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Introduction: There is considerable research focusing on the negative outcomes of psychotherapy, however, there remains an overlooked population: those who appear to show no response after treatment. This qualitative evidence synthesis aimed to review the available literature exploring client and therapist experiences of psychotherapy nonresponse., Method: Seven databases were searched for studies using qualitative approaches to explore nonresponse. Twenty-four studies met inclusion criteria and were analysed using Thematic Synthesis., Results: Six overarching client perspective themes and 18 subthemes were identified: Hopes and fears; A difficult task; A Disconnected relationship; Staying involved; Therapy was not worth the investment and, On a trajectory for improvement. Four overarching therapist perspective themes and 10 subthemes were identified: High expectations; Experiencing a disconnect; Feeling threatened and Holding onto hope., Discussion: A model of psychotherapy nonresponse is proposed. The experience of nonresponse appears to involve both clients and therapists finding therapy difficult, experiencing problems connecting and not being open in the therapeutic relationship. Importantly, nonresponse appears not to be an absence of effects, but a range of experiences that are potentially harmful, particularly to clients. However, there were varied experiences of nonresponse, which indicates the importance of qualitative outcome measurement., Competing Interests: Declaration of competing interest All authors declare that they hold no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: In patients with squamous cell carcinoma of the anus (SCCA), magnetic resonance (MR) imaging is recommended for pre-treatment staging prior to chemo-radiotherapy (CRT), but large-scale evaluation of its staging performance is lacking., Methods: We re-characterised pre-treatment MRs from 228 patients with non-metastatic SCCA treated consecutively by CRT (2006-2015) at one UK cancer centre. We derived TN staging from tumour size (mrTr) and nodal involvement (mrN), and additionally characterised novel beyond TN features such as extramural vascular invasion (mrEMVI) and tumour signal heterogeneity (mrTSH). Primary outcomes were 5-year overall survival (OS) and 3-year loco-regional failure (LRF). Time-to-event analyses used Kaplan-Meier estimates; Hazard Ratios (HRs) with confidence intervals (CIs) were derived from Cox models., Results: With a median follow up of 60.9 months, 5-year OS was 74%. Poor OS was associated with increasing mrT (HR: 1.12 per cm [95% CI: 1.07-1.33]), nodal positivity (HR 2.08 [95% CI 1.23-3.52]) and mrEMVI (HR 3.66 [95% CI: 1.88-7.41]). 3-year LRF rate was 16.5%. Increased LRF was associated with increasing mrT (HR: 1.43 per cm [95% CI: 1.26-1.63]), nodal positivity (HR 2.70 [95% CI 1.39-5.24]) and mrTSH (HR 2.66 [95% CI 1.29-5.48])., Conclusions: In SCCA, the study demonstrates that mrT and mrN stages are prognostic, while mrEMVI and mrTSH may be novel prognostic factors., (© 2024. The Author(s).)

Inequality in treatment access is a pressing issue in most healthcare systems across many medical disciplines. In mental healthcare, reduced treatment access for minorities is ubiquitous but remedies are sparse. Here we demonstrate that digital tools can reduce the accessibility gap by addressing several key barriers. In a multisite observational study of 129,400 patients within England's NHS services, we evaluated the impact of a personalized artificial intelligence-enabled self-referral chatbot on patient referral volume and diversity in ethnicity, gender and sexual orientation. We found that services that used this digital solution identified substantially increased referrals (15% increase versus 6% increase in control services). Critically, this increase was particularly pronounced in minorities, such as nonbinary (179% increase) and ethnic minority individuals (29% increase). Using natural language processing to analyze qualitative feedback from 42,332 individuals, we found that the chatbot's human-free nature and the patients' self-realization of their need for treatment were potential drivers for the observed improvement in the diversity of access. This provides strong evidence that digital tools may help overcome the pervasive inequality in mental healthcare., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Most mental health care providers face the challenge of increased demand for psychotherapy in the absence of increased funding or staffing. To overcome this supply-demand imbalance, care providers must increase the efficiency of service delivery., Objective: In this study, we examined whether artificial intelligence (AI)-enabled digital solutions can help mental health care practitioners to use their time more efficiently, and thus reduce strain on services and improve patient outcomes., Methods: In this study, we focused on the use of an AI solution (Limbic Access) to support initial patient referral and clinical assessment within the UK's National Health Service. Data were collected from 9 Talking Therapies services across England, comprising 64,862 patients., Results: We showed that the use of this AI solution improves clinical efficiency by reducing the time clinicians spend on mental health assessments. Furthermore, we found improved outcomes for patients using the AI solution in several key metrics, such as reduced wait times, reduced dropout rates, improved allocation to appropriate treatment pathways, and, most importantly, improved recovery rates. When investigating the mechanism by which the AI solution achieved these improvements, we found that the provision of clinically relevant information ahead of clinical assessment was critical for these observed effects., Conclusions: Our results emphasize the utility of using AI solutions to support the mental health workforce, further highlighting the potential of AI solutions to increase the efficiency of care delivery and improve clinical outcomes for patients., (©Max Rollwage, Johanna Habicht, Keno Juechems, Ben Carrington, Sruthi Viswanathan, Mona Stylianou, Tobias U Hauser, Ross Harper. Originally published in JMIR AI (https://ai.jmir.org), 13.12.2023.)

Pigment patterns and skin appendages are prominent features of vertebrate skin. In zebrafish, regularly patterned pigment stripes and an array of calcified scales form simultaneously in the skin during post-embryonic development. Understanding the mechanisms that regulate stripe patterning and scale morphogenesis may lead to the discovery of fundamental mechanisms that govern the development of animal form. To learn about cell types and signaling interactions that govern skin patterning and morphogenesis, we generated and analyzed single-cell transcriptomes of skin from wild-type fish as well as fish having genetic or transgenically induced defects in squamation or pigmentation. These data reveal a previously undescribed population of epidermal cells that express transcripts encoding enamel matrix proteins, suggest hormonal control of epithelial-mesenchymal signaling, clarify the signaling network that governs scale papillae development, and identify a critical role for the hypodermis in supporting pigment cell development. Additionally, these comprehensive single-cell transcriptomic data representing skin phenotypes of biomedical relevance should provide a useful resource for accelerating the discovery of mechanisms that govern skin development and homeostasis., Competing Interests: AA, LS, AC, SS, CE, BC, DW, WP, CT, DP No competing interests declared

Distinguishing individuals or small groups is essential for many experiments. The regenerative properties of zebrafish make traditional marking methods for rodent models (e.g., tattoos, tissue clipping) ineffective. Fluorescent Visible Implant Elastomer (VIE) can permanently mark juvenile and adult zebrafish but to date no marking technique has been described for larval stage zebrafish. This protocol, Zebrafish Injectable Plastic for Identification Tagging (ZIP IT), utilizes VIE in zebrafish as early as 2 days post fertilization (dpf) using standard microinjection methods and direct injection using an insulin syringe at one month. Larval zebrafish between 2 and 7 dpf were injected in the dorsal musculature. At one month, retention and visibility of the VIE was observed in 72% of the injected fish with no effect on growth; however, a variable change in mortality was observed, generally higher than the uninjected fish. This demonstrates that VIE can be used in very early stages of fish development, providing the first procedure to track individuals or groups within a larger population. Subcutaneous injection of juvenile zebrafish starting at one month has greater than 99%-mark retention and visibility with very low mortality. The combination of larvae and juvenile VIE injections also provide a powerful tool to track and gather data from marked fish throughout their lifespan.•We present a method for tagging individuals or groups of zebrafish at most life stages (larvae or juvenile/adult) with Visible Implant Elastomer (VIE).•The larvae and juvenile injection procedures can be combined so that an individual fish can be tagged for its entire lifetime. Larvae injections become significantly less visible after one month; however, the fish can be reinjected using the juvenile procedure, thus allowing continuity of the visible mark.•This protocol was empirically built on the "Evaluation of VIE tags in Zebrafish ( Danio rerio )" by Hohn and Petrie-Hanson (2013) and expanded to include larvae and a small batch elastomer mixing technique., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Generation of zebrafish ( Danio rerio ) models with targeted insertion of epitope tags and point mutations is highly desirable for functional genomics and disease modeling studies. Currently, CRISPR/Cas9-mediated knock-in is the method of choice for insertion of exogeneous sequences by providing a repair template for homology-directed repair (HDR). A major hurdle in generating knock-in models is the labor and cost involved in screening of injected fish to identify the precise knock-in events due to low efficiency of the HDR pathway in zebrafish. Thus, we developed fluorescent PCR-based high-throughput screening methods for precise knock-in of epitope tags and point mutations in zebrafish. Here, we provide a step-by-step guide that describes selection of an active sgRNA near the intended knock-in site, design of single-stranded oligonucleotide (ssODN) templates for HDR, quick validation of somatic knock-in using injected embryos, and screening for germline transmission of precise knock-in events to establish stable lines. Our screening method relies on the size-based separation of all fragments in an amplicon by fluorescent PCR and capillary electrophoresis, thus providing a robust and cost-effective strategy. Although we present the use of this protocol for insertion of epitope tags and point mutations, it can be used for insertion of any small DNA fragments (e.g., LoxP sites, in-frame codons). Furthermore, the screening strategy described here can be used to screen for precise knock-in of small DNA sequences in any model system, as PCR amplification of the target region is its only requirement. Key features This protocol expands the use of fluorescent PCR and CRISPR-STAT for screening of precise knock-in of small insertions and point mutations in zebrafish. Allows validation of selected sgRNA and HDR template within two weeks by somatic knock-in screening. Allows robust screening of point mutations by combining restriction digest with CRISPR-STAT. Graphical overview Overview of the three-phase knock-in pipeline in zebrafish (created with BioRender.com )., Competing Interests: Competing interestsThe authors declare no competing interests., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.)

Background: Oral clefts and ectrodactyly are common, heterogeneous birth defects. We performed whole-exome sequencing (WES) analysis in a Syrian family. The proband presented with both orofacial clefting and ectrodactyly but not ectodermal dysplasia as typically seen in ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome-3. A paternal uncle with only an oral cleft was deceased and unavailable for analysis., Methods: Variant annotation, Mendelian inconsistencies, and novel variants in known cleft genes were examined. Candidate variants were validated using Sanger sequencing, and pathogenicity assessed by knocking out the tp63 gene in zebrafish to evaluate its role during zebrafish development., Results: Twenty-eight candidate de novo events were identified, one of which is in a known oral cleft and ectrodactyly gene, TP63 (c.956G > T, p.Arg319Leu), and confirmed by Sanger sequencing., Conclusion: TP63 mutations are associated with multiple autosomal dominant orofacial clefting and limb malformation disorders. The p.Arg319Leu mutation seen in this patient is de novo but also novel. Two known mutations in the same codon (c.956G > A, p.(Arg319His; rs121908839, c.955C > T), p.Arg319Cys) cause ectrodactyly, providing evidence that mutating this codon is deleterious. While this TP63 mutation is the best candidate for the patient's clinical presentation, whether it is responsible for the entire phenotype is unclear. Generation and characterization of tp63 knockout zebrafish showed necrosis and rupture of the head at 3 days post-fertilization (dpf). The embryonic phenotype could not be rescued by injection of zebrafish or human messenger RNA (mRNA). Further functional analysis is needed to determine what proportion of the phenotype is due to this mutation., (© 2023 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Germ line loss-of-function heterozygous mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancies (FPDMM) characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant hematopoietic stem and progenitor cells (HSPCs) in vivo long term. We generated a rhesus macaque with an FPDMM competitive repopulation model using CRISPR/Cas9 nonhomologous end joining editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous HSPCs and tracked mutated allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared with AAVS1-edited cells. Platelet counts remained below the normal range in the long term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous-mutated HSPCs long term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse myelodysplastic syndrome/ acute myeloid leukemia predisposition and thrombopoietic defects.

Background: Genome editing using CRISPR/Cas9 has become a powerful tool in zebrafish to generate targeted gene knockouts models. However, its use for targeted knock-in remains challenging due to inefficient homology directed repair (HDR) pathway in zebrafish, highlighting the need for efficient and cost-effective screening methods.  RESULTS: Here, we present our fluorescent PCR and capillary electrophoresis based screening approach for knock-in using a single-stranded oligodeoxynucleotide donor (ssODN) as a repair template for the targeted insertion of epitope tags, or single nucleotide changes to recapitulate pathogenic human alleles. For the insertion of epitope tags, we took advantage of the expected change in size of the PCR product. For point mutations, we combined fluorescent PCR with restriction fragment length polymorphism (RFLP) analysis to distinguish the fish with the knock-in allele. As a proof-of-principle, we present our data on the generation of fish lines with insertion of a FLAG tag at the tcnba locus, an HA tag at the gata2b locus, and a point mutation observed in Gaucher disease patients in the gba gene. Despite the low number of germline transmitting founders (1-5%), combining our screening methods with prioritization of founder fish by fin biopsies allowed us to establish stable knock-in lines by screening 12 or less fish per gene., Conclusions: We have established a robust pipeline for the generation of zebrafish models with precise integration of small DNA sequences and point mutations at the desired sites in the genome. Our screening method is very efficient and easy to implement as it is PCR-based and only requires access to a capillary sequencer., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Tumor necrosis factor (TNF) is a pleiotropic cytokine belonging to a family of trimeric proteins with both proinflammatory and immunoregulatory functions. TNF is a key mediator in autoimmune diseases and during the last couple of decades several biologic drugs have delivered new therapeutic options for patients suffering from chronic autoimmune diseases such as rheumatoid arthritis and chronic inflammatory bowel disease. Attempts to design small molecule therapies directed to this cytokine have not led to approved products yet. Here we report the discovery and development of a potent small molecule inhibitor of TNF that was recently moved into phase 1 clinical trials. The molecule, SAR441566, stabilizes an asymmetrical form of the soluble TNF trimer, compromises downstream signaling and inhibits the functions of TNF in vitro and in vivo . With SAR441566 being studied in healthy volunteers we hope to deliver a more convenient orally bioavailable and effective treatment option for patients suffering with chronic autoimmune diseases compared to established biologic drugs targeting TNF., Competing Interests: AVu, JO’C, MAN, DW, TL, EH, AVe, XY, MR, BC, MM, AM, JMN, PS, SW, YF, DB, HH, MH, SR, MK and PF are either employees of UCB Pharma or Sanofi and may hold stock of the respective organizations. PF was employed by Sanofi-Aventis Deutschland GmbH at the time the work of the manuscript was conducted. He is now an employee of Boehringer Ingelheim Vetmedica GmbH. TM is employed by MilvuswoodConsultancy. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vugler, O’Connell, Nguyen, Weitz, Leeuw, Hickford, Verbitsky, Ying, Rehberg, Carrington, Merriman, Moss, Nicholas, Stanley, Wright, Bourne, Foricher, Brookings, Horsley, Herrmann, Rao, Kohlmann and Florian.)

ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in 3' splice-site recognition as a component of both the major and minor spliceosomes that mediate the splicing of U2-type (major) and U12-type (minor) introns, respectively. Studies of ZRSR2 -depleted cell lines and ZRSR2 -mutated patient samples revealed its essential role in the U12-dependent minor spliceosome. However, the role of ZRSR2 during embryonic development is not clear, as its function is compensated for by Zrsr1 in mice. Here, we utilized the zebrafish model to investigate the role of zrsr2 during embryonic development. Using CRISPR/Cas9 technology, we generated a zrsr2 -knockout zebrafish line, termed zrsr2 hg129/hg129 (p.Trp167Argfs*9) and examined embryo development in the homozygous mutant embryos. zrsr2 hg129/hg129 embryos displayed multiple developmental defects starting at 4 days post fertilization (dpf) and died after 8 dpf, suggesting that proper Zrsr2 function is required during embryonic development. The global transcriptome analysis of 3 dpf zrsr2 hg129/hg129 embryos revealed that the loss of Zrsr2 results in the downregulation of essential metabolic pathways and the aberrant retention of minor introns in about one-third of all minor intron-containing genes in zebrafish. Overall, our study has demonstrated that the role of Zrsr2 as a component of the minor spliceosome is conserved and critical for proper embryonic development in zebrafish.

The use of zebrafish in functional genomics and disease modeling has become popular due to the ease of targeted mutagenesis with genome editing nucleases, i.e., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). These nucleases, specifically CRISPR/Cas9, are routinely used to generate gene knockout mutants by causing a double stranded break at the desired site in the target gene and selecting for frameshift insertions or deletions (indels) caused by the errors during the repair process. Thus, a variety of methods have been developed to identify fish with indels during the process of mutant generation and phenotypic analysis. These methods range from PCR and gel-based low-throughput methods to high-throughput methods requiring specific reagents and/or equipment. Here, we provide a comprehensive review of currently used indel detection methods in zebrafish. By discussing the molecular basis for each method as well as their pros and cons, we hope that this review will serve as a comprehensive resource for zebrafish researchers, allowing them to choose the most appropriate method depending upon their budget, access to required equipment and the throughput needs of the projects.

Progressive pancytopenia is a common feature observed in DNA crosslink repair deficiency disorder, Fanconi anemia (FA). However, this phenotype has not been recapitulated in single FA gene knockout animal models. In this study, we analyzed hematological characteristics in zebrafish null mutants for two FA genes, fanca and fanco. In adult mutants, we demonstrate age-associated reduction in blood cell counts for all lineages, resembling progressive pancytopenia in FA patients. In larval mutants, we demonstrate vascular injury-induced thrombosis defects, particularly upon treatment with crosslinking agent diepoxybutane (DEB), indicating DNA damage induced inefficiency of thrombocytes., (Published by Elsevier Inc.)

RUNX1 is essential for the generation of hematopoietic stem cells (HSCs). Runx1-null mouse embryos lack definitive hematopoiesis and die in mid-gestation. However, although zebrafish embryos with a runx1 W84X mutation have defects in early definitive hematopoiesis, some runx1W84X/W84X embryos can develop to fertile adults with blood cells of multilineages, raising the possibility that HSCs can emerge without RUNX1. Here, using 3 new zebrafish runx1-/- lines, we uncovered the compensatory mechanism for runx1-independent hematopoiesis. We show that, in the absence of a functional runx1, a cd41-green fluorescent protein (GFP)+ population of hematopoietic precursors still emerge from the hemogenic endothelium and can colonize the hematopoietic tissues of the mutant embryos. Single-cell RNA sequencing of the cd41-GFP+ cells identified a set of runx1-/--specific signature genes during hematopoiesis. Significantly, gata2b, which normally acts upstream of runx1 for the generation of HSCs, was increased in the cd41-GFP+ cells in runx1-/- embryos. Interestingly, genetic inactivation of both gata2b and its paralog gata2a did not affect hematopoiesis. However, knocking out runx1 and any 3 of the 4 alleles of gata2a and gata2b abolished definitive hematopoiesis. Gata2 expression was also upregulated in hematopoietic cells in Runx1-/- mice, suggesting the compensatory mechanism is conserved. Our findings indicate that RUNX1 and GATA2 serve redundant roles for HSC production, acting as each other's safeguard., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental approaches using animal model systems. Zebrafish has become a powerful vertebrate genetic model system with availability due to the ease of genome editing and large-scale phenotype screening. Here, we generated zebrafish mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. Large-scale phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level., (Copyright © 2021. Published by Elsevier B.V.)