The DNA helicase FANCJ (BRIP1) functions in double strand break repair processing, but not crossover formation during prophase I of meiosis in male mice.

Meiotic recombination between homologous chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs). Approximately 10% of these DSBs result in crossovers (COs), sites of physical DNA exchange between homologs that are critical to correct chromosome segregation. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers, the latter representing the defining marks of CO sites. The regulation of CO number and position is poorly understood, but undoubtedly requires the coordinated action of multiple repair pathways. In a previous report, we found gene-trap disruption of the DNA helicase, FANCJ (BRIP1/BACH1), elicited elevated numbers of MLH1 foci and chiasmata. In somatic cells, FANCJ interacts with numerous DNA repair proteins including MLH1, and we hypothesized that FANCJ functions with MLH1 to regulate the major CO pathway. To further elucidate the meiotic function of FANCJ, we produced three new Fancj mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, truncation of the N-terminal Helicase domain, and a C-terminal dual-tagged allele. We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, none of our Fancj mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 in meiosis. Instead, FANCJ co-localizes with BRCA1 and TOPBP1, forming discrete foci along the chromosome cores beginning in early meiotic prophase I and densely localized to unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Fancj mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data indicate a role for FANCJ in early DSB repair, but they rule out a role for FANCJ in MLH1-mediated CO events.

RNF212B E3 ligase is essential for crossover designation and maturation during male and female meiosis in the mouse.

Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we functionally characterized a recently identified RING-E3 ligase, RNF212B. RNF212B colocalizes and interacts with RNF212, forming foci along chromosomes from zygonema onward in a synapsis-dependent and DSB-independent manner. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 by late pachynema. Genetically, RNF212B foci formation depends on Rnf212 but not on Msh4, Hei10, and Cntd1, while the unloading of RNF212B at the end of pachynema is dependent on Hei10 and Cntd1. Mice lacking RNF212B, or expressing an inactive RNF212B protein, exhibit modest synapsis defects, a reduction in the localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of late CO-intermediates (MLH1). This loss of most COs by diakinesis results in mostly univalent chromosomes. Double mutants for Rnf212b and Rnf212 exhibit an identical phenotype to that of Rnf212b single mutants, while double heterozygous demonstrate a dosage-dependent reduction in CO number, indicating a functional interplay between paralogs. SUMOylome analysis of testes from Rnf212b mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is an E3-ligase with Ubiquitin activity, serving as a crucial factor for CO maturation. Thus, RNF212 and RNF212B play vital, yet overlapping roles, in ensuring CO homeostasis through their distinct E3 ligase activities.

A combination of conserved and diverged responses underlies Theobroma cacao's defense response to Phytophthora palmivora.

BACKGROUND: Plants have complex and dynamic immune systems that have evolved to resist pathogens. Humans have worked to enhance these defenses in crops through breeding. However, many crops harbor only a fraction of the genetic diversity present in wild relatives. Increased utilization of diverse germplasm to search for desirable traits, such as disease resistance, is therefore a valuable step towards breeding crops that are adapted to both current and emerging threats. Here, we examine diversity of defense responses across four populations of the long-generation tree crop Theobroma cacao L., as well as four non-cacao Theobroma species, with the goal of identifying genetic elements essential for protection against the oomycete pathogen Phytophthora palmivora. RESULTS: We began by creating a new, highly contiguous genome assembly for the P. palmivora-resistant genotype SCA 6 (Additional file 1: Tables S1-S5), deposited in GenBank under accessions CP139290-CP139299. We then used this high-quality assembly to combine RNA and whole-genome sequencing data to discover several genes and pathways associated with resistance. Many of these are unique, i.e., differentially regulated in only one of the four populations (diverged 40 k-900 k generations). Among the pathways shared across all populations is phenylpropanoid biosynthesis, a metabolic pathway with well-documented roles in plant defense. One gene in this pathway, caffeoyl shikimate esterase (CSE), was upregulated across all four populations following pathogen treatment, indicating its broad importance for cacao's defense response. Further experimental evidence suggests this gene hydrolyzes caffeoyl shikimate to create caffeic acid, an antimicrobial compound and known inhibitor of Phytophthora spp. CONCLUSIONS: Our results indicate most expression variation associated with resistance is unique to populations. Moreover, our findings demonstrate the value of using a broad sample of evolutionarily diverged populations for revealing the genetic bases of cacao resistance to P. palmivora. This approach has promise for further revealing and harnessing valuable genetic resources in this and other long-generation plants.

Genome Size Changes by Duplication, Divergence, and Insertion in Caenorhabditis Worms.

Genome size has been measurable since the 1940s but we still do not understand genome size variation. Caenorhabditis nematodes show strong conservation of chromosome number but vary in genome size between closely related species. Androdioecy, where populations are composed of males and self-fertile hermaphrodites, evolved from outcrossing, female-male dioecy, three times in this group. In Caenorhabditis, androdioecious genomes are 10-30% smaller than dioecious species, but in the nematode Pristionchus, androdioecy evolved six times and does not correlate with genome size. Previous hypotheses include genome size evolution through: 1) Deletions and "genome shrinkage" in androdioecious species; 2) Transposable element (TE) expansion and DNA loss through large deletions (the "accordion model"); and 3) Differing TE dynamics in androdioecious and dioecious species. We analyzed nematode genomes and found no evidence for these hypotheses. Instead, nematode genome sizes had strong phylogenetic inertia with increases in a few dioecious species, contradicting the "genome shrinkage" hypothesis. TEs did not explain genome size variation with the exception of the DNA transposon Mutator which was twice as abundant in dioecious genomes. Across short and long evolutionary distances Caenorhabditis genomes evolved through small structural mutations including gene-associated duplications and insertions. Seventy-one protein families had significant, parallel decreases across androdioecious Caenorhabditis including genes involved in the sensory system, regulatory proteins and membrane-associated immune responses. Our results suggest that within a dynamic landscape of frequent small rearrangements in Caenorhabditis, reproductive mode mediates genome evolution by altering the precise fates of individual genes, proteins, and the phenotypes they underlie.

Control of Meiotic Crossovers: From Double-Strand Break Formation to Designation.

Meiosis, the mechanism of creating haploid gametes, is a complex cellular process observed across sexually reproducing organisms. Fundamental to meiosis is the process of homologous recombination, whereby DNA double-strand breaks are introduced into the genome and are subsequently repaired to generate either noncrossovers or crossovers. Although homologous recombination is essential for chromosome pairing during prophase I, the resulting crossovers are critical for maintaining homolog interactions and enabling accurate segregation at the first meiotic division. Thus, the placement, timing, and frequency of crossover formation must be exquisitely controlled. In this review, we discuss the proteins involved in crossover formation, the process of their formation and designation, and the rules governing crossovers, all within the context of the important landmarks of prophase I. We draw together crossover designation data across organisms, analyze their evolutionary divergence, and propose a universal model for crossover regulation.

Evaluating threshold for donor fraction cell-free DNA using clinically available assay for rejection in pediatric and adult heart transplantation.

BACKGROUND: The aims of the study were to assess the performance of a clinically available cell-free DNA (cfDNA) assay in a large cohort of pediatric and adult heart transplant recipients and to evaluate performance at specific cut points in detection of rejection. METHODS: Observational, non-interventional, prospective study enrolled pediatric and adult heart transplant recipients from seven centers. Biopsy-associated plasma samples were used for cfDNA measurements. Pre-determined cut points were tested for analytic performance. RESULTS: A total of 487 samples from 160 subjects were used for the analysis. There were significant differences for df-cfDNA values between rejection [0.21% (IQR 0.12-0.69)] and healthy samples [0.05% (IQR 0.01-0.14), p < .0001]. The pediatric rejection group had a median df-cfDNA value of 0.93% (IQR 0.28-2.84) compared to 0.09% (IQR 0.04-0.23) for healthy samples, p = .005. Overall negative predictive value was 0.94 while it was 0.99 for pediatric patients. Cut points of 0.13% and 0.15% were tested for various types of rejection profiles and were appropriate to rule out rejection. CONCLUSION: The study suggests that pediatric patients with rejection show higher levels of circulating df-cfDNA compared to adults and supports the specific cut points for clinical use in pediatric and adult patients with overall acceptable performance.

Role of EXO1 nuclease activity in genome maintenance, the immune response and tumor suppression in Exo1D173A mice.

DNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here, we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1-/- and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1-/- mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1-/- mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1-/- mice was comparably defective, switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1-/- mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1-/- mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease.

Genomic structural variants constrain and facilitate adaptation in natural populations of Theobroma cacao, the chocolate tree.

Genomic structural variants (SVs) can play important roles in adaptation and speciation. Yet the overall fitness effects of SVs are poorly understood, partly because accurate population-level identification of SVs requires multiple high-quality genome assemblies. Here, we use 31 chromosome-scale, haplotype-resolved genome assemblies of Theobroma cacao-an outcrossing, long-lived tree species that is the source of chocolate-to investigate the fitness consequences of SVs in natural populations. Among the 31 accessions, we find over 160,000 SVs, which together cover eight times more of the genome than single-nucleotide polymorphisms and short indels (125 versus 15 Mb). Our results indicate that a vast majority of these SVs are deleterious: they segregate at low frequencies and are depleted from functional regions of the genome. We show that SVs influence gene expression, which likely impairs gene function and contributes to the detrimental effects of SVs. We also provide empirical support for a theoretical prediction that SVs, particularly inversions, increase genetic load through the accumulation of deleterious nucleotide variants as a result of suppressed recombination. Despite the overall detrimental effects, we identify individual SVs bearing signatures of local adaptation, several of which are associated with genes differentially expressed between populations. Genes involved in pathogen resistance are strongly enriched among these candidates, highlighting the contribution of SVs to this important local adaptation trait. Beyond revealing empirical evidence for the evolutionary importance of SVs, these 31 de novo assemblies provide a valuable resource for genetic and breeding studies in Tcacao.

Automated Image Quality and Protocol Adherence Assessment of Examinations in Teledermatology: First Results.

Introduction: Image quality and acquisition protocol adherence assessment is a neglected area in teledermatology. We examine if it is feasible to use deep learning methods to automate the assessment of the adherence of examinations to image acquisition protocols. In this study, we focused on the quality criteria of two image acquisition protocols: (1) approximation image and (2) panoramic image, as these are present in all teledermatology examination protocols currently used by the Santa Catarina State Integrated Telemedicine and Telehealth System (STT/SC). Methods: We use a data set of 36,102 teledermatological examinations performed at the STT/SC during 2021. As our validation process, we adopted standard machine learning metrics and an inter-rater agreement (IRA) study with 11 dermatologists. For the approximation image protocol, we used the Mask-Region based Convolutional Neural Network (RCNN) Object Detection Deep Learning (DL) architecture to identify the presence of a lesion identification tag and a ruler used to provide a frame reference of the lesion. For the panoramic image protocol, we used DensePose, a pose estimation DL, architecture to assess the presence of a whole patient body and its orientation. A combination of the two approaches was additionally validated through an IRA study between specialists. Results: Mask-RCNN achieved a score of 96% mean average precision (mAP), while DensePose presented 75% mAP. IRA achieved a level of agreement of 96.68% with the Krippendorff alpha score. Conclusions: Our results show the feasibility of using deep learning to automate the image quality and protocol adherence assessment in teledermatology, before the specialist's manual analysis of the examination.

Profibrotic COVID-19 subphenotype exhibits enhanced localized ER-dependent HSP47+ expression in cardiac myofibroblasts in situ.

We recently described a subgroup of autopsied COVID-19 subjects (∼40%), termed 'profibrotic phenotype,' who exhibited clusters of myofibroblasts (Mfbs), which were positive for the collagen-specific chaperone heat shock protein 47 (HSP47+) in situ. This report identifies increased, localized (hot spot restricted) expression of αSMA, COLα1, POSTN and FAP supporting the identity of HSP47+ cells as myofibroblasts and characterizing a profibrotic extracellular matrix (ECM) phenotype. Coupled with increased GRP78 in COVID-19 subjects, these data could reflect induction of the unfolded protein response for mitigation of proteostasis (i.e., protein homeostasis) dysfunction in discrete clusters of cells. ECM shifts in selected COVID-19 subjects occur without significant increases in either global trichrome positive staining or myocardial injury based quantitively on standard H&E scoring. Our findings also suggest distinct mechanism(s) for ECM remodeling in the setting of SARS-CoV-2 infection. The ratio of CD163+/CD68+ cells is increased in hot spots of profibrotic hearts compared with either controls or outside of hot spots in COVID-19 subjects. In sum, matrix remodeling of human COVID-19 hearts in situ is characterized by site-restricted profibrotic mediated (e.g., HSP47+ Mfbs, CD163+ Mφs) modifications in ECM (i.e., COLα1, POSTN, FAP), with a strong correlation between COLα1 and HSP47+cells within hot spots. Given the established associations of viral infection (e.g., human immunodeficiency virus; HIV), myocardial fibrosis and sudden cardiac death, early screening tools (e.g., plasma biomarkers, noninvasive cardiac magnetic resonance imaging) for diagnosis, monitoring and treatment of fibrotic ECM remodeling are warranted for COVID-19 high-risk populations.

Slow Recovery from Inbreeding Depression Generated by the Complex Genetic Architecture of Segregating Deleterious Mutations.

The deleterious effects of inbreeding have been of extreme importance to evolutionary biology, but it has been difficult to characterize the complex interactions between genetic constraints and selection that lead to fitness loss and recovery after inbreeding. Haploid organisms and selfing organisms like the nematode Caenorhabditis elegans are capable of rapid recovery from the fixation of novel deleterious mutation; however, the potential for recovery and genomic consequences of inbreeding in diploid, outcrossing organisms are not well understood. We sought to answer two questions: 1) Can a diploid, outcrossing population recover from inbreeding via standing genetic variation and new mutation? and 2) How does allelic diversity change during recovery? We inbred C. remanei, an outcrossing relative of C. elegans, through brother-sister mating for 30 generations followed by recovery at large population size. Inbreeding reduced fitness but, surprisingly, recovery from inbreeding at large populations sizes generated only very moderate fitness recovery after 300 generations. We found that 65% of ancestral single nucleotide polymorphisms (SNPs) were fixed in the inbred population, far fewer than the theoretical expectation of ∼99%. Under recovery, 36 SNPs across 30 genes involved in alimentary, muscular, nervous, and reproductive systems changed reproducibly across replicates, indicating that strong selection for fitness recovery does exist. Our results indicate that recovery from inbreeding depression via standing genetic variation and mutation is likely to be constrained by the large number of segregating deleterious variants present in natural populations, limiting the capacity for recovery of small populations.

Segregation analysis identifies specific alpha-defensin (DEFA1A3) SNP-CNV haplotypes in predisposition to IgA nephropathy.

BACKGROUND: Immunoglobulin A (IgA) nephropathy is a disorder of the immune system affecting kidney function, and genome-wide association studies (GWAS) have defined numerous loci with associated variation, all implicating components of innate or adaptive immunity. Among these, single nucleotide polymorphisms (SNPs) in a region including the multiallelic copy number variation (CNV) of DEFA1A3 are associated with IgA nephropathy in both European and Asian populations. At present, the precise factors underlying the observed associations at DEFA1A3 have not been defined, although the key alleles differ between Asian and European populations, and multiple independent factors may be involved even within a single population. METHODS: In this study, we measured DEFA1A3 copy number in UK family trios with an offspring affected by IgA nephropathy, used the population distributions of joint SNP-CNV haplotypes to infer the likely segregation in trios, and applied transmission disequilibrium tests (TDT) to examine joint SNP-CNV haplotypes for over- or undertransmission into affected offspring from heterozygous parents. RESULTS AND CONCLUSIONS: We observed overtransmission of 3-copy class 2 haplotypes (raw p = 0.029) and some evidence for under-transmission of 3-copy class 1 haplotypes (raw p = 0.051), although these apparent effects were not statistically significant after correction for testing of multiple haplotypes.

A new bimodal approach for sentinel lymph node imaging in prostate cancer using a magnetic and fluorescent hybrid tracer.

PURPOSE: To obtain initial data on sentinel lymph node (SLN) visualisation by pre-operative magnetic resonance imaging (MRI) and intra-operative bimodal SLN identification using a new magnetic fluorescent hybrid tracer in prostate cancer (PCa) patients. METHODS: Ten patients at > 5% risk for lymph node (LN) invasion were included. The day before surgery, a magnetic fluorescent hybrid tracer consisting of superparamagnetic iron oxide nanoparticles (SPION) and indocyanine green was transrectally injected into the prostate. Five hours after injection, transversal pelvic MRI scans were recorded and T2*-weighed images were screened for pelvic LNs with SPION uptake. Intra-operatively, magnetically active and/or fluorescent SLNs were detected by a handheld magnetometer and near-infrared fluorescence imaging (FI). Extended pelvic lymph node dissection (PLND) and radical prostatectomy completed the surgery. All resected specimens were checked ex situ for magnetic activity and fluorescence and were histopathologically examined. RESULTS: Pre-operative MRI identified 145 pelvic LNs with SPION uptake. In total, 75 (median 6, range 3‒13) magnetically active SLNs were resected, including 14 SLNs not seen on MRI. FI identified 89 fluorescent LNs (median 8.5, range 4‒13) of which 15 LNs were not magnetically active. Concordance of the different techniques was 70% for pre-operative MRI vs. magnetometer-guided PLND and 88% for magnetic vs. fluorescent SLN detection. CONCLUSION: These are the first promising results of bimodal, magnetic fluorescent SLN detection in PCa patients. Our magnetic fluorescent hybrid approach provides the surgeon a pre-operative lymphatic roadmap by using MRI and intra-operative visual guidance through the application of a fluorescent lymphatic agent. The diagnostic accuracy of our new hybrid approach has to be evaluated in further studies. TRIAL REGISTRATION: DRKS00032808. Registered 04 October 2023, retrospectively registered.

Treatment patterns and outcomes for Hodgkin Lymphoma patients aged 60 and older: a report from the Brazilian Prospective Hodgkin Lymphoma Registry.

The treatment of older patients with Hodgkin lymphoma (HL) remains a challenge. We sought to identify the treatment patterns and outcomes in older HL patients included in the Brazilian HL registry (NCT02589548). A total of 136 patients with HIV-negative classic HL, aged ≥ 60 years, diagnosed between 2009 and 2018, were analyzed. The median age was 66 years old (60-90), 72% had advanced disease, 62% had a high IPS, and 49% had a nodular sclerosis subtype. Median follow-up was 64 months for alive patients. ABVD was the front-line treatment in 96% of patients. Twenty-one patients (15%) died during front-line treatment. The 5-year PFS and 5-year OS rates were 55% and 59%, respectively. The 5-year OS rates in localized and advanced disease were 81% and 51% (p=0.013). Lung toxicity developed in 11% of the patients treated with ABVD. Bleomycin was administered for > 2 cycles in 65% of patients. Compared with 2009-2014, there was a decrease in the use of bleomycin for > 2 cycles in 2015-2018 (88% × 45%, p<0.0001). The impact of socioeconomic status (SES) on outcomes was studied in patients treated with ABVD. After adjusting for potential confounders, lower SES remained independently associated with poorer survival (HR 2.22 [1.14-4.31] for OS and HR 2.84 [1.48-5.45] for PFS). Treatment outcomes were inferior to those observed in developed countries. These inferior outcomes were due to an excess of deaths during front-line treatment and the excessive use of bleomycin. SES was an independent factor for shorter survival.

A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome.

Protein kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through in vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 in vitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport.

Oxidation Products from the Neolignan Licarin A by Biomimetic Reactions and Assessment of in vivo Acute Toxicity.

Licarin A, a dihydrobenzofuranic neolignan presents in several medicinal plants and seeds of nutmeg, exhibits strong activity against protozoans responsible for Chagas disease and leishmaniasis. From biomimetic reactions by metalloporphyrin and Jacobsen catalysts, seven products were determined: four isomeric products yielded by epoxidation from licarin A, besides a new product yielded by a vicinal diol, a benzylic aldehyde, and an unsaturated aldehyde in the structure of the licarin A. The incubation with rat and human liver microsomes partially reproduced the biomimetic reactions by the production of the same epoxidized product of m/z 343 [M + H]+. In vivo acute toxicity assays of licarin A suggested liver toxicity based on biomarker enzymatic changes. However, microscopic analysis of tissues sections did not show any tissue damage as indicative of toxicity after 14 days of exposure. New metabolic pathways of the licarin A were identified after in vitro biomimetic oxidation reaction and in vitro metabolism by rat or human liver microsomes.

Four-year review of New Zealand laboratory infliximab and adalimumab concentration results indicating potential for improved dosing.

A review of laboratory results across New Zealand for therapeutic drug monitoring (TDM) of infliximab and adalimumab concentrations and antidrug antibodies (ADAs) over 4 years was completed. Of 6591 results, the median serum concentration for infliximab was 5.7 mg/L and for adalimumab was 5.5 mg/L. Subtherapeutic drug concentrations (<7 mg/L) were measured in 54% of samples. Drug concentrations <2 mg/L were measured in 23% of samples, with ADAs detected in 51% of these. The high number of samples with subtherapeutic drug concentrations and common ADA detection is consistent with failing therapy but could also suggest that standard dosing is frequently too low for patients. These results reinforce the value of antitumour necrosis factor drug TDM in making decisions to adjust dosing or switch agents in patients taking infliximab and adalimumab.

Somatic CAG expansion in Huntington's disease is dependent on the MLH3 endonuclease domain, which can be excluded via splice redirection.

Somatic expansion of the CAG repeat tract that causes Huntington's disease (HD) is thought to contribute to the rate of disease pathogenesis. Therefore, factors influencing repeat expansion are potential therapeutic targets. Genes in the DNA mismatch repair pathway are critical drivers of somatic expansion in HD mouse models. Here, we have tested, using genetic and pharmacological approaches, the role of the endonuclease domain of the mismatch repair protein MLH3 in somatic CAG expansion in HD mice and patient cells. A point mutation in the MLH3 endonuclease domain completely eliminated CAG expansion in the brain and peripheral tissues of a HD knock-in mouse model (HttQ111). To test whether the MLH3 endonuclease could be manipulated pharmacologically, we delivered splice switching oligonucleotides in mice to redirect Mlh3 splicing to exclude the endonuclease domain. Splice redirection to an isoform lacking the endonuclease domain was associated with reduced CAG expansion. Finally, CAG expansion in HD patient-derived primary fibroblasts was also significantly reduced by redirecting MLH3 splicing to the endogenous endonuclease domain-lacking isoform. These data indicate the potential of targeting the MLH3 endonuclease domain to slow somatic CAG repeat expansion in HD, a therapeutic strategy that may be applicable across multiple repeat expansion disorders.

Influence of different growing stages of whole-plant soybeans on their nutrient content and silage quality for cattle.

The objective of this study was to determine the effects of growing stage (GS) on morphological and chemical composition of whole-plant soybean (WPS), and fermentative profile and chemical composition of whole-plant soybean silage (SS). This study was divided into two trials conducted in a complete randomised block design. The first trial evaluated the effect of GS from R1 to R8 (59-135 d after sowing) on morphological and chemical composition of WPS and its botanical components. The second trial determined the effects of GS from R3 (71 d after sowing) to R7 (124 d after sowing) on dry matter (DM) losses, fermentative profile, chemical composition and aerobic stability of SS. The proportion of leaves in WPS was reduced, while stem and pod proportions were increased as the GS progressed. Ensiling WPS at R6 and R7 decreased the contents of acetic acid, lactic acid and branched-chain fatty acids, and ethanol, and increased the contents of propionic acid and NH3-N. However, silage butyric acid concentrations in R6 and R7 were relatively high (18.1 and 19.9 g/kg DM, respectively). Butyric acid and buffering capacity varied according to GS with the lowest values observed in silages derived from GS R3, R5 and R7, and the highest values observed in silages made from GS R5. Later GS resulted in greater contents of DM, crude protein and ether extract, and lower contents of acid detergent fibre and non-fibre carbohydrate in SS. The high fat of SS produced from later GS limits high inclusion levels in ruminant diets. Morphological components impacted chemical composition of SS, whereas the R7 stage improved fermentative profile and resulted in an SS with greater in situ degradability of DM and neutral detergent fibre.

A scoping review on hyposalivation associated with systemic conditions: the role of physical stimulation in the treatment approaches.

BACKGROUND: Several systemic conditions can result in distinct degrees of salivary gland damage and consequent hypofunction. The development of successful management schemes is highly challenging due to the complexity of saliva. This study aimed to systematically map the literature on the physical stimulation of salivary glands for hyposalivation management and the response of individuals according to different systemic conditions causing salivary impairment. METHODS: A systematic search in the literature was performed. Two reviewers independently selected clinical trials, randomized or not, that used physical stimulation to treat hyposalivation caused by systemic conditions. Studies evaluating healthy subjects without hyposalivation were included as controls. Single-arm clinical studies or case series were also included for protocol mapping (PRISMA extension for scoping reviews). RESULTS: Out of 24 included studies, 10 evaluated healthy subjects, from which 9 tested transcutaneous electrical nerve stimulation (TENS) and 1 tested acupuncture and electroacupuncture. Fourteen studies evaluated individuals with hyposalivation: 6 applied TENS, 6 applied low-level laser therapy (LLLT), and 2 applied acupuncture, carried out in post-chemotherapy, medication use, postmenopausal women, hemodialysis patients, smokers, diabetics, Sjögren's syndrome (SS). All showed increased salivation after treatment, except for two LLLT studies in individuals with SS. CONCLUSIONS: Among the different patient groups, individuals with Sjögren's syndrome (SS) exhibited the poorest responses, while those with medication-induced hyposalivation demonstrated the most favorable treatment outcomes, independently of the management strategy for saliva stimulation. It means that physical stimulation of salivary glands holds promise as an alternative for managing hyposalivation in cases of reversible gland damage. However, to make informed decisions in current practice, it is necessary to conduct new well-designed randomized clinical trials with appropriate methodologies.