Miracle-dz/UniFAR-FTUs huggingface dataset:
Towards a scalable bioprocess for rAAV production using a HeLa stable cell line

The majority of recombinant adeno‐associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale‐up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa‐based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3‐based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3‐4 months, and presented rAAV2 volumetric production (bulk) > 3 × 1011 vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity‐based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time‐efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 1011 vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.
Diverse targets of SMN2-directed splicing-modulating small molecule therapeutics for spinal muscular atrophy

Abstract Designing an RNA-interacting molecule that displays high therapeutic efficacy while retaining specificity within a broad concentration range remains a challenging task. Risdiplam is an FDA-approved small molecule for the treatment of spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. Branaplam is another small molecule which has undergone clinical trials. The therapeutic merit of both compounds is based on their ability to restore body-wide inclusion of Survival Motor Neuron 2 (SMN2) exon 7 upon oral administration. Here we compare the transcriptome-wide off-target effects of these compounds in SMA patient cells. We captured concentration-dependent compound-specific changes, including aberrant expression of genes associated with DNA replication, cell cycle, RNA metabolism, cell signaling and metabolic pathways. Both compounds triggered massive perturbations of splicing events, inducing off-target exon inclusion, exon skipping, intron retention, intron removal and alternative splice site usage. Our results of minigenes expressed in HeLa cells provide mechanistic insights into how these molecules targeted towards a single gene produce different off-target effects. We show the advantages of combined treatments with low doses of risdiplam and branaplam. Our findings are instructive for devising better dosing regimens as well as for developing the next generation of small molecule therapeutics aimed at splicing modulation.
Role of dipolar interaction in magnetic hyperthermia

The dynamic properties of magnetic nanoparticles are known to be substantially influenced by the dipole-dipole interaction. In this paper we study how this affects the efficiency of magnetic hyperthermia experiments. In particular we ask whether it is possible to use the dipolar interaction as a mechanism to increase the heat released by the nanoparticles, thus improving the application. The investigation is carried out via numerical simulations based on a mean-field model developed to include the dipolar interaction in the Fokker-Planck equation describing the time evolution of the system. Both the linear and nonlinear regimes (related to the amplitude of the external magnetic field) are studied in detail. It is shown that even moderate changes in the particle concentration may have substantial effects on the magnetization dynamics of the system, being capable of increasing or decreasing the heat released by orders of magnitude, depending on the values of other system parameters. It is found that the dipolar interaction can be used to increase the dissipation of magnetically soft particles, but should be avoided in the case of hard particles.
Toxoplasma ERK7 protects the apical complex from premature degradation

O’Shaughnessy et al. identify a putative ubiquitin E3 ligase that is required for cytoskeleton turnover during Toxoplasma division. Remarkably, knockout of the E3 ligase suppresses the loss-of-function of Toxoplasma ERK7, unveiling an essential pathway that regulates protein homeostasis during division of this ubiquitous parasite.
Wykorzystanie emisji akustycznej do diagnozowania wtryskiwacza silnika 3Al25/30

Acoustic emission method (AE) can be used for the diagnosis of machine parts such as, for example: fuel injectors. This paper presents the methodology and research results of 3Al25/30 engine fuel injector. During research was studied one injector in good condition and second with simulated failure involving the closing 2 of 9 holes of the injector tip. Research was carried out on a laboratory test stand using a set of acoustic emission Vallen System. The kit includes: 4 channel signal recorder AMSY 6, two measurement modules ASIP-2/S, preamplifier with a frequency range 20 kHz 1 MHz and the strengthening of 34dB and AE signal measurement sensor type VS 150M, with a frequency range 100 450kHz. During the study, the acoustic emission (AE) generated during the injection was recorded parameters: amplitude, number of events hits, the total injection time, the effective value of the signal (RMS). Analysis of the results showed significantly longer total time of the injection in the case of damaged injector compared to the injector in good conditions. Signal amplitude was higher, however, the RMS signal reached approximately 3-times lower value for the injector with damaged tip. This means lower quality fuel atomization. The study can be the basis for subsequent identification of possible damages of the engine equipment while the engine is running, using acoustic emission as an effective diagnostic tool.
A Hybrid System for Automatic Identification of Corneal Layers on In Vivo Confocal Microscopy Images.

Purpose Accurate identification of corneal layers with in vivo confocal microscopy (IVCM) is essential for the correct assessment of corneal lesions. This project aims to obtain a reliable automated identification of corneal layers from IVCM images. Methods A total of 7957 IVCM images were included for model training and testing. Scanning depth information and pixel information of IVCM images were used to build the classification system. Firstly, two base classifiers based on convolutional neural networks and K-nearest neighbors were constructed. Second, two hybrid strategies, namely weighted voting method and light gradient boosting machine (LightGBM) algorithm were used to fuse the results from the two base classifiers and obtain the final classification. Finally, the confidence of prediction results was stratified to help find out model errors. Results Both two hybrid systems outperformed the two base classifiers. The weighted area under the curve, weighted precision, weighted recall, and weighted F1 score were 0.9841, 0.9096, 0.9145, and 0.9111 for weighted voting hybrid system, and were 0.9794, 0.9039, 0.9055, and 0.9034 for the light gradient boosting machine stacking hybrid system, respectively. More than one-half of the misclassified samples were found using the confidence stratification method. Conclusions The proposed hybrid approach could effectively integrate the scanning depth and pixel information of IVCM images, allowing for the accurate identification of corneal layers for grossly normal IVCM images. The confidence stratification approach was useful to find out misclassification of the system. Translational Relevance The proposed hybrid approach lays important groundwork for the automatic identification of the corneal layer for IVCM images.
Is Unidirectional Drying in a Round Capillary Always Diffusive?

The unidirectional drying of water in cylindrical capillaries has been described since the discovery of Stefan’s solution as a vapor diffusion-controlled process with a square root of time kinetics. Here we show that this well-known process actually depends on the way the capillary is closed. Experiments are performed on the evaporation of water in capillaries closed at one end with a solid material or connected to a fluid reservoir. While we recover Stefan’s solution in the first case, we show that in the second situation the water plug evaporates at a constant rate with the water–air meniscus remaining pinned at the exit where evaporation proceeds. The presence of the liquid reservoir closing the capillary combined with a capillary pumping effect induces a flow of the water plug toward the evaporation front leading to a constant-rate drying, substantially faster than the prediction of Stefan’s equation. Our results show that a transition from a constant-rate evaporation regime at short times to a diffusion-driven evaporation regime at long times can be observed by increasing the viscosity of the fluid in the reservoir blocking the other end of the capillary. Such transition can also be observed by connecting the capillary end to a solidifying fluid like epoxy glue.
COMPARISON OF FEATURE SIGNIFICANCE QUANTILE APPROXIMATIONS

Curve estimates and surface estimates often contain features such as inclines, bumps or ridges which may signify an underlying structural mechanism. However, spurious features are also a common occurrence and it is important to identify those features that are statistically significant. A method has been developed recently for recognising feature significance based on the derivatives of the function estimate. It requires simultaneous confidence intervals and tests, which in turn require quantiles for the maximal deviation statistics. This paper reviews and compares various approximations to these quantiles. Applying upcrossing‐probability theory to this problem yields better quantile approximations than the use of an independent blocks method.
A Derivative-based Parser Generator for Visibly Pushdown Grammars

In this article, we present a derivative-based, functional recognizer and parser generator for visibly pushdown grammars. The generated parser accepts ambiguous grammars and produces a parse forest containing all valid parse trees for an input string in linear time. Each parse tree in the forest can then be extracted also in linear time. Besides the parser generator, to allow more flexible forms of the visibly pushdown grammars, we also present a translator that converts a tagged CFG to a visibly pushdown grammar in a sound way, and the parse trees of the tagged CFG are further produced by running the semantic actions embedded in the parse trees of the translated visibly pushdown grammar. The performance of the parser is compared with popular parsing tools, including ANTLR, GNU Bison, and other popular hand-crafted parsers. The correctness and the time complexity of the core parsing algorithm are formally verified in the proof assistant Coq.
Glucocorticoid activation by HSD11B1 limits T cell-driven interferon signaling and response to PD-1 blockade in melanoma

Background Immune responses against tumors are subject to negative feedback regulation. Immune checkpoint inhibitors (ICIs) blocking Programmed cell death protein 1 (PD-1), a receptor expressed on T cells, or its ligand PD-L1 have significantly improved the treatment of cancer, in particular malignant melanoma. Nevertheless, responses and durability are variables, suggesting that additional critical negative feedback mechanisms exist and need to be targeted to improve therapeutic efficacy. Methods We used different syngeneic melanoma mouse models and performed PD-1 blockade to identify novel mechanisms of negative immune regulation. Genetic gain-of-function and loss-of-function approaches as well as small molecule inhibitor applications were used for target validation in our melanoma models. We analyzed mouse melanoma tissues from treated and untreated mice by RNA-seq, immunofluorescence and flow cytometry to detect changes in pathway activities and immune cell composition of the tumor microenvironment. We analyzed tissue sections of patients with melanoma by immunohistochemistry as well as publicly available single-cell RNA-seq data and correlated target expression with clinical responses to ICIs. Results Here, we identified 11-beta-hydroxysteroid dehydrogenase-1 (HSD11B1), an enzyme that converts inert glucocorticoids into active forms in tissues, as negative feedback mechanism in response to T cell immunotherapies. Glucocorticoids are potent suppressors of immune responses. HSD11B1 was expressed in different cellular compartments of melanomas, most notably myeloid cells but also T cells and melanoma cells. Enforced expression of HSD11B1 in mouse melanomas limited the efficacy of PD-1 blockade, whereas small molecule HSD11B1 inhibitors improved responses in a CD8+ T cell-dependent manner. Mechanistically, HSD11B1 inhibition in combination with PD-1 blockade augmented the production of interferon-γ by T cells. Interferon pathway activation correlated with sensitivity to PD-1 blockade linked to anti-proliferative effects on melanoma cells. Furthermore, high levels of HSD11B1, predominantly expressed by tumor-associated macrophages, were associated with poor responses to ICI therapy in two independent cohorts of patients with advanced melanomas analyzed by different methods (scRNA-seq, immunohistochemistry). Conclusion As HSD11B1 inhibitors are in the focus of drug development for metabolic diseases, our data suggest a drug repurposing strategy combining HSD11B1 inhibitors with ICIs to improve melanoma immunotherapy. Furthermore, our work also delineated potential caveats emphasizing the need for careful patient stratification.
Mechanism of delayed cell death following simultaneous CRISPR-Cas9 targeting in pancreatic cancers

When we transduced pancreatic cancers with sgRNAs that targeted 2-16 target sites in the human genome, we found that increasing the number of CRISPR-Cas9 target sites produced greater cytotoxicity, with >99% growth inhibition observed by targeting only 12 sites. However, cell death was delayed by 2-3 weeks after sgRNA transduction, in contrast to the repair of double strand DNA breaks (DSBs) that happened within 3 days after transduction. To explain this discrepancy, we used both cytogenetics and whole genome sequencing to interrogate the genome. We first detected chromatid and chromosome breaks, followed by radial formations, dicentric, ring chromosomes, and other chromosomal aberrations that peaked at 14 days after transduction. Structural variants (SVs) were detected at sites that were directly targeted by CRISPR-Cas9, including SVs generated from two sites that were targeted, but the vast majority of SVs (89.4%) were detected elsewhere in the genome that arose later than those directly targeted. Cells also underwent polyploidization that peaked at day 10 as detected by XY FISH assay, and ultimately died via apoptosis. Overall, we found that the simultaneous DSBs induced by CRISPR-Cas9 in pancreatic cancers caused chromosomal instability and polyploidization that ultimately led to delayed cell death. Statement of significance Using whole genome sequencing and conventional cytogenetics, we discovered that CRISPR-Cas9 cuts led to genomic instability, including chromosomal rearrangements and polyploidization, and ultimately to delayed cell death, 2-3 weeks after the induction of CRISPR-Cas9 DSBs.
Beyond a PPR-RNA recognition code: Many aspects matter for the multi-targeting properties of RNA editing factor PPR56

The mitochondrial C-to-U RNA editing factor PPR56 of the moss Physcomitrium patens is an RNA-binding pentatricopeptide repeat protein equipped with a terminal DYW-type cytidine deaminase domain. Transferred into Escherichia coli, PPR56 works faithfully on its two native RNA editing targets, nad3eU230SL and nad4eU272SL, and also converts cytidines into uridines at over 100 off-targets in the bacterial transcriptome. Accordingly, PPR56 is attractive for detailed mechanistic studies in the heterologous bacterial setup, allowing for scoring differential RNA editing activities of many target and protein variants in reasonable time. Here, we report (i) on the effects of numerous individual and combined PPR56 protein and target modifications, (ii) on the spectrum of off-target C-to-U editing in the bacterial background transcriptome for PPR56 and two variants engineered for target re-direction and (iii) on combinations of targets in tandem or separately at the 5’- and 3’-ends of large mRNAs. The latter experimentation finds enhancement of RNA editing at weak targets in many cases, including cox3eU290SF as a new candidate mitogenome target. We conclude that C-to-U RNA editing can be much enhanced by transcript features also outside the region ultimately targeted by PPRs of a plant editing factor, possibly facilitated by its enrichment or scanning along transcripts.
Genetic variation in promoter region of the bovine LAP3 gene associated with estimated breeding values of milk production traits and clinical mastitis in dairy cattle

The purpose of this study was to identify genetic variants in the promoter and 5’UTR regions of bovine leucine amino peptidase three (LAP3) gene and analysed their associations with estimated breeding values (EBVs) of milk production traits and clinical mastitis in Sahiwal and Karan Fries cattle. Eleven SNPs were identified within the region under study of the LAP3 gene, including seven promoter variants (rs717156555: C>G, rs720373055: T>C, rs715189731: A>G, rs516876447: A>G, rs4618572 69: C>T, rs136548163: C>T, and rs720349928: G>A) and four 5’UTR variants (rs717884982: C>T, rs722359733: C>T, rs481631804: C>T and rs462932574: T>G). Out of them, 10 SNPs variants were found in both Sahiwal and Karan Fries cattle, with one SNP variant (rs481631804: C>T) being unique to Karan Fries cattle. Seven of these identified SNPs were chosen for association analyses. Individual SNP based association analysis revealed that two SNPs (rs720373055: T>C and rs7203499 28: G>A) were significantly associated with EBVs of lactation milk yield (LMY), 305-day milk yield (305dMY), and one significant association of SNP rs722359733: C>T with lactation length (LL) was observed. Haplotype based association analysis indicated that diplotypes are significantly associated with EBVs of LMY, 305dMY, and LL, individuals with H1H3 (CTACGCT/GCGTACG) being linked to higher lactation performance than other diplotypes. Further logistic regression analysis revealed that, animals with diplotype H1H3 was less susceptible to the incidence of clinical mastitis than other cows, as the odds ratio for the non-incidence of clinical mastitis was found to be low. Altogether, variations in the LAP3 gene promoter could be used as a genetic marker, most notably diplotype H1H3, may greatly benefit the simultaneous improvement of mastitis resistance and milk yield traits in dairy cattle. Moreover, bioinformatics analysis predicted that the SNPs rs720373055: T>C, rs715189731:A>G and rs720349928: G>A is located in the core promoter region and in TFBs, play key role in regulation of studied phenotypes.
Asparagine availability controls B cell homeostasis

Germinal centre (GC) B cells proliferate at some of the highest rates of any mammalian cell, yet the metabolic processes which enable this are poorly understood. We performed integrated metabolomic and transcriptomic profiling of GC B cells, and found that metabolism of the non-essential amino acid asparagine (Asn) was highly upregulated. Asn was conditionally essential to B cells, and its synthetic enzyme, asparagine synthetase (ASNS) was upregulated following their activation, particularly more markedly in the absence of Asn, through the integrated stress response sensor general control non-derepressible 2 (GCN2). When Asns is deleted B cell survival and proliferation in low Asn conditions were strongly impaired, and removal of environmental Asn by asparaginase or dietary restriction markedly compromised the GC reaction, impairing affinity maturation and the humoral response to influenza infection. Using stable isotope tracing, we found that metabolic adaptation to the absence of Asn requires ASNS, and that oxidative phosphorylation, mitochondrial homeostasis, and synthesis of nucleotides was particularly sensitive to Asn deprivation. Altogether, we reveal that Asn metabolism acts as a key regulator of B cell function and GC homeostasis.
TALLSorts: a T-cell acute lymphoblastic leukemia subtype classifier using RNA-seq expression data

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive and heterogenous haematological malignancy affecting both children and adults. T-ALL subtype identification is an emerging area of active research, with several recent studies proposing potential subtypes based on transcriptomic and genomic analyses. Here we present TALLSorts, a machine-learning bioinformatic tool which classifies T-ALL samples by using bulk RNA sequencing (RNA-seq) data. Trained on four international cohorts totalling 264 samples, TALLSorts exhibits excellent accuracy when tested on holdout and independent test sets. TALLSorts is publicly available for use and will be constantly updated as the field of T-ALL classification further develops.
PARAMETERIZATION OF N2O5 REACTION PROBABILITIES FOR INCLUSION IN CMAQ

Heterogeneous reactions of N2O5 have a substantial influence on gaseous and particulate pollutant concentrations. To accurately simulate nighttime nitrogen chemistry and the resulting impacts on ozone, particulate nitrate, and nitrogen oxides, air quality models must contain a reliable parameterization of the heterogeneous reaction probability, γ , which is defined as the fraction of collisions between gaseous N2O5 molecules and particle surfaces that lead to the production of HNO3. Bhave et al. (2006) updated the parameterization of γ  in the Community Multiscale Air Quality (CMAQ) model v4.6 based on an equation by Evans and Jacob (2005), referred to hereafter as EJ05. Later, we discovered a typographical error in that equation. The form of their equation should read γ = α×10β but the negative sign was accidentally omitted from the publication (Mathew Evans, personal communication). Correction of that typographical error led to a substantial degradation in wintertime model performance for particulate NO3. In the process of diagnosing this problem, we found a few other shortcomings in EJ05 so we began an effort to derive a new parameterization of γ . Results of our efforts are described on this poster. Our work has several advantages over previous studies: (1) our parameterization uses all published laboratory measurements of γ on ammoniated sulfate and nitrate particles; (2) rigorous statistical methods are employed (e.g., significance test of each independent variable, weighting of each data point by the measurement uncertainty, defining extrapolation limits) leading to a simple formula that captures the most important features in the laboratory data; (3) the effect of particulate nitrate to suppress γ is quantified using