The rRNA m6A methyltransferase METTL5 is involved in pluripotency and developmental programs.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N6-methyladenosine (m6A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m6A in 18S rRNA at position A1832 We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m6A in rRNA in stemness, differentiation, development, and diseases.

Overcoming Challenges of Lignin Nanoparticles: Expanding Opportunities for Scalable and Multifunctional Nanomaterials.

ConspectusThe increasing demand for polymeric materials derived from petroleum resources, along with rising concerns about climate change and global plastic pollution, has driven the development of biobased polymeric materials. Lignin, which is the second most abundant biomacromolecule after cellulose, represents a promising renewable raw material source for the preparation of advanced materials. The lucrative properties of lignin include its high carbon content (>60 atom %), high thermal stability, biodegradability, antioxidant activity, absorbance of ultraviolet radiation, and slower biodegradability compared to other wood components. Moreover, the advent of lignin nanoparticles (LNPs) over the last ten years has circumvented many well-known shortcomings of technical lignins, such as heterogeneity and poor compatibility with polymers, thereby unlocking the great potential of lignin for the development of advanced functional materials.LNPs stand out owing to their well-defined spherical shape and excellent colloidal stability, which is due to the electrostatic repulsion forces of carboxylic acid and phenolic hydroxyl groups enriched on their surface. These forces prevent their aggregation in aqueous dispersions (pH 3-9) and provide a high surface area to mass ratio that has been exploited to adsorb positively charged compounds such as enzymes or polymers. Consequently, it is not surprising that LNPs have become a prominent player in applied research in areas such as biocatalysis and polymeric composites, among others. However, like all ventures of life, LNPs also face certain challenges that limit their potential end-uses. Solvent instability remains the most challenging aspect due to the tendency of these particles to dissolve or aggregate in organic solvents and basic or acidic pH, thus limiting the window for their chemical functionalization and applications. In addition, the need for organic solvent during their preparation, the poor miscibility with hydrophobic polymeric matrices, and the nascent phase regarding their use in smart materials have been identified as important challenges that need to be addressed.In this Account, we recapitulate our efforts over the past years to overcome the main limitations mentioned above. We begin with a brief introduction to the fundamentals of LNPs and a detailed discussion of their associated challenges. We then highlight our work on: (i) Preparation of lignin-based nanocomposites with improved properties through a controlled dispersion of LNPs within a hydrophobic polymeric matrix, (ii) Stabilization of LNPs via covalent (intraparticle cross-linking) and noncovalent (hydration barrier) approaches, (iii) The development of an organic-solvent-free method for the production of LNPs, and (iv) The development of LNPs toward smart materials with high lignin content. Finally, we also offer our perspectives on this rapidly growing field.

Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction.

Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.

TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity.

The transcription factor TRPS1 is a context-dependent oncogene in breast cancer. In the mammary gland, TRPS1 activity is restricted to the luminal population and is critical during puberty and pregnancy. Its function in the resting state remains however unclear. To evaluate whether it could be a target for cancer therapy, we investigated TRPS1 function in the healthy adult mammary gland using a conditional ubiquitous depletion mouse model where long-term depletion does not affect fitness. Using transcriptomic approaches, flow cytometry and functional assays, we show that TRPS1 activity is essential to maintain a functional luminal progenitor compartment. This requires the repression of both YAP/TAZ and SRF/MRTF activities. TRPS1 represses SRF/MRTF activity indirectly by modulating RhoA activity. Our work uncovers a hitherto undisclosed function of TRPS1 in luminal progenitors intrinsically linked to mechanotransduction in the mammary gland. It may also provide new insights into the oncogenic functions of TRPS1 as luminal progenitors are likely the cells of origin of many breast cancers.

Implication of transcription factor FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.

sRNAbench and sRNAtoolbox 2022 update: accurate miRNA and sncRNA profiling for model and non-model organisms.

The NCBI Sequence Read Archive currently hosts microRNA sequencing data for over 800 different species, evidencing the existence of a broad taxonomic distribution in the field of small RNA research. Simultaneously, the number of samples per miRNA-seq study continues to increase resulting in a vast amount of data that requires accurate, fast and user-friendly analysis methods. Since the previous release of sRNAtoolbox in 2019, 55 000 sRNAbench jobs have been submitted which has motivated many improvements in its usability and the scope of the underlying annotation database. With this update, users can upload an unlimited number of samples or import them from Google Drive, Dropbox or URLs. Micro- and small RNA profiling can now be carried out using high-confidence Metazoan and plant specific databases, MirGeneDB and PmiREN respectively, together with genome assemblies and libraries from 441 Ensembl species. The new results page includes straightforward sample annotation to allow downstream differential expression analysis with sRNAde. Unassigned reads can also be explored by means of a new tool that performs mapping to microbial references, which can reveal contamination events or biologically meaningful findings as we describe in the example. sRNAtoolbox is available at: https://arn.ugr.es/srnatoolbox/.

Stress and suicide risk among adolescents: the role of problematic internet use, gaming disorder and emotional regulation.

BACKGROUND: Previous studies have associated videogame playing and social media use with suicidal behaviors together with lower stress coping or poor emotion regulation strategies. Due to the inconclusive evidence regarding the factors associated with suicidal behavior, the present study aimed to overcome the limitations of previous research and explored the relationship between adolescent stress, problematic internet use (PIU), gaming disorder (GD), and emotional regulation (ER) in a cross-section design. It was hypothesized that stress would have a direct effect on suicide risk (SR) as well as being mediated by PIU, GD, and ER. METHODS: The participants comprised 430 adolescents (58.4% male) aged between 16 and 19 years. They completed an online survey including the Mobile-Related Experiences Questionnaire, Internet Gaming Disorder Scale-Short Form, Meta-Mood Trait Repair Scale, and Spanish version of the Suicidal Behaviors Questionnaire. RESULTS: A total of 34.2% of the adolescents (N = 147) were at risk for SR. Results also indicated that 30,7% had experienced suicidal ideation at some point in their life, 12.1% had at least one plan to die by suicide, and 5.1% had attempted suicide. Results of path analysis confirmed that stress appeared to be a risk factor for suicide, but that its effects were not mediated by PIU. However, ER and GD mediated the effect of stress on SR. The results suggest that stress is a main risk factor for suicide, especially among adolescents with poor emotional regulation or problematic gaming. CONCLUSIONS: Considering the prevalence of suicide among adolescents, the results of the present study suggest that suicide prevention programs should include emotional regulation strategies, stress coping, and videogaming management skills in the early stages of high school. Providing these protective resources to adolescents will help them face the stressful and changing situations typical of adolescence and will help them to attain greater well-being and satisfaction with life.

Breathable Lignin Nanoparticles as Reversible Gas Swellable Nanoreactors.

The design of stimuli-responsive lignin nanoparticles (LNPs) for advanced applications has hitherto been limited to the preparation of lignin-grafted polymers in which usually the lignin content is low (<25 wt.%) and its role is debatable. Here, the preparation of O2 -responsive LNPs exceeding 75 wt.% in lignin content is shown. Softwood Kraft lignin (SKL) is coprecipitated with a modified SKL fluorinated oleic acid ester (SKL-OlF) to form colloidal stable hybrid LNPs (hy-LNPs). The hy-LNPs with a SKL-OlF content ranging from 10 to 50 wt.% demonstrated a reversible swelling behavior upon O2 /N2 bubbling, increasing their size - ≈35% by volume - and changing their morphology from spherical to core-shell. Exposition of hy-LNPs to O2 bubbling promotes a polarity change on lignin-fluorinated oleic chains, and consequently their migration from the inner part to the surface of the particle, which not only increases the particle size but also endows hy-LNPs with enhanced stability under harsh conditions (pH < 2.5) by the hydration barrier effect. Furthermore, it is also demonstrated that these new stimuli-responsive particles as gas tunable nanoreactors for the synthesis of gold nanoparticles. Combining a straightforward preparation with their enhanced stability and responsiveness to O2 gas these new LNPs pave the way for the next generation of smart lignin-based nanomaterials.

Knockout mouse models as a resource for the study of rare diseases.

Rare diseases (RDs) are a challenge for medicine due to their heterogeneous clinical manifestations and low prevalence. There is a lack of specific treatments and only a few hundred of the approximately 7,000 RDs have an approved regime. Rapid technological development in genome sequencing enables the mass identification of potential candidates that in their mutated form could trigger diseases but are often not confirmed to be causal. Knockout (KO) mouse models are essential to understand the causality of genes by allowing highly standardized research into the pathogenesis of diseases. The German Mouse Clinic (GMC) is one of the pioneers in mouse research and successfully uses (preclinical) data obtained from single-gene KO mutants for research into monogenic RDs. As part of the International Mouse Phenotyping Consortium (IMPC) and INFRAFRONTIER, the pan-European consortium for modeling human diseases, the GMC expands these preclinical data toward global collaborative approaches with researchers, clinicians, and patient groups.Here, we highlight proprietary genes that when deleted mimic clinical phenotypes associated with known RD targets (Nacc1, Bach2, Klotho alpha). We focus on recognized RD genes with no pre-existing KO mouse models (Kansl1l, Acsf3, Pcdhgb2, Rabgap1, Cox7a2) which highlight novel phenotypes capable of optimizing clinical diagnosis. In addition, we present genes with intriguing phenotypic data (Zdhhc5, Wsb2) that are not presently associated with known human RDs.This report provides comprehensive evidence for genes that when deleted cause differences in the KO mouse across multiple organs, providing a huge translational potential for further understanding monogenic RDs and their clinical spectrum. Genetic KO studies in mice are valuable to further explore the underlying physiological mechanisms and their overall therapeutic potential.

Biallelic loss-of-function variants in RABGAP1 cause a novel neurodevelopmental syndrome.

PURPOSE: RABGAP1 is a GTPase-activating protein implicated in a variety of cellular and molecular processes, including mitosis, cell migration, vesicular trafficking, and mTOR signaling. There are no known Mendelian diseases caused by variants in RABGAP1. METHODS: Through GeneMatcher, we identified 5 patients from 3 unrelated families with homozygous variants in the RABGAP1 gene found on exome sequencing. We established lymphoblastoid cells lines derived from an affected individual and her parents and performed RNA sequencing and functional studies. Rabgap1 knockout mice were generated and phenotyped. RESULTS: We report 5 patients presenting with a common constellation of features, including global developmental delay/intellectual disability, microcephaly, bilateral sensorineural hearing loss, and seizures, as well as overlapping dysmorphic features. Neuroimaging revealed common features, including delayed myelination, white matter volume loss, ventriculomegaly, and thinning of the corpus callosum. Functional analysis of patient cells revealed downregulated mTOR signaling and abnormal localization of early endosomes and lysosomes. Rabgap1 knockout mice exhibited several features in common with the patient cohort, including microcephaly, thinning of the corpus callosum, and ventriculomegaly. CONCLUSION: Collectively, our results provide evidence of a novel neurodevelopmental syndrome caused by biallelic loss-of-function variants in RABGAP1.

Stabilized Lignin Nanoparticles for Versatile Hybrid and Functional Nanomaterials.

Spherical lignin nanoparticles are emerging biobased nanomaterials, but instability and dissolution in organic solvents and aqueous alkali restrict their applicability. Here, we report the synthesis of hydroxymethylated lignin nanoparticles and their hydrothermal curing to stabilize the particles by internal cross-linking reactions. These colloidally stable particles contain a high biobased content of 97% with a tunable particle size distribution and structural stability in aqueous media (pH 3 to 12) and organic solvents such as acetone, ethanol, dimethylformamide, and tetrahydrofuran. We demonstrate that the free phenolic hydroxyl groups that are preserved in the cured particles function as efficient reducing sites for silver ions, giving rise to hybrid lignin-silver nanoparticles that can be used for quick and facile sensing of hydrogen peroxide. The stabilized lignin particles can also be directly modified using base-catalyzed reactions such as the ring-opening of cationic epoxides that render the particles with pH-dependent agglomeration and redispersion properties. Combining scalable synthesis, solvent stability, and reusability, this new class of lignin nanoparticles shows potential for its use in circular biobased nanomaterials.

A comprehensive database for integrated analysis of omics data in autoimmune diseases.

BACKGROUND: Autoimmune diseases are heterogeneous pathologies with difficult diagnosis and few therapeutic options. In the last decade, several omics studies have provided significant insights into the molecular mechanisms of these diseases. Nevertheless, data from different cohorts and pathologies are stored independently in public repositories and a unified resource is imperative to assist researchers in this field. RESULTS: Here, we present Autoimmune Diseases Explorer ( https://adex.genyo.es ), a database that integrates 82 curated transcriptomics and methylation studies covering 5609 samples for some of the most common autoimmune diseases. The database provides, in an easy-to-use environment, advanced data analysis and statistical methods for exploring omics datasets, including meta-analysis, differential expression or pathway analysis. CONCLUSIONS: This is the first omics database focused on autoimmune diseases. This resource incorporates homogeneously processed data to facilitate integrative analyses among studies.

RANK signaling increases after anti-HER2 therapy contributing to the emergence of resistance in HER2-positive breast cancer.

BACKGROUND: Around 15-20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance. METHODS: RANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling. RESULTS: RANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status. CONCLUSIONS: Our data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated.

Systematic identification of genetic systems associated with phenotypes in patients with rare genomic copy number variations.

Copy number variation (CNV) related disorders tend to show complex phenotypic profiles that do not match known diseases. This makes it difficult to ascertain their underlying molecular basis. A potential solution is to compare the affected genomic regions for multiple patients that share a pathological phenotype, looking for commonalities. Here, we present a novel approach to associate phenotypes with functional systems, in terms of GO categories and KEGG and Reactome pathways, based on patient data. The approach uses genomic and phenomic data from the same patients, finding shared genomic regions between patients with similar phenotypes. These regions are mapped to genes to find associated functional systems. We applied the approach to analyse patients in the DECIPHER database with de novo CNVs, finding functional systems associated with most phenotypes, often due to mutations affecting related genes in the same genomic region. Manual inspection of the ten top-scoring phenotypes found multiple FunSys connections supported by the previous studies for seven of them. The workflow also produces reports focussed on the genes and FunSys connected to the different phenotypes, alongside patient-specific reports, which give details of the associated genes and FunSys for each individual in the cohort. These can be run in "confidential" mode, preserving patient confidentiality. The workflow presented here can be used to associate phenotypes with functional systems using data at the level of a whole cohort of patients, identifying important connections that could not be found when considering them individually. The full workflow is available for download, enabling it to be run on any patient cohort for which phenotypic and CNV data are available.

Capsazepine decreases corneal pain syndrome in severe dry eye disease.

BACKGROUND: Dry eye disease (DED) is a multifactorial disease of the ocular surface accompanied by neurosensory abnormalities. Here, we evaluated the effectiveness of transient receptor potential vanilloid-1 (TRPV1) blockade to alleviate ocular pain, neuroinflammation, and anxiety-like behavior associated with severe DED. METHODS: Chronic DED was induced by unilateral excision of the Harderian and extraorbital lacrimal glands of adult male mice. Investigations were conducted at 21 days after surgery. The mRNA levels of TRPV1, transient receptor potential ankyrin-1 (TRPA1), and acid-sensing ion channels 1 and 3 (ASIC1 and ASIC3) in the trigeminal ganglion (TG) were evaluated by RNAscope in situ hybridization. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous and stimulated (cold, heat, and acid) corneal nerve responsiveness in ex vivo eye preparations. DED mice received topical instillations of the TRPV1 antagonist (capsazepine) twice a day for 2 weeks from d7 to d21 after surgery. The expression of genes involved in neuropathic and inflammatory pain was evaluated in the TG using a global genomic approach. Chemical and mechanical corneal nociception and spontaneous ocular pain were monitored. Finally, anxiety-like behaviors were assessed by elevated plus maze and black and white box tests. RESULTS: First, in situ hybridization showed DED to trigger upregulation of TRPV1, TRPA1, ASIC1, and ASIC3 mRNA in the ophthalmic branch of the TG. DED also induced overexpression of genes involved in neuropathic and inflammatory pain in the TG. Repeated instillations of capsazepine reduced corneal polymodal responsiveness to heat, cold, and acidic stimulation in ex vivo eye preparations. Consistent with these findings, chronic capsazepine instillation inhibited the upregulation of genes involved in neuropathic and inflammatory pain in the TG of DED animals and reduced the sensation of ocular pain, as well as anxiety-like behaviors associated with severe DED. CONCLUSION: These data provide novel insights on the effectiveness of TRPV1 antagonist instillation in alleviating abnormal corneal neurosensory symptoms induced by severe DED, opening an avenue for the repositioning of this molecule as a potential analgesic treatment for patients suffering from chronic DED.

Unravelling the Hydration Barrier of Lignin Oleate Nanoparticles for Acid- and Base-Catalyzed Functionalization in Dispersion State.

Lignin nanoparticles (LNPs) are promising renewable nanomaterials with applications ranging from biomedicine to water purification. However, the instability of LNPs under acidic and basic conditions severely limits their functionalization for improved performance. Here, we show that controlling the degree of esterification can significantly improve the stability of lignin oleate nanoparticles (OLNPs) in acidic and basic aqueous dispersions. The high stability of OLNPs is attributed to the alkyl chains accumulated in the shell of the particle, which delays protonation/deprotonation of carboxylic acid and phenolic hydroxyl groups. Owing to the enhanced stability, acid- and base-catalyzed functionalization of OLNPs at pH 2.0 and pH 12.0 via oxirane ring-opening reactions were successfully performed. We also demonstrated these new functionalized particles as efficient pH-switchable dye adsorbents and anticorrosive particulate coatings.

ImaGEO: integrative gene expression meta-analysis from GEO database.

SUMMARY: The Gene Expression Omnibus (GEO) database provides an invaluable resource of publicly available gene expression data that can be integrated and analyzed to derive new hypothesis and knowledge. In this context, gene expression meta-analysis (geMAs) is increasingly used in several fields to improve study reproducibility and discovering robust biomarkers. Nevertheless, integrating data is not straightforward without bioinformatics expertise. Here, we present ImaGEO, a web tool for geMAs that implements a complete and comprehensive meta-analysis workflow starting from GEO dataset identifiers. The application integrates GEO datasets, applies different meta-analysis techniques and provides functional analysis results in an easy-to-use environment. ImaGEO is a powerful and useful resource that allows researchers to integrate and perform meta-analysis of GEO datasets to lead robust findings for biomarker discovery studies. AVAILABILITY AND IMPLEMENTATION: ImaGEO is accessible at http://bioinfo.genyo.es/imageo/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Can harmonisation of outcomes bridge the translation gap for pre-clinical research? A systematic review of outcomes measured in mouse models of type 2 diabetes.

BACKGROUND: In pre-clinical research, systematic reviews have the potential to mitigate translational challenges by facilitating understanding of how pre-clinical studies can inform future clinical research. Yet their conduct is encumbered by heterogeneity in the outcomes measured and reported, and those outcomes may not always relate to the most clinically important outcomes. We aimed to systematically review outcomes measured and reported in pre-clinical in vivo studies of pharmacological interventions to treat high blood glucose in mouse models of type 2 diabetes. METHODS: A systematic review of pre-clinical in vivo studies of pharmacological interventions aimed at addressing elevated blood glucose in mouse models of type 2 diabetes was completed. Studies were screened for eligibility and outcomes extracted from the included studies. The outcomes were recorded verbatim and classified into outcome domains using an existing outcome taxonomy. Outcomes were also compared to those identified in a systematic review of registered phase 3/4 clinical trials for glucose lowering interventions in people with type 2 diabetes. RESULTS: Review of 280 included studies identified 532 unique outcomes across 19 domains. No single outcome, or domain, was measured in all studies and only 132 (21%) had also been measured in registered phase 3/4 clinical trials. A core outcome set, representing the minimum that should be measured and reported, developed for type 2 diabetes effectiveness clinical trials includes 18 core outcomes, of these 12 (71%) outcomes were measured and reported in one or more of the included pre-clinical studies. CONCLUSIONS: There is heterogeneity of outcomes reported in pre-clinical research. Harmonisation of outcomes across the research pathway using a core outcome set may facilitate interpretation, evidence synthesis and translational success, and may contribute to the refinement of the use of animals in research. Systematic review registration: The study was prospectively registered on the PROSPERO Database, registration number CRD42018106831.

Murine tissue factor disulfide mutation causes a bleeding phenotype with sex specific organ pathology and lethality.

Tissue factor is highly expressed in sub-endothelial tissue. The extracellular allosteric disulfide bond Cys186-Cys209 of human tissue factor shows high evolutionary conservation and in vitro evidence suggests that it significantly contributes to tissue factor procoagulant activity. To investigate the role of this allosteric disulfide bond in vivo, we generated a C213G mutant tissue factor mouse by replacing Cys213 of the corresponding disulfide Cys190-Cys213 in murine tissue factor. A bleeding phenotype was prominent in homozygous C213G tissue factor mice. Pre-natal lethality of 1/3rd of homozygous offspring was observed between E9.5 and E14.5 associated with placental hemorrhages. After birth, homozygous mice suffered from bleedings in different organs and reduced survival. Homozygous C213G tissue factor male mice showed higher incidence of lung bleedings and lower survival rates than females. In both sexes, C213G mutation evoked a reduced protein expression (about 10-fold) and severely reduced pro-coagulant activity (about 1000-fold). Protein glycosylation was impaired and cell membrane exposure decreased in macrophages in vivo. Single housing of homozygous C213G tissue factor males reduced the occurrence of severe bleeding and significantly improved survival, suggesting that inter-male aggressiveness might significantly account for the sex differences. These experiments show that the tissue factor allosteric disulfide bond is of crucial importance for normal in vivo expression, post-translational processing and activity of murine tissue factor. Although C213G tissue factor mice do not display the severe embryonic lethality of tissue factor knock-out mice, their postnatal bleeding phenotype emphasizes the importance of fully functional tissue factor for hemostasis.

Dual Biochemically Breakable Drug Carriers from Programmed Telechelic Homopolymers.

Well-defined hydrophilic telechelic dibromo poly(triethylene glycol monomethyl ether acrylate)s were prepared by single-electron transfer living radical polymerization employing a hydrophobic difunctional initiator containing acetal and disulfide linkages. Although the resulting homopolymers have low hydrophobic contents (<8.5 wt % of the entire structure), they are able to self-assemble in water into nanoscale micellelike particles via chain folding. Acetal and disulfide linkages were demonstrated to be "keystone" units for their dual stimuli-responsive behavior under biochemically relevant conditions. Their site-selective middle-chain cleavage under both acidic pH and reductive conditions splits the homopolymer into two equal-sized fragments and results in the breakdown of the nanoassemblies. The drug loading/delivery potential of these nanoparticles was investigated using curcumine combining in vitro drug release, cytotoxicity, and cellular uptake studies with human cancer cell lines (HT-29 and HeLa). Importantly, this strategy may be extended to prepare innovative nanoplatforms based on hydrophilic homopolymers or random copolymers for intelligent drug delivery.