A volumetric method for quantifying atherosclerosis in mice by using microCT: comparison to en face

Precise quantification of atherosclerotic plaque in preclinical models of atherosclerosis requires the volumetric assessment of the lesion(s) while maintaining in situ architecture. Here we use micro-computed tomography (microCT) to detect ex vivo aortic plaque established in three dyslipidemic mouse models of atherosclerosis. All three models lack the low-density lipoprotein receptor (Ldlr^−/−), each differing in plaque severity, allowing the evaluation of different plaque volumes using microCT technology. From clearly identified lesions in the thoracic aorta from each model, we were able to determine plaque volume (0.04–3.1 mm³), intimal surface area (0.5–30 mm²), and maximum plaque (intimal-medial) thickness (0.1–0.7 mm). Further, quantification of aortic volume allowed calculation of vessel occlusion by the plaque. To validate microCT for future preclinical studies, we compared microCT data to intimal surface area (by using en face methodology). Both plaque surface area and plaque volume were in excellent correlation between microCT assessment and en face surface area (r² = 0.99, p<0.0001 and r² = 0.95, p<0.0001, respectively). MicroCT also identified internal characteristics of the lipid core and fibrous cap, which were confirmed pathologically as Stary type III-V lesions. These data validate the use of microCT technology to provide a more exact empirical measure of ex vivo plaque volume throughout the entire intact aorta in situ for the quantification of atherosclerosis in preclinical models.     

High-throughput sequencing offers insight into mechanisms of resource partitioning in cryptic bat species

Sympatric cryptic species, characterized by low morphological differentiation, pose a challenge to understanding the role of interspecific competition in structuring ecological communities. We used traditional (morphological) and novel molecular methods of diet analysis to study the diet of two cryptic bat species that are sympatric in southern England (Plecotus austriacus and P. auritus) (Fig. 1). Using Roche FLX 454 (Roche, Basel, CH) high-throughput sequencing (HTS) and uniquely tagged generic arthropod primers, we identified 142 prey Molecular Operational Taxonomic Units (MOTUs) in the diet of the cryptic bats, 60% of which were assigned to a likely species or genus. The findings from the molecular study supported the results of microscopic analyses in showing that the diets of both species were dominated by lepidopterans. However, HTS provided a sufficiently high resolution of prey identification to determine fine-scale differences in resource use. Although both bat species appeared to have a generalist diet, eared-moths from the family Noctuidae were the main prey consumed. Interspecific niche overlap was greater than expected by chance (O(jk) = 0.72, P < 0.001) due to overlap in the consumption of the more common prey species. Yet, habitat associations of nongeneralist prey species found in the diets corresponded to those of their respective bat predator (grasslands for P. austriacus, and woodland for P. auritus). Overlap in common dietary resource use combined with differential specialist prey habitat associations suggests that habitat partitioning is the primary mechanism of coexistence. The performance of HTS is discussed in relation to previous methods of molecular and morphological diet analysis. By enabling species-level identification of dietary components, the application of DNA sequencing to diet analysis allows a more comprehensive comparison of the diet of sympatric cryptic species, and therefore can be an important tool for determining fine-scale mechanisms of coexistence.     

Contrasting factors associated with COVID-19-related ICU admission and death outcomes in hospitalised patients by means of Shapley values

Identification of those at greatest risk of death due to the substantial threat of COVID-19 can benefit from novel approaches to epidemiology that leverage large datasets and complex machine-learning models, provide data-driven intelligence, and guide decisions such as intensive-care unit admission (ICUA). The objective of this study is two-fold, one substantive and one methodological: substantively to evaluate the association of demographic and health records with two related, yet different, outcomes of severe COVID-19 (viz., death and ICUA); methodologically to compare interpretations based on logistic regression and on gradient-boosted decision tree (GBDT) predictions interpreted by means of the Shapley impacts of covariates. Very different association of some factors, e.g., obesity and chronic respiratory diseases, with death and ICUA may guide review of practice. Shapley explanation of GBDTs identified varying effects of some factors among patients, thus emphasising the importance of individual patient assessment. The results of this study are also relevant for the evaluation of complex automated clinical decision systems, which should optimise prediction scores whilst remaining interpretable to clinicians and mitigating potential biases.     

Polyfunctional CD4(+) T Cell Responses to Immunodominant Epitopes Correlate with Disease Activity of Virulent Salmonella

Salmonella enterica serovars are intracellular bacteria capable of causing typhoid fever and gastroenteritis of significant morbidity and mortality worldwide. Current prophylactic and therapeutic treatment is hampered by the emergence of multidrug-resistant (MDR) strains of Salmonella, and vaccines provide only temporal and partial protection in vaccinees. To develop more effective Salmonella vaccines, it is important to understand the development of protective adaptive immunity to virulent Salmonella. Here we report the identification of novel CD4(+) T cell peptide epitopes, which are conserved among Salmonella serovars. Immunization of Salmonella-infected mice with these peptide epitopes reduces the burden of Salmonella disease. Furthermore, we show that distinct polyfunctional (interferon-γ(+), tumor necrosis factor(+), and interleukin-2(+)) Salmonella-specific CD4(+) T cell responses develop with respect to magnitude and kinetics. Moreover, we found that CD4(+) T cell responses against immunodominant epitopes are predictive for active Salmonella disease. Collectively, these data could contribute to improved diagnosis of Salmonella-related diseases and rational design of Salmonella vaccines.     

4-Phenyl tetrahydroisoquinolines as dual norepinephrine and dopamine reuptake inhibitors

Novel 4-phenyl tetrahydroisoquinolines that inhibit both dopamine and norepinephrine transporters were designed and prepared. In this Letter, we describe the synthesis, in vitro activity and associated structure–activity relationships of this series. We also report the ex vivo NET occupancy of a representative compound, 41.     

IFT proteins accumulate during cell division and localize to the cleavage furrow in Chlamydomonas

Intraflagellar transport (IFT) proteins are well established as conserved mediators of flagellum/cilium assembly and disassembly. However, data has begun to accumulate in support of IFT protein involvement in other processes elsewhere in the cell. Here, we used synchronous cultures of Chlamydomonas to investigate the temporal patterns of accumulation and localization of IFT proteins during the cell cycle. Their mRNAs showed periodic expression that peaked during S and M phase (S/M). Unlike most proteins that are synthesized continuously during G1 phase, IFT27 and IFT46 levels were found to increase only during S/M phase. During cell division, IFT27, IFT46, IFT72, and IFT139 re-localized from the flagella and basal bodies to the cleavage furrow. IFT27 was further shown to be associated with membrane vesicles in this region. This localization pattern suggests a role for IFT in cell division.     

Sources of pre-analytical variations in yield of DNA extracted from blood samples: analysis of 50,000 DNA samples in EPIC

The European Prospective Investigation into Cancer and nutrition (EPIC) is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS) and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an automated DNA extraction platform. Here we have evaluated the pre-analytical factors affecting DNA yield, including anthropometric, epidemiological and technical factors such as center of subject recruitment, age, gender, body-mass index, disease case or control status, tobacco consumption, number of aliquots of buffy coat used for DNA extraction, extraction machine or procedure, DNA quantification method, degree of haemolysis and variations in the timing of sample processing. We show that the largest significant variations in DNA yield were observed with degree of haemolysis and with center of subject recruitment. Age, gender, body-mass index, cancer case or control status and tobacco consumption also significantly impacted DNA yield. Feedback from laboratories which have analyzed DNA with different SNP genotyping technologies demonstrate that the vast majority of samples (approximately 88%) performed adequately in different types of assays. To our knowledge this study is the largest to date to evaluate the sources of pre-analytical variations in DNA extracted from peripheral leucocytes. The results provide a strong evidence-based rationale for standardized recommendations on blood collection and processing protocols for large-scale genetic studies.     

Crosstalk between endogenous and synthetic components--synthetic signaling meets endogenous components

Synthetic biology uses biological components to engineer new functionality in living organisms. We have used the tools of synthetic biology to engineer detector plants that can sense man-made chemicals, such as the explosive trinitrotoluene, and induce a response detectable by eye or instrumentation. A goal of this type of work is to make the designed system orthogonal, that is, able to function independently of systems in the host. In this review, the design and function of two partially synthetic signaling pathways for use in plants is discussed. We describe observed interactions (crosstalk) with endogenous signaling components. This crosstalk can be beneficial, allowing the creation of hybrid synthetic/endogenous signaling pathways, or detrimental, resulting in system noise and/or false positives. Current approaches in the field of synthetic biology applicable to the design of orthogonal signaling systems, including the design of synthetic components, partially synthetic systems that utilize crosstalk to signal through endogenous components, computational redesign of proteins, and the use of heterologous components, are discussed.     

Reconstructing spatial vulnerability to forest loss by fire in pre‐historic New Zealand

Aim Despite small and transient populations, early Māori transformed large areas of New Zealand's forest landscapes. We sought to isolate the biophysical predictors that explain forest loss in the pre‐historic (i.e. pre‐European) period in New Zealand. Location New Zealand. Methods We used resampled boosted regression trees to isolate the key predictors of forest loss from a suite of 19 topographic, climatic, soil‐related and archaeological predictors at a 1‐km spatial resolution across New Zealand. Results The key predictors of fire‐driven forest loss during New Zealand's pre‐history relate to moisture and elevation gradients, with sites characterized by low moisture levels and gentle slopes being most vulnerable. Proxies for human activity were important in the North Island, where Māori population densities were higher, but not the South Island. The predicted pattern of forest loss and its relationship with biophysical variables suggest that early Māori neither deliberately protected fire‐prone regions nor systematically burnt less fire‐prone ones. Main conclusions Before Māori settlement of New Zealand fire was naturally rare, despite biophysical conditions being conducive to fire spread. The introduction of an ignition source by humans made widespread forest loss inevitable, even in the absence of sustained and deliberate use of fire. Rapid forest loss at the time of human settlement is recurrent across eastern Polynesia, so understanding this dynamic in New Zealand has implications for the region as a whole.