Correction: Correction: Hypoxia inducible factor (HIF) as a model for studying inhibition of protein–protein interactions

Correction for ‘Correction: Hypoxia inducible factor (HIF) as a model for studying inhibition of protein–protein interactions’ by George M. Burslem et al., Chem. Sci., 2017, 8, 5214–5215

Selective Affimers Recognise the BCL‐2 Family Proteins BCL‐xL and MCL‐1 through Noncanonical Structural Motifs

The BCL‐2 family is a challenging group of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL‐2 family regulators of apoptosis are useful as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non‐antibody‐binding proteins based on a conserved scaffold) to identify ligands for MCL‐1, BCL‐xL, BCL‐2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL‐2 protein. For anti‐apoptotic targets BCL‐xL and MCL‐1, competitive inhibition of their canonical protein‐protein interactions is demonstrated. Co‐crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug‐bound‐like conformation. These proof‐of‐concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL‐2 family modulators and more generally other protein‐protein interaction inhibitors

Development of an Affimer-antibody combined immunological diagnosis kit for glypican-3

Glypican-3 (GPC3) is a promising new marker for hepatocellular carcinoma, but the reported values for serum GPC3 differ markedly between currently available kits. Here we isolated Affimer non-antibody binding proteins against GPC3 by phage display and developed a new sandwich chemiluminescence immunoassay (CLIA) combining an Affimer with a monoclonal antibody (Affimer-MAb CLIA). The proposed CLIA assay demonstrated a wide linear range  0.03–600 ng/mL) with a good linear correlation coefficient (0.9999), a high detection limitation (0.03 ng/mL) and specificity (0–0.002%) for detection of GPC3. The accuracy, hook effect and stability were demonstrated to be satisfactory. The mean level of GPC3 in serum was higher (>8.5 fold, P < 0.001) in hepatocellular carcinoma patients compared to healthy and other liver disease individuals. A poor correlation (correlation coefficients ranged from −0.286 to 0.478) was observed through pairwise comparison within different kits. However, only this newly developed CLIA test showed high specificity and correlated with the “gold standard” GPC3-immunohistochemistry. This study indicates that Affimer-MAb CLIA can be used to generate a sensitive immunodiagnostic kit, which offers the potential for a highly specific clinically-relevant detection system

Racial/ethnic and sexual behavior disparities in rates of sexually transmitted infections, San Francisco, 1999-2008

<p>Abstract</p> <p>Background</p> <p>Racial/ethnic minorities and men who have sex with men (MSM) represent populations with disparate sexually transmitted infection (STI) rates. While race-specific STI rates have been widely reported, STI rates among MSM is often challenging given the absence of MSM population estimates. We evaluated the race-specific rates of chlamydia and gonorrhea among MSM and non-MSM in San Francisco between 1999-2008.</p> <p>Methods</p> <p>2000 US Census data for San Francisco was used to estimate the number of African-American, Asian/Pacific Islander, Hispanic, and white males. Data from National HIV Behavioral Surveillance (NHBS) MSM 1, conducted in 2004, was used to estimate the total number of MSM in San Francisco and the size of race/ethnic sub-populations of MSM. Non-MSM estimates were calculated by subtracting the number of estimated MSM from the total number of males residing in San Francisco. Rates of MSM and non-MSM gonorrhea and chlamydia reported between 1999 and 2008 were stratified by race/ethnicity. Ratios of MSM and non-MSM rates of morbidity were calculated by race/ethnicity.</p> <p>Results</p> <p>Between 1999-2008, MSM accounted for 72% of gonorrhea cases and 51% of chlamydia cases. Throughout the study period, African-American MSM had the highest chlamydia rate with 606 cases per 100,000 in 1999 increasing to 2067 cases per 100,000 in 2008. Asian/Pacific Islander MSM consistently had the lowest rate among MSM with1003 cases per 100,000 in 2008. The ratio of MSM/non-MSM for chlamydia was highest among whites 11.6 (95% CI: 8.8-14.4) and Asian/Pacific Islanders 8.6 (95% CI: 6.2-11), and lowest among African-Americans 1.53 (95% CI: 1.2-1.9) and Hispanics 4.43 (95% CI: 2.8-6.0). Gonorrhea rates were similar for African-American, white, and Hispanic MSM between 2137-2441 cases per 100,000 in 2008. Asian/Pacific Islander MSM had the lowest gonorrhea rate with 865 cases per 100,000 in 2008. The ratio of MSM/non-MSM for gonorrhea was highest among whites 11.6 (95% CI: 8.8-14.4) and Asian/Pacific Islanders 8.6 (95% CI: 6.2-11), and lowest among African-Americans 1.53 (95% CI: 1.2-1.9) and Hispanics 4.43 (95% CI: 2.8-6.0).</p> <p>Conclusions</p> <p>For all racial/ethnic groups in San Francisco, MSM carried a substantially higher burden of STIs compared to non-MSM except among African-American men. These racial and sexual behavior disparities warrant further public health attention and resources.</p

Within-host evolution of Staphylococcus aureus during asymptomatic carriage

Background Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of healthy adults are carriers. Recent evidence suggests that evolution of S. aureus during nasal carriage may be associated with progression to invasive disease. However, a more detailed understanding of within-host evolution under natural conditions is required to appreciate the evolutionary and mechanistic reasons why commensal bacteria such as S. aureus cause disease. Therefore we examined in detail the evolutionary dynamics of normal, asymptomatic carriage. Sequencing a total of 131 genomes across 13 singly colonized hosts using the Illumina platform, we investigated diversity, selection, population dynamics and transmission during the short-term evolution of S. aureus. Principal Findings We characterized the processes by which the raw material for evolution is generated: micro-mutation (point mutation and small insertions/deletions), macro-mutation (large insertions/deletions) and the loss or acquisition of mobile elements (plasmids and bacteriophages). Through an analysis of synonymous, non-synonymous and intergenic mutations we discovered a fitness landscape dominated by purifying selection, with rare examples of adaptive change in genes encoding surface-anchored proteins and an enterotoxin. We found evidence for dramatic, hundred-fold fluctuations in the size of the within-host population over time, which we related to the cycle of colonization and clearance. Using a newly-developed population genetics approach to detect recent transmission among hosts, we revealed evidence for recent transmission between some of our subjects, including a husband and wife both carrying populations of methicillin-resistant S. aureus (MRSA). Significance This investigation begins to paint a picture of the within-host evolution of an important bacterial pathogen during its prevailing natural state, asymptomatic carriage. These results also have wider significance as a benchmark for future systematic studies of evolution during invasive S. aureus disease

Vasodilator factors in the systemic and local adaptations to pregnancy

We postulate that an orchestrated network composed of various vasodilatory systems participates in the systemic and local hemodynamic adaptations in pregnancy. The temporal patterns of increase in the circulating and urinary levels of five vasodilator factors/systems, prostacyclin, nitric oxide, kallikrein, angiotensin-(1–7) and VEGF, in normal pregnant women and animals, as well as the changes observed in preeclamptic pregnancies support their functional role in maintaining normotension by opposing the vasoconstrictor systems. In addition, the expression of these vasodilators in the different trophoblastic subtypes in various species supports their role in the transformation of the uterine arteries. Moreover, their expression in the fetal endothelium and in the syncytiotrophoblast in humans, rats and guinea-pigs, favour their participation in maintaining the uteroplacental circulation. The findings that sustain the functional associations of the various vasodilators, and their participation by endocrine, paracrine and autocrine regulation of the systemic and local vasoactive changes of pregnancy are abundant and compelling. However, further elucidation of the role of the various players is hampered by methodological problems. Among these difficulties is the complexity of the interactions between the different factors, the likelihood that experimental alterations induced in one system may be compensated by the other players of the network, and the possibility that data obtained by manipulating single factors in vitro or in animal studies may be difficult to translate to the human. In addition, the impossibility of sampling the uteroplacental interface along normal pregnancy precludes obtaining longitudinal profiles of the various players. Nevertheless, the possibility of improving maternal blood pressure regulation, trophoblast invasion and uteroplacental flow by enhancing vasodilation (e.g. L-arginine, NO donors, VEGF transfection) deserves unravelling the intricate association of vasoactive factors and the systemic and local adaptations to pregnancy

A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis

Stem cells reside in 'niches', where support cells provide critical signalling for tissue renewal. Culture methods mimic niche conditions and support the growth of stem cells in vitro. However, current functional assays preclude statistically meaningful studies of clonal stem cells, stem cell-niche interactions, and genetic analysis of single cells and their organoid progeny. Here, we describe a 'microraft array' (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for quantitative PCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell-niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices