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Abstract To assess if SARS-CoV-2 (COVID-19) systemic disease can be determined by available nucleoprotein assays, we compared the performance of three commercial SARS-CoV-2 nucleoprotein (N) assays in plasma. A total of 272 plasma samples collected in the period November–December 2021 were analyzed by the methods Simoa SARS CoV‐2 N Protein Advantage Kit [Quanterix Simoa], Solsten SARS-CoV-2 Antigen enzyme immunosorbent assay (ELISA) [Solsten ELISA], and Elecsys SARS‐CoV‐2 Antigen electrochemiluminescence immunoassay [Elecsys ECLIA]. Additionally, a dilution series of inactivated virus culture was analyzed by the three assays. The SARS CoV-2 PCR-status was not known for the patients. Linear correlation in the pairwise correlation between assays as well as linearity of dilution series of inactivated virus culture was estimated by Spearman score. Sensitivity and specificity were estimated by pairwise comparison. The three assays showed poor agreement on patient samples with regards to concentration. Performance on virus culture was excellent but with different level of detection (LOD). Positive vs negative results show comparable sensitivity and specificity of Quanterix Simoa and Solsten ELISA, with a higher LOD in Elecsys ECLIA and thus lower sensitivity and high specificity. N by all tested assays can be used as a marker for systemic COVID-19 disease.

Abstract This study aimed to develop a highly sensitive SARS-CoV-2 nucleocapsid antigen assay using the single molecule array (Simoa) technology and compare it with real time RT-PCR as used in routine clinical practice with the ambition to achieve a comparative technical and clinical sensitivity. Samples were available from 148 SARS-CoV-2 real time RT-PCR positive and 73 SARS-CoV-2 real time RT-PCR negative oropharyngeal swabs. For determination of technical sensitivity SARS-CoV-2 virus culture material was used. The samples were treated with lysis buffer and analyzed using both an in-house and a pre-commercial SARS-CoV-2 nucleocapsid antigen assay on Simoa. Both nucleocapsid antigen assays have a technical sensitivity corresponding to around 100 SARS-CoV-2 RNA molecules/mL. Using a cut-off at 0.1 pg/mL the pre-commercial SARS-CoV-2 nucleocapsid antigen assay had a sensitivity of 96% (95% CI 91.4–98.5%) and specificity of 100% (95% CI 95.1–100%). In comparison the in-house nucleocapsid antigen assay had sensitivity of 95% (95% CI 89.3–98.1%) and a specificity of 100% (95% CI 95.1–100%) using a cut-off at 0.01 pg/mL. The two SARS-CoV-2 nucleocapsid antigen assays correlated with r = 0.91 (P

According to current recommendations, blood samples should be taken in the morning after 15 minutes' resting time. Some components exhibit diurnal variation and in response to pressures to expand opening hours and reduce waiting time, the aims of this study were to investigate the impact of resting time prior to blood sampling and diurnal variation on biochemical components, including albumin, thyrotropin (TSH), total calcium and sodium in plasma.All patients referred to an outpatient clinic for blood sampling were included in the period Nov 2011 until June 2014 (opening hours: 7am-3pm). Each patient's arrival time and time of blood sampling were registered. The impact of resting time and the time of day for all components was analysed using simple linear regression. The "maximum allowable bias" was used as quality indicator for the change in reference interval.Significant diurnal variation was found for albumin (n = 15,544; p

Next-generation sequencing technology has increased the capacity to generate molecular data for plant biological research, including phylogenetics, and can potentially contribute to resolving complex phylogenetic problems. The evolutionary history of Medicago L. (Leguminosae: Trifoliae) remains unresolved due to incongruence between published phylogenies. Identification of the processes causing this genealogical incongruence is essential for the inference of a correct species phylogeny of the genus and requires that more molecular data, preferably from low-copy nuclear genes, are obtained across different species. Here we report the development of 50 novel LCN markers in Medicago and assess the phylogenetic properties of each marker. We used the genomic resources available for Medicago truncatula Gaertn., hybridisation-based gene enrichment (sequence capture) techniques and Next-Generation Sequencing to generate sequences. This alternative proves to be a cost-effective approach to amplicon sequencing in phylogenetic studies at the genus or tribe level and allows for an increase in number and size of targeted loci. Substitution rate estimates for each of the 50 loci are provided, and an overview of the variation in substitution rates among a large number of low-copy nuclear genes in plants is presented for the first time. Aligned sequences of major species lineages of Medicago and its sister genus are made available and can be used in further probe development for sequence-capture of the same markers.

Abstract Diagnosing patients in the medical emergency department is complex and this is expected to increase in many countries due to an ageing population. In this study we investigate the feasibility of training machine learning algorithms to assist physicians handling the complex situation in the medical emergency departments. This is expected to reduce diagnostic errors and improve patient logistics and outcome. We included a total of 9,190 consecutive patient admissions diagnosed and treated in two hospitals in this cohort study. Patients had a biochemical workup including blood and urine analyses on clinical decision totaling 260 analyses. After adding nurse-registered data we trained 19 machine learning algorithms on a random 80% sample of the patients and validated the results on the remaining 20%. We trained algorithms for 19 different patient outcomes including the main outcomes death in 7 (Area under the Curve (AUC) 91.4%) and 30 days (AUC 91.3%) and safe-discharge(AUC 87.3%). The various algorithms obtained areas under the Receiver Operating Characteristics -curves in the range of 71.8–96.3% in the holdout cohort (68.3–98.2% in the training cohort). Performing this list of biochemical analyses at admission also reduced the number of subsequent venipunctures within 24 h from patient admittance by 22%. We have shown that it is possible to develop a list of machine-learning algorithms with high AUC for use in medical emergency departments. Moreover, the study showed that it is possible to reduce the number of venipunctures in this cohort.

Abstract To improve prostate cancer (PCa) diagnosis, it is imperative to identify novel biomarkers and establish effective screening techniques. Here, we introduce electrochemical biosensing of β-2-Microglobulin (β2M) in urine as a potential diagnostic tool for PCa. The immunosensor is composed of a screen-printed graphene electrode coated with anti β2M antibodies. The sensor is capable of detecting the protein directly in urine without any sample pretreatment within 45 min including sample incubation and a lower limit of detection of 204 µg/L. The sensor demonstrated a significant difference in the β2M-creatinine ratio in urine between control and both local- and metastatic PCa (mPCa) (P = 0.0302 and P = 0.0078 respectively), and between local- and mPCa (P = 0.0302). This first example of electrochemical sensing of β2M for the diagnosis of PCa may set the stage for an affordable, on-site screening technique for PCa.

Abstract Neoadjuvant treatment of breast cancer is applied to an increasing extent, but treatment response varies and side effects pose a challenge. The vitamin E isoform delta-tocotrienol might enhance the efficacy of chemotherapy and reduce the risk of side effects. The aim of this study was to investigate the clinical effect of delta-tocotrienol combined with standard neoadjuvant treatment and the possible association between detectable circulating tumor DNA (ctDNA) during and after neoadjuvant treatment with pathological treatment response. This open-label, randomized phase II trial included 80 women with newly diagnosed, histologically verified breast cancer randomized to standard neoadjuvant treatment alone or in combination with delta-tocotrienol. There was no difference in the response rate or frequency of serious adverse events between the two arms. We developed a multiplex digital droplet polymerase chain reaction (ddPCR) assay for the detection of ctDNA in breast cancer patients that targets a combination of two methylations specific for breast tissue (LMX1B and ZNF296) and one cancer specific methylation (HOXA9). The sensitivity of the assay increased when the cancer specific marker was combined with the ones specific to breast tissue (p

The TYK2 gene is associated with development of type 1 diabetes. Here the authors show that TYK2 regulates β-cell development, but at the same time TYK2 inhibition in the islets prevents IFNα responses and enhances their survival against CD8+ T-cell cytotoxicity; representing a potent therapeutic target to halt T1D progression.

Insulin gene mutations are a leading cause of neonatal diabetes. They can lead to proinsulin misfolding and its retention in endoplasmic reticulum (ER). This results in increased ER-stress suggested to trigger beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. Here we show that misfolded proinsulin impairs developing beta-cell proliferation without increasing apoptosis. We generated induced pluripotent stem cells (iPSCs) from people carrying insulin (INS) mutations, engineered isogenic CRISPR-Cas9 mutation-corrected lines and differentiated them to beta-like cells. Single-cell RNA-sequencing analysis showed increased ER-stress and reduced proliferation in INS-mutant beta-like cells compared with corrected controls. Upon transplantation into mice, INS-mutant grafts presented reduced insulin secretion and aggravated ER-stress. Cell size, mTORC1 signaling, and respiratory chain subunits expression were all reduced in INS-mutant beta-like cells, yet apoptosis was not increased at any stage. Our results demonstrate that neonatal diabetes-associated INS-mutations lead to defective beta-cell mass expansion, contributing to diabetes development.

Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2-6.8% and 10.5-31.4%, respectively. Using droplet digital PCR the intra-assay variation was 4.4-20.1%, and the inter-assay variation 5.7-26.7%. Plasma preparation and microRNA purification were found to account for 39-73% of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability.

beta-Catenin is a multifunctional protein involved in both signalling by secreted factors of Wnt family and regulation of the cellular architecture. We show that beta-catenin stabilization in mouse midbrain-rhombomere 1 region leads to robust up-regulation of several Wnt signalling target genes, including Fgf8. Suggestive of direct transcriptional regulation of the Fgf8 gene, beta-catenin stabilization resulted in Fgf8 up-regulation also in other tissues, specifically in the ventral limb ectoderm. Interestingly, stabilization of beta-catenin rapidly caused down-regulation of the expression of Wnt1 itself, suggesting a negative feedback loop. The changes in signal molecule expression were concomitant with deregulation of anterior-posterior and dorso-ventral patterning. The transcriptional regulatory functions of beta-catenin were confirmed by beta-catenin loss-of-function experiments. Temporally controlled inactivation of beta-catenin revealed a cell-autonomous role for beta-catenin in the maintenance of cell-type specific gene expression in the progenitors of midbrain dopaminergic neurons. These results highlight the role of beta-catenin in establishment of neuroectodermal signalling centers, promoting region-specific gene expression and regulation of cell fate determination.

Conditional gene targeting strategies are progressively used to study gene function tissue-specifically and/or at a defined time period. Instrumental to all of these strategies is the generation of targeting vectors, and any methodology that would streamline the procedure would be highly beneficial. We describe a comprehensive transposition-based strategy to produce gene-targeting vectors for the generation of mouse conditional alleles. The system employs a universal cloning vector and two custom-designed mini-Mu transposons. It produces targeting constructions directly from BAC clones, and the alleles generated are modifiable by Cre and Flp recombinases. We demonstrate the applicability of the methodology by modifying two mouse genes, Chd22 and Drapc1. This straightforward strategy should be readily suitable for high-throughput targeting vector production.

The thioredoxin-dependent system is an essential regulator of cellular redox balance. Since oxidative stress has been linked with neurodegenerative disease, we studied the roles of thioredoxin reductases in brain using mice with nervous system (NS)-specific deletion of cytosolic (Txnrd1) and mitochondrial (Txnrd2) thioredoxin reductase. While NS-specific Txnrd2 null mice develop normally, mice lacking Txnrd1 in the NS were significantly smaller and displayed ataxia and tremor. A striking patterned cerebellar hypoplasia was observed. Proliferation of the external granular layer (EGL) was strongly reduced and fissure formation and laminar organisation of the cerebellar cortex was impaired in the rostral portion of the cerebellum. Purkinje cells were ectopically located and their dendrites stunted. The Bergmann glial network was disorganized and showed a pronounced reduction in fiber strength. Cerebellar hypoplasia did not result from increased apoptosis, but from decreased proliferation of granule cell precursors within the EGL. Of note, neuron-specific inactivation of Txnrd1 did not result in cerebellar hypoplasia, suggesting a vital role for Txnrd1 in Bergmann glia or neuronal precursor cells.