Functional Evidence Research Articles

Age-related unstable transient states and imbalanced activation proportion of brain networks in people with autism spectrum disorder: A resting-state fMRI study using coactivation pattern analyses

ABSTRACT The atypical static brain functions related to the executive control network (ECN), default mode network (DMN), and salience network (SN) in people with autism spectrum disorder (ASD) has been widely reported. However, their transient functions in ASD are not clear. We aim to identify transient network states (TNSs) using coactivation pattern (CAP) analysis to characterize the age-related atypical transient functions in ASD. CAP analysis was performed on a resting-state fMRI dataset (78 ASD and 78 healthy control (CON) juveniles, 54 ASD and 54 CON adults). Six TNSs were divided into the DMN-TNSs, ECN-TNSs, and SN-TNSs. The DMN-TNSs were major states with the highest stability and proportion, and the ECN-TNSs and SN-TNSs were minor states. Age-related abnormalities on spatial stability and TNS proportion were found in ASD. The spatial stability of DMN-TNSs was found increasing with age in CON, but was not found in ASD. A lower proportion of DMN-TNSs was found in ASD compared with CON of the same age, and ASD juveniles had a higher proportion of SN-TNSs while ASD adults had a higher proportion of ECN-TNSs. The abnormalities on spatial stability and TNS proportion were related to social deficits. Our results provided new evidence for atypical transient brain functions in people with ASD.

Brain metabolic response to repetitive transcranial magnetic stimulation to lesion network in cervical dystonia

BackgroundA previous study identified a brain network underlying cervical dystonia (CD) based on causal brain lesions. This network was shown to be abnormal in idiopathic CD and aligned with connections mediating treatment response to deep brain stimulation, suggesting generalizability across etiologies and relevance for treatment. The main nodes of this network were located in the deep cerebellar structures and somatosensory cortex (S1), the latter of which can be easily reached via non-invasive brain stimulation. To date, there are no studies testing brain stimulation to networks identified using lesion network mapping. ObjectivesTo assess target engagement by stimulating the S1 and testing the brain's acute metabolic response to repetitive transcranial magnetic stimulation in CD patients and healthy controls. MethodsThirteen CD patients and 14 controls received a single session of continuous theta burst (cTBS) and sham to the right S1. Changes in regional brain glucose metabolism were measured using [18F]FDG-PET. ResultscTBS increased metabolism at the stimulation site in CD (P = 0.03) but not in controls (P = 0.15; group difference P = 0.01). In subcortical regions, cTBS increased metabolism in the brainstem in CD only (PFDR = 0.04). The remote activation was positively associated with dystonia severity and efficacy of sensory trick phenomenon in CD patients. ConclusionsOur results provide further evidence of abnormal sensory system function in CD and show that a single session of S1 cTBS is sufficient to induce measurable changes in brain glucose metabolism. These findings support target engagement, motivating therapeutic trials of cTBS to the S1 in CD.

Deep-sea fish reveal alternative pathway for vertebrate visual development.

Vertebrate vision is accomplished by two phenotypically distinct types of photoreceptors in the retina: the saturation-resistant cones for the detection of bright light and the highly sensitive rods for dim light conditions [1]. The current dogma is that, during development, all vertebrates initially feature a cone-dominated retina, and rods are added later [2, 3]. By studying the ontogeny of vision in three species of deep-sea fishes, we show that their larvae express cone-specific genes in photoreceptors with rod-like morphologies. Through development, these fishes either retain this rod-like cone retina (Maurolicus mucronatus) or switch to a retina with true rod photoreceptors with expression of rod-specific genes and transcription factors (Vinciguerria mabahiss and Benthosema pterotum). In contrast to the larvae of most marine fishes, which inhabit the bright upper layer of the open ocean, the larvae of deep-sea fishes occur deeper, exposing them to a dimmer light environment [4-7]. Spectral maxima predictions from molecular dynamics simulations and environmental light estimations suggest that using transmuted photoreceptors that combine the characteristics of both cones and rods maximises visual performance in these dimmer light conditions. Our findings provide molecular, morphological, and functional evidence for the evolution of an alternative developmental pathway for vertebrate vision.

Decreased mitochondrial creatine kinase 2 impairs skeletal muscle mitochondrial function independently of insulin in type 2 diabetes.

Increased plasma creatine concentrations are associated with the risk of type 2 diabetes, but whether this alteration is associated with or causal for impairments in metabolism remains unexplored. Because skeletal muscle is the main disposal site of both creatine and glucose, we investigated the role of intramuscular creatine metabolism in the pathophysiology of insulin resistance in type 2 diabetes. In men with type 2 diabetes, plasma creatine concentrations were increased, and intramuscular phosphocreatine content was reduced. These alterations were coupled to reduced expression of sarcomeric mitochondrial creatine kinase 2 (CKMT2). In C57BL/6 mice fed a high-fat diet, neither supplementation with creatine for 2 weeks nor treatment with the creatine analog β-GPA for 1 week induced changes in glucose tolerance, suggesting that increased circulating creatine was associated with insulin resistance rather than causing it. In C2C12 myotubes, silencing Ckmt2 using small interfering RNA reduced mitochondrial respiration, membrane potential, and glucose oxidation. Electroporation-mediated overexpression of Ckmt2 in skeletal muscle of high-fat diet-fed male mice increased mitochondrial respiration, independent of creatine availability. Given that overexpression of Ckmt2 improved mitochondrial function, we explored whether exercise regulates CKMT2 expression. Analysis of public data revealed that CKMT2 content was up-regulated by exercise training in both humans and mice. We reveal a previously underappreciated role of CKMT2 in mitochondrial homeostasis beyond its function for creatine phosphorylation, independent of insulin action. Collectively, our data provide functional evidence for how CKMT2 mediates mitochondrial dysfunction associated with type 2 diabetes.

Transcriptomics identify the triggering of citrate export as the key event caused by manganese deficiency in Aspergillus niger.

Citric acid is produced on industrial scale by batch fermentation of the filamentous fungus Aspergillus niger. High-yield citric acid production requires a low (<5 ppb) manganese(II) ion concentration in the culture broth. However, the requirement for this deficiency has not been investigated on a functional genomics level. Here, we compared the transcriptome of a citric acid hyper-producer A. niger strain grown under citric acid-producing conditions in 6-L scale bioreactors at Mn2+ ion-deficient (5 ppb) and Mn2+ ion-sufficient (100 ppb) conditions at three early time points of cultivation. We observed that Mn2+ deficiency triggers an upregulation of the citrate exporter gene cexA and provides functional evidence that this event is responsible for citrate overflow. In addition to the industrial relevance, this is the first study that examined the role of Mn2+ ion deficiency in a heterotrophic eukaryotic cell on a genome-wide scale.

Revisiting the y-ome of Escherichia coli.

The model organism Escherichia coli K-12 has one of the most extensively annotated genomes in terms of functional characterization, yet a significant number of genes, ∼35%, are still considered poorly characterized. Initially genes without known functional understanding were given 'y' gene names. However, due to inconsistency in changing 'y' names to non-'y' names over the years, gene name alone does not provide sufficient information as to the characterization level of genes. Attempts to characterize y-ome genes, i.e. those that lack experimental evidence for function, are ongoing, and recent categorization based on the level of experimental evidence has helped clarify those genes that are well characterized versus uncharacterized. EcoCyc, the most comprehensive, curated genome database for E.coli K-12 substr. MG1655, has updated this approach by expanding the categories to include Partially characterized genes using a set of computational rules that includes keywords, experimental evidence codes and Gene Ontology terms. Approximately half of the previously categorized y-ome genes are now categorized as Partially characterized, leaving 15.5% (738) as Uncharacterized genes in EcoCyc. This new categorization scheme is searchable in the EcoCyc database, will be updated as new experimental evidence is curated and provides important information for research decisions.

Omics Analysis of Odorant-Binding Proteins and Cuticle-Enriched SfruOBP18 Confers Multi-Insecticide Tolerance in Spodoptera frugiperda.

Spodoptera frugiperda is a notorious pest that develops a high resistance to many insecticides. Recently, insect odorant-binding proteins (OBPs) have been proven to participate in insecticide resistance. However, the functional evidence supporting the cross-link between OBPs and insecticide resistance remains unexplored. Here, we identified 50 SfruOBPs from the larval transcriptome and genome. Notably, SfruOBP18 was highly expressed in the larval cuticle and could be induced to upregulate its expression by multi-insecticides. Ligand-binding assays revealed that SfruOBP18 bound strongly with four insecticides; RNAi and insecticide bioassay demonstrated that the knockdown of SfruOBP18 did not affect larval survival and development. However, it can significantly increase the larval susceptibility to multi-insecticides, suggesting an uncommon role of SfruOBP18 in multi-insecticide susceptibility. Our study provides a comprehensive understanding of SfruOBPs and furthermore proves that a larval cuticle-enriched OBP can bind with and confer larval tolerance to multi-insecticides. SfruOBP18 could be a new insecticidal target for controlling Lepidoptera pests.

Biosynthesis and Characterization of Iron Oxide Nanoparticles Using Chenopodium quinoa Extract.

In this study, we achieved the biosynthesis of novel 7-8 nm iron-oxide nanoparticles in the presence of different concentrations (5 to 50% w/v) of commercial white quinoa extract. Initially, quinoa extract was prepared at various concentrations by a purification route. The biosynthesis optimization was systematically monitored by X-ray diffraction, and the Rietveld quantitative analysis showed the presence of goethite (5 to 10 wt.%) and maghemite phases. The first phase disappeared upon increasing the organic loading (40 and 50% w/v). The organic loading was corroborated by thermogravimetric measurements, and it increased with quinoa extract concentration. Its use reduces the amount of precipitation agent at high quinoa extract concentrations with the formation of magnetic nanoparticles with hard ferrimagnetic character (42 and 11 emu g-1). The enrichment of hydroxyl groups and the negative zeta potential above pH = 7 were corroborated by a reduction in the point of zero charge in all the samples. For alkaline values, the zeta potential values were above the stability range, indicating highly stable chemical species. The evidence of hydroxyl and amide functionalization was qualitatively observed using infrared analysis, which showed that the carboxyl (quercetin/kaempferol), amide I, and amide III chemical groups are retained after biosynthesis. The resultant biosynthesized samples can find applications in environmental remediation due to the affinity of the chemical agents present on the particle surfaces and easy-to-handle them magnetically.

The utility of the dilute prothrombin time in the interpretation of antiphospholipid syndrome testing.

To evaluate the utility of the dilute prothrombin time (DPT) in diagnosing antiphospholipid syndrome (APS), alone and when paired with the dilute Russell viper venom time (DRVVT). Dilute prothrombin time and DRVVT testing was performed on plasma samples spiked with apixaban or rivaroxaban, or depleted of vitamin K-dependent clotting factors. A retrospective analysis of all functional APS testing results over a 44-month period at the University of Chicago Medical Center was performed. In spiking studies, the screening clotting time in the DPT (DPTS) is more sensitive to deficiency of vitamin K-dependent factors than is the screening clotting time in the DRVVT (DRVVTS). The converse is true for factor Xa direct oral anticoagulant (DOAC)-spiked plasma. In a 44-month retrospective analysis, only 2.6% of clinical APS panels showed isolated positivity in the DPT-based system. Comparing the DPT-based system with the DRVVT-based system showed utility in identifying false-positive DRVVT results due to anticoagulation. A DRVVTS/DPTS ratio of 0.785 or lower predicted an international normalized ratio of 1.5 or higher (sensitivity, 86.3%; specificity, 60.4%; likelihood ratio, 2.18). Conversely, a DRVVTS/DPTS ratio of 1.165 or higher was the optimal cutoff for predicting the identification of factor Xa DOAC (sensitivity, 61.8%; specificity, 77.8%; likelihood ratio, 2.78). Within the data set that had full DRVVT and DPT results, parameters were identified that could further improve identification of samples with anticoagulation interference. Dilute prothrombin time lupus anticoagulant assay is rarely the sole laboratory functional evidence for APS, but when combined with the DRVVT, the DPT can serve as an effective screen for common anticoagulant interference.

Allelic Variation of BnaFTA2 and BnaFTC6 Is Associated With Flowering Time and Seasonal Crop Type in Rapeseed (Brassica napus L.).

Different ecological types of rapeseed (Brassica napus L.), including winter, spring, and semi-winter cultivars, exhibit varying flowering times and cannot be planted in the same cultivation areas. FLOWERING LOCUS T (FT) plays a key role in regulating flowering. In allotetraploid B. napus six copies of FT (BnaFT) have been reported. However, there is uncertainty about how the translated products of each paralog, as well as cis-allelic variations at each locus, contribute functionally to flowering time and define specific crop types. In this study, we confirm that BnaFT exhibit distinct expression patterns in different crop types of rapeseed. Using the CRISPR/Cas9 gene editing system, we provide functional evidence that the mutants between Bnaft paralogues affects the regulation of flowering time. Furthermore, we identify a new haplotype of BnaFT.A2 that is associated with early flowering time, although this appears necessary but not sufficient to confer a spring type phenotype. Three haplotypes of BnaFT.C6 were further identified and associated with both flowering time and crop types. We speculate that variations in both BnaFT.A2 and BnaFT.C6 may have undergone diversifying selection during the divergence of seasonal crop types in rapeseed.

A-102 Clinical Evaluation of Novel Markers in Brain Injury

Abstract Background Traumatic brain injury (TBI) is a significant health concern affecting individuals of all ages, demanding precise diagnostic methodologies to guide effective clinical management. According to the 2010 definition, a TBI results from an external force causing an alteration in brain function or other evidence of brain pathology. Current diagnostic tools, i.e. CT scans and the Glasgow Coma Scale (GCS), are inefficient because they are time and labor intensive. The need for rapid diagnostic tools is underscored by the drawbacks of existing methods, such as CT scans, which are expensive, expose patients to large amounts of radiation, and lead to delayed treatment. Recent advances in TBI diagnosis involve novel biomarkers, such as Glial Fibrillary Acidic Protein (GFAP) and Ubiquitin C-Terminal Hydrolase L1 (UCH-L1), which have shown promise in revolutionizing the assessment of head traumas. GFAP, a glial-specific protein, is elevated in circulation after a TBI, distinguishing it from multi-trauma patients without brain injuries. UCH-L1, found in neurons, is significantly elevated in TBI patients compared to uninjured controls. A 2021 study further supports the clinical effectiveness of these biomarkers, demonstrating their capacity to exclude CT-positive injury in acute TBI cases. The primary aim of this study is to explore the clinical utility of the Abbott Alinity TBI assay, specifically biomarkers GFAP and UCH-L1, in emergency department patients with a history of head trauma. Methods A retrospective study involving 238 emergency department patients with a history of head trauma and noncontrast CT scans was conducted using the Abbott Alinity TBI assay. Serum samples were collected, and biomarker values were established and compared to CT scan results. The study was comprised of 94 females and 144 males spanning various age groups. The GFAP and UCH-L1 cutoffs used were FDA approved, at 35.0 ng/L and 400.0 ng/L, respectively. If either marker was above the cutoff the patient ruled at risk for TBI. Results Results demonstrated the assay correctly identified all CT-positive cases (100% sensitivity), placing them in the rule-in group, while identifying 44 patients with a 0 chance of TBI. The negative predictive value for the TBI overall was 100% (95% CI: 91.96%-100%). The GFAP assay alone identified 30/31 positive CT scans, reporting a 98.77% (95% CI: 92.03%-99.82%) negative predictive value. The UCH-L1 assay reported 26/31 positive CT scans with a negative predictive value of 94.68% (95% CI: 88.71-97.58%). Analyzing the data revealed the TBI assay would have reduced CT scans by 18.5%. Conclusions The results of this study did show a potential reduction in the number of patients sent to CT making it a valuable tool for triaging patients presenting with head trauma; however, the complexity of neurological problems may lead to increased false positives, reducing assay specificity. Despite this limitation, this study concludes that the GFAP and UCH-L1 markers could be used to triage patients into CT rule-out and rule-in groups, thereby streamlining clinical management. In conclusion, this assay, incorporating GFAP and UCH-L1 biomarkers, presents as a promising diagnostic tool for emergency departments.

Motor function and gait decline in individuals with cerebral palsy during adulthood: a narrative review of potential physiological determinants.

Cerebral palsy (CP) is the most common childhood-onset disability. The evolution of gait according to severity is well known amongst children and thought to peak between 8 and 12years of age among those walking without assistive devices. However, among adults, clinical experience as well as scientific studies report, through clinical assessments, questionnaires and interviews, increasing walking difficulties leading to an increased dependency of assistive devices in everyday ambulation. For many individuals with CP, this change will occur around 30-40years, with the risk of losing mobility increasing with age. This narrative review aims to first provide objective evidence of motor function and gait decline in adults with CP when ageing, and then to offer mechanistic hypotheses to explain those alterations. Many studies have compared individuals with CP to the typically developing population, yet the evolution with ageing has largely been understudied. Comorbid diagnoses comprise one of the potential determinants of motor function and gait decline with ageing in people with CP, with the first manifestations happening at an early age and worsening with ageing. Similarly, ageing appears to cause alterations to the neuromuscular and cardiovascular systems at an earlier age than their typically developing (TD) peers. Future studies should, however, try to better understand how the physiological particularities of CP change with ageing that could pave the way for better strategies for maintaining function and quality of life in people with CP.

An Empirical Study on the Effect of Blended Scents in Driving Environments From a Neuro-Cognitive Perspective.

An effective method that is easy to implement and widely applicable for improving driving performance and reducing driving risks remains a challenge. Although fragrances are widely used in daily driving, there is a gap between empirical research on everyday blended fragrances and functional fragrances clinical reports. In this study, a deliberately chosen blend of scent without overtly stimulating or functional proven evidence was tested for its potential to enhance performance in a driving environment. Thirty qualified young drivers were recruited to participate in the experiment. They were asked to watch a 15-min first-person perspective driving video to simulate a driving environment and then complete questionnaires and three sets of behavioral experiments while their brain activity was monitored by EEG. Participants in the scented environment exhibited statistically significant advantages in two cognitive tasks during behavioral measures. These findings were effectively supported by the EEG data, showing that beta waves exhibited more activity in the occipital and prefrontal cortex, enhanced theta waves were observed in the prefrontal cortex, and the TAB index characterizing driving fatigue was suppressed in the prefrontal cortex. This empirical evidence highlights the potential of pleasant, natural, and blended scents in enhancing driving performance, suggesting that promoting the aromatherapy while driving as an easily applicable approach in daily life seems justified and expands the application of aromatherapy in daily life.

Functional and radiological sinonasal outcomes of CFTR modulators for sinus disease in cystic fibrosis: A meta-analysis.

Cystic fibrosis transmembrane conductance regulator (CFTR) modulators improve pulmonary outcomes in cystic fibrosis (CF) by stabilizing the CFTR protein on respiratory epithelial surfaces. To determine the efficacy of CFTR modulators on sinonasal outcomes in patients with CF, we performed a meta-analysis of clinical trials to date that include functional and radiographic evidence of sinus disease. English full-text articles were searched in PubMed, Embase, and Scopus databases. Two reviewers screened articles and a third reviewer resolved disagreements. Articles were included if they reported functional or radiological sinonasal outcomes in patients with CF before and after CFTR modulator therapies. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed, and the risk of bias in non-randomized studies of interventions tool was used for quality assessment. The generic inverse variance method with random effects model was used for meta-analysis. Standardized mean difference (SMD) and mean difference (MD) were used as effect measurements. Seven prospective and two retrospective studies representing 248 patients were included in this analysis. There was a significant improvement in sinonasal outcome test-22 scores on elexacaftor‒tezacaftor‒ivacaftor (MD=12.80, [95% confidence interval, CI: 10.46‒15.13], p<0.001, n=222), with no heterogeneity detected (I2=0%, p=0.820). There was also a significant improvement in Lund‒Mackay scores (SMD=1.25, [95% CI: 0.58‒1.91], p<0.001, n=88), with heterogeneity detected (I2=67%, p=0.030). CFTR modulators improve functional and radiologic sinonasal outcomes. Given the utility of CFTR modulators, the treatment paradigm for CF-related chronic rhinosinusitis promises to evolve.

Phosphorus-doped carbon nitride for enhanced supercapacitor performance and energy storage applications

Exploiting the advantages of graphitic carbon nitride doped with phosphorus (xP-CN) electrodes were designed for high-performance hybrid asymmetric supercapacitor via facile hydrothermal and thermal decomposition techniques. The structural, surface chemistry, functional and morphological characterizations evidence depict successful phosphorus incorporation in Carbon Nitride (CN). X-ray photoelectron spectroscopy analysis shows that phosphorus (P) atoms have replaced corner carbon atoms in the CN structure. These findings clearly demonstrate that the presence of phosphorus doping improves the electrochemical activity of the electrodes. The designed three-electrode system has a specific capacitance of 189.36 F/g with a current density of 1 A/g. Phosphorus doping has been discovered to play a significant role in improving both specific capacitance and cycling stability. The capacitance retention of the two-electrode solid-state device has 97.2 % after 5000 cycles. These electrochemical evaluations reveal that the xP-CN electrode exhibits superior electrochemical performances than pristine CN. This doping strategy for CN presents a novel and efficient approach for crafting high-performance supercapacitors.

Preserved Spontaneous Mentalizing amid Reduced Intersubject Variability in Autism during a Movie Narrative

While individuals with autism often face challenges in everyday social interactions, they may demonstrate proficiency in structured Theory of Mind (ToM) tasks that assess their ability to infer others' mental states. Using functional MRI and pupillometry, we investigated whether these discrepancies stem from diminished spontaneous mentalizing or broader difficulties in unstructured contexts. Fifty-two adults diagnosed with autism and 52 neurotypical controls viewed 'Partly Cloudy', a nonverbal animated film with a dynamic social narrative known to engage the ToM brain network during specific scenes. Analysis focused on comparing brain and pupil responses to these ToM events. Additionally, dynamic intersubject correlations explored the variability of these responses throughout the film. Both groups showed similar brain and pupil responses to ToM events and provided comparable descriptions of the characters' mental states. However, participants with autism exhibited significantly stronger correlations in their responses across the film's social narrative, indicating reduced inter-individual variability. This distinct pattern emerged well before any ToM events and involved brain regions beyond the ToM network. Our findings provide functional evidence of spontaneous mentalizing in autism, demonstrating this capacity in a context affording but not requiring mentalizing. Rather than responses to ToM events, a novel neurocognitive signature - inter-individual variability in brain and pupil responses to evolving social narratives - differentiated neurotypical individuals from those with autism. These results suggest that idiosyncratic narrative processing in unstructured settings, a common element of everyday social interactions, may offer a more sensitive scenario for understanding the autistic mind.

A heterozygous SPRY4 variant identified in female infertility characterized by reduced oocyte potential and early embryonic arrest.

Can novel genetic factors contributing to early embryonic arrest in infertile patients be identified, along with the underlying mechanisms of the pathogenic variant? We identified a heterozygous variant in the SPRY4 (sprouty RTK signaling antagonist 4) in infertile patients and conducted in vitro and in vivo studies to investigate the effects of the variant/deletion, highlighting its critical role in female reproductive health. SPRY4 acts as a negative regulator of receptor tyrosine kinases (RTKs) and functions as a tumor suppressor. Its abnormal expression can lead to recurrent miscarriage by affecting trophoblast function. In mice, Spry4 knockout (KO) leads to craniofacial anomalies and growth defects. A human study links the SPRY4 variant to a male patient with isolated hypogonadotropic hypogonadism (IHH), hypothetically impacting gonadotropin-releasing hormone (GnRH) neurons, and causing reproductive dysfunctions. SPRY4 is thus potentially integral in regulating endocrine homeostasis and reproductive function. To date, no study has reported SPRY4 variants associated with female fertility, and a causal relationship has not been established with functional evidence. Whole-exome sequencing (WES) was performed in 392 infertile women who suffered from primary infertility of unknown reason, and the heterozygous SPRY4 variant were identified in one independent family. The infertile patients presenting were recruited from July 2017 to November 2023. Women diagnosed with primary infertility were recruited from the Reproduction Center of Zhongshan Hospital, Fudan University. Genomic DNA was extracted from peripheral blood for WES analysis. The SPRY4 variant were identified through WES, in silico analysis, and variant screening. All variants were confirmed by Sanger sequencing. The effects of the variants were investigated in human embryonic kidney (HEK) 293T (HEK293T) cells via western blotting, and in mouse oocytes and embryos through complementary RNA (cRNA) injection, RNA sequencing, fluorescence, absorbance, and RT-qPCR assays. Gene function was further examined in Spry4 KO mice via histology, western blotting, ELISA, and RT-qPCR assays. We identified a missense heterozygous pathogenic variant in SPRY4 (GRCh38, GenBank: NM_030964.5, c.157C>T p.(Arg53Trp), rs200531302) that reduces SPRY4 protein levels in HEK293T cells and disrupts the redox system and mitochondrial function in mouse oocyte, and perturbs developmental potential in mouse embryos. These phenotypes could be partially reversed by the exogenous addition of Nrf1 cRNA. Additionally, Spry4-/- mice exhibit ovarian oxidative stress and decreased ovarian function. Due to the limited WES data and population, we identified only one family with a SPRY4 mutation. The deeper mechanism and therapeutic strategy should be further investigated through mutant mice and recovery experiment. Our study has identified a pathogenic variant in SPRY4 associated with early embryonic arrest in humans. These findings enhance our understanding of the role of SPRY4 in early embryonic development and present a new genetic marker for female infertility. This work was supported by the National Natural Science Foundation of China (82071643 and 82171655) and Natural Science Foundation of Shanghai (22ZR1456200). None of the authors have any competing interests. N/A.

Identification and functional characteristics of CHD1L gene variants implicated in human Müllerian duct anomalies

BackgroundMüllerian duct anomalies (MDAs) are congenital developmental disorders that present as a series of abnormalities within the reproductive tracts of females. Genetic factors are linked to MDAs and recent advancements in whole-exome sequencing (WES) provide innovative perspectives in this field. However, relevant mechanism has only been investigated in a restricted manner without clear elucidation of respective observations.MethodsOur previous study reported that 2 of 12 patients with MDAs harbored the CHD1L variant c.348-1G>C. Subsequently, an additional 85 MDAs patients were recruited. Variants in CHD1L were screened through the in-house database of WES performed in the cohort and two cases were identified. One presented with partial septate uterus with left renal agenesis and the other with complete septate uterus, duplicated cervices and longitudinal vaginal septum. The pathogenicity of the discovered variants was further assessed by molecular dynamics simulation and various functional assays.ResultsUltimately, two novel heterozygous CHD1L variants, including a missense variant c.956G>A (p.R319Q) and a nonsense variant c.1831C>T (p.R611*) were observed. The variants were absent in 100 controls. Altogether, the contribution yield of CHD1L to MDAs was calculated as 4.12% (4/97). All three variants were assessed as pathogenic through various functional analysis. The splice-site variant c.348-1G>C resulted in a 11 bp sequence skipping in exon 4 of CHD1L and led to nonsense mediated decay of its transcripts. Unlike WT CHD1L, the truncated R611* protein mislocalized to the cytoplasm, abolish the ability of CHD1L to promote cell migration and failed to interact with PARP1 owing to the loss of macro domain. The R319Q variant exhibited conformational disparities and showed abnormal protein recruitment behavior through laser microirradiation comparing with the WT CHD1L. All these variants impaired the CHD1L function in DNA damage repair, thus participating in MDAs.ConclusionsThe current study not only expands the mutational spectrum of CHD1L in MDAs but determines three variants as pathogenic according to ACMG guidelines with reliable functional evidence. Additionally, the impairment in DNA damage repair is an underlying mechanism involved in MDAs.

Identification and functional validation of P450 genes associated with pyrethroid resistance in the malaria vector Anopheles sinensis (Diptera Culicidae)

Cytochrome P450 monooxygenases (P450s), a multifunctional protein superfamily, are one of three major classes of detoxification enzymes. However, the diversity and functions of P450 genes from pyrethroid-resistant populations of Anopheles sinensis have not been fully explored. In this study, P450 genes associated with pyrethroid resistance were systematically screened using RNA-seq in three field pyrethroid-resistant populations (AH-FR, CQ-FR, YN-FR) and one laboratory resistant strain (WX-LR) at developmental stages, tissues, and post blood-meal in comparison to the laboratory susceptible strain (WX-LS) in An. sinensis. Importantly, the expression of significantly upregulated P450s was verified using RT-qPCR, and the function of selected P450s in pyrethroid detoxification was determined with RNA interference using four laboratory pyrethroid-resistant strains (WX-LR, AH-LR, CQ-LR, YN-LR). Sixteen P450 genes were significantly upregulated in at least one field-resistant population, and 44 were significantly upregulated in different developmental stages, tissues or post blood-meal. A total of 19 P450s were selected to verify their association with pyrethroid resistance, and four of them (AsCYP6P3v1, AsCYP6P3v2, AsCYP9J10, and AsCYP9K1) demonstrated significant upregulation in laboratory pyrethroid-resistant strains using RT-qPCR. Knockdown of these four genes all significantly reduced pyrethroid resistance and increased the mortality by 57.19% (AsCYP6P3v1 and AsCYP6P3v2 knockdown group), 38.39% (AsCYP9K1 knockdown group) and 48.87% (AsCYP9J10 knockdown group) in An. sinensis by RNAi, which determined the pyrethroid detoxification function of these four genes. This study revealed the diversity of P450 genes and provided functional evidence for four P450s in pyrethroid detoxification in An. sinensis for the first time, which increases our understanding of the pyrethroid resistance mechanism, and is of potential value for pyrethroid resistance detection and surveillance.

A Novel Human SDHA-Knockout Cell Line Model for the Functional Analysis of Clinically Relevant SDHA Variants.

SDHA mutations are the most common cause of succinate dehydrogenase (SDH)-deficient GIST. Enhanced cancer surveillance of individuals carrying a known pathogenic germline SDHA mutation has the potential to detect early-stage tumors, allowing for improved patient outcomes. However, more than 95% of the >1,000 SDHA missense variants listed in ClinVar are variants of uncertain significance. Our ability to interpret the significance of SDHA variants must improve before genetic sequencing can be utilized to its full potential. SDHA variants were introduced into a clonal SDHA-knockout cell line via Bxb1-mediated recombination. SDH activity and SDHA abundance were determined for each variant, and logistic regression analysis was used to derive functional evidence for clinical variant interpretation. Our analysis revealed that cancer-associated SDHA missense variants can be clearly distinguished from noncancer variants according to the extent of SDH dysfunction caused. As such, SDH activity data can be used to predict cancer pathogenicity with strong performance metrics, exceeding those of computational prediction tools. From these data, we obtained functional evidence for clinical variant interpretation from 21 of 22 assayed variants of uncertain significance, with 19 in favor of cancer pathogenicity and two against pathogenicity. Lastly, simulating the addition of our functional evidence with limited preexisting evidence allowed for 18 of 22 variants to be reclassified. We describe a novel pipeline for investigating the functional consequences of SDHA missense variants. In total, we characterized 72 variants, developed criteria for obtaining functional evidence, and demonstrated the potential of this evidence for clinical variant interpretation.