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Fluorescent Cell Barcoding (FCB) is a flow cytometric technique which has been used for assessing signaling proteins. This FCB technique has the potential to be applied in other multiparameter analyses. Since data on antigen (Ag)-specific T-cell immune responses, like intracellular cytokine production, are still lacking in infants because limited blood volumes can be obtained for analysis, the FCB technique could be very useful for this purpose. The objectives of this study were to modify the FCB method to be able to measure multiple Ag-specific cytokine reponses in T-cells upon simultaneous stimulation by various antigens and mitogens in small amounts of blood and to investigate the cytokine pattern of T-cell subsets in healthy infants aged six and twelve months. Blood samples, collected from 20 healthy infants aged six and twelve months, were stimulated in vitro with the antigens: phorbol-myristate-acetate (PMA), purified-protein-derivative (PPD), Tetanus-toxoid (TT), Staphylococcal-enterotoxin-B (SEB), and phytohemagglutinin (PHA). Each stimulus was barcoded by labelling with different intensities of fluorescent cell barcoding (FCB) markers. Intracellular production of interleukin-2, interferon-gamma, and tumor necrosis factor-alpha was measured simultaneously in just one blood sample of 600 µl whole blood. Significant age-related differences in cytokine production were shown for PMA, PHA, and TT in CD4(+) T-cells, and for PMA, PHA, SEB, and TT in CD8(+) T-cells. The intracellular cytokine production by CD4(+) and CD8(+) T-cells was higher at twelve months compared to six months of age for all antigens, except for PMA, which was lower at the age of twelve months. Based on the consistency in both T-cell subsets, we conclude that the new FCB method is a promising tool to investigate the age-related development of intracellular cytokine production in infants.

This is a follow up study of a multicenter randomised placebo-controlled trial in seven centres in five West European countries. The RCT assessed the effect of infant formula supplemented with a mixture of prebiotics (with neutral short-chain and long-chain oligosaccharides and pectin-derived acidic oligosaccharides) during infancy in term-born children (n=1130). In the follow-up study 672 children (60% of the study population) participated: 232 (56%) from the prebiotics group (PG), 243 (58%) from the control group (CG), and 197 (66%) from the non-randomised breast-fed group (BG). The primary outcome was the occurrence of febrile episodes at three to five years of age prospectively documented by the parents: in the PG 1.17 (interquartile range 0.50-2.08) episodes per year versus 1.20 (0.52-2.57) in the CG; and 1.48 (0.65-2.60) in the BG. This specific prebiotics mixture given during infancy in healthy non-atopic subjects does not decrease febrile episodes and therefore seems not to prevent infection between their third and fifth birthday.

► We studied 164 infants in a RCT who were randomized to prebiotics or placebo added to formula feeding. ► We examined their immunoglobulin-G concentrations specific to Hib and tetanus immunizations. ► Prebiotics supplementation has no effect on vaccination specific antibody levels in young infants. Previous studies have shown, that prebiotics can modulate the immune response in infants at risk for allergy, leading to a lower incidence of atopic dermatitis. Few studies have evaluated the effect of prebiotic carbohydrates alone on the vaccine-specific antibody response as a marker for the development of the immune system in healthy infants not at risk for allergy. This study evaluates the effect of adding a specific prebiotic mixture of short chain galacto-oligosaccharides (scGOS)/long chain fructo-oligosaccharides (lcFOS) ratio 9:1 and pectin-derived acidic oligosaccharides (pAOS) to formula feeding on the specific immunoglobulin responses to Haemophilus influenza type b (Hib) and tetanus immunization in healthy non-atopic infants during the first year of life. This substudy has been embedded in a multinational multicenter RCT (n=1130 children) to evaluate the effect of study prebiotics on the incidence of fever episodes during the first year of life. The study prebiotics were administered throughout the first year of life. This is a substudy on the vaccine-specific immunoglobulin responses to Hib and tetanus immunizations. Only data of the Dutch children, 80 in the prebiotics group and 84 in the control group, were used for this substudy. They all followed the national vaccination schedule leading to a homogeneous group. Blood was sampled at 6 and 12months of age. Hib immunizations: median values did not differ between groups at the age of 6 and 12months. At the age of 12months, 34 out of 37 (91.9%) infants in the prebiotics group and 31 out of 34 infants (91.2%) in the control group had Hib antibody levels >1.0μg/ml. Tetanus immunizations: median values did not differ between groups at the age of 6 and 12months and were above the cut-off value of 0.1IU/ml in all infants in both the prebiotics and the control group. No effect of prebiotics supplementation on vaccination specific antibody levels was found in children up to the age of 12months; the vaccine specific antibody levels in infants fed the study prebiotics or a control diet were similar during the first year of life. We hypothesize that this specific prebiotic mixture, which resembles the composition of oligosaccharides in human milk, mainly promotes Th1 and Treg dependent immune responses and induces a down regulation of IgE-mediated allergic responses, while the desired vaccine-specific serum antibody responses remain intact.

Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment. Fusion gene partner choice and breakpoint-position promiscuity restricts diagnostic detection, even for known and recurrent configurations. Here, we develop FUDGE (FUsion Detection from Gene Enrichment) to accurately and impartially identify fusions. FUDGE couples target-selected and strand-specific CRISPR-Cas9 activity for fusion gene driver enrichment — without prior knowledge of fusion partner or breakpoint-location — to long read nanopore sequencing with the bioinformatics pipeline NanoFG. FUDGE has flexible target-loci choices and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in one sequencing run. We observe on-average 665 fold breakpoint-site enrichment and identify nucleotide resolution fusion breakpoints within 2 days. The assay identifies cancer cell line and tumor sample fusions irrespective of partner gene or breakpoint-position. FUDGE is a rapid and versatile fusion detection assay for diagnostic pan-cancer fusion detection. Fusion genes are a hallmarks of cancer, though breakpoint-position promiscuity restricts diagnostic detection. Here, the authors present FUDGE, a CRISPR-Cas9-based enrichment strategy for nanopore sequencing to identify target fusions irrespective of genomic breakpoint or fusion partner.

Wild tomato species, like Solanum chilense, are important germplasm resources for enhanced biotic and abiotic stress resistance in tomato breeding. S. chilense also serves as a model to study adaptation of plants to drought and the evolution of seed banks. The absence of a well-annotated reference genome in this compulsory outcrossing, very diverse species limits in-depth studies on the genes involved. We generated ∼134 Gb of DNA and 157 Gb of RNA sequence data for S chilense, which yielded a draft genome with an estimated length of 914 Mb, encoding 25,885 high-confidence predicted gene models, which show homology to known protein-coding genes of other tomato species. Approximately 71% of these gene models are supported by RNA-seq data derived from leaf tissue samples. Benchmarking with Universal Single-Copy Orthologs (BUSCO) analysis of predicted gene models retrieved 93.3% of BUSCO genes. To further verify the genome annotation completeness and accuracy, we manually inspected the NLR resistance gene family and assessed its assembly quality. We find subfamilies of NLRs unique to S. chilense. Synteny analysis suggests significant degree of the gene order conservation between the S. chilense, S. lycopersicum and S. pennellii genomes. We generated the first genome and transcriptome sequence assemblies for the wild tomato species Solanum chilense and demonstrated their value in comparative genomics analyses. These data are an important resource for studies on adaptation to biotic and abiotic stress in Solanaceae, on evolution of self-incompatibility and for tomato breeding.

Abstract Background Large-scale use of mRNA COVID-19 vaccines during the pandemic was associated with enhanced safety monitoring to ensure accurate and timely review of safety. We reviewed the mRNA-1273 (original strain) safety profile following 2 years of use (>772 million administered doses), primarily focusing on predefined safety topics (ie, adverse events of special interest [AESIs]) proposed in advance of COVID-19 vaccine use. Methods Cumulative mRNA-1273 safety data were included from spontaneous adverse event (AE) cases reported to Moderna’s global safety database between 18 December 2020 and 17 December 2022. Reporting rates of AESIs were calculated per 1 million doses of mRNA-1273 administered. Observed-to-expected (OE) ratios were computed by comparing observed rates of AESIs with the background/expected rate for these events to evaluate potential associations with mRNA-1273. Results There were 658 759 identified case reports associated with 2 517 669 AEs. Most AEs were nonserious (83.4%; 2 098 954/2 517 669). Overall 0.7% (17 751/2 517 669) were fatal. AESIs represented 13.7% of all AEs (344 921/2 517 669), with reporting rates for most AESIs below the expected background incidence. Exceptions included anaphylaxis (OE ratio 3 days after vaccination, 2.09; 95% CI, 1.93–2.25) and, among individuals aged 12 to 40 years, myocarditis (OE ratio 7 days after any dose, 3.89 [3.50–4.32]; among men after dose 2, 8.57 [6.88–10.68]) and pericarditis (OE ratio 7 days after vaccination, 3.47; 2.89–4.16). Conclusions This safety analysis of mRNA-1273 identified evidence of increased risk for anaphylaxis, myocarditis, and pericarditis but not for other AESIs identified for enhanced monitoring ahead of COVID-19 vaccine use. This analysis evaluated observed-to-expected ratios for adverse events of special interest that were proposed in advance of COVID-19 vaccine use. Following administration of an estimated 772 908 958 doses of mRNA-1273, meaningful associations were found only for anaphylaxis, myocarditis, and pericarditis.

Relapse remains a major challenge in the clinical management of acute myeloid leukemia (AML) and is driven by rare therapy‐resistant leukemia stem cells (LSCs) that reside in specific bone marrow niches. Hypoxia signaling maintains cells in a quiescent and metabolically relaxed state, desensitizing them to chemotherapy. This suggests the hypothesis that hypoxia contributes to the chemoresistance of AML‐LSCs and may represent a therapeutic target to sensitize AML‐LSCs to chemotherapy. Here, we identify HIFhigh and HIFlow specific AML subgroups (inv(16)/t(8;21) and MLLr, respectively) and provide a comprehensive single‐cell expression atlas of 119,000 AML cells and AML‐LSCs in paired diagnostic‐relapse samples from these molecular subgroups. The HIF/hypoxia pathway signature is attenuated in AML‐LSCs compared with more differentiated AML cells but is more expressed than in healthy hematopoietic cells. Importantly, chemical inhibition of HIF cooperates with standard‐of‐care chemotherapy to impair AML growth and to substantially eliminate AML‐LSCs in vitro and in vivo. These findings support the HIF pathway in the stem cell‐driven drug resistance of AML and unravel avenues for combinatorial targeted and chemotherapy‐based approaches to specifically eliminate AML‐LSCs.

Allo-HSCT with CCR5Δ32/Δ32 donor cells is the only curative HIV-1 intervention. We investigated the impact of allo-HSCT on the viral reservoir in PBMCs and post-mortem tissue in two patients. IciS-05 and IciS-11 both received a CCR5Δ32/Δ32 allo-HSCT. Before allo-HSCT, ultrasensitive HIV-1 RNA quantification; HIV-1-DNA quantification; co-receptor tropism analysis; deep-sequencing and viral characterization in PBMCs and bone marrow; and post-allo-HSCT, ultrasensitive RNA and HIV-1-DNA quantification were performed. Proviral quantification, deep sequencing, and viral characterization were done in post-mortem tissue samples. Both patients harbored subtype B CCR5-tropic HIV-1 as determined genotypically and functionally by virus culture. Pre-allo-HSCT, HIV-1-DNA could be detected in both patients in bone marrow, PBMCs, and T-cell subsets. Chimerism correlated with detectable HIV-1-DNA LTR copies in cells and tissues. Post-mortem analysis of IciS-05 revealed proviral DNA in all tissue biopsies, but not in PBMCs. In patient IciS-11, who was transplanted twice, no HIV-1-DNA could be detected in PBMCs at the time of death, whereas HIV-1-DNA was detectable in the lymph node. In conclusion, shortly after CCR5Δ32/Δ32, allo-HSCT HIV-1-DNA became undetectable in PBMCs. However, HIV-1-DNA variants identical to those present before transplantation persisted in post-mortem-obtained tissues, indicating that these tissues play an important role as viral reservoirs.

Familial hemiplegic migraine is a rare autosomal dominant subtype of migraine with aura. Three genes have been identified, all involved in ion transport. There is considerable clinical variation associated with FHM mutations. Genotype–phenotype correlation studies are needed, but are challenging mainly because the number of carriers of individual mutations is low. One exception is the recurrent T666M mutation in the FHM1 CACNA1A gene that was identified in almost one-third of FHM families and showed variable associated clinical features and severity, both within and among FHM families. Similar studies in the FHM2 ATP1A2 gene have not been performed because of the low number of carriers with individual mutations. Here we report on the recurrence of ATP1A2 mutations M731T and T376M that affect sodium–potassium pump functioning in two Portuguese FHM families. Considerably increasing the number of mutation carriers with these mutations indicated a clear genotype–phenotype correlation: both mutations are associated with pure FHM. In addition, we show that recurrent mutations for ATP1A2 are more frequent than previously thought, which has implications for genotype–phenotype correlations and genetic testing.

Background: Around 15% of patients die or become dependent after cerebral vein and dural sinus thrombosis (CVT). Method: We used the International Study on Cerebral Vein and Dural Sinus Thrombosis (ISCVT) sample (624 patients, with a median follow-up time of 478 days) to develop a Cox proportional hazards regression model to predict outcome, dichotomised by a modified Rankin Scale score >2. From the model hazard ratios, a risk score was derived and a cut-off point selected. The model and the score were tested in 2 validation samples: (1) the prospective Cerebral Venous Thrombosis Portuguese Collaborative Study Group (VENOPORT) sample with 91 patients; (2) a sample of 169 consecutive CVT patients admitted to 5 ISCVT centres after the end of the ISCVT recruitment period. Sensitivity, specificity, c statistics and overall efficiency to predict outcome at 6 months were calculated. Results: The model (hazard ratios: malignancy 4.53; coma 4.19; thrombosis of the deep venous system 3.03; mental status disturbance 2.18; male gender 1.60; intracranial haemorrhage 1.42) had overall efficiencies of 85.1, 84.4 and 90.0%, in the derivation sample and validation samples 1 and 2, respectively. Using the risk score (range from 0 to 9) with a cut-off of ≥3 points, overall efficiency was 85.4, 84.4 and 90.1% in the derivation sample and validation samples 1 and 2, respectively. Sensitivity and specificity in the combined samples were 96.1 and 13.6%, respectively. Conclusions: The CVT risk score has a good estimated overall rate of correct classifications in both validation samples, but its specificity is low. It can be used to avoid unnecessary or dangerous interventions in low-risk patients, and may help to identify high-risk CVT patients.