WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.

Phylogenomic reconstruction of Cryptosporidium spp. captured directly from clinical samples reveals extensive genetic diversity.

Cryptosporidium is a leading cause of severe diarrhea and mortality in young children and infants in Africa and southern Asia. More than twenty Cryptosporidium species infect humans, of which C. parvum and C. hominis are the major agents causing moderate to severe diarrhea. Relatively few genetic markers are typically applied to genotype and/or diagnose Cryptosporidium. Most infections produce limited oocysts making it difficult to perform whole genome sequencing (WGS) directly from stool samples. Hence, there is an immediate need to apply WGS strategies to 1) develop high-resolution genetic markers to genotype these parasites more precisely, 2) to investigate endemic regions and detect the prevalence of different genotypes, and the role of mixed infections in generating genetic diversity, and 3) to investigate zoonotic transmission and evolution. To understand Cryptosporidium global population genetic structure, we applied Capture Enrichment Sequencing (CES-Seq) using 74,973 RNA-based 120 nucleotide baits that cover ~92% of the genome of C. parvum. CES-Seq is sensitive and successfully sequenced Cryptosporidium genomic DNA diluted up to 0.005% in human stool DNA. It also resolved mixed strain infections and captured new species of Cryptosporidium directly from clinical/field samples to promote genome-wide phylogenomic analyses and prospective GWAS studies.

WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. ( C. cuniculus , C. hominis , C. meleagridis , C. parvum , C. tyzzeri , and C. viatorum ) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.

Social Inference from Middle to Older Adulthood.

OBJECTIVE: Inferring the emotional state or the true meaning of another person's utterance is a key aspect of social cognition and critical for successful social interactions. In this study, we assess age-related differences in emotion recognition and sincere and sarcastic social exchanges in the context of cognitive and demographic factors. METHOD: One hundred and eighty-seven adults ranging from middle to older adulthood completed the Mini-SEA Emotion Recognition test and Part B of The Awareness of Social Inference Test - Short Form (TASIT-S). Fluid intelligence and executive abilities were also assessed. Sex differences and the relationship with education level were also investigated. Regression models were used to assess age-related differences controlling for baseline cognitive and demographic factors. RESULTS: Age was negatively associated with accuracy for inferring sincere social exchanges. No differences were identified for accuracy for inferring sarcastic exchanges. Likewise, no age differences were identified for emotion recognition (Mini-SEA). Fluid intelligence was associated with accuracy for inferring sincere exchanges, but this was independent of age-related effects. A female advantage was identified for emotion recognition. CONCLUSION: Age is associated with difficulty in inferring sincere exchanges, which is not explained by fluid intelligence, verbal abstract reasoning, or auditory verbal attention. A female advantage in emotion recognition is consistent with findings reported in younger adults. Both age and sex should be considered in clinical assessments using the Mini-SEA and the TASIT-S.

Diagnostic accuracy of an automated artificial intelligence derived right ventricular to left ventricular diameter ratio tool on CT pulmonary angiography to predict pulmonary hypertension at right heart catheterisation.

AIM: To determine the diagnostic accuracy of an automated artificial intelligence derived right ventricle/left ventricle diameter ratio (RV/LV) computed tomography pulmonary angiography (CTPA) analysis tool to detect pulmonary hypertension (PH) in patients with suspected PH referred to a specialist centre. MATERIALS AND METHODS: The present study was a retrospective analysis of a prospectively maintained database of 202 consecutive patients with suspected PH, who underwent CTPA within 12 months of right heart catheterisation (RHC). Automated ventricular segmentation and RV/LV calculation (Imbio LLC, Minneapolis, MN, USA) was undertaken on the CTPA images. PH diagnosis was made using the RHC reference standard. RESULTS: The automated RV/LV correlated more strongly with RHC metrics than main pulmonary artery (MPA) diameter and MPA to ascending aorta diameter ratio (MPA/AA) measured manually (mean pulmonary arterial pressure [mPAP] r=0.535, R2 = 0.287 p<0.001; pulmonary vascular resistance [PVR] r=0.607, R2 = 0.369 p<0.001). In the derivation cohort (n=100), the area under the receiver-operating curve for automated RV/LV discriminating PH was 0.752 (95% confidence interval [CI] 0.677-0.827, p<0.001). Using an optimised Youden's Index of ≥1.12 classified from derivation, automated RV/LV ratio analysis was more sensitive for the detection of PH with higher positive predictive value (PPV) when compared with manual MPA and MPA/AA in the validation cohort (n=102). Automated RV/LV compromise (1.12) and specific (1.335) thresholds were strongly predictive of mortality (log-rank 7.401, p=0.007 and log-rank 16.075, p<0.001 respectively). CONCLUSION: In suspected PH, automated RV/LV diameter thresholds have high sensitivity for PH, outperform manual MPA and MPA/AA and can predict survival.

Pretreatment Normal WM Magnetization Transfer Ratio Predicts Risk of Radiation Necrosis in Patients with Medulloblastoma.

BACKGROUND AND PURPOSE: Radiation necrosis, for which abnormal WM enhancement is a hallmark, is an uncommon complication of craniospinal irradiation in children with medulloblastoma. The magnetization transfer ratio measures macromolecular content, dominated by myelin in the WM. We investigated whether the pretreatment supratentorial (nonsurgical) WM magnetization transfer ratio could predict patients at risk for radiation necrosis after radiation therapy for medulloblastoma. MATERIALS AND METHODS: Ninety-five eligible patients with medulloblastoma (41% female; mean age, 11.0 [SD, 5.4] years) had baseline balanced steady-state free precession MR imaging before proton or photon radiation therapy. Associations among baseline supratentorial magnetization transfer ratio, radiation necrosis (spontaneously resolving/improving parenchymal enhancement within the radiation field)3, age, and the presence of visible brain metastases were explored by logistic regression and parametric/nonparametric techniques as appropriate. RESULTS: Twenty-three of 95 (24.2%) children (44% female; mean age, 10.7 [SD, 6.7] years) developed radiation necrosis after radiation therapy (19 infratentorial, 1 supratentorial, 3 both). The mean pretreatment supratentorial WM magnetization transfer ratio was significantly lower in these children (43.18 versus 43.50, P = .03). There was no association between the supratentorial WM magnetization transfer ratio and age, sex, risk/treatment stratum, or the presence of visible brain metastases. CONCLUSIONS: A lower baseline supratentorial WM magnetization transfer ratio may indicate underlying structural WM susceptibility to radiation necrosis and may identify children at risk for developing radiation necrosis after craniospinal irradiation for medulloblastoma.

Oculomotor nerve guidance and terminal branching requires interactions with differentiating extraocular muscles.

Muscle function is dependent on innervation by the correct motor nerves. Motor nerves are composed of motor axons which extend through peripheral tissues as a compact bundle, then diverge to create terminal nerve branches to specific muscle targets. As motor nerves approach their targets, they undergo a transition where the fasciculated nerve halts further growth then after a pause, the nerve later initiates branching to muscles. This transition point is potentially an intermediate target or guidepost to present specific cellular and molecular signals for navigation. Here we describe the navigation of the oculomotor nerve and its association with developing muscles in mouse embryos. We found that the oculomotor nerve initially grew to the eye three days prior to the appearance of any extraocular muscles. The oculomotor axons spread to form a plexus within a mass of cells, which included precursors of extraocular muscles and other orbital tissues and expressed the transcription factor Pitx2. The nerve growth paused in the plexus for more than two days, persisting during primary extraocular myogenesis, with a subsequent phase in which the nerve branched out to specific muscles. To test the functional significance of the nerve contact with Pitx2+ cells in the plexus, we used two strategies to genetically ablate Pitx2+ cells or muscle precursors early in nerve development. The first strategy used Myf5-Cre-mediated expression of diphtheria toxin A to ablate muscle precursors, leading to loss of extraocular muscles. The oculomotor axons navigated to the eye to form the main nerve, but subsequently largely failed to initiate terminal branches. The second strategy studied Pitx2 homozygous mutants, which have early apoptosis of Pitx2-expressing precursor cells, including precursors for extraocular muscles and other orbital tissues. Oculomotor nerve fibers also grew to the eye, but failed to stop to form the plexus, instead grew long ectopic projections. These results show that neither Pitx2 function nor Myf5-expressing cells are required for oculomotor nerve navigation to the eye. However, Pitx2 function is required for oculomotor axons to pause growth in the plexus, while Myf5-expressing cells are required for terminal branch initiation.

Analysis of Cryptosporidium spp. from clinical samples by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

AIM: To purify Cryptosporidium spp. oocysts from clinical stool samples and evaluate using an up-to-date mass spectrometry protocol producing high-quality reference spectra. METHODS AND RESULTS: A refined purification protocol was developed for oocysts from stools, involving salt flotation and potassium bromide density centrifugation. Purified oocysts were prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) by formic acid extraction, and the extracts analysed using the Bruker MALDI Biotyper system. Individual spectral markers were identified by their specific mass peaks. Cryptosporidium parvum oocysts (Iowa strain) propagated in vivo, and C. parvum (n = 2) and Cryptosporidium hominis (n = 1) oocysts from clinical stool samples produced distinct spectra that were considered specific to Cryptosporidium spp. with no evidence of contamination. CONCLUSIONS: The production of distinct spectra demonstrated the utility of the purification method for oocysts from clinical stool samples and provided reference spectra. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of MALDI-TOF MS and other mass spectrometry techniques has been limited previously to C. parvum oocysts propagated in vivo. Appropriate purification of oocysts can achieve sufficient biomass, enabling analysis by MALDI-TOF MS and potentially other mass spectrometry platforms, facilitating peptide and protein discovery and identification of biomarkers from a much wider range of Cryptosporidium spp. from natural infections.

Response initiation and inhibition and the relationship with fluid intelligence across the adult lifespan.

BACKGROUND: Cognitive processes associated with frontal lobe functioning are often termed "executive functions." Two such processes are initiation and inhibition or the starting and stopping of responses. It has recently been claimed dysfunction of executive abilities can be explained by a single measure of fluid intelligence. Here, we test this claim, specifically for the executive abilities of response initiation and inhibition, across the healthy lifespan. METHOD: In a cohort of 336 healthy adults (18-89 years), initiation and inhibition were assessed with the Hayling test, Stroop test, and phonemic and semantic verbal fluency. All participants also completed a measure of fluid intelligence. The relationship between fluid intelligence and executive measures was explored across the lifespan using a continuous approach. Mediation models were computed to assess whether age-related decline across the four initiation/inhibition tasks could be fully explained by a single measure of fluid intelligence. RESULTS: Age was negatively correlated with response initiation/inhibition and fluid intelligence. The mediation analyses identified only partial mediation of fluid intelligence for age and Hayling performance. By contrast, fluid intelligence did not mediate performance on the Stroop test or phonemic and semantic verbal fluency. CONCLUSIONS: Response initiation/inhibition are not able to be explained by fluid intelligence. The results support a multifactorial theory of executive functions and provide evidence for the inclusion of multiple specific executive measures in a thorough neuropsychological assessment of age-related cognitive decline.

COVID-19: looking beyond the peak. Challenges and tips for radiologists in follow-up of a novel patient cohort.

As the coronavirus pandemic evolves, the focus of radiology departments has begun to change. The acute phase of imaging a new disease entity whilst rationalising radiology services in the face of lockdown has passed. Radiologists are now becoming familiar with the complications of COVID-19, particularly the lung parenchymal and pulmonary vascular sequelae and are considering the impact follow-up imaging may have on departments already struggling with a backlog of suspended imaging in the face of reduced capacity. This review from the British Society of Thoracic Imaging explores both the thoracic and extra-thoracic complications of COVID-19, recognising the importance of a holistic approach to patient follow-up. The British Thoracic Society guidelines for respiratory follow-up of COVID-19 will be discussed, together with newly developed reporting templates, which aim to provide consistency for clinicians as well as an opportunity for longer-term data collection.

Improving pulse crops as a source of protein, starch and micronutrients.

Pulse crops have been known for a long time to have beneficial nutritional profiles for human diets but have been neglected in terms of cultivation, consumption and scientific research in many parts of the world. Broad dietary shifts will be required if anthropogenic climate change is to be mitigated in the future, and pulse crops should be an important component of this change by providing an environmentally sustainable source of protein, resistant starch and micronutrients. Further enhancement of the nutritional composition of pulse crops could benefit human health, helping to alleviate micronutrient deficiencies and reduce risk of chronic diseases such as type 2 diabetes. This paper reviews current knowledge regarding the nutritional content of pea (Pisum sativum L.) and faba bean (Vicia faba L.), two major UK pulse crops, and discusses the potential for their genetic improvement.