Low copy CRISPR-Cas13d mitigates collateral RNA cleavage.

While CRISPR-Cas13 systems excel in accurately targeting RNA, the potential for collateral RNA degradation poses a concern for therapeutic applications and limits broader adoption for transcriptome perturbations. We evaluate the extent to which collateral RNA cleavage occurs when Rfx Cas13d is delivered via plasmid transfection or lentiviral transduction and find that collateral activity only occurs with high levels of Rfx Cas13d expression. Using transcriptome-scale and combinatorial CRISPR pooled screens in cell lines with low-copy Rfx Cas13d, we find high on-target knockdown, without extensive collateral activity regardless of the expression level of the target gene. In contrast, transfection of Rfx Cas13d, which yields higher nuclease expression, results in collateral RNA degradation. Further, our analysis of a high-fidelity Cas13 variant uncovers a marked decrease in on-target efficiency, suggesting that its reduced collateral activity may be due to an overall diminished nuclease capability.

"Candidatus Siderophilus nitratireducens": a putative nap-dependent nitrate-reducing iron oxidizer within the new order Siderophiliales.

Nitrate leaching from agricultural soils is increasingly found in groundwater, a primary source of drinking water worldwide. This nitrate influx can potentially stimulate the biological oxidation of iron in anoxic groundwater reservoirs. Nitrate-dependent iron-oxidizing (NDFO) bacteria have been extensively studied in laboratory settings, yet their ecophysiology in natural environments remains largely unknown. To this end, we established a pilot-scale filter on nitrate-rich groundwater to elucidate the structure and metabolism of nitrate-reducing iron-oxidizing microbiomes under oligotrophic conditions mimicking natural groundwaters. The enriched community stoichiometrically removed iron and nitrate consistently with the NDFO metabolism. Genome-resolved metagenomics revealed the underlying metabolic network between the dominant iron-dependent denitrifying autotrophs and the less abundant organoheterotrophs. The most abundant genome belonged to a new Candidate order, named Siderophiliales. This new species, "Candidatus Siderophilus nitratireducens," carries genes central genes to iron oxidation (cytochrome c cyc2), carbon fixation (rbc), and for the sole periplasmic nitrate reductase (nap). Using thermodynamics, we demonstrate that iron oxidation coupled to nap based dissimilatory reduction of nitrate to nitrite is energetically favorable under realistic Fe3+/Fe2+ and NO3-/NO2- concentration ratios. Ultimately, by bridging the gap between laboratory investigations and nitrate real-world conditions, this study provides insights into the intricate interplay between nitrate and iron in groundwater ecosystems, and expands our understanding of NDFOs taxonomic diversity and ecological role.

Artificial Intelligence in Cataract Surgery: A Systematic Review.

Purpose: The purpose of this study was to assess the current use and reliability of artificial intelligence (AI)-based algorithms for analyzing cataract surgery videos. Methods: A systematic review of the literature about intra-operative analysis of cataract surgery videos with machine learning techniques was performed. Cataract diagnosis and detection algorithms were excluded. Resulting algorithms were compared, descriptively analyzed, and metrics summarized or visually reported. The reproducibility and reliability of the methods and results were assessed using a modified version of the Medical Image Computing and Computer-Assisted (MICCAI) checklist. Results: Thirty-eight of the 550 screened studies were included, 20 addressed the challenge of instrument detection or tracking, 9 focused on phase discrimination, and 8 predicted skill and complications. Instrument detection achieves an area under the receiver operator characteristic curve (ROC AUC) between 0.976 and 0.998, instrument tracking an mAP between 0.685 and 0.929, phase recognition an ROC AUC between 0.773 and 0.990, and complications or surgical skill performs with an ROC AUC between 0.570 and 0.970. Conclusions: The studies showed a wide variation in quality and pose a challenge regarding replication due to a small number of public datasets (none for manual small incision cataract surgery) and seldom published source code. There is no standard for reported outcome metrics and validation of the models on external datasets is rare making comparisons difficult. The data suggests that tracking of instruments and phase detection work well but surgical skill and complication recognition remains a challenge for deep learning. Translational Relevance: This overview of cataract surgery analysis with AI models provides translational value for improving training of the clinician by identifying successes and challenges.

Blood-based assessment of oxidative stress, inflammation, endocrine and metabolic adaptations in eventing horses accounting for plasma volume shift after exercise.

BACKGROUND: After submaximal exercise, blood values of eventing horses show physiological reactions. OBJECTIVES: This prospective longitudinal study investigated blood parameters in 20 elite eventing horses before and after two-four-star cross-country rides. METHODS: Using a mixed model adjusting for plasma volume shift, we assessed exercise-dependent parameters and compared blood values with reference ranges for healthy horses at rest. RESULTS: Following exercise, cortisol, triiodothyronine (T3) and thyroxine (T4) showed short-term increases, and superoxide-dismutase showed a small short-term increase. Hepatic values showed short-term (haemoglobin [HGB], globulins) or sustained increases (bilirubin, glutamate dehydrogenase, alanine aminotransferase). Digestion-related parameters showed small short-term increases (α-amylase, triglycerides) or decreases (cholesterol, DGGR-lipase), apparent through plasma shift adjustment. Zinc decreased in the short term, and iron showed a delayed decrease. White blood cell count increased persistently after training, whereas serum amyloid A remained unchanged. CONCLUSIONS: Exercised eventing horses had consistently elevated HGB and cortisol levels 10 and 30 min after submaximal exercise, exceeding the reference ranges for healthy horses at rest. Exercise activates the hypothalamic-pituitary-adrenocortical and hypothalamic-pituitary-thyroid axes. Antioxidant activity was observed. Increased energy requirements led to the mobilization of energy reserves, and a sustained increase in liver enzymes indicated hepatocellular injury. Mild haemolysis suggested increased muscle metabolism, whereas signs of inflammation were subtle. Further research is needed to identify which horses deviate from mean values.

RAVER1 hinders lethal EMT and modulates miR/RISC activity by the control of alternative splicing.

The RAVER1 protein serves as a co-factor in guiding the polypyrimidine tract-binding protein (PTBP)-dependent control of alternative splicing (AS). Whether RAVER1 solely acts in concert with PTBPs and how it affects cancer cell fate remained elusive. Here, we provide the first comprehensive investigation of RAVER1-controlled AS in cancer cell models. This reveals a pro-oncogenic role of RAVER1 in modulating tumor growth and epithelial-mesenchymal-transition (EMT). Splicing analyses and protein-association studies indicate that RAVER1 guides AS in association with other splicing regulators, including PTBPs and SRSFs. In cancer cells, one major function of RAVER1 is the stimulation of proliferation and restriction of apoptosis. This involves the modulation of AS events within the miR/RISC pathway. Disturbance of RAVER1 impairs miR/RISC activity resulting in severely deregulated gene expression, which promotes lethal TGFB-driven EMT. Among others, RAVER1-modulated splicing events affect the insertion of protein interaction modules in factors guiding miR/RISC-dependent gene silencing. Most prominently, in all three human TNRC6 proteins, RAVER1 controls AS of GW-enriched motifs, which are essential for AGO2-binding and the formation of active miR/RISC complexes. We propose, that RAVER1 is a key modulator of AS events in the miR/RISC pathway ensuring proper abundance and composition of miR/RISC effectors. This ensures balanced expression of TGFB signaling effectors and limits TGFB induced lethal EMT.

Dermatology teaching for undergraduate medical students in clinical routine - a structured four-week curriculum.

BACKGROUND: Dermatology teaching is fundamental for the promotion of young colleagues in our specialty. However, traditional teaching methods are being scrutinized by students of the 'Generation Y and Z', which can pose new challenges for teaching institutions. We therefore aimed to assess the motivational impact and reception of a newly created four-week curriculum containing modernized teaching methods integrated into clinical routine. METHODS: In this single-center study, 67 medical students completed this curriculum composed of weekly learning objectives including knowledge of morphological terms, 10 common dermatoses, communication and presentation skills. The participants provided information on their level of interest in dermatology each week as well as positive and negative aspects of the curriculum. RESULTS: During the curriculum a significant median increase in interest in dermatology was reported with no differences between the genders. Low initial interest could be improved, high initial interest maintained. Participants with an interest in scientific work (20.9%) were more motivated during the curriculum. The variety, quality of teaching and structure were the main aspects rated positively. Suggestions for improvement included the need for more teaching by senior doctors, transfer of responsibility, and a working environment updated to the latest technology standards. CONCLUSION: The presented curriculum was well received by the participants and allowed to better define learning preferences of new generations which can be helpful to modernize traditional teaching methods. Interest in scientific work could be a factor to identify students with a particularly strong interest in dermatology.

The Pink Doughnut Sign-A Clinical Clue to Recognize Basal Cell Carcinoma Presenting as Chronic Leg Ulcer.

Chronic lower leg ulcers (LLUs) are a very common and associated with a high subjective and economic burden. They can be caused by a variety of factors. We have observed a common clinical feature in patients suffering from LLUs, which ultimately proved to be basal cell carcinomas (BCCs). We have nicknamed this sign "the pink doughnut sign." The pink doughnut sign can aid in the early recognition of such BCCs, which is crucial to prevent unnecessary suffering, treatment costs, and poor local outcomes.

Fibroblast Growth Factor-23 (FGF-23) in Dogs-Reference Interval and Correlation with Hematological and Biochemical Parameters.

Fibroblast growth factor-23 (FGF-23) is a phosphaturic hormone used to monitor chronic kidney disease (CKD) in humans. The aims of this study were (1) to determine the intra- and interassay precision of the FGF-23 concentrations in dogs as measured via the Kainos ELISA FGF-23 kit, (2) to calculate a reference interval, and (3) to assess the correlation of the FGF-23 concentration with the hematological and biochemical parameters. The coefficient of variation was below 15% for both the intra- and interassay precision, indicating good reproducibility. The reference interval ranged between 95.8 (90% confidence interval: 44.6; 139.2) and 695.1 pg/mL (598.7; 799.1) based on 136 clinically healthy dogs, classified as such according to the information of treating veterinarians as well as the unremarkable results of hematology and biochemistry. The FGF-23 concentration differed significantly between dogs aged <9 and ≥9 years (p = 0.045). Four groups of 10 dogs each were retrospectively formed based on the creatinine concentration classification according to the IRIS staging. Correlation was the strongest for the renal parameters. Statistically significant differences in the FGF-23 concentration were demonstrated between the study groups I and III (p < 0.001), I and IV (p < 0.001), and II and IV (p = 0.005). There was a trend for a rising FGF-23 concentration in older dogs. Due to the wide reference interval, diagnostic cut-offs and/or subject-based FGF-23 reference values in each dog are needed for monitoring and clinical interpretation.

Blood-Based Markers for Skeletal and Cardiac Muscle Function in Eventing Horses before and after Cross-Country Rides and How They Are Influenced by Plasma Volume Shift.

Horses competing in cross-country tests are subjected to high physical demands. Within the scope of this prospective longitudinal study, blood values of 20 elite eventing horses were examined before and after two- to four-star cross-country rides. The aim was to find out whether blood-based markers for skeletal muscle and cardiac muscle function change after cross-country exercise. Parameters that provide information about fluid balance, muscle enzymes, metabolites and cardiac muscle-specific markers were investigated. We developed an approach to eliminate the concentration changes caused by reduced plasma volume. Parameters were measured pre, 10 and 30 min post exercise and the next morning and were evaluated using a mixed model. Thirty minutes after exercise, most parameter concentrations changed in an exercise-dependent manner. The next morning, most exercise-related markers recovered rapidly, while creatine kinase (CK) (26% increase; p = 0.008) and lactate dehydrogenase (LDH) (15% increase; p < 0.001) showed a declining but sustained increase. Cardiac troponin I (cTnI) increased above the reference range in 40 of the 55 rides (73%) and in 18 of 20 horses in the morning after exercise.

Comparison of Three Different Diagnostic Assays for Fibroblast Growth Factor-23 (FGF-23) Measurements in Cats: A Pilot Study.

Fibroblast growth factor-23 (FGF-23) is a phosphaturic hormone used to monitor chronic kidney disease (CKD) in humans. The aim of this pilot study was to compare three diagnostic assays and to assess how the results correlate with parameters of renal dysfunction in cats. Four groups of 10 cats each were formed retrospectively according to creatinine, based on IRIS staging. FGF-23 was measured using two different ELISAs (MyBioSource and Kainos ELISA FGF-23 Kit) and an automated assay on the DiaSorin Liaison platform. Measurements were performed in 40 cats. Spearman's rank correlation coefficient showed a strong correlation between the Kainos and DiaSorin assays (ρ = 0.742/p < 0.001) and a low correlation (ρ = 0.443/p = 0.005) between the Kainos and MyBioSource assays. The measurements with the Kainos assay strongly correlated with urea (ρ = 0.835/p < 0.001) and creatinine (ρ = 0.764/p < 0.001), and moderately correlated with SDMA (ρ = 0.580/p < 0.001) and phosphorus (ρ = 0.532/p < 0.001). The results of the MyBioSource and DiaSorin assays only showed a moderate correlation with urea (ρ = 0.624/0.572) and creatinine (ρ = 0.622/0.510) concentrations (p = 0.001 each). The Kainos assay showed the strongest correlation (ρ = 0.806) with the various creatinine concentrations according to the IRIS, followed by the MyBioSource (ρ = 0.663/p < 0.001) and DiaSorin assays (ρ = 0.580/p < 0.001). Overall, the Kainos assay demonstrated the best correlations with both biomarkers and various creatinine concentrations according to the IRIS. Individual assay-based reference values should be established to make a reliable interpretation of FGF-23 levels possible to diagnose or monitor feline CKD.

Acceptance of Telemedicine Compared to In-Person Consultation From the Providers' and Users' Perspectives: Multicenter, Cross-Sectional Study in Dermatology.

BACKGROUND: Teledermatology is currently finding its place in modern health care worldwide as a rapidly evolving field. OBJECTIVE: The aim of this study was to investigate the acceptance of teledermatology compared to in-person consultation from the perspective of patients and professionals. METHODS: This multicenter, cross-sectional pilot study was performed at secondary and tertiary referral centers of dermatology in Switzerland from August 2019 to January 2020. A customized questionnaire addressing demographics and educational data, experience with telemedicine, and presumed willingness to replace in-patient consultations with teledermatology was completed by dermatological patients, dermatologists, and health care workers in dermatology. RESULTS: Among a total of 664 participants, the ones with previous telemedicine experience (171/664, 25.8%) indicated a high level of overall experience with it (patients: 73/106, 68.9%, dermatologists: 6/8, 75.0%, and health care workers: 27/34, 79.4%). Patients, dermatologists, and health care workers were most likely willing to replace in-person consultations with teledermatology for minor health issues (353/512, 68.9%; 37/45, 82.2%; and 89/107, 83.2%, respectively). We observed a higher preference for telemedicine among individuals who have already used telemedicine (patients: P<.001, dermatologists: P=.03, and health care workers, P=.005), as well as among patients with higher educational levels (P=.003). CONCLUSIONS: This study indicates that the preference for teledermatology has a high potential to increase over time since previous experience with telemedicine and a higher level of education were associated with a higher willingness to replace in-patient consultations with telemedicine. We assume that minor skin problems are the most promising issue in teledermatology. Our findings emphasize the need for dermatologists to be actively involved in the transition to teledermatology. TRIAL REGISTRATION: ClinicalTrials.gov NCT04495036; https://classic.clinicaltrials.gov/ct2/show/NCT04495036.

Human epidermis organotypic cultures, a reproducible system recapitulating the epidermis in vitro.

The translatability of research is highly dependent on models that recapitulate human tissues and organs. Here, we describe a procedure for the generation of human epidermis organotypic cultures (HEOCs) from primary keratinocytes isolated from foreskin and adult skin as well as from an immortalized keratinocyte cell line (KerTr). We tested several media conditions to develop a defined HEOC growing and expansion media. We characterized the HEOCs and show that in optimal culture conditions they express the proliferation marker Ki67, the basement membrane protein collagen 17 (col17) and the epidermal differentiation markers keratin 15 (K15), keratin 14 (K14), keratin 5 (K5), keratin 10 (K10), keratin 1 (K1), transglutaminase 1 (TGM1), transglutaminase 3 (TGM3) and filaggrin (FLG). Thus, they recapitulate the human epidermis and are stratified from the basal layer to the stratum corneum. These HEOC can be generated reproducibly on a large scale, making it an invaluable model for screening therapeutic compounds and also for the study of pathologies affecting the epidermis.

RNA-binding proteins in cancer drug discovery.

RNA-binding proteins (RBPs) are crucial players in tumorigenesis and, hence, promising targets in cancer drug discovery. However, they are largely regarded as 'undruggable', because of the often noncatalytic and complex interactions between protein and RNA, which limit the discovery of specific inhibitors. Nonetheless, over the past 10 years, drug discovery efforts have uncovered RBP inhibitors with clinical relevance, highlighting the disruption of RNA-protein networks as a promising avenue for cancer therapeutics. In this review, we discuss the role of structurally distinct RBPs in cancer, and the mechanisms of RBP-directed small-molecule inhibitors (SMOIs) focusing on drug-protein interactions, binding surfaces, potency, and translational potential. Additionally, we underline the limitations of RBP-targeting drug discovery assays and comment on future trends in the field.

Meta-omics profiling of full-scale groundwater rapid sand filters explains stratification of iron, ammonium and manganese removals.

Rapid sand filters (RSF) are an established and widely applied technology for groundwater treatment. Yet, the underlying interwoven biological and physical-chemical reactions controlling the sequential removal of iron, ammonia and manganese remain poorly understood. To resolve the contribution and interactions between the individual reactions, we studied two full-scale drinking water treatment plant configurations, namely (i) one dual-media (anthracite and quartz sand) filter and (ii) two single-media (quartz sand) filters in series. In situ and ex situ activity tests were combined with mineral coating characterization and metagenome-guided metaproteomics along the depth of each filter. Both plants exhibited comparable performances and process compartmentalization, with most of ammonium and manganese removal occurring only after complete iron depletion. The homogeneity of the media coating and genome-based microbial composition within each compartment highlighted the effect of backwashing, namely the complete vertical mixing of the filter media. In stark contrast to this homogeneity, the removal of the contaminants was strongly stratified within each compartment, and decreased along the filter height. This apparent and longstanding conflict was resolved by quantifying the expressed proteome at different filter heights, revealing a consistent stratification of proteins catalysing ammonia oxidation and protein-based relative abundances of nitrifying genera (up to 2 orders of magnitude difference between top and bottom samples). This implies that microorganisms adapt their protein pool to the available nutrient load at a faster rate than the backwash mixing frequency. Ultimately, these results show the unique and complementary potential of metaproteomics to understand metabolic adaptations and interactions in highly dynamic ecosystems.

Colchicine - Renaissance of an "ancient" drug.

Colchicine, which was already used by the ancient Egyptians, has recently experienced a renaissance in various medical disciplines, including dermatology. However, due to the potentially significant side effects of systemic use, many clinicians are cautious in their use of colchicine. This review provides a practical overview of the data on the established and emerging use of systemic and topical colchicine in dermatologic diseases.

CSF1R-Mediated Myeloid Cell Depletion Prolongs Lifespan But Aggravates Distinct Motor Symptoms in a Model of Multiple System Atrophy.

As the CNS-resident macrophages and member of the myeloid lineage, microglia fulfill manifold functions important for brain development and homeostasis. In the context of neurodegenerative diseases, they have been implicated in degenerative and regenerative processes. The discovery of distinct activation patterns, including increased phagocytosis, indicated a damaging role of myeloid cells in multiple system atrophy (MSA), a devastating, rapidly progressing atypical parkinsonian disorder. Here, we analyzed the gene expression profile of microglia in a mouse model of MSA (MBP29-hα-syn) and identified a disease-associated expression profile and upregulation of the colony-stimulating factor 1 (Csf1). Thus, we hypothesized that CSF1 receptor-mediated depletion of myeloid cells using PLX5622 modifies the disease progression and neuropathological phenotype in this mouse model. Intriguingly, sex-balanced analysis of myeloid cell depletion in MBP29-hα-syn mice revealed a two-faced outcome comprising an improved survival rate accompanied by a delayed onset of neurological symptoms in contrast to severely impaired motor functions. Furthermore, PLX5622 reversed gene expression profiles related to myeloid cell activation but reduced gene expression associated with transsynaptic signaling and signal release. While transcriptional changes were accompanied by a reduction of dopaminergic neurons in the SNpc, striatal neuritic density was increased upon myeloid cell depletion in MBP29-hα-syn mice. Together, our findings provide insight into the complex, two-faced role of myeloid cells in the context of MSA emphasizing the importance to carefully balance the beneficial and adverse effects of CSF1R inhibition in different models of neurodegenerative disorders before its clinical translation.SIGNIFICANCE STATEMENT Myeloid cells have been implicated as detrimental in the disease pathogenesis of multiple system atrophy. However, long-term CSF1R-dependent depletion of these cells in a mouse model of multiple system atrophy demonstrates a two-faced effect involving an improved survival associated with a delayed onset of disease and reduced inflammation which was contrasted by severely impaired motor functions, synaptic signaling, and neuronal circuitries. Thus, this study unraveled a complex role of myeloid cells in multiple system atrophy, which indicates important functions beyond the previously described disease-associated, destructive phenotype and emphasized the need of further investigation to carefully and individually fine-tune immunologic processes in different neurodegenerative diseases.

Tyrosine 146 of the Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) Is Essential for Its Hepatitis B Virus (HBV) Receptor Function and HBV Entry into Hepatocytes.

Na+/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1) is a hepatic bile acid uptake carrier participating in the enterohepatic circulation of bile acids. Apart from its transporter function, NTCP acts as the high-affinity liver-specific receptor for the hepatitis B virus (HBV), which attaches via its preS1-peptide domain of the large surface protein to NTCP, subsequently leading to endocytosis of the virus/NTCP-receptor complex. Although the process of NTCP-dependent HBV infection of hepatocytes has received much attention over the last decade, the precise molecular sites of the virus/NTCP interaction have not been fully identified. Inspection of the primary protein sequence of human NTCP revealed 139YIYSRGIY146 as a highly conserved tyrosine-rich motif. To study the role of Y139, Y141 and Y146 amino acids in NTCP biology, the aforementioned residues were substituted with alanine, phenylalanine or glutamate (mimicking phosphorylation) using site-directed mutagenesis. Similar to wt NTCP, the Y139A, Y141A, Y146A, Y141F, Y146F, and Y146E mutants were expressed at the plasma membrane of HEK293 cells and exhibited intact bile acid transport function. Y146A, Y146E, and Y146F demonstrated transport kinetics comparable to wild-type NTCP with Km values of 57.3-112.4 µM and Vmax values of 6683-7579 pmol/mg protein/min. Only Y141E was transport deficient, most likely due to an intracellular accumulation of the mutant protein. Most importantly, Y146A and Y146E mutation completely abrogated binding of the viral preS1-peptide to NTCP, while the Y146F mutant of NTCP showed some residual binding competence for preS1. Consequently, the NTCP mutants Y146A and Y146E, when expressed in HepG2 hepatoma cells, showed complete loss of susceptibility for in vitro HBV infection. In conclusion, tyrosine 146, and to some extent tyrosine 141, both belonging to the tyrosine-rich motif 139YIYSRGIY146 of human NTCP, are newly identified amino acid residues that play an essential role in the interaction of HBV with its receptor NTCP and, thus, in the process of virus entry into hepatocytes.

HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins.

The biological role of RNA-binding proteins in the secretory pathway is not well established. Here, we describe that human HDLBP/Vigilin directly interacts with more than 80% of ER-localized mRNAs. PAR-CLIP analysis reveals that these transcripts represent high affinity HDLBP substrates and are specifically bound in their coding sequences (CDS), in contrast to CDS/3'UTR-bound cytosolic mRNAs. HDLBP crosslinks strongly to long CU-rich motifs, which frequently reside in CDS of ER-localized mRNAs and result in high affinity multivalent interactions. In addition to HDLBP-ncRNA interactome, quantification of HDLBP-proximal proteome confirms association with components of the translational apparatus and the signal recognition particle. Absence of HDLBP results in decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in model cell lines, as well as decreased tumor growth in a lung cancer mouse model. These results highlight a general function for HDLBP in the translation of ER-localized mRNAs and its relevance for tumor progression.