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STUDY QUESTIONIs nanopore sequencing for PGT-A analysis of pooled polar bodies a reliable, fast, and cost-effective method and applicable for routine diagnostics in human reproductive care?SUMMARY ANSWERNanopore sequencing of pooled polar bodies (PB) revealed high concordance rate with traditional array comparative genomic hybridization (aCGH) analysis and the nanopore sequencing workflow was fast (feasible in under 5 hours) and cost-effective (100-150€ per sample), allowing fresh embryo transfer.WHAT IS KNOWN ALREADYPGT-A using PB biopsy derives a clinical benefit by reducing number of embryo transfers and miscarriage rates but is currently not cost-efficient. Results are often unclear and require expert review. Nanopore sequencing technology opens possibilities by providing cost-efficient, fast sequencing results with uncomplicated sample preparation workflows. Interrogating the polar bodies avoids harming the embryo itself and is the only option for PGT-A in some jurisdictions.STUDY DESIGN, SIZE, DURATIONIn this prospective clinical trial, 102 pooled PB samples from 20 patients treated for infertility between March and December 2022 were analyzed for aneuploidy using nanopore sequencing technology and compared with aCGH results generated as part of the clinical routine. All patients participating in this trial were treated for infertility at ‘Wunschbaby Institut Feichtinger’ (WIF) in Vienna and chose aneuploidy screening of their polar bodies. All patients provided written informed consent.PARTICIPANTS/MATERIALS, SETTING, METHODSPB samples were analyzed by aCGH for routine PGT-A. Aliquots of whole-genome amplified DNA were anonymized and prepared for sequencing by end-prepping, barcoding and adapter ligation. Samples were pooled equimolar for sequencing on a Nanopore MinION machine. Samples were sequenced for up to 9 hours for 6 pooled PB samples. Whole-chromosome copy-numbers were called by a custom bioinformatic analysis software after alignment and pre-processing. Automatically called results were compared to aCGH results.MAIN RESULTS AND THE ROLE OF CHANCEIn total, 99 pooled polar body samples were compared, three samples were excluded because of failed or uninterpretable results. Overall, the Nanopore sequencing workflow showed high concordance rates with aCGH: 96 of 99 samples were consistently detected as euploid or aneuploid (concordance=97%, specificity = 0.957, sensitivity = 1.0, PPV = 0.906, NPV = 1.0) and 91 samples showed a fully concordant karyotype (92%). Chromosomal aneuploidies analyzed in this trial covered all 23 chromosomes with 98 trisomies, 97 monosomies in 70 aCGH samples.Detailed calculation of time and cost for the nanopore sequencing workflow was performed for different scenarios. Time calculation revealed that the whole nanopore workflow is feasible in under 5 hours (for one sample) with maximum time of 16 hours (for 12 samples in parallel). This enables fresh PB euploid embryo transfer. Material cost for the whole workflow range between 100€ and 150€, including sequencing cost of only 40€ per sample, resulting in cost-efficient aneuploidy screening.LIMITATIONS, REASONS FOR CAUTIONFor some samples, the reference result remained unclear, due to increased noise and limited resolution of the aCGH method. In particular, the small chromosomes show higher variability in both platforms and manual review is often required. A larger study with follow-up data or a clinical non-selection trial would be beneficial for increased confidence.WIDER IMPLICATIONS OF THE FINDINGSThis is the first clinical study, systematically comparing nanopore sequencing for aneuploidy of pooled polar bodies with standard detection methods. High concordance rates confirmed feasibility of nanopore technology for this application. Additionally, the fast and cost-efficient sequencing workflow reveals clinical utility of this technology, making polar body PGT-A clinically attractive.STUDY FUNDING/COMPETING INTEREST(S)The study was funded by the Wunschbaby Institut Feichtinger, Dr. Wilfried Feichtinger GmbH. The authors declare no competing conflict of interest.TRIAL REGISTRATION NUMBERNot available

The wood-water interactions of modified beech wood (Fagus sylvatica L.) were studied. Specimens were thermally modified at 180 (TM1), 200 (TM2) and 220 °C (TM3), acetylated (Acet), and melamine formaldehyde (MF) resin (Mel) modified. Afterwards, the water vapour characteristics, i.e. water vapour sorption isotherms, equilibrium moisture content (EMC), dimensional stability of specimens conditioned at 30, 65 and 90% RH and liquid water characteristics, i.e. water absorption, maximum moisture content (MC), leachability and swelling kinetics, were determined and the results compared with reference (Ref) specimens. From the results, it is evident that the scale of wood-water interactions was highly dependent on the thermal modification temperature and type of chemical modification. The water vapour isotherms of thermally modified wood decreased, whereas more severe treatment exhibited more distinct reduction. The EMC values of the Mel and TM1 specimens decreased only at high RH, whereas the most significant decrease, within the whole range of observation, was found in the Acet group. The maximum MC reduction was achieved by acetylation. As a consequence of swelling reduction, dimensional stability expressed as anti-swelling efficiency (ASE) was considerably improved. A relatively high initial linear-phase swelling rate was found for the Ref specimens, whereas modified wood exhibited comparatively slow and gradual swelling.

Basalt fabric with manufacturer-made silane sizing was additionally treated by propane-butane enriched atmospheric pressure nitrogen plasma for 15 and 30 s. The wetting improvement of treated fabric was demonstrated by faster soaking of K2CO3 solution. FTIR and XPS analyses confirmed the presence of new nitrogen-containing chemical groups. The impact on adhesion was investigated via a shear strength test of basalt fabric inlay between two beech wooden boards. A short 15 s long treatment led to almost 50% increase in melamine-urea formaldehyde (MUF), polyvinyl acetate (PVAC) and epoxy resin bond strength. Phenol-resorcinol formaldehyde (PRF) needed longer treatment to achieve a bond strength of 11.2 MPa. Only the polyurethane (PUR) showed reduced adhesion to plasma-treated fibers. However, from all tested resins, PUR exhibited the highest bond strength (15.7 MPa) with untreated basalt fabric. The SEM of tested specimens confirmed the changes in the mechanical adherence of resins to plasma treated fibers. Based on these results, PRF and PUR seem to be the most suitable polymers for basalt fibers reinforcement of structural wood-based materials.

Tannins and their toxic effects against various decay organisms have been interrelated for centuries. As natural products have gained more interest, waste from several production fields abundant in tannins has yielded promising components for wood preservation. In this work, the main approach was to split condensed tannins into smaller fractions by chemical hydrolysis and evaluate their suitability for beech wood impregnation. Commercial extract from black wattle was treated with mineral acid of low concentration. The volatile hydrochloric acid was completely removed after reaction by evaporation in the course of freeze-drying. The modified extract was then applied as aqueous solution into beech wood. The water resistance of the modified beech wood was not increased significantly, and scanning electron microscopy (SEM) showed no evidence of tannin successful bonding within wood cells. Nevertheless, after strong leaching cycles, part of the extract accumulated in fibers. Moreover, lyophilization was found to be a suitable technique to eliminate volatile acids from temperature-sensitive extracts, such as tannins. These findings could help in the development of water-borne tannin formulations for wood protection, while using phlobaphene formation as a potential natural pathway of tannin autocondensation.

Background and aims Autoimmune hepatitis (AIH) is a chronic liver disease that regularly relapses when immunosuppression is tapered. It is thought to be driven by T-cells, whereas the etiologic impact of an apparently deregulated B lineage system, as evidenced by hypergammaglobulinemia and autoantibodies, remains elusive. We set out to investigate T and B cell repertoires supporting chronic inflammation in AIH. Approach and results T and B cell receptor (TCR/BCR) and human leukocyte antigen (HLA) next-generation immunosequencing were used to record immune signatures from a cohort of 60 patients with AIH and disease controls. Blood and liver B lineage immune metrics were not indicative of a dominant directional antigen selection apart from a slight skewing of IGHV-J genes. More importantly, we found strong AIH-specific TRBV-J skewing not attributable to the HLA-DRB1 specificities of the cohort. This TCR repertoire bias was generated as a result of peripheral T cell (de)selection and persisted in disease remission. Using a clustering algorithm according to antigenic specificity, we identified liver TCR clusters that were shared between patients with AIH but were absent or deselected in patients with other liver pathologies. Conclusions Patients with AIH show profound and persisting T-cell architectural changes that may explain high relapse rates after tapering immunosuppression. Liver T-cell clusters shared between patients may mediate liver damage and warrant further study.

The aim of this paper was to analyze selected properties of beech wood (Fagus sylvatica L.) treated by one-sided surface charring. Specimens were one-side charred with a hot plate using several time-temperature combinations (from 200 to 400 °C). Characteristics such as colour, discoloration, surface roughness, fire resistance, total carbohydrate content at several wood layers and decay resistance were evaluated. Surface charring was applied to the radial and tangential surfaces. Colour measurements showed that the surface of the wood turned grey due to charring. In addition to colour measurements, other experiments showed significant differences between radial and tangential specimens due to their different structures. The higher the temperature used in treating them, the lower the roughness values for radial specimens, while the trend for tangential specimens was the opposite. A smoother surface is more fire resistant, so radial specimens are generally better in this regard. Tangential specimens are more susceptible during preparation to forming cracks that impair flame resistance because a continuous protective densified layer is not formed. The determination of total carbohydrates revealed significant changes at various wood depths after surface charring. These changes were more predictable in radial specimens due to the annual ring orientation, because each layer consisted of a similar earlywood/latewood ratio. Finally, when decay resistance was assessed, weight loss was found to be lower in all specimens than in the references. The results suggest that charring at a particular combination of temperature and time improved the investigated properties of the surface-modified beech.

Research question Full-length 16S rRNA gene sequencing using nanopore technology is a fast alternative to conventional short-read 16S rRNA gene sequencing with low initial investment costs that has been used for various microbiome studies but has not yet been investigated as an alternative approach for endometrial microbiome analysis. Is in-situ 16S rRNA gene long-read sequencing using portable nanopore sequencing technology feasible and reliable for endometrial microbiome analysis? Design A prospective experimental study based on 33 patients seeking infertility treatment between January and October 2019. A 16S rRNA gene long-read nanopore sequencing protocol for analysing endometrial microbiome samples was established, including negative controls for contamination evaluation and positive controls for bias evaluation. Contamination caused by kit and exterior sources was identified and excluded from the analysis. Endometrial samples from 33 infertile patients were sequenced using the optimized long-read nanopore sequencing protocol and compared with conventional short-read sequencing carried out by external laboratories. Results Of the 33 endometrial patient samples, 23 successfully amplified (69.7%) and their microbiome was assessed using nanopore sequencing. Of those 23 samples, 14 (60.9%) were Lactobacillus-dominated (>80% of reads mapping to Lactobacillus), with 10 samples resulting in more than 90% Lactobacillus reads. Our long-read nanopore sequencing revealed results similar to two conventional short-read sequencing approaches and to long-read sequencing validation carried out in external laboratories. Conclusion In this pilot study, 16S rRNA gene long-read nanopore sequencing was established to analyse the endometrial microbiome in situ that could be widely applied owing to its cost efficiency and portable character.

Recent studies suggest that circulating tumor cells and cell-free DNA may represent powerful non-invasive tools for monitoring disease in patients with solid and hematologic malignancies. Here, we conducted a pilot study in 27 myeloma patients to explore the clonotypic V(D)J rearrangement for monitoring circulating myeloma cells and cell-free myeloma DNA. Next-generation sequencing was used to define the myeloma V(D)J rearrangement and for subsequent peripheral blood tracking after treatment initiation. Positivity for circulating myeloma cells/cell-free myeloma was associated with conventional remission status (P

One-sided surface charred European beech wood (Fagus sylvatica L.) was studied. Radial and tangential specimens of dimensions of 50 × 25 × 350 mm were one-sided surface charred at 200, 250, 300, 350, and 400 °C for various times using a contact heating system. Specimens of dimensions of 50 × 25 × 50 mm3 were prepared for treatment intensity analysis and its effect on surface wettability. Density profiles of the radial and tangential charred specimens were determined using X-ray densitography with a resolution of 0.05 mm. The wettability of the one-sided surface charred wood specimens was analyzed via contact angle, measured using the sessile drop method and determined over 10 to 150 s. The oven-dried specimens were partially submerged in water and water uptake was recorded after 2, 4, 6, 8, 10, 48, and 72 h according to EN-927-5. The surface density of the radial specimen groups charred at 200 °C for 6 min and 250 °C for 4 min decreased by about 4.5 to 8.2%. With increasingly severe charring, the surface density decreased by about 15.5 to 33.5%. A mild charring process produced a surface charred layer of approx. 2 mm, while higher temperatures and longer times affected the density up to 4–6 mm beneath the surface. Differences were found between the water uptake of the radial and tangential charred beech specimens. The most significant decrease of 56% in water uptake was recorded for the radial group prepared at 200 °C for 20 min after floating in water for 72 h. Water uptake in the radial groups modified at 250 °C for 4 and 6 min after 72 h decreased by 38% and 36%, respectively. The tangential groups did not show any statistically significant decrease. The average water uptake of the groups charred at 200 °C for 20 min, 350 °C for 2 min and 400 °C for 1 min was greater than that of the reference, the variability of the measured data was significantly greater due to the highly anisotropic character of the tangential specimens. Micro cracks were also visible on the surface. Concluding from the results of this study, one-sided surface charred beech wood exhibits increased potential in terms of wood–water related properties when a temperature range of 200–400 °C is applied.

Using next-generation immunoglobulin (IGH) sequencing and flow cytometry, we characterized the composition, diversity and dynamics of non-malignant B cells in patients undergoing treatment with the Bruton tyrosine kinase (BTK) inhibitor ibrutinib or chemo-immunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR). During ibrutinib therapy, non-malignant B cell numbers declined, but patients maintained stable IGH diversity and constant fractions of IGH-mutated B cells. This indicates partial preservation of antigen-experienced B cells during ibrutinib therapy, but impaired replenishment of the normal B cell pool with naïve B cells. In contrast, after FCR we noted a recovery of normal B cells with a marked predominance of B cells with unmutated IGH. This pattern is compatible with a deletion of pre-existing antigen-experienced B cells followed by repertoire renewal with antigen-naïve B cells. These opposite patterns in B cell dynamics may result in different responses towards neoantigens versus recall antigens, which need to be further defined.

The cell surface ecto-enzyme CD38 is a promising target antigen for the treatment of hematological malignancies, as illustrated by the recent approval of daratumumab for the treatment of multiple myeloma. Our aim was to evaluate the potential of CD38-specific nanobodies as novel diagnostics for hematological malignancies. We successfully identified 22 CD38-specific nanobody families using phage display technology from immunized llamas. Crossblockade analyses and in-tandem epitope binning revealed that the nanobodies recognize three different non-overlapping epitopes, with four nanobody families binding complementary to daratumumab. Three nanobody families inhibit the enzymatic activity of CD38 in vitro, while two others were found to act as enhancers. In vivo, fluorochrome-conjugated CD38 nanobodies efficiently reach CD38 expressing tumors in a rodent model within 2 hours after intravenous injection, thereby allowing for convenient same day in vivo tumor imaging. These nanobodies represent highly specific tools for modulating the enzymatic activity of CD38 and for diagnostic monitoring CD38-expressing tumors.

The cell surface ecto-enzyme CD38 is a promising target antigen for the treatment of hematological malignancies, as illustrated by the recent approval of daratumumab for the treatment of multiple myeloma. Our aim was to evaluate the potential of CD38-specific nanobodies as novel diagnostics for hematological malignancies. We successfully identified 22 CD38-specific nanobody families using phage display technology from immunized llamas. Crossblockade analyses and in-tandem epitope binning revealed that the nanobodies recognize three different non-overlapping epitopes, with four nanobody families binding complementary to daratumumab. Three nanobody families inhibit the enzymatic activity of CD38 in vitro, while two others were found to act as enhancers. In vivo, fluorochrome-conjugated CD38 nanobodies efficiently reach CD38 expressing tumors in a rodent model within 2 hours after intravenous injection, thereby allowing for convenient same day in vivo tumor imaging. These nanobodies represent highly specific tools for modulating the enzymatic activity of CD38 and for diagnostic monitoring CD38-expressing tumors. Fil: Fumey, William. University Medical Center Hamburg-Eppendorf; Alemania Fil: Koenigsdorf, Julia. University Medical Center Hamburg-Eppendorf; Alemania Fil: Kunick, Valentin. University Medical Center Hamburg-Eppendorf; Alemania Fil: Menzel, Stephan. University Medical Center Hamburg-Eppendorf; Alemania Fil: Schütze, Kerstin. University Medical Center Hamburg-Eppendorf; Alemania Fil: Unger, Mandy. University Medical Center Hamburg-Eppendorf; Alemania Fil: Schriewer, Levin. University Medical Center Hamburg-Eppendorf; Alemania Fil: Haag, Friedrich. University Medical Center Hamburg-Eppendorf; Alemania Fil: Adam, Gerhard. University Medical Center Hamburg-Eppendorf; Alemania Fil: Oberle, Anna. University Medical Center Hamburg-Eppendorf; Alemania Fil: Binder, Mascha. University Medical Center Hamburg-Eppendorf; Alemania Fil: Fliegert, Ralf. University Medical Center Hamburg-Eppendorf; Alemania Fil: Guse, Andreas. University Medical Center Hamburg-Eppendorf; Alemania Fil: Zhao, Yong Juan. Peking University; China Fil: Lee, Hon Cheung. Peking University; China Fil: Malavasi, Fabio. Università di Torino; Italia Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Instituto Leloir; Argentina Fil: Van Hegelsom, Rob. Ablynx nv; Bélgica Fil: Stortelers, Catelijne. Ablynx nv; Bélgica Fil: Bannas, Peter. University Medical Center Hamburg-Eppendorf; Alemania Fil: Koch Nolte, Friedrich. University Medical Center Hamburg-Eppendorf; Alemania