE if they received prophylaxis other than low molecular weight heparin and similarly there was a trend in increased risk of DVT in the presence of metastatic disease. However, using univariate analysis, these results were not statistically significant (P = 0.18 and 0.11, respectively)., Discussion: The use of thromboprophylaxis was high in our centre, and the incidence of VTE was low when patients received a median of 8 days pharmacological prophylaxis combined with mechanical prophylaxis. The VTE incidence of 4% is similar to previous studies using extended prophylaxis. Our study findings do not support changing local protocol to extended prophylaxis., (© 2013 Royal Australasian College of Surgeons.)

Introduction. Acalculous cholecystitis in the setting of typhoid fever in adults is an infrequent clinical encounter, reported sparsely in the literature. In this case report we review the presentation and management of enteric fever involving the biliary system and consider the literature surrounding this topic. The aim of this case report is to alert clinicians to the potential diagnosis of extraintestinal complications in the setting of typhoid fever in the returned traveller, requiring surgical intervention. Presentation of Case. We report the case of a 23-year-old woman with acalculous cholecystitis secondary to Salmonella Typhi. Discussion. There is scarce evidence surrounding the optimal treatment and prognosis of typhoidal acalculous cholecystitis. In the current case, surgical invention was favoured due to failure of medical management. Conclusion. Clinical judgement dictated surgical intervention in this case of typhoidal acute acalculous cholecystitis, and cholecystectomy was safely performed.

Surgeons commonly need to treat patients receiving anticoagulant and anti-platelet therapy. This requires risk assessment and management to balance minimization of bleeding complications and avoidance of further ischaemic or thrombotic events. This review considers the evidence available to guide management of patients on anti-platelet and anticoagulant therapy, including some of the new classes of anti-platelets and anticoagulants which clinicians may be less familiar with., (© 2013 The Authors. ANZ Journal of Surgery © 2013 Royal Australasian College of Surgeons.)

Human polymorphonuclear leucocytes (PMN) are thought to be immunosuppressive. The suppressive mechanism(s) used by PMN are, however, not well defined and in this study they were analysed using T-cell responses to CD3(+) CD28 monoclonal antibodies (mAb) as a readout. We demonstrate that in vitro activated PMN (PMN(act)) can, without any T-cell interaction, induce apparent T-cell suppression by inhibiting the stimulatory capacity of the CD3 mAb. However, a cell-directed suppression of T-cell proliferation was observed when PMN(act) were added to pre-activated T cells that are already committed to polyclonal proliferation. This suppression was partially reversed by catalase addition (P < 0·01) and largely reversed by addition of exogenous interleukin-2 (P < 0·001) but was not significantly reduced by nitric oxide synthase inhibition, myeloperoxidase inhibition or addition of excess arginine. Following removal of PMN(act) , suppressed T cells could respond normally to further stimulation. In addition to suppressing proliferation, co-culture with PMN(act) also induced a significant decrease in T-cell viability that was reversed by catalase addition (P < 0·05). The addition of the arginase inhibitor N-hydroxy-nor-l-arginine induced both a further significant, catalase-sensitive, loss in T-cell viability and increased nitrite release (P < 0·001). These data demonstrate that PMN, when activated, can both induce T-cell death and reversibly inhibit proliferation of activated T cells. The mechanisms underlying these distinct processes and the effects of arginase inhibitors on PMN induced cytotoxicity merit further investigation., (© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.)

The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequence-specificity of four VapC proteins from two different organisms (VapC(PAE0151) and VapC(PAE2754) from Pyrobaculum aerophilum, and VapC(Rv0065) and VapC(Rv0617) from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms.

The largest family of toxin-antitoxin (TA) modules are encoded by the vapBC operons, but their roles in bacterial physiology remain enigmatic. Microarray analysis in Mycobacterium smegmatis overexpressing VapC/VapBC revealed a high percentage of downregulated genes with annotated roles in carbon transport and metabolism, suggesting that VapC was targeting specific metabolic mRNA transcripts. To validate this hypothesis, purified VapC was used to identify the RNA cleavage site in vitro. VapC had RNase activity that was sequence specific, cleaving single-stranded RNA substrates at AUAU and AUAA in vitro and in vivo (viz., MSMEG&#95;2121 to MSMEG&#95;2124). A bioinformatic analysis of these regions suggested that an RNA hairpin 3' of the AUA(U/A) motif is also required for efficient cleavage. VapC-mediated regulation in vivo was demonstrated by showing that MSMEG&#95;2124 (dhaF) and MSMEG&#95;2121 (dhaM) were upregulated in a ΔvapBC mutant growing on glycerol. The ΔvapBC mutant had a specific rate of glycerol consumption that was 2.4-fold higher than that of the wild type during exponential growth. This increased rate of glycerol consumption was not used for generating bacterial biomass, suggesting that metabolism by the ΔvapBC mutant was uncoupled from growth. These data suggest a model in which VapC regulates the rate of glycerol utilization to match the anabolic demands of the cell, allowing for fine-tuning of the catabolic rate at a posttranscriptional level.

Background: Cancer, particularly cutaneous squamous cell carcinoma (SCC), is a major cause of mortality in renal transplant recipients (RTRs). Myeloid-derived suppressor cells (MDSC) play a central role in suppressing cancer immunosurveillance but their potential mobilisation in RTRs and levels relative to those of other immunoregulatory dendritic cell (DC) populations have not been analysed., Methods: The circulating frequencies of MDSC and DC were analysed by multicolour flow cytometry in immunocompetent patients without (n = 13) or with (ICI-SCC(Pos), n = 14) current SCC, normal donors (NDs, n = 34), chronic kidney disease patients (CKD patients, n = 22) and RTRs (n = 31)., Results: Compared to NDs, RTRs had significantly elevated levels of both CD14(Neg) and CD14(Pos) MDSC subsets (P < 0.001), while CKD patients and ICI-SCC(Pos) had significantly elevated levels of only the CD14(Neg)-MDSC subset. DC frequencies were significantly decreased in RTRs and CKD patients but were at normal levels in ICI-SCC(Pos). The MDSC/DC ratio was significantly elevated (P < 0.05) in RTRs (median = 5.7), CKD patients (median = 3.2) and ICI-SCC(Pos) (median = 3.5) relative to NDs (median = 0.7). The use of immunosuppressive drugs in CKD patients and past/current occurrence of SCC in RTRs was associated with significantly increased CD14(Neg)-MDSC frequencies. MDSC enriched from RTRs, when co-cultured with activated NDs T cells significantly suppressed extracellular IL-10 levels and can, when activated with formyl-methionyl-leucyl-phenylalanine, inhibit T-cell proliferation., Conclusions: RTRs, CKD patients and ICI-SCC(Pos) have increased MDSC frequencies and MDSC/DC ratios. These changes may impact on cancer immunosurveillance. Therefore, MDSC represent both a potential therapeutic target and prognostic marker in these patients, with respect to the development of SCC and other malignancies.

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as 'non-toxic'. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of 'non-toxic' VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617--VapC proteins with similarity to Rv0549c and Rv3320c, respectively--these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.

The PIN-domains are small proteins of ~130 amino acids that are found in bacteria, archaea and eukaryotes and are defined by a group of three strictly conserved acidic amino acids. The conserved three-dimensional structures of the PIN-domains cluster these acidic residues in an enzymatic active site. PIN-domains cleave single-stranded RNA in a sequence-specific, Mg²+- or Mn²+-dependent manner. These ribonucleases are toxic to the cells which express them and to offset this toxicity, they are co-expressed with tight binding protein inhibitors. The genes encoding these two proteins are adjacent in the genome of all prokaryotic organisms where they are found. This sequential arrangement of inhibitor-RNAse genes conforms to that of the so-called toxin-antitoxin (TA) modules and the PIN-domain TAs have been named VapBC TAs (virulence associated proteins, VapB is the inhibitor which contains a transcription factor domain and VapC is the PIN-domain ribonuclease). The presence of large numbers of vapBC loci in disparate prokaryotes has motivated many researchers to investigate their biochemical and biological functions. For example, the devastating human pathogen Mycobacterium tuberculosis has 45 vapBC loci encoded in its genome whereas its non-pathogenic relative, Mycobacterium smegmatis has just one vapBC operon. On another branch of the prokaryotic tree, the nitrogen-fixing symbiont of legumes, Sinorhizobium meliloti has 21 vapBC loci and at least one of these loci have been implicated in the regulation of growth in the plant nodule. A range of biological functions has been suggested for these operons and this review sets out to survey the PIN-domains and summarise the current knowledge about the vapBC TA systems and their roles in diverse bacteria.

Aim: The aim of this study is to determine whether the analysis of CD38 expression by chronic lymphocytic leukaemia (CLL) cells provides useful additional prognostic information., Methods: Clinical, laboratory, overall survival (OS) and treatment-free survival (TFS) data were collected on 130 CLL patients who had CD38 expression analysed at Canterbury Health Laboratories, New Zealand (NZ) during 1998-2008., Results: The detection of any level of CD38 expression by CLL cells was associated with a significantly shorter OS and TFS. When analysis was restricted to Binet stage A patients, CD38 expression identified a subset of patients (21%) who, in common with Binet stage B/C patients, had a significantly shorter OS and TFS (P<0.0015), and a TFS at 4 years of <10%. In contrast, CD38-negative Binet stage A patients had an OS that was not significantly different from that of an age/sex-matched NZ population and a 5-year TFS of 77%., Conclusion: This study indicates that, when combined with clinical staging, the presence of any detectable CD38 expression can be used to further improve the identification of CLL patients with more aggressive disease (i.e. Binet stage B/C or Binet stage A and CD38 positive). This will allow better identification of those patients requiring more intensive monitoring and also allow improved patient counselling regarding prognosis., (© 2010 The Authors. Internal Medicine Journal © 2010 Royal Australasian College of Physicians.)

The human mitochondrial DNA polymerase (pol γ) is nuclearly encoded and is solely responsible for the replication and repair of the mitochondrial genome. The progressive accumulation of mutations within the mitochondrial genome is thought to be related to aging, and mutations in the pol γ gene are responsible for numerous heritable disorders including progressive external opthalmoplegia, Alpers syndrome, and parkinsonism. Here we investigate the kinetic effect of H932Y, a mutation associated with opthalmoplegia. Mutations H932Y and H932A reduce the specificity constant governing correct nucleotide incorporation 150- and 70-fold, respectively, without significantly affecting fidelity of incorporation or the maximum rate of incorporation. However, this leads to only a 2-fold reduction in rate of incorporation at a physiological nucleotide concentration (∼100 μm). Surprisingly, incorporation of T:T or C:T mismatches catalyzed by either H932Y or H932A mutants was followed by slow pyrophosphate release (or fast pyrophosphate rebinding). Also, H932Y readily catalyzed incorporation of multiple mismatches, which may have a profound physiological impact over time. His-932 is thought to contact the β-phosphate of the incoming nucleotide, so it is perhaps surprising that H932Y appears to slow rather than accelerate pyrophosphate release.

Soluble CD83 (sCD83), a potent immunosuppressive agent, circulates at elevated levels in some chronic lymphocytic leukemia (CLL) patients. We report that CLL patients with elevated plasma sCD83 levels had significantly shorter (P=0.038) treatment free survival. Culture of CLL cells with solid phase CD83 mAb+IL-4 significantly increases sCD83 release (23-117-fold, P=0.013) and ligation of normal donor PBMC with solid phase CD83 mAb alone induces similar significant increases in sCD83 release (P=0.003). RT-PCR analysis detected the presence of a transcript for sCD83 in 2/3 CLL samples. These results suggest sCD83 release may play a regulatory role in CLL progression.

The largest family of bacterial toxin-antitoxin (TA) modules is formed by the vapBC operons, and these are grouped together by virtue of their toxin components belonging to the PilT N-terminal domain family of proteins that are thought to function as ribonucleases. We have identified a single vapBC operon in the genome of Mycobacterium smegmatis and herein report the molecular and biochemical characterisation of this TA module. In M. smegmatis, the vapBC genes are transcribed as a leaderless mRNA that is constitutively synthesised throughout the growth cycle. The vapBC operon is autoregulated by the VapBC protein complex as demonstrated by a threefold increase in vapBC expression (promoter-vapB-lacZ) in a DeltavapBC mutant. Electrophoretic mobility shift assays using purified VapBC protein complex show that the complex binds to inverted repeat DNA sequences in the vapBC promoter region that overlap the -35 and -10 promoter elements, thus explaining the autoregulation and the low-level constitutive expression of this operon in M. smegmatis. Neither a DeltavapBC nor a DeltavapB mutant strain exhibited any phenotypic deviation to that of the isogenic wild-type parent strain under normal laboratory growth conditions, but conditional overexpression of VapC in M. smegmatis inhibited growth by a bacteriostatic mechanism and this phenotype is exacerbated in a DeltavapBC mutant. This effect is mediated through VapC-dependent inhibition of translation, not inhibition of DNA replication or transcription. The growth inhibitory effect of VapC was neutralised when co-expressed with its cognate antitoxin VapB. Western blot analysis revealed the overproduction of VapC under inducing conditions and that the VapC protein is not produced in the DeltavapB mutant despite the presence of mRNA transcript. Taken together, these data demonstrate that VapBC from M. smegmatis has all the hallmarks of a TA module with the capacity to cause growth inhibition by regulating translation.