Ultra-fast deep-learned CNS tumour classification during surgery.

Central nervous system tumours represent one of the most lethal cancer types, particularly among children1. Primary treatment includes neurosurgical resection of the tumour, in which a delicate balance must be struck between maximizing the extent of resection and minimizing risk of neurological damage and comorbidity2,3. However, surgeons have limited knowledge of the precise tumour type prior to surgery. Current standard practice relies on preoperative imaging and intraoperative histological analysis, but these are not always conclusive and occasionally wrong. Using rapid nanopore sequencing, a sparse methylation profile can be obtained during surgery4. Here we developed Sturgeon, a patient-agnostic transfer-learned neural network, to enable molecular subclassification of central nervous system tumours based on such sparse profiles. Sturgeon delivered an accurate diagnosis within 40 minutes after starting sequencing in 45 out of 50 retrospectively sequenced samples (abstaining from diagnosis of the other 5 samples). Furthermore, we demonstrated its applicability in real time during 25 surgeries, achieving a diagnostic turnaround time of less than 90 min. Of these, 18 (72%) diagnoses were correct and 7 did not reach the required confidence threshold. We conclude that machine-learned diagnosis based on low-cost intraoperative sequencing can assist neurosurgical decision-making, potentially preventing neurological comorbidity and avoiding additional surgeries.

Targeted locus amplification reveals heterogeneity between and within CFTR genotypes and association with CFTR function in patient-derived intestinal organoids.

BACKGROUND: Cystic fibrosis (CF) disease severity can be highly variable, even between people with CF (pwCF) with similar genotypes. Here we use patient-derived intestinal organoids to study the influence of genetic variation within the cystic fibrosis transmembrane conductance regulator (CFTR) gene on CFTR function. METHODS: Organoids of F508del/class I, F508del/S1251N and pwCF with only one detected CF-causing mutation were cultured. Allele-specific CFTR variation was investigated using targeted locus amplification (TLA), CFTR function was measured using the forskolin-induced swelling assay and mRNA levels were quantified using RT-qPCR. RESULTS: We were able to distinguish CFTR genotypes based on TLA data. Additionally, we observed heterogeneity within genotypes, which we were able to link to CFTR function for S1251N alleles. CONCLUSIONS: Our results indicate that the paired analysis of CFTR intragenic variation and CFTR function can gain insights in the underlying CFTR defect for individuals where the disease phenotype does not match the CFTR mutations detected during diagnosis.

Production of zirconium-88 via proton irradiation of metallic yttrium and preparation of target for neutron transmission measurements at DICER.

A process for the production of tens to hundreds of GBq amounts of zirconium-88 (88Zr) using proton beams on yttrium was developed. For this purpose, yttrium metal targets (≈20 g) were irradiated in a ~16 to 34 MeV proton beam at a beam current of 100-200 µA at the Los Alamos Isotope Production Facility (IPF). The 88Zr radionuclide was produced and separated from the yttrium targets using hydroxamate resin with an elution yield of 94(5)% (1σ). Liquid DCl solution in D2O was selected as a suitable 88Zr sample matrix due to the high neutron transmission of deuterium compared to hydrogen and an even distribution of 88Zr in the sample matrix. The separated 88Zr was dissolved in DCl and 8 µL of the obtained solution was transferred to a tungsten sample can with a 1.2 mm diameter hole using a syringe and automated filling station inside a hot cell. Neutron transmission of the obtained 88Zr sample was measured at the Device for Indirect Capture Experiments on Radionuclides (DICER).

Large-scale production of 88Y and 88Zr/88Y generators: A proof of concept study for a 70 MeV H- cyclotron.

The large-scale production of 88Y with proton-induced reactions has been investigated from the perspective of new generation 70 MeV H- cyclotrons. Tandem target configurations are presented for both the direct production of 88Y as well as for producing 88Zr/88Y generators. Based on the relevant excitation functions, physical yields have been derived for 88Y production with Y2O3/SrCO3 tandem targets and 88Zr production with Zr/Y2O3 tandem targets. Yields are presented for optimized targets (i.e. optimum yield) as well as for balanced thermal loads on the individual targets. Liquid 88Zr/88Y generators have been produced using both natural Zr and Nb target materials, the former for dedicated productions and the latter as a byproduct by processing spent irradiated Nb capsules which normally would constitute radioactive waste. These stock solutions, which contain both the target material and 88Zr precursor, are retained virtually unchanged after processing except for the removal of 88Y on AG MP-50 macroporous cation-exchange resin. Methods are presented for the preparation of Nb stock solutions in hydrofluoric acid and Zr stock solutions in sulphuric acid. It is shown that multi-Ci productions of 88Y are feasible at a 70 MeV cyclotron facility, suitable for the needs of fracking applications. In addition, 88Zr/88Y generators can provide 88Y with very high specific activity, suitable for labelling of biomolecules. LA-UR-20-24305.

Age-related gene and miRNA expression changes in airways of healthy individuals.

Knowledge on age-related miRNA changes in healthy individuals and their interaction with mRNAs is lacking. We studied age-related mRNA and miRNA expression changes and their interactions in normal airways. RNA and small RNA sequencing was performed on bronchial biopsies of 86 healthy individuals (age: 18-73) to determine age-related expression changes. Per age-related miRNA we determined the enrichment of age-related predicted targets and their correlation. We identified 285 age-related genes and 27 age-related miRNAs. Pathway enrichment showed that genes higher expressed with age were involved in synapse-related processes. Genes lower expressed with age were involved in cell cycle regulation, the immune system and DNA damage/repair. MiR-146a-5p, miR-146b-5p and miR-142-5p were lower expressed with increasing age and we found a significant enrichment for predicted targets of these miRNAs among genes that were higher expressed with age. The expression levels of the enriched predicted targets RIMS2 and IGSF1 were negatively correlated with both miR-146a-5p and miR-146b-5p. RIMS2 was present in the enriched process, i.e. positive regulation of synaptic transmission. In conclusion, genes decreased with ageing are involved in several of the ageing hallmarks. Genes higher expressed with ageing were involved in synapse-related processes, of which RIMS2 is potentially regulated by two age-related miRNAs.

Individual Differences in Heparin Sensitivity and Their Effect on Heparin Anticoagulation During Arterial Vascular Surgery.

OBJECTIVES: To investigate whether a fixed heparin dose results in adequate heparinisation levels and consequent inhibition of haemostatic activation in all patients. METHODS: This prospective clinical pilot study included 24 patients undergoing arterial vascular surgery. Individual heparin responsiveness was assessed using the Heparin Dose Response (HDR) test, while the activated clotting time (ACT) and heparin concentration were measured to monitor the peri-procedural degree of anticoagulation. Finally, peri-operative haemostasis was evaluated with rotational thromboelastometry (ROTEM). RESULTS: Eight patients were identified with reduced heparin sensitivity (RS group) and 16 patients with normal heparin sensitivity (NS group). Compared with the NS group, the RS group showed less prolonged ACTs after heparinisation with heparin concentrations below the calculated target heparin concentration. ROTEM revealed shorter clot formation times in the intrinsically activated coagulation test (INTEM) 3 min (114 ± 48 s vs. 210 ± 128 s) and 30 min after the initial heparin bolus (103 ± 48 s vs. 173 ± 81 s) in the RS group compared with the NS group. In the RS group, one patient developed a major thromboembolic complication. CONCLUSIONS: This study shows that a third of the study population had reduced heparin sensitivity, which was associated with lower levels of heparinisation, and lower inhibition levels of clot initiation and clot formation. Identifying patients with reduced heparin sensitivity by monitoring the anticoagulant effect of heparin could decrease the risk of thrombotic complications after arterial vascular surgery.

Concurrent spectrometry of annihilation radiation and characteristic gamma-rays for activity assessment of selected positron emitters.

A method is described to determine the activity of non-pure positron emitters in a radionuclide production environment by assessing the 511keV annihilation radiation concurrently with selected γ-lines, using a single High-Purity Germanium (HPGe) detector. Liquid sources of 22Na, 52Fe, 52mMn, 61Cu, 64Cu, 65Zn, 66Ga, 68Ga, 82Rb, 88Y, 89Zr and 132Cs were prepared specifically for this study. Acrylic absorbers surrounding the sources ensured that the emitted ß+-particles could not escape and annihilate away from the source region. The absorber thickness was matched to the maximum ß+ energy for each radionuclide. The effect on the 511keV detection efficiency by the non-homogeneous distribution of annihilation sites inside the source and absorber materials was investigated by means of Monte Carlo simulations. It was found that no self-absorption corrections other than those implicit to the detector calibration procedure needed to be applied. The medically important radionuclide, 64Cu, is of particular interest as its strongest characteristic γ-ray has an intensity of less than 0.5%. In spite of the weakness of its emission intensity, the 1346keV γ-line is shown to be suitable for quantifying the 64Cu production yield after chemical separation from the target matrix has been performed.

Production of (28)Mg by bombardment of (nat)Cl with 200MeV protons: Proof-of-concept study for a stacked LiCl target.

A stacked target consisting of ten Al-encapsulated LiCl discs, for producing (28)Mg via the (nat)Cl(p,X)(28)Mg process in the energy region 50-200MeV, is described. This target was irradiated with a 200MeV beam at an intensity of 100nA, providing information on both yield and outscattering losses. Results of a Monte Carlo modelling of the beam and target, by means of the code MCNPX, are also presented. Similar Al-encapsulated LiCl discs were individually irradiated with 66MeV proton beams of 65 and 90µA, respectively, to study their behaviour under high-intensity bombardment. Once removed from the Al encapsulation, the (28)Mg can be separated from the LiCl target material efficiently, using a 12.5cm x 1cm(2) column containing Purolite S950 chelating resin. The eluate contains (7)Be but no other measurable radio-contaminants. The removal of the (7)Be contaminant is performed by cation exchange chromatography in malate media, with (28)Mg being retained by the resin and (7)Be eluted.

Shared Decision Making in Vascular Surgery: An Exploratory Study.

OBJECTIVES: Shared decision making (SDM) is a process in which patients and their doctors collaborate in choosing a suitable treatment option by incorporating patient values and preferences, as well as the best available evidence. Particularly in vascular surgery, several conditions seem suitable for SDM because there are multiple treatment options. The objective of this study was to assess the degree of SDM behaviour in vascular surgery. METHODS: Vascular surgeons of four Dutch hospitals selected consultations with patients who were facing a treatment decision. Immediately after the consultation, patients and surgeons completed the (subjective) SDM Q-9 and SDM Q-doc questionnaires respectively, to appreciate the perceived level of SDM behaviour. Two evaluators independently and objectively rated SDM behaviour in the audiotaped consultations, using the Observing Patient Involvement (OPTION-12) scale. RESULTS: Nine vascular surgeons and three vascular surgeons in training conducted 54 consultations. The patients' median SDM Q-9 score was high, 93% (IQR 79-100%), and 16/54 (29.6%) of them gave the maximum score. The surgeons' median score was also high, 84% (IQR 73-92%), while 4/54 (7.4%) gave the maximum score. In contrast, mean OPTION score was 31% (SD 11%). Surgeons hardly ever asked the patients for their preferred approach to receive information, whether they had understood the provided information, and how they would like to be involved in SDM. CONCLUSIONS: Currently, objective SDM behaviour among vascular surgeons is limited, even though the presented disorders allow for SDM. Hence, SDM in vascular surgical consultations could be improved by increasing the patients' and surgeons' awareness and knowledge about the concept of SDM.

Flies who cannot take the heat: genome-wide gene expression analysis of temperature-sensitive lethality in an inbred line of Drosophila melanogaster.

Fitness decreases associated with inbreeding depression often become more pronounced in a stressful environment. The functional genomic causes of these inbreeding-by-environment (I × E) interactions, and of inbreeding depression in general, are poorly known. To further our understanding of I × E interactions, we performed a genome-wide gene expression study of a single inbred line that suffers from temperature-sensitive lethality. We confirmed that increased differential expression between the thermosensitive line and the control line occurs at the restrictive temperature. This demonstrates that I × E interactions in survival are reflected in similar I × E interactions at the gene expression level. To make an impression of the cellular response associated with the lethal effect, we analysed all functional annotation terms that were overrepresented among the differentially expressed genes. Some sets of differentially expressed genes function in the general stress response, and these are more likely to also be differentially expressed in other studies of inbreeding, inbreeding depression, immunity and heat stress. Other sets of differentially expressed genes are shared with studies of gene expression in inbred lines, but not studies of the response to extrinsic stress, and represent a general transcriptomic signature of inbreeding. Finally, some sets of genes have an annotation that is not reported in other studies. These we consider to be candidates for the genes harbouring the mutations responsible for the thermosensitive phenotype, as these mutations are expected to be unique to this line. These genes may also serve as candidate QTL in studies of thermal tolerance and heat resistance.

Transcriptomic analysis of inbreeding depression in cold-sensitive Drosophila melanogaster shows upregulation of the immune response.

In sexually reproducing species, increased homozygosity often causes a decline in fitness, called inbreeding depression. Recently, researchers started describing the functional genomic changes that occur during inbreeding, both in benign conditions and under environmental stress. To further this aim, we have performed a genome-wide gene expression study of inbreeding depression, manifesting as cold sensitivity and conditional lethality. Our focus was to describe general patterns of gene expression during inbreeding depression and to identify specific processes affected in our line. There was a clear difference in gene expression between the stressful restrictive environment and the benign permissive environment in both the affected inbred line and the inbred control line. We noted a strong inbreeding-by-environment interaction, whereby virtually all transcriptional differences between lines were found in the restrictive environment. Functional annotation showed enrichment of transcripts coding for serine proteases and their inhibitors (serpins and BPTI/Kunitz family), which indicates activation of the innate immune response. These genes have previously been shown to respond transcriptionally to cold stress, suggesting the conditional lethal effect is associated with an exaggerated cold stress response. The set of differentially expressed genes significantly overlapped with those found in three other studies of inbreeding depression, demonstrating that it is possible to detect a common signature across different genetic backgrounds.

Proteomic characterization of a temperature-sensitive conditional lethal in Drosophila melanogaster.

Genetic variation that is expressed only under specific environmental conditions can contribute to additional adverse effects of inbreeding if environmental conditions change. We present a proteomic characterization of a conditional lethal found in an inbred line of Drosophila melanogaster. The lethal effect is apparent as a large increase in early mortality at the restrictive temperature (29 degrees C) as opposed to normal survival at the permissive temperature (20 degrees C). The increased mortality in response to the restrictive temperature is probably caused by a single recessive major locus. A quantitative trait locus (QTL) region segregating variation affecting the lethal effect has been identified, allowing for a separation of primary/causal effects and secondary consequences in the proteome expression patterns observed. In this study, the proteomic response to the restrictive temperature in the lethal-line (L-line) was compared with the response in an inbred-control-line (IC-line) and an outbred-control-line (OC-line). Quantitative protein changes were detected using isobaric tags for relative and absolute quantitation (iTRAQ) two-dimensional liquid chromatography-tandem mass spectrometry. In all, 45 proteins were found to be significantly differently regulated in response to the restrictive temperature in the L-line as compared with the IC-line. No proteins were significantly differently regulated between the IC-line and the OC-line, verifying that differential protein regulation was specific to a genetic defect in the L-line. Proteins associated with oxidative phosphorylation and mitochondria were significantly overrepresented within the list of differentially expressed proteins. Proteins related to muscle contraction were also found to be differentially expressed in the L-line in response to the restrictive temperature, supporting phenotypic observations of moribund muscle hyper-contraction.

Cutaneous lymphoid hyperplasia (pseudolymphoma) in tattoos: a case series of seven patients.

BACKGROUND: Cutaneous lymphoid hyperplasia (CLH or pseudolymphoma) is considered a rare complication occurring in tattoos. We analysed the demographics, clinical features, histological aspects, allergy tests, outcome and therapeutic data of CLH in tattoos performed by a professional in a series of patients. METHODS: We conducted a retrospective chart review of seven patients managed in private practice of dermatology all over France from 2001 to 2007. RESULTS: Reactions occurred within 2 years after tattooing, were non-specific (pruritus, swelling, infiltration) and mainly restricted to red. Photosensitivity was noted in two cases. Histology was characteristic of pseudolymphoma with a T-cell predominance in all cases. Surgery (2 cases) and laser removal (2 cases, CO2 and QS-532 nm) were efficient, while local corticosteroids were not. One patient had spontaneous clinical remission after 2 years of evolution. CONCLUSIONS: This is the largest series of CLH occurring in tattoos. This delayed hypersensitivity reaction may be underestimated and triggered by a specific immunogenic compound of the ink. Its management remains difficult.

Stress specific correlated responses in fat content, Hsp70 and dopamine levels in Drosophila melanogaster selected for resistance to environmental stress.

Studies of adaptation to stressful environments have frequently encountered cross resistance. This has prompted the hypothesis that certain adaptations confer resistance to multiple stressors. Some of the genes and mechanisms conferring stress resistance have been identified, however, the generality and basis of stress adaptation and cross resistance is still unclear. We investigated several physiological traits that have been previously linked to increased stress resistance: Hsp70 expression, fat content and dopamine levels. Additionally, we studied a behavioural trait, locomotor activity, as a proxy for the physiological state of the organisms. Physiology is the mechanistic link between resistance phenotype and underlying genetic background, and provides insights into the background and generality of cross resistance and correlated responses to selection for stress resistance. We assessed the relationship between the measured traits and stress resistance in a set of lines selected for increased resistance to several environmental stressors. We found that, although all physiological traits displayed significant differentiation among selection regimes, none were consistently associated with increased general stress resistance. This demonstrates that directional changes in Hsp70 expression level, dopamine level and fat content occur in response to the specific requirements of the different stress regimes, rather than as a general response to stress.

QTL mapping of inbreeding-related cold sensitivity and conditional lethality in Drosophila melanogaster.

Inbreeding depression is a central theme within genetics, and is of specific interest for researchers within evolutionary and conservation genetics and animal and plant breeding. Inbreeding effects are thought to be caused by the joint expression of conditional and unconditional deleterious alleles. Whenever the expression of deleterious alleles is conditional, this can result in extreme environmental sensitivity in certain inbred lineages. Analysis of conditional lethal effects can reveal some of the loci that are sensitive to inbreeding. We performed a QTL (quantitative trait locus) mapping study of inbreeding-related and conditionally expressed lethality in Drosophila melanogaster. The lethal effect was triggered by exposure to a cold shock. We used a North Carolina crossing Design 3 to establish the mapping population, as well as to estimate the average dominance ratio and heritability. We found two QTL on the major autosomes carrying recessive lethals that caused male mortality, one of which also affected female mortality. More detailed study of these loci will provide information on the mechanistic basis and environmental sensitivity of inbreeding depression.

Decreased prevalence of dysplasia in high-risk population immigrants in a low-risk area for cervical cancer.

Incidence rates of cervical cancer and its precursors vary considerably, with the highest rates found in developing countries. Differences are influenced by endogenous and exogenous factors. Comparing cytologic abnormality incidence rates from a high-risk population in the original high-risk area, with those of women from this high-risk population who have immigrated to a low-risk area could give insight in the significance of endogenous versus environmental factors. Smears collected from Surinamese women attending the Surinamese screening program and smears collected from immigrant Surinamese women attending the Dutch screening program were cytologically analyzed using the Dutch microscopical coding system KOPAC. Statistical analysis was performed by using logistic regression to calculate (age-adjusted) odds ratios (ORs). The age-adjusted ORs of having dysplasia were higher for Surinamese women living in Suriname versus Surinamese immigrant women and increased with increasing P-scores: 0.77 (0.31-1.91) for borderline changes, 1.62 (0.58-4.57) for mild dysplasia, and 3.20 (1.55-6.60) for moderate to severe dysplasia/neoplasia. We conclude that fewer cases with dysplasia are present in a high-risk population that has immigrated to a low-risk area for cervical cancer than in the high-risk population continuously living in a high-risk area. This finding emphasizes the importance of environmental factors.

Longevity and the stress response in Drosophila.

The concept that lifespan is a function of the capacity to withstand extrinsic stress is very old. In concordance with this, long-lived individuals often have increased resistance against a variety of stresses throughout life. Genes underlying the stress response may therefore have the ability to affect lifespan. The progress in modern genetic techniques has allowed researchers to test this idea. The general stress response involves the expression of stress proteins, such as chaperones and antioxidative proteins, downregulation of genes involved in energy metabolism and the release of protective substances. Do these same changes in patterns of expression have the ability to mitigate ageing and prolong lifespan? It appears that parts of this response indeed are also associated with extended longevity, whereas some elements are not, due to their high cost or long-term deleterious consequences. Here we briefly review the state of the art of research on ageing and longevity in the model organism Drosophila, with focus on the role of the general stress response. We will conclude by contemplating some of the implications of the findings in this research and will suggest several directions for future research.

Developmental and age-specific effects of selection on divergent virgin life span on fat content and starvation resistance in Drosophila melanogaster.

Investigations into the genetic basis of longevity variation have shown life span to be positively correlated with starvation resistance and negatively with female fecundity, both of which rely on lipid content. To assess the firmness of this relation, we assayed correlated responses in age-specific relative fat content (RFC) and starvation resistance in lines successfully selected for divergent virgin life span. We have previously demonstrated that genetic differentiation in female fecundity between our selection lines had disappeared during relaxation of selection. Therefore, we also expected genetic differences in lipid content and starvation resistance to have disappeared. However, RFC and starvation resistance were still significantly lower in short-lived flies than in control flies. Surprisingly, also in long-lived flies RFC and starvation resistance were mostly, but not invariably, found to be significantly lower than in control flies. These results indicate that the genetic correlation of RFC and starvation resistance with reproduction has broken down. Furthermore, the relationship between life span and starvation resistance appears to be more complex than previously anticipated. Also, we could demonstrate that differences in RFC were not brought about by differences in lipid accumulation during adult life, but were already present at eclosion. These findings suggest that pre-adult developmental pathways already impact on the rate of ageing of the adult fly.

Candida and squamous (pre)neoplasia of immigrants and Dutch women as established in population-based cervical screening.

The objective of this study was to establish the relationship between Candida vaginalis and (pre)neoplasia and the prevalence of Candida and (pre)neoplasia related to age and ethnicity. Data were collected from 445,671 asymptomatic women invited for mass screening between 1995 and 2002 and coded according to the Dutch cervical smear coding system (KOPAC) with six grades for (pre)neoplastic changes. Prevalence and relative risks (RRs) were established for Candida and squamous abnormalities in Dutch women and four groups of immigrants. The prevalence of Candida is significantly higher in the cohort of 30-year-old women and lower in the cohorts of 45-, 50-, 55-, and 60-year-old women. The RR of having Candida was higher for Surinamese women (1.24; CI 1.08-1.42). Furthermore, the RR of having mild dysplasia was higher for Surinamese women (1.47; CI 1.14-1.89) and for women born in other countries than in The Netherlands, Turkey, and Morocco (1.36; CI 1.13-1.62). No statistically significant relationship between (pre)neoplasia and Candida was observed. C. vaginalis is more frequent among Surinamese women. Presence of Candida is not associated with an increased risk for squamous abnormalities; therefore, women carrying Candida are not at an increased risk of developing cervical cancer.

Occurrence of polyfunctional thiols in fresh lager beers.

Polyfunctional thiols are known to have a strong impact on the overall aroma of many fermented foods. Surprisingly, very little data is available on their occurrence in beer. A specific extraction with p-hydroxymercuribenzoic acid was performed on four different fresh light-protected lager beers. gas chromatography-olfactometry, gas chromatography-mass spectrometry, and gas chromatography-pulsed-flame photometer detector analyses of the extracts revealed the presence of more than 10 polyfunctional thiols. All of them were absent from wort, suggesting a key role of the H(2)S excreted by yeasts. 3-Methyl-2-buten-1-thiol, 2-mercapto-3-methylbutanol, 3-mercapto-3-methylbutanol seem to be created from hop allylic alcohols via four different mechanisms: nucleophilic substitution, addition-elimination, and radical anti-Markovnikov or electrophilic Markovnikov additions. 1,4 Addition of hydrogen sulfide to wort alpha,beta-unsaturated aldehydes or ketones may explain the synthesis of 1-mercapto-3-pentanol, 3-mercaptohexanol, and 4-mercapto-4-methyl-2-pentanone through fermentation. Finally, 2-mercaptoethanol, 3-mercaptopropanol, and their corresponding acetates may derive from Ehrlich degradation of sulfur amino acids, while 2-methyl-3-furanthiol should be logically issued from Maillard reactions.