Synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors.     

The Leishmania major BBSome subunit BBS1 is essential for parasite virulence in the mammalian host

Bardet–Biedl syndrome (BBS) is a human genetic disorder with a spectrum of symptoms caused by primary cilium dysfunction. The disease is caused by mutations in one of at least 17 identified genes, of which seven encode subunits of the BBSome, a protein complex required for specific trafficking events to and from the primary cilium. The molecular mechanisms associated with BBSome function remain to be fully elucidated. Here, we generated null and complemented mutants of the BBSome subunit BBS1 in the protozoan parasite, Leishmania. In the absence of BBS1, extracellular parasites have no apparent defects in growth, flagellum assembly, motility or differentiation in vitro but there is accumulation of vacuole‐like structures close to the flagellar pocket. Infectivity of these parasites for macrophages in vitro is reduced compared with wild‐type controls but the null parasites retain the ability to differentiate to the intracellular amastigote stage. However, infectivity of BBS1 null parasites is severely compromised in a BALB/c mouse footpad model. We hypothesize that the absence of BBS1 in Leishmania leads to defects in specific trafficking events that affect parasite persistence in the host. This is the first report of an association between the BBSome complex and pathogen infectivity.     

Detection and Strain Typing of Ancient Mycobacterium leprae from a Medieval Leprosy Hospital

Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP) in Winchester, UK, showing skeletal signs of lepromatous leprosy (LL) have been studied using a multidisciplinary approach including osteological, geochemical and biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from control skeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae with detection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, the preservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP) and multiple locus variable number tandem repeat analysis (MLVA). Three of the five cases proved to be infected with SNP type 3I-1, ancestral to contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From the remaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotope analysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all three individuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as the precursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains. Thus we show that type 2F had also spread westwards to Britain by the early medieval period.     

Cancer incidence and mortality in people aged less than 75 years: Changes in Australia over the period 1987–2007

BackgroundAustralia has one of the highest rates of cancer incidence worldwide and, despite improving survival, cancer continues to be a major public health problem. Our aim was to provide simple summary measures of changes in cancer mortality and incidence in Australia so that progress and areas for improvement in cancer control can be identified.MethodsWe used national data on cancer deaths and newly registered cancer cases and compared expected and observed numbers of deaths and cases diagnosed in 2007. The expected numbers were obtained by applying 1987 age–sex specific rates (average of 1986–1988) directly to the 2007 population. The observed numbers of deaths and incident cases were calculated for 2007 (average of 2006–2008). We limited the analyses to people aged less than 75 years.ResultsThere was a 28% fall in cancer mortality (7827 fewer deaths in 2007 vs. 1987) and a 21% increase in new cancer diagnoses (13,012 more diagnosed cases in 2007). The greatest reductions in deaths were for cancers of the lung in males (?2259), bowel (?1797), breast (?773) and stomach (?577). Other notable falls were for cancers of the prostate (?295), cervix (?242) and non-Hodgkin lymphoma (?240). Only small or no changes occurred in mortality for cancers of the lung (female only), pancreas, brain and related, oesophagus and thyroid, with an increase in liver cancer (267). Cancer types that showed the greatest increase in incident cases were cancers of the prostate (10,245), breast (2736), other cancers (1353), melanoma (1138) and thyroid (1107), while falls were seen for cancers of the lung (?1705), bladder (?1110) and unknown primary (?904).ConclusionsThe reduction in mortality indicates that prevention strategies, improvements in cancer treatment, and screening programmes have made significant contributions to cancer control in Australia since 1987. The rise in incidence is partly due to diagnoses being brought forward by technological improvements and increased coverage of screening and early diagnostic testing.     

An HIV-1 Envelope Glycoprotein Trimer with an Embedded IL-21 Domain Activates Human B Cells

Broadly neutralizing antibodies (bNAbs) that target the HIV-1 envelope glycoproteins (Env) can prevent virus acquisition, but several Env properties limit its ability to induce an antibody response that is of sufficient quantity and quality. The immunogenicity of Env can be increased by fusion to co-stimulatory molecules and here we describe novel soluble Env trimers with embedded interleukin-4 (IL-4) or interleukin-21 (IL-21) domains, designed to activate B cells that recognize Env. In particular, the chimeric Env_IL-21 molecule activated B cells efficiently and induced the differentiation of antibody secreting plasmablast-like cells. We studied whether we could increase the activity of the embedded IL-21 by designing a chimeric IL-21/IL-4 (ChimIL-21/4) molecule and by introducing amino acid substitutions in the receptor binding domain of IL-21 that were predicted to enhance its binding. In addition, we incorporated IL-21 into a cleavable Env trimer and found that insertion of IL-21 did not impair Env cleavage, while Env cleavage did not impair IL-21 activity. These studies should guide the further design of chimeric proteins and Env_IL-21 may prove useful in improving antibody responses against HIV-1.     

Tissue-specific patterns of allelically-skewed DNA methylation

While DNA methylation is usually thought to be symmetrical across both alleles, there are some notable exceptions. Genomic imprinting and X chromosome inactivation are two well-studied sources of allele-specific methylation (ASM), but recent research has indicated a more complex pattern in which genotypic variation can be associated with allelically-skewed DNA methylation in cis. Given the known heterogeneity of DNA methylation across tissues and cell types we explored inter- and intra-individual variation in ASM across several regions of the human brain and whole blood from multiple individuals. Consistent with previous studies, we find widespread ASM with > 4% of the ～220,000 loci interrogated showing evidence of allelically-skewed DNA methylation. We identify ASM flanking known imprinted regions, and show that ASM sites are enriched in DNase I hypersensitivity sites and often located in an extended genomic context of intermediate DNA methylation. We also detect examples of genotype-driven ASM, some of which are tissue-specific. These findings contribute to our understanding of the nature of differential DNA methylation across tissues and have important implications for genetic studies of complex disease. As a resource to the community, ASM patterns across each of the tissues studied are available in a searchable online database: http://epigenetics.essex.ac.uk/ASMBrainBlood.     

Central Coherence in Eating Disorders: A Synthesis of Studies Using the Rey Osterrieth Complex Figure Test

BackgroundLarge variability in tests and differences in scoring systems used to study central coherence in eating disorders may lead to different interpretations, inconsistent findings and between study discrepancies. This study aimed to address inconsistencies by collating data from several studies from the same research group that used the Rey Osterrieth Complex Figure Test (Rey Figure) in order to produce norms to provide benchmark data for future studies.MethodData was collated from 984 participants in total. Anorexia Nervosa, Bulimia Nervosa, recovered Anorexia Nervosa, unaffected family members and healthy controls were compared using the Rey Figure.ResultsPoor global processing was observed across all current eating disorder sub-groups and in unaffected relatives. There was no difference in performance between recovered AN and HC groups.ConclusionsThis is the largest dataset reported in the literature and supports previous studies implicating poor global processing across eating disorders using the Rey Figure. It provides robust normative data useful for future studies.     

Using simulations to evaluate Mantel‐based methods for assessing landscape resistance to gene flow

Mantel‐based tests have been the primary analytical methods for understanding how landscape features influence observed spatial genetic structure. Simulation studies examining Mantel‐based approaches have highlighted major challenges associated with the use of such tests and fueled debate on when the Mantel test is appropriate for landscape genetics studies. We aim to provide some clarity in this debate using spatially explicit, individual‐based, genetic simulations to examine the effects of the following on the performance of Mantel‐based methods: (1) landscape configuration, (2) spatial genetic nonequilibrium, (3) nonlinear relationships between genetic and cost distances, and (4) correlation among cost distances derived from competing resistance models. Under most conditions, Mantel‐based methods performed poorly. Causal modeling identified the true model only 22% of the time. Using relative support and simple Mantel r values boosted performance to approximately 50%. Across all methods, performance increased when landscapes were more fragmented, spatial genetic equilibrium was reached, and the relationship between cost distance and genetic distance was linearized. Performance depended on cost distance correlations among resistance models rather than cell‐wise resistance correlations. Given these results, we suggest that the use of Mantel tests with linearized relationships is appropriate for discriminating among resistance models that have cost distance correlations <0.85 with each other for causal modeling, or <0.95 for relative support or simple Mantel r. Because most alternative parameterizations of resistance for the same landscape variable will result in highly correlated cost distances, the use of Mantel test‐based methods to fine‐tune resistance values will often not be effective.     

Quantifying and Understanding Voltage Losses Due to Nonradiative Recombination in Bulk Heterojunction Organic Solar Cells with Low Energetic Offsets

Open‐circuit voltage (V_OC) losses in organic photovoltaics (OPVs) inhibit devices from reaching V_OC values comparable to the bandgap of the donor–acceptor blend. Specifically, nonradiative recombination losses (?V_nr) are much greater in OPVs than in silicon or perovskite solar cells, yet the origins of this are not fully understood. To understand what makes a system have high or low loss, an investigation of the nonradiative recombination losses in a total of nine blend systems is carried out. An apparent relationship is observed between the relative domain purity of six blends and the degree of nonradiative recombination loss, where films exhibiting relatively less pure domains show lower ?V_nr than films with higher domain purity. Additionally, it is shown that when paired with a fullerene acceptor, polymer donors which have bulky backbone units to inhibit close π–π stacking exhibit lower nonradiative recombination losses than in blends where the polymer can pack more closely. This work reports a strategy that ensures ?V_nr can be measured accurately and reports key observations on the relationship between ?V_nr and properties of the donor/acceptor interface.     

Colistin heteroresistance in Enterobacter cloacae is regulated by PhoPQ‐dependent 4‐amino‐4‐deoxy‐l‐arabinose addition to lipid A

The Enterobacter cloacae complex (ECC) consists of closely related bacteria commonly associated with the human microbiota. ECC are increasingly isolated from healthcare‐associated infections, demonstrating that these Enterobacteriaceae are emerging nosocomial pathogens. ECC can rapidly acquire multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the highly conserved lipid A component of the Gram‐negative outer membrane. Many Enterobacteriaceae fortify their outer membrane with cationic amine‐containing moieties to prevent CAMP binding, which can lead to cell lysis. The PmrAB two‐component system (TCS) directly activates 4‐amino‐4‐deoxy‐l ‐arabinose (l ‐Ara4N) biosynthesis to result in cationic amine moiety addition to lipid A in many Enterobacteriaceae such as E. coli and Salmonella. In contrast, PmrAB is dispensable for CAMP resistance in E. cloacae. Interestingly, some ECC clusters exhibit colistin heteroresistance, where a subpopulation of cells exhibit clinically significant resistance levels compared to the majority population. We demonstrate that E. cloacae lipid A is modified with l ‐Ara4N to induce CAMP heteroresistance and the regulatory mechanism is independent of the PmrAB_Ecl TCS. Instead, PhoP_Ecl binds to the arnB_Ecl promoter to induce l ‐Ara4N biosynthesis and PmrAB‐independent addition to the lipid A disaccharolipid. Therefore, PhoPQ_Ecl contributes to regulation of CAMP heteroresistance in some ECC clusters.