The OCT2/MATE1 Interaction Between Trifluridine, Metformin and Cimetidine: A Crossover Pharmacokinetic Study.

BACKGROUND AND OBJECTIVES: Trifluridine/tipiracil, registered for the treatment of patients with metastatic gastric and colorectal cancer, is a substrate and inhibitor for the organic cation transporter 2 (OCT2) and the multidrug and toxin extrusion protein 1 (MATE1), which raises the potential for drug-drug interactions with other OCT2/MATE1 modulators. Therefore, we prospectively examined the effect of an OCT2/MATE1 inhibitor (cimetidine) and substrate (metformin) on the pharmacokinetics of trifluridine. METHODS: In this three-phase crossover study, patients with metastatic colorectal or gastric cancer were sequentially treated with trifluridine/tipiracil alone (phase A), trifluridine/tipiracil concomitant with metformin (phase B) and trifluridine/tipiracil concomitant with cimetidine (phase C). The primary endpoint was the relative difference in exposure of trifluridine assessed by the area under the curve from timepoint zero to infinity. A > 30% change in exposure was considered clinically relevant. A p-value of < 0.025 was considered significant because of a Bonferroni correction. RESULTS: Eighteen patients were included in the analysis. Metformin did not significantly alter the exposure to trifluridine (- 12.6%; 97.5% confidence interval - 25.0, 1.8; p = 0.045). Cimetidine did alter the exposure to trifluridine significantly (+ 18.0%; 97.5% confidence interval 4.5, 33.3; p = 0.004), but this increase did not meet our threshold for clinical relevance. Metformin trough concentrations were not influenced by trifluridine/tipiracil. CONCLUSIONS: Our result suggests that the OCT2/MATE1 modulators cimetidine and metformin can be co-administered with trifluridine/tipiracil without clinically relevant effects on drug exposure. CLINICAL TRIAL REGISTRATION: NL8067 (registered 04-10-2019).

Global adrenal insufficiency in two independent patients carrying the same homozygous c.172A>G, p.(Thr58Ala) mutation in the TBX19 gene.

Introduction TBX19 mutations cause isolated ACTH-deficiency. While this classically results in severe hypocortisolism, potential consequences for mineralocorticoid biosynthesis have not been described to date. Liquid chromatography mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS) allow novel insights into the steroid metabolism of pediatric endocrine diseases. Case presentation Patient 1 (female) presented right after birth with hypoglycemia and hyponatremia (minimum sodium 126 mmol/l). She recovered under therapy with hydrocortisone, fludrocortisone and initial NaCl. Patient 2 (male) presented after birth with prolonged cholestatic jaundice. Only at the age of 3.5 months, repeated episodes of hypoglycemia occurred. Both patients showed severely reduced ACTH. LC-MS/MS analyses on plasma samples demonstrated combined reduced glucocorticoid- and mineralocorticoid biosynthesis confirmed by GC-MS analyses on spot urine. In contrast to patient 1, patient 2 (currently 8 years old) never suffered from hyponatremia. Both patients carry the same homozygous c.172A>G, p.(Thr58Ala) mutation in the TBX19 gene proving isolated ACTH-deficiency Conclusion Isolated ACTH-deficiency can be associated with reduced mineralocorticoids and hyponatremia. We hypothesize that sufficient pituitary ACTH-secretion is an important predisposition for regular adrenal mineralocorticoid biosynthesis.

Central slip defect reconstruction utilizing partial ulnar side of flexor digitorum superficial tendon for chronic boutonniere deformity: A case report.

Background: Post-traumatic central slip injuries, resulting in boutonniere deformity, are a complex issue that can significantly impact hand function and quality of life. The deformity should be easily reducible in the acute phase, but if left untreated, it shortens the oblique retinacular ligament, leading to chronic contracture. This is a challenging issue in hand surgery, as chronic central slip defects cannot be sutured like other tendon. Various methods for reconstructing central slips have been discussed, but the best method for subacute or chronic injuries remains unclear. This case presents a case of chronic traumatic boutonniere deformity with central slip defect reconstruction. Case report: A 65-year-old male patient presented with swelling and boutonniere deformity on the digiti III of the right hand. The patient had previously fallen from a motorcycle, and the patient's right middle finger got was by a motorcycle six months ago. After the incident, the patient's right middle finger cannot be fully extended. The patient's right hand showed edema with flexion of the interphalangeal (PIP) joint and hyperextension of the distal interphalangeal (DIP) joint. The Range of Motion (ROM) of the PIP joint right middle finger was 45-110 degrees. The X-ray of the right hand AP/oblique showed no bone involvement in the deformity. The patient underwent central slip defect reconstruction utilizing the partial ulnar side of the flexor digitorum superficial tendon. A PIP joint extension splint was applied for 2 weeks. Active and passive exercise of the ROM of the PIP joint began after 2 weeks of PIP extension joint splinting. The patient's ROM of the PIP joint (0-90 degrees) significantly improved 1 month after surgery. The patient's ROM of the PIP joint returned to normal after 2 months after surgery. The function of the patient's right hand is evaluated with the DASH score, which improves significantly from 50 to 4.2. Conclusion: Central slip defect reconstruction utilizing the partial ulnar side of the flexor digitorum superficial tendon is a reliable method for traumatic chronic boutonniere deformity and results in great functional outcomes after 2 months of follow-up. Central slip defect reconstruction utilizing the partial ulnar side of the flexor digitorum superficial tendon is a simple and cost-effective method compared to other methods.

Branched polymer grafted graphene oxide (GO) as a 2D template for calcium phosphate growth.

HYPOTHESIS: Graphene Oxide (GO)-templated deposition of inorganic materials through synthesis on dispersed single sheets of GO is often complicated by the loss of the desired 2D morphology owing to the coagulation of GO sheets at high salt concentrations and non-templated homogenous nucleation. Modifying GO with anionic polymer is expected to solve both problems by i) enhancing electrostatic(steric) stabilization upon exposure to high concentrations of the ionic precursors, and ii) offering additional nucleation sites at the grafted anionic moieties to avoid homogeneous secondary nucleation. EXPERIMENTS: GO was grafted with branched copolymers of poly(ethylene glycol) methacrylate (PEGMA 500) and diethylene glycol dimethacrylate (DEGDMA) and ω-vinyl terminated methacrylic acid macromonomer (P(MAA)), the latter serving as an addition-fragmentation chain transfer agent. The colloidal stability of GO dispersions in water toward salt was evaluated before and after modification. Precipitation of calcium phosphate (CaP) was performed by incubating modified GO in the precursor solutions. The conditions were optimized to maximize the nucleation selectively onto GO without homogeneous CaP nucleation and coagulation of the GO-sheets. FINDINGS: The copolymer grafted GO-sheets shows superior colloidal stability when dispersed in water. No aggregation occurs in the incubating ionic CaP precursor solutions. The optimum templated deposition of CaP onto the GO sheets by precipitation is to add a second shot of precursors after the nucleation stage to obtain GO sheets fully decorated with calcium phosphate nanorods without self-nucleation. Via the careful design on the GO modification and incubation process, the growth of calcium phosphate nanorods were confined in the desired 2D order exclusively, hereby achieving the goal of an efficient GO-templated synthesis.

Impact of Guideline-Directed Drug Therapy after ST-Elevation Myocardial Infarction on Outcome in Young Patients-Age and Sex-Specific Factors.

Background: Specifically young women are at risk for a poor outcome after ST-elevation myocardial infarction (STEMI). We aimed to investigate sex- and age-specific differences in outcome and associate these results with adherence to a guideline-directed optimal medical therapy (OMT). Methods: Administrative insurance data (≈26 million insured) were screened for patients aged 18-60 years with STEMI. Patient demographics, details on in-hospital treatment, adherence to OMT and its effect on mortality were assessed. Adherence to OMT was analyzed using multistate models and an association of those with death was fitted using multivariable Cox regression models with time-dependent co-variables. Results: Overall, 59,401 patients (19.3% women), median age 52 (interquartile range 48, 56) presented with STEMI. Female sex was associated with a poor outcome early after STEMI (90-day mortality: odds ratio 1.22, 95% confidence interval (CI) 1.12-1.32, p < 0.001). Overall survival was reduced in women compared to same-aged men. The ten-year survival rate was 19.7% (18.1-21.2%) versus 19.6% (18.9-20.4%) in men (p < 0.001). Although long-term drug adherence was low, its intake was associated with a better outcome. Specifically younger women showed a markedly lower mortality when on OMT (hazard ratio (HR) 0.22 (95% CI 0.19-0.26) versus HR 0.31 (95% CI 0.28-0.33) in men, pint < 0.001). Conclusions: Specifically young women were at risk for a poor outcome in the early phase after STEMI. Although long-term adherence to OMT was low, it was generally associated with a lower mortality, specifically in women. Our findings emphasize on early and long-term preventive measures in all patients after STEMI.

Pathlength-selective, interferometric diffuse correlation spectroscopy (PaLS-iDCS).

Diffuse correlation spectroscopy (DCS) is an optical method that offers non-invasive assessment of blood flow in tissue through the analysis of intensity fluctuations in diffusely backscattered coherent light. The non-invasive nature of the technique has enabled several clinical applications for deep tissue blood flow measurements, including cerebral blood flow monitoring as well as tumor blood flow mapping. While a promising technique, in measurement configurations targeting deep tissue hemodynamics, the standard DCS implementations suffer from insufficient signal-to-noise ratio (SNR), depth sensitivity, and sampling rate, limiting their utility. In this work, we present an enhanced DCS method called pathlength-selective, interferometric DCS (PaLS-iDCS), which improves upon both the sensitivity of the measurement to deep tissue hemodynamics and the SNR of the measurement using pathlength-specific coherent gain. Through interferometric detection, PaLS-iDCS can provide time-of-flight (ToF) specific blood flow information without the use of expensive time-tagging electronics and low-jitter detectors. The new technique is compared to time-domain DCS (TD-DCS), another enhanced DCS method able to resolve photon ToF in tissue, through Monte Carlo simulation, phantom experiments, and human subject measurements. PaLS-iDCS consistently demonstrates improvements in SNR (>2x) for similar measurement conditions (same photon ToF), and the SNR improvements allow for measurements at extended photon ToFs, which have increased sensitivity to deep tissue hemodynamics (~50% increase). Further, like TD-DCS, PaLS-iDCS allows direct estimation of tissue optical properties from the sampled ToF distribution without the need for a separate spectroscopic measurement. This method offers a relatively straightforward way to allow DCS systems to make robust measurements of blood flow with greatly enhanced sensitivity to deep tissue hemodynamics, enabling further applications of this non-invasive technology.

Synchronization of Neurophysiological and Biomechanical Data in a Real-Time Virtual Gait Analysis System (GRAIL): A Proof-of-Principle Study.

The investigation of gait and its neuronal correlates under more ecologically valid conditions as well as real-time feedback visualization is becoming increasingly important in neuro-motor rehabilitation research. The Gait Real-time Analysis Interactive Lab (GRAIL) offers advanced opportunities for gait and gait-related research by creating more naturalistic yet controlled environments through immersive virtual reality. Investigating the neuronal aspects of gait requires parallel recording of brain activity, such as through mobile electroencephalography (EEG) and/or mobile functional near-infrared spectroscopy (fNIRS), which must be synchronized with the kinetic and /or kinematic data recorded while walking. This proof-of-concept study outlines the required setup by use of the lab streaming layer (LSL) ecosystem for real-time, simultaneous data collection of two independently operating multi-channel EEG and fNIRS measurement devices and gait kinetics. In this context, a customized approach using a photodiode to synchronize the systems is described. This study demonstrates the achievable temporal accuracy of synchronous data acquisition of neurophysiological and kinematic and kinetic data collection in the GRAIL. By using event-related cerebral hemodynamic activity and visually evoked potentials during a start-to-go task and a checkerboard test, we were able to confirm that our measurement system can replicate known physiological phenomena with latencies in the millisecond range and relate neurophysiological and kinetic data to each other with sufficient accuracy.

Clinical Value of Fine Needle Aspiration Cytology and Core Needle Biopsy in Head and Neck Pathology - A Nationwide Survey in Germany.

BACKGROUND/AIM: Fine-needle aspiration cytology (FNA) and core needle biopsy (CNB) of the thyroid gland, salivary glands, and lymph nodes are considered simple and rapid methods for minimally invasive tissue collection. We performed a postal survey to analyse the diagnostic value and complication rate of FNA and CNB in Germany. PATIENTS AND METHODS: A questionnaire comprising 11 questions was sent to all 161 German ENT departments in September 2015. RESULTS: The response rate was 45%. In 33 of the 73 responding clinics neither FNA nor CNB were carried out. Of the 26 clinics that provided detailed reasons, the majority (n=18) cited a lack of expertise among the collaborating pathologists. Overall, FNA was used more often, regardless of the anatomical region investigated. The study was based on a total of 36,684 FNAs and 9,624 CNBs. The rate of estimated meaningful and correct findings was 63% (10%-90%) for FNA, and 83% (50%-100%) for CNB. In eight cases (<0.001%) a potential tumor cell spread was reported. CONCLUSION: This is the first nationwide survey in Germany to investigate the utility of FNA and CNB across different localizations in the head and neck region. This study revealed comparable results to the literature regarding the diagnostic value of FNA and CNB. Cell spreading was only observed in individual cases. The appraisal of needle biopsies in the head and neck area seems to be rather inhomogeneous in Germany.

Whole pelvis vs. hemi pelvis elective nodal radiotherapy in patients with PSMA-positive nodal recurrence after radical prostatectomy - a retrospective multi-institutional propensity score analysis.

PURPOSE: Despite growing evidence for bilateral pelvic radiotherapy (whole pelvis RT, WPRT) there is almost no data on unilateral RT (hemi pelvis RT, HPRT) in patients with nodal recurrent prostate cancer after prostatectomy. Nevertheless, in clinical practice HPRT is sometimes used with the intention to reduce side effects compared to WPRT. Prostate-specific membrane antigen positron emission tomography / computed tomography (PSMA-PET/CT) is currently the best imaging modality in this clinical situation. This analysis compares PSMA-PET/CT based WPRT and HPRT. METHODS: A propensity score matching was performed in a multi-institutional retrospective dataset of 273 patients treated with pelvic RT due to nodal recurrence (214 WPRT, 59 HPRT). In total, 102 patients (51 in each group) were included in the final analysis. Biochemical recurrence-free survival (BRFS) defined as prostate specific antigen (PSA) < post-RT nadir + 0.2ng/ml, metastasis-free survival (MFS) and nodal recurrence-free survival (NRFS) were calculated using the Kaplan-Meier method and compared using the log rank test. RESULTS: Median follow-up was 29 months. After propensity matching, both groups were mostly well balanced. However, in the WPRT group there were still significantly more patients with additional local recurrences and biochemical persistence after prostatectomy. There were no significant differences between both groups in BRFS (p = .97), MFS (p = .43) and NRFS (p = .43). After two years, BRFS, MFS and NRFS were 61%, 86% and 88% in the WPRT group and 57%, 90% and 82% in the HPRT group, respectively. Application of a boost to lymph node metastases, a higher RT dose to the lymphatic pathways (> 50 Gy EQD2α/ß=1.5 Gy) and concomitant androgen deprivation therapy (ADT) were significantly associated with longer BRFS in uni- and multivariate analysis. CONCLUSIONS: Overall, this analysis presents the outcome of HPRT in nodal recurrent prostate cancer patients and shows that it can result in a similar oncologic outcome compared to WPRT. Nevertheless, patients in the WPRT may have been at a higher risk for progression due to some persistent imbalances between the groups. Therefore, further research should prospectively evaluate which subgroups of patients are suitable for HPRT and if HPRT leads to a clinically significant reduction in toxicity.

Long days induce adaptive secondary dormancy in the seeds of the Mediterranean plant Aethionema arabicum.

Secondary dormancy is an adaptive trait that increases reproductive success by aligning seed germination with permissive conditions for seedling establishment. Aethionema arabicum is an annual plant and member of the Brassicaceae that grows in environments characterized by hot and dry summers. Aethionema arabicum seeds may germinate in early spring when seedling establishment is permissible. We demonstrate that long-day light regimes induce secondary dormancy in the seeds of Aethionema arabicum (CYP accession), repressing germination in summer when seedling establishment is riskier. Characterization of mutants screened for defective secondary dormancy demonstrated that RGL2 mediates repression of genes involved in gibberellin (GA) signaling. Exposure to high temperature alleviates secondary dormancy, restoring germination potential. These data are consistent with the hypothesis that long-day-induced secondary dormancy and its alleviation by high temperatures may be part of an adaptive response limiting germination to conditions permissive for seedling establishment in spring and autumn.

A Targeted LC-MRM3 Proteomic Approach for the Diagnosis of SARS-CoV-2 Infection in Nasopharyngeal Swabs.

Since its first appearance, severe acute respiratory syndrome coronavirus 2 quickly spread around the world and the lack of adequate PCR testing capacities, especially during the early pandemic, led the scientific community to explore new approaches such as mass spectrometry (MS). We developed a proteomics workflow to target several tryptic peptides of the nucleocapsid protein. A highly selective multiple reaction monitoring-cubed (MRM3) strategy provided a sensitivity increase in comparison to conventional MRM acquisition. Our MRM3 approach was first tested on an Amsterdam public health cohort (alpha-variant, 760 participants) detecting viral nucleocapsid protein peptides from nasopharyngeal swabs samples presenting a cycle threshold value down to 35 with sensitivity and specificity of 94.2% and 100.0%, without immunopurification. A second iteration of the MS-diagnostic test, able to analyze more than 400 samples per day, was clinically validated on a Leiden-Rijswijk public health cohort (delta-variant, 2536 participants) achieving 99.9% specificity and 93.1% sensitivity for patients with cycle threshold values up to 35. In this manuscript, we also developed and brought the first proof of the concept of viral variant monitoring in a complex matrix using targeted MS.

An efficient pyrrolysyl-tRNA synthetase for economical production of MeHis-containing enzymes.

Genetic code expansion has emerged as a powerful tool in enzyme design and engineering, providing new insights into sophisticated catalytic mechanisms and enabling the development of enzymes with new catalytic functions. In this regard, the non-canonical histidine analogue Nδ-methylhistidine (MeHis) has proven especially versatile due to its ability to serve as a metal coordinating ligand or a catalytic nucleophile with a similar mode of reactivity to small molecule catalysts such as 4-dimethylaminopyridine (DMAP). Here we report the development of a highly efficient aminoacyl tRNA synthetase (G1PylRSMIFAF) for encoding MeHis into proteins, by transplanting five known active site mutations from Methanomethylophilus alvus (MaPylRS) into the single domain PylRS from Methanogenic archaeon ISO4-G1. In contrast to the high concentrations of MeHis (5-10 mM) needed with the Ma system, G1PylRSMIFAF can operate efficiently using MeHis concentrations of ∼0.1 mM, allowing more economical production of a range of MeHis-containing enzymes in high titres. Interestingly G1PylRSMIFAF is also a 'polyspecific' aminoacyl tRNA synthetase (aaRS), enabling incorporation of five different non-canonical amino acids (ncAAs) including 3-pyridylalanine and 2-fluorophenylalanine. This study provides an important step towards scalable production of engineered enzymes that contain non-canonical amino acids such as MeHis as key catalytic elements.

Patterns and inferred causes of liana mortality in a tropical forest.

Lianas are major contributors to tropical forest dynamics, yet we know little about their mortality. Using overlapping censuses of the lianas and trees across a 50 ha stand of moist tropical forest, we contrasted community-wide patterns of liana mortality with relatively well-studied patterns of tree mortality to quantify patterns of liana death and identify contributing factors. Liana mortality rates were 172% higher than tree mortality rates, but species-level mortality rates of lianas were similar to trees with 'fast' life-history strategies and both growth forms exhibited similar spatial and size-dependent patterns. The mortality rates of liana saplings (<2.1 cm in diameter), which represent about 50% of liana individuals, decreased with increasing disturbance severity and remained consistently low during post-disturbance stand thinning. In contrast, larger liana individuals and trees of all sizes had elevated mortality rates in response to disturbance and their mortality rates decreased over time since disturbance. Within undisturbed forest patches, liana mortality rates increased with increasing soil fertility in a manner similar to trees. The distinct responses of liana saplings to disturbance appeared to distinguish liana mortality from that of trees, whereas similarities in their patterns of death suggest that there are common drivers of woody plant mortality.

Myelinated peripheral axons are more vulnerable to mechanical trauma in a model of enlarged axonal diameters.

The velocity of axonal impulse propagation is facilitated by myelination and axonal diameters. Both parameters are frequently impaired in peripheral nerve disorders, but it is not known if the diameters of myelinated axons affect the liability to injury or the efficiency of functional recovery. Mice lacking the adaxonal myelin protein chemokine-like factor-like MARVEL-transmembrane domain-containing family member-6 (CMTM6) specifically from Schwann cells (SCs) display appropriate myelination but increased diameters of peripheral axons. Here we subjected Cmtm6-cKo mice as a model of enlarged axonal diameters to a mild sciatic nerve compression injury that causes temporarily reduced axonal diameters but otherwise comparatively moderate pathology of the axon/myelin-unit. Notably, both of these pathological features were worsened in Cmtm6-cKo compared to genotype-control mice early post-injury. The increase of axonal diameters caused by CMTM6-deficiency thus does not override their injury-dependent decrease. Accordingly, we did not detect signs of improved regeneration or functional recovery after nerve compression in Cmtm6-cKo mice; depleting CMTM6 in SCs is thus not a promising strategy toward enhanced recovery after nerve injury. Conversely, the exacerbated axonal damage in Cmtm6-cKo nerves early post-injury coincided with both enhanced immune response including foamy macrophages and SCs and transiently reduced grip strength. Our observations support the concept that larger peripheral axons are particularly susceptible toward mechanical trauma.

How Charges Separate when Surfaces Are Dewetted.

Charge separation behind moving water drops occurs in nature and technology. Yet, the physical mechanism has remained obscure, as charge deposition is energetically unfavorable. Here, we analyze how a part of the electric double layer charge remains on the dewetted surface. At the contact line, the chemical equilibrium of bound surface charge and diffuse charge in the liquid is influenced by the contact angle and fluid flow. We summarize the mechanism in an analytical model that compares well with experiments and simulations. It correctly predicts that charge separation increases with increasing contact angle and decreases with increasing velocity.

More than the clock: distinct regulation of muscle function and metabolism by PER2 and RORα.

Circadian rhythms, governed by the dominant central clock, in addition to various peripheral clocks, regulate almost all biological processes, including sleep-wake cycles, hormone secretion and metabolism. In certain contexts, the regulation and function of the peripheral oscillations can be decoupled from the central clock. However, the specific mechanisms underlying muscle-intrinsic clock-dependent modulation of muscle function and metabolism remain unclear. We investigated the outcome of perturbations of the primary and secondary feedback loops of the molecular clock in skeletal muscle by specific gene ablation of Period circadian regulator 2 (Per2) and RAR-related orphan receptor alpha (Rorα), respectively. In both models, a dampening of core clock gene oscillation was observed, while the phase was preserved. Moreover, both loops seem to be involved in the homeostasis of amine groups. Highly divergent outcomes were seen for overall muscle gene expression, primarily affecting circadian rhythmicity in the PER2 knockouts and non-oscillating genes in the RORα knockouts, leading to distinct outcomes in terms of metabolome and phenotype. These results highlight the entanglement of the molecular clock and muscle plasticity and allude to specific functions of different clock components, i.e. the primary and secondary feedback loops, in this context. The reciprocal interaction between muscle contractility and circadian clocks might therefore be instrumental to determining a finely tuned adaptation of muscle tissue to perturbations in health and disease. KEY POINTS: Specific perturbations of the primary and secondary feedback loop of the molecular clock result in specific outcomes on muscle metabolism and function. Ablation of Per2 (primary loop) or Rorα (secondary loop) blunts the amplitude of core clock genes, in absence of a shift in phase. Perturbation of the primary feedback loop by deletion of PER2 primarily affects muscle gene oscillation. Knockout of RORα and the ensuing modulation of the secondary loop results in the aberrant expression of a large number of non-clock genes and proteins. The deletion of PER2 and RORα affects muscle metabolism and contractile function in a circadian manner, highlighting the central role of the molecular clock in modulating muscle plasticity.

Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics.

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.

Post-transcriptional (re)programming of B lymphocyte development: From bench to bedside?

Hematopoiesis, a process which generates blood and immune cells, changes significantly during mammalian development. Definitive hematopoiesis is marked by the emergence of long-term hematopoietic stem cells (HSCs). Here, we will focus on the post-transcriptional differences between fetal liver (FL) and adult bone marrow (ABM) HSCs. It remains unclear how or why exactly FL HSCs transition to ABM HSCs, but we aim to leverage their differences to revive an old idea: in utero HSC transplantation. Unexpectedly, the expression of certain RNA-binding proteins (RBPs) play an important role in HSC specification, and can be employed to convert or reprogram adult HSCs back to a fetal-like state. Among other features, FL HSCs have a broad differentiation capacity that includes the ability to regenerate both conventional B and T cells, as well as innate-like or unconventional lymphocytes such as B-1a and marginal zone B (MzB) cells. This chapter will focus on RNA binding proteins, namely LIN28B and IGF2BP3, that are expressed during fetal life and how they promote B-1a cell development. Furthermore, this chapter considers a potential clinical application of synthetic co-expression of LIN28B and IGF2BP3 in HSCs.

A Humanized and Viable Animal Model for Congenital Adrenal Hyperplasia-CYP21A2-R484Q Mutant Mouse.

Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder impairing cortisol synthesis due to reduced enzymatic activity. This leads to persistent adrenocortical overstimulation and the accumulation of precursors before the blocked enzymatic step. The predominant form of CAH arises from mutations in CYP21A2, causing 21-hydroxylase deficiency (21-OHD). Despite emerging treatment options for CAH, it is not always possible to physiologically replace cortisol levels and counteract hyperandrogenism. Moreover, there is a notable absence of an effective in vivo model for pre-clinical testing. In this work, we developed an animal model for CAH with the clinically relevant point mutation p.R484Q in the previously humanized CYP21A2 mouse strain. Mutant mice showed hyperplastic adrenals and exhibited reduced levels of corticosterone and 11-deoxycorticosterone and an increase in progesterone. Female mutants presented with higher aldosterone concentrations, but blood pressure remained similar between wildtype and mutant mice in both sexes. Male mutant mice have normal fertility with a typical testicular appearance, whereas female mutants are infertile, exhibit an abnormal ovarian structure, and remain in a consistent diestrus phase. Conclusively, we show that the animal model has the potential to contribute to testing new treatment options and to prevent comorbidities that result from hormone-related derangements and treatment-related side effects in CAH patients.

Comparison of the performance of two targeted metagenomic virus capture probe-based methods using reference control materials and clinical samples.

Viral enrichment by probe hybridization has been reported to significantly increase the sensitivity of viral metagenomics. This study compares the analytical performance of two targeted metagenomic virus capture probe-based methods: (i) SeqCap EZ HyperCap by Roche (ViroCap) and (ii) Twist Comprehensive Viral Research Panel workflow, for diagnostic use. Sensitivity, specificity, and limit of detection were analyzed using 25 synthetic viral sequences spiked in increasing proportions of human background DNA, eight clinical samples, and American Type Culture Collection (ATCC) Virome Virus Mix. Sensitivity and specificity were 95% and higher for both methods using the synthetic and reference controls as gold standard. Combining thresholds for viral sequence read counts and genome coverage [respectively 500 reads per million (RPM) and 10% coverage] resulted in optimal prediction of true positive results. Limits of detection were approximately 50-500 copies/mL for both methods as determined by ddPCR. Increasing proportions of spike-in cell-free human background sequences up to 99.999% (50 ng/mL) did not negatively affect viral detection, suggesting effective capture of viral sequences. These data show analytical performances in ranges applicable to clinical samples, for both probe hybridization metagenomic approaches. This study supports further steps toward more widespread use of viral metagenomics for pathogen detection, in clinical and surveillance settings using low biomass samples. IMPORTANCE: Viral metagenomics has been gradually applied for broad-spectrum pathogen detection of infectious diseases, surveillance of emerging diseases, and pathogen discovery. Viral enrichment by probe hybridization methods has been reported to significantly increase the sensitivity of viral metagenomics. During the past years, a specific hybridization panel distributed by Roche has been adopted in a broad range of different clinical and zoonotic settings. Recently, Twist Bioscience has released a new hybridization panel targeting human and animal viruses. This is the first report comparing the performance of viral metagenomic hybridization panels.