Insertion Allele Research Articles

LRR1 involved in the abscisic acid signaling pathway to regulate the early growth and development of Arabidopsis thaliana.

Living organisms possess the remarkable capacity to swiftly adapt to fluctuations in their environment. In the context of cell signal transduction, a significant challenge lies in ensuring the effective perception of external signals and the execution of appropriate responses. To investigate this phenomenon, a recent study utilized Arabidopsis thaliana as a model plant and induced stress by administering abscisic acid (ABA), a plant hormone, to elucidate the involvement of leucine-rich repeat receptor-like kinase1 (LRR1) in ABA signaling pathways. Homozygous T-DNA insertion alleles for LRR1 and KIN7 were isolated. Quantitative real-time PCR (qRT-PCR) was performed to confirm the expression of the LRR1 gene. Subcellular localization and beta-glucuronidase (GUS) tissue labeling techniques were utilized to determine the expression pattern of the LRR1 gene in cells and tissues. Yeast two-hybrid complementation, bimolecular fluorescence complementation assay, and GST pull-down assays were conducted to validate the interaction of LRR1 proteins. Phenotypic analyses revealed that lrr1 and kin7 mutants are less sensitive to the inhibitory effects of ABA on germination and cotyledon greening that is seen in WT. Mutants LRR1 and kinase 7 (KIN7) exhibited resistance to ABA and displayed normal growth patterns under control conditions. The double mutant lrr1kin7 showed reduced responsiveness to ABA. Conversely, overexpression lines LRR1ox2 and LRR1ox10 demonstrated heightened sensitivity to ABA, resulting in severe growth reduction. qRT-PCR assay indicated that exogenous application of ABA led to significant down-regulation of ABI3, ABI4, and ABI5 transcription factors in LRR1 material compared to wild-type WT material. An investigation was conducted to determine the expression pattern and transcriptional level of LRR1 in Arabidopsis. The results revealed ubiquitous expression of LRR1 across all developmental stages and tissue tested. Subcellular localization assays confirmed the presence of LRR1 on the plasma membrane of cells. Furthermore, BiFC assay, yeast two-hybrid complementation, and GST pull-down assays demonstrated an interaction between LRR1 and PYL6 in vitro. These findings provide substantial insights into the involvement of LRR1 in the ABA signaling pathway while regulating seed germination and cotyledon greening during early development in Arabidopsis. This study significantly advances our understanding regarding the correlation between LRR1 and ABA signaling pathways with potential applications for enhancing crop stress resistance.

Association Study of rs1632947, rs1233334, and rs371194629 Polymorphisms in Human Leukocyte Antigen GGene Expression and soluble Human Leukocyte Antigen Gwith Lupus.

Systemic lupus erythematosus is a chronic autoimmune disease that has been associated with human leukocyte antigen G (HLA-G) in previous studies on immunological diseases. This study aimed to investigate the association between three HLA-G gene polymorphisms (rs1632947, rs1233334, and rs371194629) and their impact on HLA-G mRNA expression and soluble HLA-G levels in serum. Genotyping was performed using TaqMan probe PCR. RNA extraction, reverse transcription PCR, and real-time PCR assays were conducted to assess the expression of the HLA-G gene in tissue samples. Soluble HLA-G was measured using enzyme-linked immunosorbent assay in serum. Results show a significant difference in the frequency of the G allele for two 5'-untranslated region (UTR) polymorphisms of the HLA-G gene (rs1632947 and rs1233334) located at positions -964 and -725, respectively, between lupus patients and controls, with p-values of 0.009 and 0.040, respectively. In addition, the study identified the 14 bp insertion allele of the rs371194629 polymorphism located in the 3' UTR of the gene as a risk factor for lupus, with a p-value of 0.001. Our results also indicate that lupus-related alleles may increase the risk of developing the disease by upregulating the expression of HLA-G and increasing soluble HLA-G levels in serum. The findings of the study suggest that the identified genetic variants may play a role in the development of lupus and could be useful in identifying individuals at risk for the disease. These results are important for advancing our understanding of the genetic basis of lupus and may have implications for the development of new treatments and diagnostic tools for the disease.

Association of nuclear factor-kappa B1 (NF-κB1) Ins/Del gene polymorphism with Hepatitis C virus outcomes.

To explore the association of nuclear factor-kappa B1 polymorphism in the promotor area of the gene with hepatitis C virus infection outcomes. The case-control study was conducted at the Hepatology and Gastroenterology Teaching Hospital, Baghdad, Iraq, from Dec 1, 2020, to Aug 30, 2021, and comprised individuals ages 20-68 years. Group 1 had patients with persistent hepatitis C virus infection, group 2 had subjects with spontaneous hepatitis C virus clearance, group 3 had subjects treated with direct-acting antiviral drugs, and group 4 had healthy controls. Venous blood was collected for polymorphism genetic analysis of nuclear factor-kappa B1 insertion/deletion ATTG (Adenine-Thymine- Thymine-Guanine) at rs28362491 using a high-resolution melting technique. Data was analysed using SPSS 27. Of the 88 subjects, there were 22(25%) in each of the 4 groups. Overall, there were 55(62.5%) females and 33(37.5%) males, and 40(45.45%) were aged 20-39 years while 48(54.54%) were aged 40-68 years (p>0.05). The Ins allele of rs28362491 was significantly more frequent in the patients than in controls (p=0.0053). The carriage of rs28362491 insertion/insertion and insertion/deletion genotypes, compared to wild-type homozygous deletion/deletion, had a significantly higher risk of developing hepatitis C virus infection (p=0.0013). No association was found between rs28362491 and spontaneous hepatitis C virus clearance (p>0.05). The insertion allele of rs28362491 was found to be associated with increased susceptibility to developing hepatitis C virus infection.

A novel codominant plumage color pattern of white breast patches in WugangTong geese was controlled by EDNRB2

Two basic plumage color patterns are observed in adult geese: solid grey (G) or colorless white (W). However, a Chinese indigenous breed, the Wugangtong goose (WGT), continues to be subject to selective breeding efforts as it displays segregation of plumage colors, including G, W, and a novel color pattern designated Wb (G with white breast circles). The underlying genetic mechanisms responsible for the Wb phenotype are yet to be determined. The current study employed the population differentiation index (FST) to analyze 90 geese exhibiting diverse plumage colors, identifying the fifth intron of EDNRB2 as a particularly noteworthy region with the highest FST values. Sanger sequencing of the region surrounding the EDNRB2 gene identified a 14-bp insertion within exon 3 as the causal mutation. The heterozygosity of this 14-bp insertion and wild-type alleles was completely associated with the Wb phenotype, thereby substantiating the codominant nature of the G and W phenotypes. An inter-species corroborated this finding cross between the graylag (no 14-bp insertion) and the swan goose (homozygous for the 14-bp insertion) breeds, as hybrids from this cross exhibited the Wb phenotype. Transcriptomes from white breast patches and gray dorsal skins of 4 Wb geese were compared. A significant downregulation of genes involved in melanin synthesis and melanocyte development was observed, including EDRNB2 and MLANA. The downregulation of MLANA indicated that the mutated EDNRB2 resulted in melanocyte loss in specific body regions, as MLANA is a marker gene for melanocytes. The findings were corroborated by melanin staining using the Mansson-Fontana method, which revealed no melanin particles deposited in the white breast patches. In summary, the gray plumage color was codominant to the white color in WGT geese, and plumage color variations were controlled by EDNRB2. The findings of our study offer valuable and practical guidance for the purification of plumage colors among WGT, whether through traditional phenotype selection or molecular breeding methods.

Association of ACE and AGTR1 variants with retinopathy of prematurity: a case-control study and meta-analysis.

Retinopathy of prematurity (ROP) is a major cause of childhood blindness worldwide, linked to gene variants in the renin-angiotensin-aldosterone system, including angiotensin-converting enzyme (ACE) and angiotensin II receptor type 1 (AGTR1). This study aims to evaluate the association between ACE insertion/deletion (I/D) and AGTR1 rs5186A > C variants with the occurrence and progression of ROP in a Polish cohort. A total of 377 premature infants were enrolled in the study. The ACE variant was evaluated using PCR, and AGTR1 was assessed using TaqMan probes. Clinical characteristics, including risk factors and comorbidities, were documented. A meta-analysis of the effects of the studied variants on ROP was also conducted. The AGTR1 rs5186C allele was significantly associated with both the progression of ROP and treatment outcomes. Homozygotes exhibited a 2.47-fold increased risk of developing proliferative ROP and a 4.82-fold increased risk of treatment failure. The impact of this allele increased at low birth weight. A meta-analysis, including 191 cases and 1661 controls, indicated an overall risk of 1.7 (95%CI 1.02-2.84) for the recessive effect of the rs5186C allele. The ACE variant did not show a significant association with ROP in our population; however, a meta-analysis of 996 cases and 2787 controls suggested a recessive effect of the insertion allele (an odds ratio of 1.21 (95%CI 1.00-1.60)). These results indicate that gain-of-function AGTR1 variants may play a crucial role in the development of ROP, potentially by promoting angiogenesis and pro-inflammatory effects. Screening for these variants could facilitate the development of personalized risk assessment and treatment strategies for ROP.

Mutations in nuclear genes encoding mitochondrial ribosome proteins restore pollen fertility in S male-sterile maize.

The interaction of plant mitochondrial and nuclear genetic systems is exemplified by mitochondria-encoded cytoplasmic male sterility (CMS) under the control of nuclear restorer-of-fertility genes. The S type of CMS in maize is characterized by a pollen collapse phenotype and a unique paradigm for fertility restoration in which numerous nuclear restorer-of-fertility lethal mutations rescue pollen function but condition homozygous-lethal seed phenotypes. Two nonallelic restorer mutations recovered from Mutator transposon-active lines were investigated to determine the mechanisms of pollen fertility restoration and seed lethality. Mu Illumina sequencing of transposon-flanking regions identified insertion alleles of nuclear genes encoding mitochondrial ribosomal proteins RPL6 and RPL14 as candidate restorer-of-fertility lethal mutations. Both candidates were associated with lowered abundance of mitochondria-encoded proteins in developing maize pollen, and the rpl14 mutant candidate was confirmed by independent insertion alleles. While the restored pollen functioned despite reduced accumulation of mitochondrial respiratory proteins, normal-cytoplasm plants heterozygous for the mutant alleles showed a significant pollen transmission bias in favor of the nonmutant Rpl6 and Rpl14 alleles. CMS-S fertility restoration affords a unique forward genetic approach to investigate the mitochondrial requirements for, and contributions to, pollen and seed development.

Nonphotochemical quenching kinetics GWAS in sorghum identifies genes that may play conserved roles in maize and Arabidopsis thaliana photoprotection.

Photosynthetic organisms must cope with rapid fluctuations in light intensity. Nonphotochemical quenching (NPQ) enables the dissipation of excess light energy as heat under high light conditions, whereas its relaxation under low light maximizes photosynthetic productivity. We quantified variation in NPQ kinetics across a large sorghum (Sorghum bicolor) association panel in four environments, uncovering significant genetic control for NPQ. A genome-wide association study (GWAS) confidently identified three unique regions in the sorghum genome associated with NPQ and suggestive associations in an additional 61 regions. We detected strong signals from the sorghum ortholog of Arabidopsis thaliana Suppressor Of Variegation 3 (SVR3) involved in plastid-nucleus signaling. By integrating GWAS results for NPQ across maize (Zea mays) and sorghum-association panels, we identified a second gene, Non-yellowing 1 (NYE1), originally studied by Gregor Mendel in pea (Pisum sativum) and involved in the degradation of photosynthetic pigments in light-harvesting complexes. Analysis of nye1 insertion alleles in A. thaliana confirmed the effect of this gene on NPQ kinetics in eudicots. We extended our comparative genomics GWAS framework across the entire maize and sorghum genomes, identifying four additional loci involved in NPQ kinetics. These results provide a baseline for increasing the accuracy and speed of candidate gene identification for GWAS in species with high linkage disequilibrium.

A sequence of SVA retrotransposon insertions in ASIP shaped human pigmentation

Retrotransposons comprise about 45% of the human genome1, but their contributions to human trait variation and evolution are only beginning to be explored2,3. Here, we find that a sequence of SVA retrotransposon insertions in an early intron of the ASIP (agouti signaling protein) gene has probably shaped human pigmentation several times. In the UK Biobank (n = 169,641), a recent 3.3-kb SVA insertion polymorphism associated strongly with lighter skin pigmentation (0.22 [0.21–0.23] s.d.; P = 2.8 × 10−351) and increased skin cancer risk (odds ratio = 1.23 [1.18–1.27]; P = 1.3 × 10−28), appearing to underlie one of the strongest common genetic influences on these phenotypes within European populations4–6. ASIP expression in skin displayed the same association pattern, with the SVA insertion allele exhibiting 2.2-fold (1.9–2.6) increased expression. This effect had an unusual apparent mechanism: an earlier, nonpolymorphic, human-specific SVA retrotransposon 3.9 kb upstream appeared to have caused ASIP hypofunction by nonproductive splicing, which the new (polymorphic) SVA insertion largely eliminated. Extended haplotype homozygosity indicated that the insertion allele has risen to allele frequencies up to 11% in European populations over the past several thousand years. These results indicate that a sequence of retrotransposon insertions contributed to a species-wide increase, then a local decrease, of human pigmentation.

SYNAPTOTAGMIN 4 is expressed mainly in the phloem and participates in abiotic stress tolerance in Arabidopsis.

Plant synaptotagmins structurally resemble animal synaptotagmins and extended-synaptotagmins. Animal synaptotagmins are well-characterized calcium sensors in membrane trafficking, and extended-synaptotagmins mediate lipid transfer at the endoplasmic reticulum-plasma membrane contact sites. Here, we characterize SYNAPTOTAGMIN 4 (SYT4), which belongs to the six-member family in Arabidopsis. Fluorometric GUS assay showed that the SYT4 promoter was strongest in roots and the least active in rosettes and cauline leaves, which was confirmed by qPCR. In seedlings, promoter activity was influenced by several factors, such as plant growth regulators, mannitol, sucrose, polyethylene glycol and cold. GUS histochemistry revealed SYT4 promoter activity in the phloem of all organs and even almost exclusively in sieve element precursors and differentiating sieve elements. Accordingly, the SYT-GFP fusion protein also accumulated in these cells with maximal abundance in sieve element precursors. The protein formed a network in the cytoplasm, but during sieve tube differentiation, it deposited at the cell periphery and disappeared from mature tubes. Using photoconvertible fluorescence technology, we showed that a high abundance of SYT4 protein in meristematic protophloem cells was due to its extensive synthesis. SYT4 protein synthesis was interrupted in differentiating sieve elements, but protein degradation was also reduced. In addition to phloem, the fusion protein was detected in shoot and root stem cell niche as early as the late heart stage of the embryo. We isolated and molecularly and biologically characterized five syt4 T-DNA insertion alleles and subjected them to phenotype analysis. The allele with the C2B domain interrupted by an T-DNA insertion exhibits increased sensitivity to factors such as auxins, osmotics, salicylic acid, sodium chloride, and the absence of sucrose in the root growth test.

Identification and Characterization of the miRNA Transcriptome Controlling Green Pigmentation of Chicken Eggshells

Green eggs are mainly caused by inserting an avian endogenous retrovirus (EVA-HP) fragment into the SLCO1B3 gene. Although the genotypes for this insertion allele are consistent, eggshell color (ESC) may vary after a peak laying period; light-colored eggs are undesired by consumers and farmers and result in financial loss, so it is necessary to resolve this problem. miRNAs are small non-coding RNAs that exert essential functions in animal development and diseases. However, the regulatory miRNAs and detailed molecular mechanisms regulating eggshell greenness remain unclear. In the present study, we determined the genotype of green-eggshell hens through the detection of a homozygous allele insertion in the SLCO1B3 gene. The shell gland epithelium was obtained from green-eggshell hens that produced white and green shell eggs to perform transcriptome sequencing and investigate the important regulatory mechanisms that influence the ESC. Approximately 921 miRNAs were expressed in these two groups, which included 587 known miRNAs and 334 novel miRNAs, among which 44 were differentially expressed. There were 22 miRNAs that were significantly upregulated in the green and white groups, respectively, which targeted hundreds of genes, including KIT, HMOX2, and several solute carrier family genes. A Gene Ontology enrichment analysis of the target genes showed that the differentially expressed miRNA-targeted genes mainly belonged to the functional categories of homophilic cell adhesion, gland development, the Wnt signaling pathway, and epithelial tube morphogenesis. A KEGG enrichment analysis showed that the Hedgehog signaling pathway was significantly transformed in this study. The current study provides an overview of the miRNA expression profiles and the interaction between the miRNAs and their target genes. It provides valuable insights into the molecular mechanisms underlying green eggshell pigmentation, screening more effective hens to produce stable green eggs and obtaining higher economic benefits.

A novel insertional allele of the CG18135 gene is associated with severe mutant phenotypes in Drosophila melanogaster.

Drosophila melanogaster has been at the forefront of genetic studies and biochemical modeling for over a century. Yet, the functions of many genes are still unknown, mainly because no phenotypic data are available. Herein, we present the first evidence data regarding the particular molecular and other quantifiable phenotypes, such as viability and anatomical anomalies, induced by a novel P{lacW} insertional mutant allele of the CG18135 gene. So far, the CG18135 functions have only been theorized based on electronic annotation and presumptive associations inferred upon high-throughput proteomics or RNA sequencing experiments. The descendants of individuals harboring the CG18135 P{lacW}CG18135 allele were scored in order to assess mutant embryonic, larval, and pupal viability versus Canton Special (CantonS). Our results revealed that the homozygous CG18135 P{lacW}CG18135 /CG18135 P{lacW}CG18135 genotype determines significant lethality both at the inception of the larval stage and during pupal development. The very few imago escapers that either breach or fully exit the puparium exhibit specific eye depigmentation, wing abnormal unfolding, strong locomotor impairment with apparent spasmodic leg movements, and their maximum lifespan is shorter than 2days. Using the quantitative real-time PCR (qRT-PCR) method, we found that CG18135 is upregulated in male flies, but an unexpected gene upregulation was also detected in heterozygous mutants compared to wild-type flies, probably because of regulatory perturbations induced by the P{lacW} transposon. Our work provides the first phenotypic evidence for the essential role of CG18135, a scenario in accordance with the putative role of this gene in carbohydrate-binding processes.

Fatty acid desaturase insertion-deletion polymorphism rs66698963 predicts colorectal polyp prevention by the n–3 fatty acid eicosapentaenoic acid: a secondary analysis of the seAFOod polyp prevention trial

BackgroundA fatty acid desaturase (FADS) insertion-deletion (Indel) polymorphism (rs66698963) influences the expression of FADS1, which controls the synthesis of n–6 highly unsaturated fatty acid (HUFA) arachidonic acid (AA). The anti-inflammatory activity of the n–3 HUFA eicosapentaenoic acid (EPA) may be explained by competition with AA for proinflammatory lipid mediator synthesis. A precision medicine approach based on stratification by FADS Indel genotype could identify individuals, who benefit from greatest disease risk reduction by n–3 HUFAs. ObjectivesWe tested the hypothesis that the FADS insertion (I) allele predicts colorectal polyp risk reduction in a secondary analysis of the randomized, placebo-controlled, 2×2 factorial seAFOod polyp prevention trial of EPA 2000 mg daily and aspirin 300 mg daily for 12 mo (ISRCTN05926847). MethodsParticipant Indel genotype was determined by polymerase chain reaction (PCR) blind to trial outcomes. Colorectal polyp outcomes were included in negative binomial (polyp number) and logistic (polyp detection rate [PDR; percentage with one or more polyps]) regression models comparing each active intervention with its placebo. Presence of ≥1 Indel I allele and an interaction term (I allele × active intervention) were covariates. ResultsIn 528 participants with colonoscopy and FADS Indel data, EPA use irrespective of Indel genotype, was not associated with reduced colorectal polyp number (incidence rate ratio [IRR]: 0.92; 95% confidence interval: 0.74, 1.16), mirroring original seAFOod trial analysis. However, the presence of ≥1 I allele identified EPA users with a significant reduction in colorectal polyp number (IRR: 0.50 [0.28, 0.90]), unlike aspirin, for which there was no interaction. Similar findings were obtained for the PDR. ConclusionsThe FADS Indel I allele identified individuals, who displayed colorectal polyp prevention by EPA with a similar effect size to aspirin. Assessment of rs66698963 as a biomarker of therapeutic response to n–3 HUFAs in other populations and healthcare settings is warranted.The seAFOod polyp prevention trial and STOP-ADENOMA study were registered at International Standard Randomised Controlled Trial Number registry as ISRCTN05926847.

The association of the ACE gene I/D polymorphism and in-game performance in elite Rugby Union players

Genetic variation has been proposed as a factor in human athletic performance. The angiotensin converting enzyme (ACE) gene has been studied extensively albeit with heterogeneous results. The insertion (I) and deletion (D) alleles have been associated with altered serum and tissue ACE, as well as endurance- and power-based performance respectively. It is suggested the distinction in rugby union performance can be partly attributed to variations in the ACE gene. The aim of this study was to identify associations between the ACE I/D genetic polymorphism and in-game performance in elite (≥ 5 games at top-flight) rugby union athletes. The athlete cohort consisted of 347 Caucasian men, divided into positional groups (backs and forwards) and positional subgroups (front five, back row, halves, centres, and back three). Ethical approval was provided by Manchester Metropolitan University with written informed consent obtained from all participants. Genotyping and genotype analysis was performed using a combination of the X9 High-Throughput Genomics System (Standard BioTools) and the StepOnePlus™ Real-Time PCR System (Applied Biosystems) and their respective corresponding software. IBM SPSS Statistics 28 (SPSS, Chicago, IL) software was used to conduct Pearson's Chi-square (χ2) test of independence comparing genotype and allelic frequencies and the Kruskal-Wallis H and Mann-Whitney U tests to investigate differences between genotypes. Genotypic and allelic frequencies did not differ at the population level, nor at the positional group/subgroup levels. Performance did not differ between genotypes nor alleles at the population and positional group levels. Back row II genotypes beat more defenders than IDs (P = 0.008) but completed fewer tackles than both IDs (P = 0.05) and DDs (P = 0.013). Halfback DDs missed fewer tackles than both IDs (P = 0.003) and IIs (P = 0.01). Centre DD genotypes made more carries than IIs (P = 0.006). Back three IIs scored more tries than both IDs (P = 0.005) and DDs (P = 0.007) and made more clean breaks than IDs (P = 0.004). Most of the existing literature has been concerned with identifying the ACE gene as a marker of elite performance. To our knowledge, this is the first study to elucidate how the inter-personal differences in rugby union performance are influenced by the ACE I/D polymorphism. The findings from this study may provide novel insight into the specialisation of training and rehabilitation modalities for athletes, based on their genetic makeup.

A chemical genetic screen with the EXO70 inhibitor Endosidin2 uncovers potential modulators of exocytosis in Arabidopsis.

Exocytosis plays an essential role in delivering proteins, lipids, and cell wall polysaccharides to the plasma membrane and extracellular spaces. Accurate secretion through exocytosis is key to normal plant development as well as responses to biotic and abiotic stresses. During exocytosis, an octameric protein complex named the exocyst facilitates the tethering of secretory vesicles to the plasma membrane. Despite some understanding of molecular and cellular aspects of exocyst function obtained through reverse genetics and direct interaction assays, knowledge about upstream modulators and genetic interactors remains limited. Traditional genetic screens encounter practical issues in exocyst subunit mutant backgrounds, such as lethality of certain knockout mutants and/or potential redundancy of EXO70 homologs. To address these challenges, this study leverages the tunable and reversible nature of chemical genetics, employing Endosidin2 (ES2)-a synthetic inhibitor of EXO70-for a large-scale chemical genetic mutant screen in Arabidopsis. This approach led to the identification of 70 ES2-hypersensitive mutants, named es2s. Through a whole-genome sequencing-based mapping strategy, 14 nonallelic es2s mutants were mapped and the candidate mutations reported here. In addition, T-DNA insertion lines were tested as alternative alleles to identify causal mutations. We found that T-DNA insertion alleles for DCP5, VAS1/ISS1, ArgJ, and MEF11 were hypersensitive to ES2 for root growth inhibition. This research not only offers new genetic resources for systematically identifying molecular players interacting with the exocyst in Arabidopsis but also enhances understanding of the regulation of exocytosis.

Genetic interaction between TTG2 and AtPLC1 reveals a role for phosphoinositide signaling in a co-regulated suite of Arabidopsis epidermal pathways

The TTG2 transcription factor of Arabidopsis regulates a set of epidermal traits, including the differentiation of leaf trichomes, flavonoid pigment production in cells of the inner testa (or seed coat) layer and mucilage production in specialized cells of the outer testa layer. Despite the fact that TTG2 has been known for over twenty years as an important regulator of multiple developmental pathways, little has been discovered about the downstream mechanisms by which TTG2 co-regulates these epidermal features. In this study, we present evidence of phosphoinositide lipid signaling as a mechanism for the regulation of TTG2-dependent epidermal pathways. Overexpression of the AtPLC1 gene rescues the trichome and seed coat phenotypes of the ttg2-1 mutant plant. Moreover, in the case of seed coat color rescue, AtPLC1 overexpression restored expression of the TTG2 flavonoid pathway target genes, TT12 and TT13/AHA10. Consistent with these observations, a dominant AtPLC1 T-DNA insertion allele (plc1-1D) promotes trichome development in both wild-type and ttg2-3 plants. Also, AtPLC1 promoter:GUS analysis shows expression in trichomes and this expression appears dependent on TTG2. Taken together, the discovery of a genetic interaction between TTG2 and AtPLC1 suggests a role for phosphoinositide signaling in the regulation of trichome development, flavonoid pigment biosynthesis and the differentiation of mucilage-producing cells of the seed coat. This finding provides new avenues for future research at the intersection of the TTG2-dependent developmental pathways and the numerous molecular and cellular phenomena influenced by phospholipid signaling.

Novel indel variation of LTBP4 gene associates with risk of sudden cardiac death in Chinese populations with coronary artery disease

The objective of this study is to investigate whether common genetic variants of the LTBP4 gene are linked to the susceptibility of sudden cardiac death in individuals who have atherosclerotic coronary artery disease (SCD-CAD) in Chinese populations. A total of 208 SCD-CAD cases and 638 controls were included in the analysis, and logistic regression was employed to assess the association between a 4-bp insertion/deletion polymorphism (rs34005443) within LTBP4 and the susceptibility to SCD-CAD among Chinese individuals. Logistic regression analysis demonstrated a notable association between the insertion allele of rs34005443 and an escalated susceptibility to SCD-CAD [odds ratio (OR) = 1.434; 95 % confidence interval:1.14–1.80; P = 1.79 × 10−3]. Genotype-phenotype correlation analysis was performed using Genotype-Tissue expression (GTEx) database and further validated by human myocardium using qPCR. Correlation analysis revealed that LTBP4 expression level was lower in samples with the insertion allele. Furthermore, the dual-luciferase activity assays indicated that rs34005443 may play a regulatory role. Additionally, we predicted 30 transcription factors that are likely to bind to rs34005443 and its highly linked genetic variants via 3DSNP database. Subsequent GO and KEGG analysis indicated that these transcription factors have a significant function in regulating gene expression. Finally, PPI network analysis suggested a tight connection between LTBP4 proteins and TGFβs, highlighting these genes as potential hub genes in the context of SCD-CAD. In summary, our study revealed that rs34005443 might contribute to SCD-CAD susceptibility by regulating LTBP4 expression. These findings revealed that this indel could be a potentially functional marker for molecular diagnosis and risk stratification of SCD-CAD.

Activation of the erythropoietin system in exhaustively exercise muscle relates to ACE gene polymorphism-modulated metabolic signalling and mitochondrial transcript expression

Introduction
 Introduction A fall in muscle oxygen saturation is a potent activator of mitochondrial biogenesis in exercised skeletal muscle which is subject to the training-modulated influence of the insertion/deletion polymorphism of the angiotensin converting enzyme gene (ACE-I/D; Gasser et al 2022) and may involve paracrine erythropoietin (EPO) signaling (Desplanches et al 2014, Nijholt et al 2021). We tested the hypothesis that erythropoietin expression and signaling in fatiguingly exercised muscle would correspond to the expression of hypoxia-regulated mitochondrial genes and altered metabolic signaling and would be subject to a genetic influence by ACE-I/D.
 Methods
 Methods 22 healthy, male white Caucasian men (27.0 +/- 1.4 years; BMI 23.6 +/- 0.6 kg m-2) completed a session of fatiguing one-legged exercise in the fasted state. Microbiopsies were collected from m. vastus lateralis of the non-exercised leg immediately before exercise, and ½, 3, and 8 hours thereafter from the exercising leg. Levels of the hypoxia-regulated cytochrome c oxidase subunit 4 isoforms 1 and 2 (COX4I1 and COX4I2), ACE and EPO transcript, glycogen concentration in m. vastus lateralis, the ACE-I/D genotype and aerobic fitness state were assessed as described (Desplanches et al 2014; Gasser et al 2022). EPO protein concentration and phosphorylation of intracellular transducers of EPO signaling were quantified in muscle homogenates with validated enzyme-linked immune sorbent and a phospho-kinase assays. Effects and Pearson correlations were assessed with analysis of variance and declared significant at p < 0.05.
 Results
 One-legged exercise produced metabolic fatigue as indicated by the voluntary cessation of exercise and a reduced glycogen concentration in m. vastus lateralis in all subjects ½ hour after exercise (-0.044 mg mg-1). Concomitantly, EPO protein levels were 4-fold lowered; and subsequently increased 3-8 hours after cessation of exercise alike EPO transcripts levels.
 Aerobically fit ACE I-allele carriers demonstrated a sparing of muscle glycogen, exaggerated EPO transcript response, and higher phosphorylation levels of EPO signal transducers [STAT5a-Y694 (+31%), STAT5b-Y699 (+40%)]. The phosphorylation level of the metabolic signal transducer AMPKa2-T172 correlated to EPO transcript levels 3 hours post exercise (r = 0.61). EPO protein levels correlated to ACE and COX4I2 transcript levels (r = -0.79; -0.54).
 Discussion
 The findings highlight that a paracrine loop of metabolically-regulated EPO signaling exists in exercised human skeletal muscle which variability is associated with the ACE-I/D gene polymorphism in fair correspondence with a mitochondrial marker of local hypoxia.
 References
 Desplanches, D., Amami, M., Mueller, M., Hoppeler, H., Kreis, R., & Flück, M. (2014). Hypoxia refines plasticity of mitochondrial respiration to repeated muscle work. European Journal of Applied Physiology, 114, 405-417. https://doi.org/10.1007/s00421-013-2783-8
 Gasser, B., Franchi, M. V., Ruoss, S., Frei, A., Popp, W. L., Niederseer, D., Catuogno, S., Frey, W. O., & Flück, M. (2022). Accelerated muscle deoxygenation in aerobically fit subjects during exhaustive exercise is associated with the ACE insertion allele. Frontiers in Sports and Active Living, 4, Article 814975. https://doi.org/10.3389/fspor.2022.814975
 Nijholt, K. T., Meems, L. M. G., Ruifrok, W. P. T., Maass, A. H., Yurista, S. R., Pavez-Giani, M. G., Mahmoud, B., Wolters, A. H. G., van Veldhuisen, D. J., van Gilst, W. H., Silljé, H. H. W., de Boer, R. A., & Westenbrink, B. D. (2021). The erythropoietin receptor expressed in skeletal muscle is essential for mitochondrial biogenesis and physiological exercise. Pflügers Archiv - European Journal of Physiology, 473, 1301-1313. https://doi.org/10.1007/s00424-021-02577-4

Genetic Interactions Between BEN1‐ and Cytochrome P450‐Mediated Brassinosteroid Inactivation

AbstractBrassinosteroids (BRs) are essential plant growth‐promoting hormones involved in many processes throughout plant development, from seed germination to flowering time. Since BRs do not undergo long‐distance transport, cell‐ and tissue‐specific regulation of hormone levels involves both biosynthesis and inactivation. To date, ten BR‐inactivating enzymes, with at least five distinct biochemical activities, have been experimentally identified in the model plant Arabidopsis thaliana. Epigenetic interactions between T‐DNA insertion alleles and genetic linkage have hindered analysis of higher‐order null mutants in these genes. A previous study demonstrated that the bas1‐2 sob7‐1 ben1‐1 triple‐null mutant could not be characterized due to epigenetic interactions between the exonic T‐DNA insertions in bas1‐2 and sob7‐1, causing the intronic T‐DNA insertion of ben1‐1 to revert to a partial loss‐of‐function allele. We used CRISPR‐Cas9 genome editing to avoid this problem and generated the bas1‐2 sob7‐1 ben1‐3 triple‐null mutant. This triple‐null mutant resulted in an additive seedling long‐hypocotyl phenotype. We also uncovered a role for BEN1‐mediated BR‐inactivation in seedling cotyledon petiole elongation that was not observed in the single ben1‐2 null mutant but only in the absence of both BAS1 and SOB7. In addition, genetic analysis demonstrated that BEN1 does not contribute to the early‐flowering phenotype, which BAS1 and SOB7 redundantly regulate. Our results show that BAS1, BEN1, and SOB7 have overlapping and independent roles based on their differential spatiotemporal tissue expression patterns

Naming internal insertion alleles created using CRISPR in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is a powerful model organism, partly because of the ease of genome alterations due to the combination of a fast generation time and many molecular genetic tools. Recent advances in CRISPR-based systems allow for the easier creation of alleles with internally inserted sequences within the coding regions of genes, such as the internal insertion of sequences that code for epitopes or fluorescent proteins. Here we briefly summarize some exisiting nomenclature standards and suggest nomenclature guidelines for internal insertion alleles which are informative, consistent, and computable.

Spermatozoa in mice lacking the nucleoporin NUP210L show defects in head shape and motility but not in nuclear compaction or histone replacement.

Biallelic loss-of-function mutation of NUP210L, encoding a testis-specific nucleoporin, has been reported in an infertile man whose spermatozoa show uncondensed heads and histone retention. Mice with a homozygous transgene intronic insertion in Nup210l were infertile but spermatozoa had condensed heads. Expression from this insertion allele is undefined, however, and residual NUP210L production could underlie the milder phenotype. To resolve this issue, we have created Nup210lem1Mjmm , a null allele of Nup210l, in the mouse. Nup210lem1Mjmm homozygotes show uniform mild anomalies of sperm head morphology and decreased motility, but nuclear compaction and histone removal appear unaffected. Thus, our mouse model does not support that NUP210L loss alone blocks spermatid nuclear compaction. Re-analyzing the patient's exome data, we identified a rare, potentially pathogenic, heterozygous variant in nucleoporin gene NUP153 (p.Pro485Leu), and showed that, in mouse and human, NUP210L and NUP153 colocalize at the caudal nuclear pole in elongating spermatids and spermatozoa. Unexpectedly, in round spermatids, NUP210L and NUP153 localisation differs between mouse (nucleoplasm) and human (nuclear periphery). Our data suggest two explanations for the increased phenotypic severity associated with NUP210L loss in human compared to mouse: a genetic variant in human NUP153 (p.Pro485Leu), and inter-species divergence in nuclear pore function in round spermatids.