Candidate gene is a powerful approach to study gene-trait association and offers valuable information for genetic improvement using marker-assisted selection. The current work aimed to study the polymorphisms of four single nucleotide polymorphism (SNPs) at located growth hormone (GH), insulin-like growth factor-II (IGF-II), fat mass and obesity-associated (FTO), and insulin receptor substrate-1 (IRS-1) genes, and their association with the carcass, and meat quality traits in rabbits. The SNPs were genotyped using RFLP-PCR in New Zealand White and local Baladi rabbits. The results revealed that the heterozygous genotype was the most frequent in all cases, except for the FTO SNP in LB rabbits. There was a significant effect for GH genotypes on meat lightness after slaughter and hind-part weight. While, IGF-II mutation significantly affected slaughter, hot carcass, commercial carcass, and hind-part weights. The FTO SNP was associated with cooking loss and intramuscular fat weight, and the IRS-1 SNP was significantly associated with drip loss and intramuscular fat. Specific-breed effects were obtained for IGF-II SNP on cooking loss, and for the intramuscular fat. Although the results suggested that these mutations are useful candidate genes for selection, more research for detecting more variants associated with carcass and meat quality traits in rabbits are recommended. Oryctolagus cuniculus) are prolific animals that help fill the gap between the production and consumption of animal protein [Rabbits , which is located on chromosome 19 in rabbits, and comprises 4 introns and 5 exons, coding for a protein of 216 amino acids and 190 amino acids mature peptides [GH gene have shown significant associations with body weight in rabbits [GH polymorphisms with carcass or meat quality traits in rabbits [Like other species, growth hormone (GH) in rabbits is essential for growth and development. The GH hormone is the product of the peptides . The GH peptides . The pol rabbits , 19. How rabbits .IGF-II gene (NM_001171406) that is located on chromosome 4 in rabbits. It has a function similar to insulin and IGF-1 hormones, as it involves mediating growth and development, and promotes the growth of different tissue by binding to the IGFs receptor [IGF-II gene have been associated with different traits in different animals, such as body weights in rabbits [Insulin-like growth factor 2 (IGF-2) is the product of the receptor . Both IG rabbits , 22, bac rabbits , growth  rabbits , and mil rabbits .FTO gene (ENSOCUG00000001741) is located on chromosome 5 in rabbits and encodes the FTO protein. Loss of the FTO function leads to postnatal growth retardation with a significant reduction in adipose tissue, and lean body mass [FTO shared 36.36–91.88% identity with the FTO gene from other species, and is also closely related to humans [FTO gene in rabbits are scarce. Only one study investigated the mutations of the FTO gene in rabbits and reported a missense mutation  associated with body weight in New Zealand rabbits [FTO intron 1 SNPs may be associated with increased energy intake [The fat mass and obesity-associated (FTO) protein also play an essential role in postnatal growth. The ody mass . Rabbit o humans . Studies rabbits . Neverthy intake , 30.IRS-1) gene is another important gene that is associated with the previous ones [IRS-1 gene in rabbits is located on chromosome 7 (ENSOCUG00000007187) [IRS-1 gene and detected nine synonymous mutations, and only two SNPs (c.189G>T and c.2574G>A) were associated with growth traits. Further analysis for the c.189G>T mutation was performed [IRS-1 gene were also associated with growth in sheep [The insulin receptor substrate 1  . This ge0007187) . In rabb0007187)  sequencein sheep .GH (c.-78C>T), IGF-2 (c.156+61delA), FTO (c.499G> A), and IRS-1 (c.189G>T) genes with carcass and meat quality traits in two rabbit breeds.In recent years, the candidate-gene approach has proven effective in identifying several SNPs associated with meat rabbits –39. HoweAll procedures of the current study were approved from the institutional animal care and use committee at Cairo University (CU-IACUC), approval number is CU-IACUC/ I /F/46/22.ad libitum commercial pellets fattening diet , and water was provided ad libitum via an automatic nipple system.In the present study, data from 120 rabbits were analyzed: 71 (33 ♂♂ and 38 ♀♀) were New Zealand White (NZW) rabbits as a commercial line, and 49 (25 ♂♂ and 24 ♀♀) were improved local Baladi rabbits (LB). Rabbits were collected from different places and sources based on their age (5 weeks of age) and body weights . All rabbits were housed under the same conditions (summer season) during the fattening period in a semi-closed housing system, ambient temperatures ranged from 17 (low) to 33 (high) °C with a light period of about 16h/day, and relative humidity ranged from 40 to 60%. Rabbits were raised in grouping cages (60 × 50 × 40 cm) and received the same management practices, where they were fed At marketing age (12 weeks), rabbits were slaughtered after 12 hours of fasting with free access to water. Rabbits were anesthetized by intravenous injection of Ketamine (10 mg/kg) and Xylazine (3 mg/ kg), followed by exsanguination. Carcasses were then dissected according to that previously reported . The recLongissimus dorsi muscle carcass surface. Meat color was also determined two times (0 h and 24 h after slaughtering) using Chroma meter  at different locations of the Longissimus dorsi muscle, and expressed by lightness (L*), redness (a*), and yellowness (b*) color system. Cooking loss was calculated according to Pascual and Pla [Meat quality traits included measuring the pH of the muscles two times (0 h and 24 h after slaughtering) on the  and Pla .GH , IGF-2 , FTO (233 bp of exon 3), and IRS1 (CDs and 5’-untranslated region of exon 1) genes. The mutations were then genotyped using the restriction fragment length polymorphism (RFLP) technique as shown in GH [IGF2 [IRS-1 genes [FTO gene mutation, primers were designed using Primer Designing Tool of NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify a fragment of 233 bp of exon 3 that was previously sequenced [Individual blood samples were collected from rabbits before slaughter. DNA was extracted using WizPrep genomic DNA kit . Polymerase chain reaction (PCR) and amplifications of the different gene sequences were performed using a thermal cycler machine . The PCR reaction was adjusted to 20 μl, including 2 μl of DNA template, 1 μl of each of the forward and reverse primers, 8 μl of mater mix, and 8 μl of nuclease-free water. The PCR program included the initial denaturation step (94°C for 5 min), 35 cycles of amplification , and the final elongation step was set to 72°C for 5 min. The primers were designed to amplify fragments that included one mutation in each of the wn in GH , IGF2 [1GH [IGF2 , 22, andequenced .The association analysis between the different mutations and studied traits was performed using General Linear Model (GLM) procedure of SAS . The folBi), sex (Sj), litter size (Lk), and genotype (Gl). Hardy-Weinberg equilibrium of the different SNPs was tested using χ2. Differences with p-value less than 0.05 were considered statistically significant. The additive and dominance effects were calculated according to Russo et al. [The model included the fixed effects of breed , meat color after 24 hours of slaughtering , CL, IMF, DOP, and HPW traits.The descriptive statistics of the studied traits are presented in FTO gene in LB rabbits. For the GH gene, the allelic frequencies differed between the breeds, where allele T was the predominant allele in NZW rabbits. While in LB rabbits, allele C was the predominant. A similar situation was observed for IGF-2 mutation. However, the G and G alleles were predominant in the two breeds for the mutations of FTO and IRS-1 genes, respectively. GH mutation, 0.484 to 0.500 for IGF-2 mutation, 0.490 to 0.493 for FTO mutation, and 0.480 to 0.495 for IRS-1 mutation. The number of effective alleles was relatively high and ranged from 2.000 to 2.083 for all mutations in the two breeds, and the polymorphic information content ranged from 0.367 to 0.375. All frequency distributions were consistent with Hardy-Weinberg’s law.The allelic and genotypic frequencies of the different SNPs in the two rabbit breeds are presented in GH mutation (c.-78C>T) and different traits in the two breeds. The obtained results indicated significant associations for the genotypes with the meat lightness after slaughtering  and HPW (p < 0.001) in both breeds. The heterozygous genotype (TC) was superior to the homozygous genotype (CC) in the two breeds, for meat lightness after slaughtering. Regarding HPW, the trait was influenced by the mutation in the two breeds, where rabbits with TT genotype had significantly higher HPW than the two other genotypes. Also, there was a significant (p < 0.001) breed specific effect on yellowness after 24 hours of slaughtering (b*24) in LB rabbits, where the heterozygous genotype had significantly higher values of b*24 compared to the CC genotype, which was not observed in NZW rabbits. The results also denoted that the estimated dominant genetic effects of GH gene were higher than the additive genetic effects.IGF-2 gene and different traits in the two breeds are presented in The associations between the mutation of the FTO gene with carcass and meat quality traits in the two breeds. There was a significant effect of the SNP in the rabbits of the two breeds on CL (p < 0.0001), IMF (p < 0.011), and HPW (p < 0.001), where the AA genotype in both breeds had higher CL and IMF, and lower HPW than the two other genotypes (GG and AG). In NZW rabbits, the mutant genotype (GG) had significantly lower (p < 0.0002) DL than the other genotypes. In local Baladi rabbits, the AG genotype had a significantly higher meat lightness value after slaughtering  and b*0 (p < 0.0007) than the AA genotypes. As noted for the GH gene, the estimated dominant genetic effects were higher than the additive genetic effects.IRS-1 gene and different traits in the two breeds (IRS-1 mutation and DL in the two breeds (p < 0.008), where the GG genotype had higher DL than the TT genotype. While, in Baladi rabbits, there was a significant (p < 0.0005) reduction in IMF of GG rabbits compared to TT and GT rabbits. The results showed that the estimated dominant genetic effects were also higher than the additive genetic effects.Regarding the association between the mutation of the o breeds , there wAssociation analysis between traits and polymorphisms in animals is important because it can help to identify genetic variants that are associated with economic traits . This caThe evaluation of the studied traits revealed significant differences between the two breeds in four carcass traits, including slaughter weight (SW), hot carcass weight (HCW), commercial carcass weight (CCW), and drip loss (DL). The difference in slaughter weight may be attributed mainly to the difference in growth rates between the two breeds. The differences in finishing weight (12 weeks of age) between NZW and Egyptian Baladi rabbits were previously reported . These dGH gene was not greatly different between the NEZ and LB rabbits, the predominant allele was not the same in the two breeds, which implies the existence of breed effect for this locus. Ramadan et al. [IGF2 c.156+61delA mutation. The deletion mutation frequency on the same locus in Gabali rabbits was as high as 0.62 [FTO mutation was genotyped using re-sequencing technique in different rabbit breeds, and the frequency of the G allele was high in all breeds and ranged between 0.81 in NZW rabbits and 0.99 in Tianfu black rabbits [The genotypic frequencies obtained for all SNPs were consistent with HW equilibrium indicating that the two breeds used are under circumstances of random mating. Also, this may be because the rabbits used for the current work were gathered from different source, and all individuals were genetically unrelated. Although the allelic frequency of the c.-78C>T of the n et al.  reportedn et al. , however as 0.62  genotype rabbits . In the  rabbits . Helal eGH gene plays a very important role in postnatal growth and muscle deposition [IGF-2 gene is insulin-like growth factor-II (IGF-II), which belongs to the same family as IGF-I [IGF-II promoter-specific transcription [FTO gene is not only an obesity-susceptibility gene, it also affects growth, particularly postnatal growth [FTO gene and body mass index [IRS-1 gene is the IRS1 protein that has a role in the signal transudation of both GH and IGF-2 genes [In the current study, the four studied genes have important roles in controlling growth and muscle development. The position . It has as IGF-I . Furthercription . The FTOl growth . Moreovess index . Also, t-2 genes . TherefoGH gene were previously associated with carcass traits, the same SNP (c.-78C>T) genotyped in the current study was previously associated with slaughter weight in Belgian Giant Gray (BGG) rabbits, and was also associated with HCW, HPW, and meat in hind parts in BGG × NZW crossbred rabbits [GH gene in V-line and Alexandria-line rabbits, but they did not detect any mutations.The polymorphisms of the  rabbits . However rabbits  reported rabbits . In Egyp rabbits , and wit rabbits . El-Sabr rabbits  also genIGF-2 gene with carcass traits were likely to be similar to that of the GH gene SNP as expected, as the association was only observed in the case of HPW. Although there is a strong relationship between GH and IGF-2, as previously stated, there is a limited number of studies that explored the polymorphism of IGF2 in rabbits. No previous study addressed the association of IGF2 mutations with carcass or meat quality traits in rabbits. Fontanesi et al. [IGF2 gene polymorphisms were associated with carcass and meat quality traits in other animals such as rib-eye in cattle [The relationship between GH and IGFs is well documented, where GH had anti-insulin activities . For thii et al.  studied i et al.  reportedn cattle , marblinn cattle , inter-mn cattle , and carn cattle .FTO is another important gene that was not deeply subjected to association studies in rabbits. Results of the current study suggested the association between the SNP of the FTO gene with CL, HPW, IMF, and L*0. Zhang et al. [FTO gene is expressed in adipose tissues and the skeletal muscles, and has a critical function in regulating appetite and energy metabolism [FTO gene can be used as a candidate gene for IMF content in rabbits.The g et al.  genotypeg et al. . Intramug et al. . The FTOtabolism . The pattabolism . TherefoIRS-1 protein has important roles in signal transduction of both IGF2 and GH genes and has a contributory role in controlling body weight [IRS-1 gene is located between two tyrosine phosphorylation sites (Tyr46 and Tyr87) and was reported to affect the body weights at 70 and 84 days of age as well as average daily gain in rabbits [FTO and IRS-1 genes was studied [IRS-1 gene and some growth traits including body weight at 42 days of age in NZW, V-line, and Baladi Red rabbits were reported [IRS-1 gene polymorphism with carcass and meat quality traits in rabbits.The y weight . The gen rabbits . The ass studied  and it rreported . NeverthL*0, L*24, and IMF. However, there were associations with carcass traits including SW, HCW, CCW, CL, and HPW. The limited association results with meat quality may be attributed to many reasons such as the small variability at those loci in rabbits, and it may be attributed to the low heritability estimates for many meat quality traits compared with carcass and growth traits [The associations between the polymorphism of SNPs and phenotypic traits are essential for the genetic improvement of rabbit breeds and the development of new lines, which can be achieved using a marker-assisted selection approach. Such an approach is important particularly when the breeding program aims at improving traits that are difficult to be measured on live animals or traits with low levels of variation . Carcassh traits . Furtherh traits . OverallGH genotypes on meat lightness after slaughter and hind-part weight. While, the IGF2 mutation significantly affected slaughter, hot carcass, commercial carcass, and hind-part weights. The FTO SNP was associated with cooking loss and intramuscular fat weight, and the IRS-1 SNP was significantly associated with drip loss and intramuscular fat. Novel associations of FTO c.499G> A, and IRS-1 c.189G>T SNPs with intramuscular fat were detected and they have not been addressed before. Collectively, the results underlined the role of SNPs in candidate genes as sources of variability that can affect quantity and quality in rabbit meat production that are under polygenic control and highlighted the need for more research for validating the current findings and identifying more candidate/causative mutations.The results revealed a significant effect for S1 Data(XLSX)Click here for additional data file.


The Golgi apparatus of plants is the central cellular organelle for glycan processing and polysaccharide biosynthesis. These essential processes are catalyzed by a large number of Golgi-resident glycosyltransferases and glycosidases whose organization within the Golgi is still poorly understood.cis/medial Golgi enzyme N-acetylglucosaminyltransferase I (GNTI) in homomeric complex formation in the Golgi of Nicotiana benthamiana using biochemical approaches and confocal microscopy.Here, we examined the role of the stem region of the in planta complexes with GNTI, results in a similar block in N-glycan processing, while GNTI with altered subcellular localization or N-glycan processing enzymes located further downstream in the Golgi did not affect complex N-glycan processing. The GNTI-CTS-dependent alteration in N-glycan processing is caused by a specific nine-amino acid sequence motif in the stem that is required for efficient GNTI-GNTI interaction.Transient expression of the N-terminal cytoplasmic, transmembrane, and stem (CTS) regions of GNTI leads to a block in N-glycan processing on a co-expressed recombinant glycoprotein. Overexpression of the CTS region from Golgi α-mannosidase I, which can form Taken together, we have identified a conserved motif in the stem region of the key N-glycan processing enzyme GNTI. We propose that the identified sequence motif in the GNTI stem region acts as a dominant negative motif that can be used in transient glycoengineering approaches to produce recombinant glycoproteins with predominantly mannosidic N-glycans. The attached N-glycans subsequently undergo trimming and processing from mannosidic-type ones to complex-type ones. The complex N-glycans are generated in the Golgi apparatus, and distinct modifications on these N-glycans contribute to specific functions of the glycoproteins . N-acety5GlcNAc2 . The att5GlcNAc2 , N-acety5GlcNAc2 , or β1,25GlcNAc2   GNTI is mediated by its N-terminal CTS region in plants (in vivo heterodimerization with Golgi α-mannosidase I (MNS1) and possible other cis/medial Golgi enzymes is a common feature that is found in mammalian cells , a single transmembrane helix (T domain), a stem or spacer region (S region) (together referred to as the CTS region), and a large luminal catalytic domain . While dn plants . The tobn plants . The sinn plants , and then plants . While Gn plants , in vivoan cells  and in pan cells . Here, w5GlcNAc2 N-glycan on recombinant glycoproteins such as human IgG. To examine this effect in more detail and to avoid possible interference from the enzymatic activity of the attached catalytic domain, we fused the tobacco GNTI-CTS region (amino acids 1 to 77 – GNTI-CTS77) to RFP and transiently co-expressed GNTI-CTS77-RFP with an IgG antibody in N. benthamiana leaves. When expressed alone, IgG displayed almost exclusively the commonly found complex N-glycan GlcNAc2XylFucMan3GlcNAc2 (GnGnXF) in wild-type (2Man3GlcNAc2 (GnGn) in glycoengineered ΔXT/FT plants  gene expression. When GNTI-CTS77-RFP was co-expressed, the levels of complex N-glycans were drastically reduced in both wild-type and ΔXT/FT plants, while mannosidic N-glycans increased. The main peak was assigned to Man5GlcNAc2, indicating that overexpression of the GNTI-CTS77 region affects the processing of mannosidic N-glycans into complex ones. A similar N-glycan profile was obtained when the MNS1-CTS (amino acids 1-88) region was co-expressed with IgG  or XYLT   and N. benthamiana are highly conserved and differ only by two amino acid residues (Y4N in the cytoplasmic tail and Q59H in the stem region)   , chimeri(CAAATS) , and theN. benthamiana caused the accumulation of ACE2-Fc with exclusively EndoH-sensitive N-glycans  , human αand A1AT . To confN. benthamiana leaf epidermal cells   (AAATS-RFP (GNTI-CAAATS-RFP) (The truncated  and GFP . The CTSNTI-RFP) . The p31sted p31 . p31-GNTNTI-GFP) , p49-RRRNTI-GFP) , p46-NNNNTI-GFP) , p49-NNNNTI-GFP) , p31-NNRNTI-GFP) , p31-NNNNTI-GFP) , p20-MNSNTI-GFP) , p20-XYLNTI-GFP) , p31-MnINTI-GFP) , p31-GNTATS-RFP)  and pVKHATS-RFP)  were desATS-RFP) , human aATS-RFP) , pEAQ-RBATS-RFP) , pEAQ-ACATS-RFP) , and 2G1ATS-RFP)  were avaAgrobacterium tumefaciens (strain UIA143) (600 = 0.2) was used for transient expression in the leaves of 5-week-old N. benthamiana grown on soil under long-day conditions (16 h light/8 h dark) at 24°C. At the indicated time points, leaves were harvested from infiltrated plants, total soluble protein extracts were prepared, and they were subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining or immunoblotting as previously described in detail (All plant expression vectors were transformed into  UIA143) . Syringen detail . Fc-contn detail .N. benthamiana leaves with agrobacterial suspensions carrying the different expression plasmids . Two days after infiltration, the GFP-tagged proteins were purified using GFP-Trap-A beads (Proteintech), and the co-purified protein was analyzed by immunoblotting with anti-RFP antibodies. GNTI-RFP was co-expressed with GNTI-GFP stem deletion variants and analyzed in the same manner. GNTI-GFP was co-expressed with p31-CTS68 (GNTI-CTS68-RFP) and p31-NNN (GNTI-CTS77-RFP), respectively, and RFP-tagged proteins were purified using RFP-Trap-A beads (Proteintech) and subjected to immunoblotting.For the analysis of GNTI-GNTI protein interactions, GNTI-GFP was co-expressed with GNTI-RFP, 48-GNTI-RFP, and ST-GNTI-RFP, respectively, by infiltrating Plant-expressed IgG1 was purified from the protein extract by binding to rProtein A Sepharose™ Fast Flow (Cytiva) . For A1AN. benthamiana were infiltrated with agrobacterium suspensions carrying binary plant expression vectors for expression of GFP- or RFP-tagged proteins . Confocal images were acquired 2 days post-infiltration on a Leica SP5 II confocal microscope (http://www.leica.com). Dual-color image acquisition of cells expressing both GFP and RFP was performed simultaneously. Post-acquisition image processing was performed in Adobe Photoshop CS5.Leaves of 5-week-old croscope  using thSurface plasmon resonance (SPR) experiments were performed using a Biacore T200 (Cytiva). All assays were performed in HBS-EP running buffer (Cytiva) at 25°C. To determine the binding kinetics between the RBD-215 variants (MW = ∼24 kDa without glycans) and ACE2-Fc  , a CM5 cThe original contributions presented in the study are included in the article/JS: Conceptualization, Formal Analysis, Investigation, Writing – original draft. SI: Investigation, Formal Analysis, Writing – review & editing. CK: Investigation, Formal Analysis, Writing – review & editing. UV: Investigation, Formal Analysis, Writing – review & editing. JK-B: Investigation, Formal Analysis, Writing – review & editing. VR: Formal Analysis, Investigation, Writing – review & editing. CG-G: Investigation, Methodology, Formal Analysis, Writing – review & editing. AC: Conceptualization, Formal Analysis, Funding acquisition, Resources, Writing – original draft. RS: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Supervision, Writing – original draft.


Metabolomics has identified many metabolite associations with cardiovascular disease. In particular, the fat metabolism intermediates, long‐chain acyl carnitines (LCACs), have been associated with heart failure2+ release channel, the ryanodine receptor (RyR2).2+ sparks,2+ release (SCR) in HEK293 cells expressing RyR2.2+ accumulation, independently of Ca2+ released by RyR2.2+ level,2+ presence rather than a direct effect on RyR2.LCACs have been reported to affect many aspects of cardiac physiology.Properly addressing this mechanism requires research that accounts for factors that have long limited the translational impact of the field.To do this, we have exposed RyR2‐expressing HEK293 cells to LCACs 16:0 and 18:1 at concentration ranges spanning physiological (0.1 μM) and pathological (1.0 μM) levels,Compared to vehicle controls or the short‐chain acylcarnitine C3 (propionylcarnitine), LCACs 16:0 and 18:1 acutely increased SCR propensity and the average SCR frequency in our cell model Figure . This wa2+ fluorescence (‘peak area’)  Figure . Note th2+ passage from the extracellular environment, we assessed passive cytosolic Ca2+ influx in Fluo‐4‐AM loaded HEK293 cells not induced to express RyR2 prior to experimentation, which are unable to undergo SCR. Excessive LCAC 16:0 and 18:1 concentrations markedly increased passive Ca2+ influx (peak [Ca2+]c) in a fatty acyl chain length dependent manner, while the physiological and pathological concentrations did not  and perfused with 1 mM Zn2+. Consistent with the results found with Ca2+ entry, excessive, but not physiological or pathological, LCAC concentrations markedly increased Zn2+ influx proportionally to acyl chain length  and SCR termination (greater Ca2+ store depletion required to terminate a SCR event)  Figure . Given tn Figure , these d2+ release by RyR2.Collectively, the outcomes of this brief communication have implications for methodology of future research of LCACs or other amphipathic metabolites. Moreover, as a by‐product of our using it as our example system, we have also gained insight into the relationship between LCACs and pro‐arrhythmic Ca2+ homeostasis,2+/calmodulin‐dependent kinase II, which has been linked to RyR2 gating previously.First, regarding the latter, we have learned that LCACs promote RyR2‐mediated SCR at concentrations that do not overtly affect membrane integrity. Mechanistically, this raises the question of how LCACs alter RyR2 gating. Given that LCACs alter cytosolic CaSecond, and pertaining to the rationale for this study, we have found that excessive LCAC concentrations commonly used in the field do increase non‐specific intracellular ion influx in a manner dependent on the LCAC's amphipathic structure. This is suggestive of cell membrane disruption that could mask other physiologically relevant effects in the cell. Because of this, future research must consider the concentration of LCAC used to characterize effects in the heart or, alternatively, provide evidence that membrane integrity is maintained.By accounting for these important aspects of LCAC methodology, the growing body of contemporary LCAC research can better identify cellular processes affected by LCAC dysregulation in cardiovascular disease. Without doing so, efforts to determine the translatability of targeting LCAC dysregulation for new therapies will lag behind the risk and prognosis associations drawn from clinical metabolomic studies.Hamish M Aitken‐Buck: Conceptualization (lead); data curation ; formal analysis (lead); funding acquisition ; investigation (lead); methodology ; project administration ; visualization (lead); writing – original draft (lead); writing – review and editing (lead). Ei Phyo Khaing: Data curation ; formal analysis (supporting); investigation (supporting); writing – original draft (supporting); writing – review and editing (supporting). Regis R Lamberts: Conceptualization ; data curation (supporting); methodology ; project administration ; supervision (supporting); writing – original draft ; writing – review and editing . Peter P Jones: Conceptualization ; data curation ; funding acquisition ; methodology ; project administration ; resources ; software ; supervision ; writing – original draft ; writing – review and editing .The authors declare that they have no conflicts of interest to disclose.


Post-transcriptional methylation modifications, such as the N7-methylguanosine (m7G) modification, are increasingly acknowledged for their role in the development and resistance to chemotherapy in acute myeloid leukemia (AML). This study employed MeRIP-seq technology to investigate the m7G sites within circular RNAs (circRNAs) derived from human AML cells and drug-resistant AML cells, in order to identify these sites more comprehensively. In addition, a detailed analysis of the relationship between m7G and drug-resistant AML was conducted. The bioinformatics analysis was utilized to predict the functions of specific methylated transcripts. The findings revealed a significant difference in m7G level between AML cells and drug-resistant AML cells, suggesting a potentially critical role of m7G in circRNAs in drug-resistant AML development. The methylation of M7G could affect the circRNA-miRNA-mRNA co-expression during the development of AML resistance, which could further influence the regulation of resistance-associated target genes in AML. Furthermore, gene ontology analysis indicated that the distinct distribution pattern of circRNAs with m7G methylation in drug-resistant AML cells was correlated with metabolism-related pathways. These results suggested a potential association between drug-resistant AML and m7G methylation of circRNAs. Moreover, the results revealed a novel role of m7G RNA methylation in circRNAs in the progression of AML chemoresistance. The treatment of AML has traditionally relied on strong chemotherapy drugs, killing cancer cells. However, the prognosis of AML treatment fluctuates depending on the molecular biology expressed by the patient2. Therefore, understanding the molecular and phenotypic characteristics of AML plays a crucial role in its initial presentation and requires a thorough understanding of its underlying molecular mechanisms. This knowledge is essential for identifying novel prognostic biomarkers and identifying therapeutic targets, thereby preventing disease recurrence and improving long-term outcomes.Acute myeloid leukemia (AML) is a hematological tumor with a relatively poor prognosis3. Abnormal activation of drug-resistant signaling pathways caused by genetic alterations can lead to drug resistance of AML4. Initially believed to be a byproduct of faulty alternative splicing, circRNAs, also known as non-coding RNAs (ncRNAs), have emerged as a distinct class6. Due to significant advancements in high-throughput sequencing methods, it has been found that circRNAs play a vital role in various types of cancer, including AML. For instance, circRNAs have emerged as valuable biomarkers for the diagnosis and prognosis of AML. Previous studies have demonstrated that circRNAs can exert their influence on cancer progression by regulating translational, post-transcriptional, and transcriptional gene expressions8. Notably, a recent study revealed that a subset of circRNAs, previously deemed untranslatable, can undergo translation following RNA methylation9. Nevertheless, our understanding of the extent of RNA methylation, including circRNA methylation, in resistant cells of AML is limited. To address this knowledge gap, scholars have explored the involvement of ncRNAs, particularly circRNAs, in drug resistance of AML, facilitating the development of innovative therapeutic approaches to overcome this challenge5.Primary or secondary resistance, including both intrinsic and acquired resistance, plays a crucial role in limiting the prognosis of patients with AMLIn the present study, a comprehensive analysis was performed on N7-methylguanosine (m7G) modifications of circRNAs in drug-resistant AML cells. The findings indicated distinct differences in the methylation levels and patterns between drug-resistant AML cells and AML cells. Specifically, the methylation levels in drug-resistant AML cells were significantly higher compared to those in AML cells. Moreover, the differentially methylated genes were derived from various chromosomes, highlighting the widespread impact of these modifications. The results strongly indicated a potential association between AML drug-resistance and m7G modifications of circRNAs.2 and high humidity (15).Cells were provided by the American Type Culture Collection (ATCC). Cell culture was performed using an RPMI-1640 medium supplemented with thermally inactivated fetal bovine serum (10%) and antibiotics (1% penicillin–streptomycin in v/v). The cultures were maintained at a temperature of 37 °C in an atmosphere consisting of 5% CO4 cells/mL) were cultured in 96-well plates and treated with 200 μL of mitoxantrone solutions (range 0.01–2.4 μg/mL) for 24 h. After incubation, 10 μL of CCK-8 reagent was added to each well and the plates were further incubated at ambient temperature with 5% CO2 for 120 min. The absorbance at 450 nm was then measured to determine the inhibitory ratio and half-maximal inhibitory concentration (IC50) for each cell line based on their respective concentration/response curves.The cell sensitivity to mitoxantrone was assessed using Cell Count Kit-8 (CCK-8) reagent. HL60 and HL60/MX2 cells  on an Illumina NovaSeq 6000 sequencer.Following sequencing, a collection of two-end reads was obtained. To ensure data quality, Cutadapt was employed with a Q30 threshold to remove low-quality reads, retaining high-quality ones. In the investigation of circRNAs within cloud sequence organisms, a comprehensive analysis was conducted with a special concentration on the potential role of m7G. Notably, m7G is a modified form of guanosine, which is characterized by a methyl group at the 7th position. It is commonly present in the 5′ cap structure of RNA molecules and plays a crucial role in various aspects of RNA metabolism, stability, and translation initiation. The presence of m7G modification can influence the interactions of circRNAs with RNA-binding proteins and microRNAs, potentially modulating their functions. The STAR software (ver. 2.5.1b) facilitated the alignment of these high-quality reads, encompassing m7G modification, to the reference transcriptome/genome. Concurrently, the identification and detection of circRNAs were carried out using DCC software (ver. 0.4.4). Leveraging the Circ2Traits and circBase databases, the identified circRNAs, including those possibly bearing m7G-modified bases, underwent annotation. Subsequent steps involved data normalization and differential expression analysis of circRNAs using edgeR software (ver. 3.16.5). The gene set derived from differentially expressed circRNAs was subjected to GO and KEGG pathway enrichment analyses, shedding light on potential functional pathways influenced by m7G-modified circRNAs. This comprehensive analysis provides valuable insights into the intricate roles of m7G modification in the context of circRNA regulation.−ΔΔCT method. The primers that were used in RT-qPCR were summarized as follows: METTL1 forward (F): 5ʹ-AGCTACACGACTGGATGTGC-3ʹ and reverse (R): 5ʹ-TGAGGTGCCTAGATGTCCCA-3ʹ, WDR4 F: 5ʹ-AGAGTTTGTGAGCCGTATCTC-3ʹ and R: 5ʹ-GAAGATGTAGACCACAGGAGTG-3ʹ, RNMT F: 5ʹ-TGGCTGCAAATATGACTTCAAC-3ʹ and R: 5ʹ-CCTGCATTCGTTTTAAGAGCAT-3ʹ, RAM F: 5ʹ-GTCGTATCCAGTGCGTGTCGTG-3ʹ and R: 5ʹ-CACTGATTGGATCGATTGTCAC-3ʹ, WBSCR22 F: 5ʹ-AGCTGTTTTATGACGAGACAGA-3ʹ and R: 5ʹ-GGCAGATAAAGAAGCTCCAATG-3ʹ, ACTB F: 5ʹ-GTGGATCAGCAAGCAGGAGT-3ʹ and R: 5ʹ-AAAGCCATGCCAATCTCATC-3ʹ.Total RNAs were extracted from the cells using TRIzol reagent . The extracted RNAs were then used to generate cDNAs using SuperScriptTM III Reverse Transcriptase . For RT-qPCR analysis, the cDNAs were subjected to qPCR using the qPCR SYBR Green master mix (GenSeq) in the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). In different cases, ACTB was utilized as the internal control. Data analysis was performed using the 2The methylome analysis incorporated an in-depth approach to understand the epigenetic landscape. High-quality reads obtained from the sequencing data were meticulously mapped to the human genome (human gencode v32) utilizing the STAR software (ver. 2.5.1b). This process not only facilitated the precise alignment of the reads, but also allowed for the exploration of potential methylation patterns across the genome. Detection and identification of circRNAs, crucial players in the epigenetic landscape, were carried out using the DCC software (ver. 0.4.4). The subsequent stages of analysis involved comprehensive annotation of the identified circRNAs. This annotation process drew from the rich resources of the Circ2Traits and circBase databases. It allowed for a more nuanced understanding of the potential roles of the circRNAs and their interactions with other genetic elements. Following annotation, data standardization was conducted, and differential expression of circRNAs screening was performed using the edgeR software (ver. 3.16.5). This analysis aimed to not only quantify the abundance of circRNAs, but also to ascertain the differences in their expression levels in various conditions. To provide visual insights into the differential expressions of methylated genes, a scatter plot was generated based on the average logCPM (counts per million) of the two groups. This plot served as a graphical representation of the methylation-based differences in gene expression.P values were carefully evaluated, and those less than 0.05 were indicative of statistically significant findings.Statistical analysis was robustly conducted using GraphPad Prism  and SPSS  software. The two groups were rigorously compared using Student’s t-test, which is a widely recognized method for assessing the significance of differences between two groups. The resulting The miRNA binding sites and target mRNAs were visualized using miRanda (ver. 3.3a) and Target Scan (ver. 8.0) software. By comparing the m7G fold enrichment between HL60 and HL60/MX2 cells, a correlation map was generated to investigate the relationship between M7G status and expression. Cytoscape software was utilized to construct a CMM network, consisting of 2 downregulated hypo-methylated circRNAs and 5 upregulated hypermethylated circRNAs.HL-60/MX2, a multidrug resistant derivative of HL-60, exhibits resistance to various drugs, including mitoxantrone. In this study, both drug-naive HL60 and drug-resistant HL60/MX2 cells were exposed to different concentrations of mitoxantrone for 24 h. The IC 50 values were then determined. It was revealed that the IC 50 values of HL60 and HL60/MX2 in response to mitoxantrone were 0.15 ± 0.05 and 3.02 ± 0.64 μg/mL, respectively  in both cell lines . Moreover, HL60/MX2 cells demonstrated a higher abundance of circRNAs with at least two methylation peaks versus HL60 cells (P < 0.0001).Statistical analysis was conducted on methylation peaks and corresponding circRNAs in order to quantify m7G peaks on each circRNA Fig. C. It wasThe data on methylated circRNAs were analyzed and summarized, and the findings were presented in the form of a pie chart Fig. A,B. The Furthermore, the utilization of Circos enabled visualization of the methylation sites of circRNAs across the chromosomes Fig. C. The reThe Dreme was used to scan the sequence of the methylated peak (fifty bp on each side of the apex) in each sample group, and the relevant motif sequences were identified and presented in Fig. Cluster analysis and methylation heatmap analysis were conducted on the total data Fig. C. The reTo clarify the effects of methylation on transcriptional expression, combined analysis of methylation and transcriptome was conducted Fig. . Table 2Using the data obtained from sequencing, GO analysis was performed to investigate the molecular functions of differentially methylated circRNAs and biological processes in two different cell types. In terms of biological processes (BP), the genes corresponding to up-methylated m7G sites in HL60/MX2 cells were primarily involved in nitrogen compound metabolic processes, cellular metabolic processes, and primary metabolic processes. On the other hand, the genes corresponding to down-methylated m7G sites were primarily involved in cellular component organization or biogenesis, cellular macromolecule metabolic processes, and metabolic processes. When considering cellular components (CC), the upregulated methylation sites in HL60/MX2 were enriched in membrane-bounded organelles, intracellular membrane-bounded organelles, and intracellular organelles, similar to the pattern observed in HL60 cells. In terms of molecular functions (MF), it was found that genes with hypermethylated m7G were primarily associated with heterocyclic compound binding, organic cyclic compound binding, and protein binding. Conversely, genes with downmethylated m7G were primarily associated with heterocyclic compound binding, organic cyclic compound binding, and protein binding. The top 10 prominent categories are depicted in Fig. The KEGG pathway analysis was employed to identify pathways potentially associated with genes exhibiting differential methylation Fig. A,B. The In order to further assess the effects of methyl transferase on resistant AML cells, RT-qPCR assay was employed for detecting the mRNA levels of methyl transferase in HL60/MX2 and HL60 cell lines. Compared with HL60 cells, the mRNA levels of METTL1, WDR4, RNMT, RAM, and WBSCR22 were significantly reduced in HL60/MX2 cells Fig. .Figure 8To construct the CMM network, seven circRNAs that exhibited differential expression of m7G methylated RNAs were identified. Among these circRNAs, five were found to be hyper-upregulated and two were hypo-downregulated. The specific circRNAs are summarized in Table 3. A key contributor to drug resistance in cancer is m7G, which is recognized as a specific type of methylation pathway10. Notably, the quantity of m7G sites in drug-resistant AML cells is significantly greater than that in AML cells, and its down-methylated m7G modification is specifically enriched in ABC transporter-related mRNAs, which are known to contribute to multidrug resistance11. Despite m7G’s crucial role in cells, the presence and function of its circRNAs in AML cells with drug resistance have not been explored, leaving a gap in understanding of the mechanism underlying AML drug resistance.Drug-resistant AML cells display distinct cytogenetic and molecular characteristics, mainly resulting from the deletion or activation of multiple pathways associated with drug resistanceIn the present study, MeRIP-seq was employed to sequence the m7G methylation of circRNAs in HL60/MX2 and HL60 cells, comparing the results between the two groups. The findings revealed the identification of various methylation peaks, with a significant disparity in both the quantity and distribution of these peaks. Notably, it was found that the genes and methylation frequency in HL60/MX2 cells were substantially higher than those in HL60 cells, indicating a clear association between m7G and resistant AML. Furthermore, the analysis at the whole chromosome level demonstrated significant discrepancies in circRNAs from different chromosomes in both cell types, particularly the X chromosome. This suggested extensive alterations in m7G methylation in HL60/MX2 cells, potentially involving multiple pathways that could influence drug resistance of AML cells. Moreover, the results of motif analysis revealed that the sequences close to the methylation sites were consistent and relatively conserved in both groups, demonstrating that the types of methylation were relatively constant, whereas their quantities varied.12. Moreover, ZRANB1, a downregulated circRNA, has been found to be closely associated with cancer. Suppression of expression level of ZRANB1 has been found to inhibit the growth and metastasis of hepatocellular carcinoma both in vitro and in vivo. Higher expression level of ZRANB1 was associated with poorer survival13. Thus, we hypothesized that these altered circRNAs, influenced by changes in m7G methylation levels, might play a role in chemoresistance in AML.In the present study, it was revealed that PHF14, among the upregulated circRNAs, was associated with the development and progression of various malignancies. Knockdown of PHF14 in colorectal cancer cells may potentially inhibit the carcinogenic process in vivo15. Analysis of bone marrow samples from patients diagnosed with refractory and recurrent AML revealed the upregulated expression level of circPAN3. It was found that downregulation of circPAN3 could decrease the expression level of X-linked inhibitor of apoptosis protein (XIAP). However, this effect was counteracted by specific inhibitors of miR-183-5p or miR-153-3p, suggesting that circPAN3 could be a crucial mediator of chemoresistance in AML cells16. In addition, silencing of circMYBL2 was found to enhance differentiation and suppress proliferation of FLT3-ITD AML cells both in vitro and in vivo. CircMYBL2 was noted to increase the translational efficiency of FLT3 kinase by facilitating the binding of polypyrimidine tract-binding protein 1 to FLT3 messenger RNA17. Furthermore, the present study demonstrated that only a small number of methylated circRNAs in AML drug-resistant cell lines were derived from exons. Therefore, it can be concluded that methylation-induced decrease of exon-derived circRNAs leads to suppression of important proteins, ultimately contributing to the development of drug resistance in AML.Previous studies have demonstrated that chromosomal translocation in cancer could lead to the production of abnormal fused circRNAs, which were strongly associated with drug resistance18. Another gene, ZCCHC2, exhibited differential expression in a contradictory manner (up-down regulation). Previous studies have linked ZCCHC2 to immunity and resistance in lymphoma, acute lymphoblastic leukemia, AML, acute promyelocytic leukemia (APL), and chronic lymphocytic leukemia (CLL)20. ZCCHC2 has exhibited to inhibit retinoblastoma tumorigenesis by suppressing HectH9-mediated K63-linked polyubiquitination and activating c-Myc. Knockout of ZCCHC2, on the other hand, promoted the proliferation of retinoblastoma cells21. It was reported that the upregulated gene CCDC7 was intimately associated with the development of cervical cancer. Its expression was found to promote proliferation of cervical cancer cells through the JAK-STAT3 pathway22. In the present study, a CMM network was established by integrating expression profiles of matched mRNAs, miRNAs, and circRNAs, demonstrating a positive correlation between mRNA and circRNAs expressions. Additionally, M7G was identified as a mediator of circRNA expression through post-transcriptional modification. By influencing the CMM co-expression networks, it was noted that m7G could play a role in AML resistance, providing a potential mechanism for circRNA activity in AML resistance. Therefore, these circRNAs with differential methylation and expression patterns warrant further investigation to elucidate their molecular function in the emergence and development of drug resistance in AML.Additionally, a correlation was identified between the degree of methylation and gene expression in drug-resistant AML cell lines. In the present study, significant genes were identified that exhibited differential expression in both m7G and the RNA itself. One such gene, FATI, was found to be overexpressed in oxaliplatin-resistant breast cancer tissues and cells. Knockdown of circRNA FATI led to the enhanced apoptosis, decreased migration, and reduced invasion in oxaliplatin-resistant breast cancer cells. This effect was mediated through activation of the Notch and Wnt signaling pathwaysNotably, the results of the present study suggested a correlation among m7G methylations, circRNAs, genes, and their expression with chemoresistance in AML. The expression profiles of circRNAs in two cell lines (HL60 and HL60/MX2) were investigated, and 2045 circRNAs were found to be differentially expressed in both cell lines. Further analysis revealed that the majority of circRNAs in both cell lines had one methylation peak, while the quantity of circRNAs with at least two methylation peaks was higher in HL60/MX2 cells. The source data showed that most of methylated circRNAs were derived from sense overlapping and exonic regions, with a higher quantity of circRNAs derived from exons in HL60/MX2 cells. Motif analysis identified conserved motifs in the methylated peaks in both cell lines. Cluster analysis and methylation heatmap showed differences in methylation degree between HL60/MX2 and HL60 cells, with a higher methylation frequency in HL60/MX2 cells. Methylation was found to affect the transcriptional expression levels of circRNAs, with seven differentially methylated circRNAs showing differential expression. The GO and KEGG pathway enrichment analyses revealed the molecular functions, biological processes, and pathways associated with the differentially methylated circRNAs. Furthermore, mRNA levels of methyl transferase were reduced in HL60/MX2 cells compared with those in HL60 cells. Finally, a co-expression network of circRNAs, miRNAs, and mRNAs involved in AML resistance was constructed, providing insights into the underlying mechanisms of circRNAs in AML chemoresistance. The observed correlation points towards the significance of m7G methylations and circRNAs in influencing gene expression patterns associated with chemoresistance in AML. The altered expression and methylation patterns of circRNAs could contribute to the reduced effectiveness of chemotherapy in AML cells. Overall, the results suggested a complex interplay between m7G methylations, circRNAs, gene expression, and chemoresistance in AML. The differential expression and methylation patterns of circRNAs could potentially be linked to altered gene expressions and cellular responses, contributing to chemoresistance in AML cells. Further research is essential to fully understand the mechanisms underlying these correlations and their implications for AML treatment.24. In the present study, it was discovered that proteolysis mediated by ubiquitin could play a critical role in the resistant process. The ubiquitin–proteasome system (UPS) contributes to regulate the abundance and activity of a wide range of proteins. It also plays a key role in tightly controlling cell cycles, gene expression, cell survival, cell proliferation, and apoptosis25. Therefore, bortezomib, a proteasome inhibitor, was approved by the FDA in 2003 for the treatment of multiple myeloma26. As ubiquitination has been implicated in oncogenesis, researchers have attempted to explore its role in cancer chemoresistance by investigating the underlying mechanisms. Previous studies have demonstrated that the ubiquitination process is involved in chemoresistance27. Blocking the UPS can have several effects, which are summarized as follows: (1) reducing the levels of anti-apoptotic proteins  and nuclear factor-κB (NF-κB)), (2) increasing the expression level of the pro-apoptotic protein Noxa, (3) activating the caspases-mediated apoptotic pathway, (4) degrading pro-survival proteins and inducing myeloid leukemia cell differentiation protein, and (5) inhibiting drug efflux transporters. In the present study, downregulation of m7G methylation expression was found in ubiquitylation-associated genes , demonstrating that m7G methylation in ubiquitylation plays a crucial role in AML resistance.After conducting further bioinformatics analysis and examining the results of GO and KEGG pathway enrichment analyses, differentially methylated genes were identified in the two groups. In particular, it was found that methylated genes in drug-resistant AML cell lines were associated with various cell functions, such as endocytosis, protein metabolism, and energy. This suggests that methylation has a widespread impact on cell function and underscores the need for comprehensive investigation into its underlying mechanisms29. Although studies on m7G are still in their preliminary stages, they have provided sufficient evidence regarding its critical role in tumor development. The role of m7G methyltransferase is to introduce m7G modifications in specific positions of target RNA molecules, thereby impacting the production, structure, and maturation of RNA molecules, including mRNAs, miRNAs, and rRNAs. Ultimately, this process regulates the translation process28. In AML patients, there is a significant increase in the expression levels of the m7G regulators (METTL1 and WDR4), both at mRNA and protein levels30. Besides, stable knockdown of METTL1 could effectively inhibit the growth of leukemia stem cells30. Further observations revealed that METTL1-knockout mice had a lower tumor burden and a longer survival than wild-type controls. The mechanism underlying these effects involves the METTL1/WDR4 complex, which enhances the abundance of tRNAs through m7G modification, particularly affecting Arg-TCT-4-1. This modification influences the translation of AGA codon-rich mRNAs by reducing ribosomal pausing efficacy. Thus, the METTL1/WDR4-dominated M7G-modified tRNA drives oncogenic transformation via remodeling mRNA translational activity. This ultimately leads to an amplification in the expression levels of cell-cycle progression genes, making it a potential therapeutic target for AML30. Consequently, further research is necessary to fully comprehend the role of m7G in AML.According to previous research, m7G has been implicated in various cell functions, such as stress response, stem cell function, and cell differentiationIn conclusion, the present study clarified the differences in distribution and quantity of m7G between drug-resistant AML cell lines and AML cell lines. The bioinformatics analysis was employed to reveal the distribution and potential function of m7G in drug-resistant AML. Furthermore, the findings demonstrated that m7G methylation could influence the co-expression of CMM in the drug-resistant process of AML, as well as regulating target genes associated with resistance in AML. However, understanding of m7G’s function in cells is still in its early stages. Hence, further research is required to fully explore the role and mechanism of m7G and to identify new therapeutic targets for drug-resistant AML.Supplementary Table S1.


Our work suggests that, while there may be an evolved predisposition to use language as a signal of group identity, our developmental plasticity allows us to respond adaptively to social information around us, leading to psychological and behavioural variation within and across populations. How people reason about others based on their linguistic profiles will affect the payoffs of acquiring different languages and ultimately the long-term sustainability of linguistic diversity.Most human variation is structured around symbolically marked cultural (‘ethnic’) groups that require common codes of communication. Consequently, many have hypothesised that using others’ linguistic competences as markers of their descent is part of an evolved human psychology. However, there is also evidence that the use of language as ethnic markers is not universally applied, but context specific. We explore the tension between these views by studying responses to bilingualism among 121 adults living in Mayan communities undergoing rapid socioeconomic changes involving increased contact with Spanish-speaking towns. We show that, although competences in Mayan were strongly tied to perceiving others as having a Mayan ethnic identity, ethnolinguistic category membership was not seen as stable through life, vertically transmitted, nor regarded as incompatible with competences in Spanish. Moreover, we find variation in how people reasoned about ethnolinguistic identities depending on their  Media summary: Evolutionary approach to language use shows how Mayan identity is both maintained and altered by bilingualism following market integration.Humans are the only species whose social organisation is structured around symbolically marked cultural (ethnic) groups, where members tend to share norms, expectations, skills and beliefs  groups, one hypothesis is that humans have evolved to use linguistic features as predictive of ethnic category membership Moya, . KinzlerHowever, many societies worldwide are undergoing rapid socioeconomic changes that involve exposure to foreign languages, and so the greater occurrence of bi- and multilingualism. While these opportunities have probably always occurred, the scale of socioeconomic change is now much greater . The fieldwork investigated whether linguistic competences are regarded by the Mayan communities as a source of useful social information, concretely about cultural or ethnic identity and/or relatedness. Key research questions are whether different language competences, such as degree of bilingualism, influence perceptions of identity; how these are inherited; how stable they are through life; and how these vary with individual attributes such as sex, age or status. The focus is on whether, and if so, how, people can flexibly adapt their way of reasoning about ethnolinguistic identities to rapidly changing social landscapes, and how inter-individual differences in language competences are themselves a source of intra-population variation in reasoning about linguistic identity.ejido system, set up after the Mexican Revolution, establishes that ejido lands cannot be owned, inherited or sold and that their dominion resides within the village collective, which distributes them among married males .The sample of participants comprised 121 adults . All were fluent Yucatec Mayan speakers. Their Spanish competences are summarised in We fitted Bayesian mixed models with a cumulative link function in order to examine how a Mayan or Mayera identity was related to language acquisition and language competences in Yucatec Mayan and Spanish (see ESM5 for a justification of the use of Bayesian methods).Since participants responses were given in the form of ratings on a 10-point scale, instead of treating ordered categorical outcomes as continuous predictors, this type of model does not assume that the distance between two values is necessarily the same: it may take more ‘effort’ to move from a Maya rating of 9 to one of 10 than from one of 4 to 5 . These included a null model (intercept only), and a control model including the age of the interviewee and whether they were assigned to the ‘Mayera’ condition or not . In addition, we also built a model including whether the interviewee spoke Spanish or not, and a last one with an interaction between Spanish level and ‘Scenario’ (i.e. scenarios αVILLAGE) were included in all models to account for the nested structure of the data and associated clustering  how participants perceived they had themselves acquired competences in each of the languages; and (b) the subjective value they placed on being fluent on each of them.Since all but one of the participants were native Yucatec Mayan speakers, they all stated that they had learnt to speak Mayan either from their parents at home or in the village.In line with most participants’ belief that their competences in Spanish had been mainly acquired in school (Table S3), the gendered difference in Spanish competence can be attributed to men's disproportionate access to education: the mean number of years spent in education were 6.14 (SD = 3.74) and 8.33 (SD = 4.01) for females and males respectively.All of the respondents believed competences in both languages to be equally valuable. The reasons why interviewees ascribed importance to each of the languages are summarised in Table S4.Our aim here was twofold: first, we wanted to evaluate the extent to which people perceived linguistic differences to denote differences in ethnic identities, and how they reasoned about their inheritance, stability through life and mutual exclusivity with other such identities. That would allow us to determine whether people were using others’ linguistic competences as cues of ethnic category membership and/or relatedness. Second, we wanted to assess whether individual attributes, in particular one's own linguistic repertoire was a source of intra-population variation in reasoning about ethnolinguistic phenomena. To facilitate interpretation of the results reported in the text below and Ethnic category membership was not perceived as being vertically inherited, genetically or otherwise . InsteadRegarding our second aim, the model with the interaction between speaking Spanish and Scenario significantly outperformed all other models . This memore Mayan someone who was bilingual as opposed to monolingual in Mayan  transmitted nor incompatible with acquiring competences in Spanish.These data show that, although competences in Yucatec Mayan were strongly tied to perceiving others as having a Mayan ethnic identity, ethnolinguistic category membership was evolved to inevitably use linguistic boundaries as reliable signals of biological descent, because they are hard-to-fake, relatively stable through life or mutually exclusive with one another   of the women in the sample mentioned that they required the assistance of bilinguals to accompany them whenever they needed to go to the doctor in order to help them communicate. This, in turn, may make them be seen as deserving of such a high rating in their Mayan identity. This is also supported by the fact that all respondents valued bilingual acquisition .The flexible nature of human cognitive systems was also illustrated by intra-population variation in the way people reasoned about ethnolinguistic identities according to their Mayan monolinguals also appeared to essentialise ethnolinguistic identity with respect to its stability through life as they gave higher ratings to those who had forgotten how to speak Mayan compared with those who had never learnt it at all . SimilarLast, understanding the power of socioeconomic changes to redefine the relationship between ethnic and linguistic identity is also particularly important in order to assess the prospect of linguistic diversity persisting in the future (Pietikäinen, In conclusion, we found evidence that speaking Mayan was not regarded as a proxy of biological relatedness but was regarded as a badge of membership to the cultural group – that is, as marker of a Mayan ethnic identity. This was not incompatible with acquiring competences in Spanish, which was instead simply seen as a functional tool to access the new opportunities offered by increased connectedness with urban centres, including wage labour, healthcare and education. By highlighting the plasticity of the psychological underpinnings of our way of reasoning about linguistic category membership, our study suggests that when different languages serve different yet adaptive social functions and encompass different cultural norm clusters, individuals may reason about them differently. In turn, this means that we should expect the way people to reason about what linguistic affiliation means to be plastic and context specific even if built on an evolved universal human psychology . Such pl


We have recently shown that NaV1.8 contributes to arrhythmogenesis by inducing a INaL. Genome-wide association studies indicate that mutations in the SCN10A gene (NaV1.8) are associated with increased risk for arrhythmias, Brugada syndrome, and sudden cardiac death. However, the mediation of these NaV1.8-related effects, whether through cardiac ganglia or cardiomyocytes, is still a subject of controversial discussion. We used CRISPR/Cas9 technology to generate homozygous atrial SCN10A-KO-iPSC-CMs. Ruptured-patch whole-cell patch-clamp was used to measure the INaL and action potential duration. Ca2+ measurements (Fluo 4-AM) were performed to analyze proarrhythmogenic diastolic SR Ca2+ leak. The INaL was significantly reduced in atrial SCN10A KO CMs as well as after specific pharmacological inhibition of NaV1.8. No effects on atrial APD90 were detected in any groups. Both SCN10A KO and specific blockers of NaV1.8 led to decreased Ca2+ spark frequency and a significant reduction of arrhythmogenic Ca2+ waves. Our experiments demonstrate that NaV1.8 contributes to INaL formation in human atrial CMs and that NaV1.8 inhibition modulates proarrhythmogenic triggers in human atrial CMs and therefore NaV1.8 could be a new target for antiarrhythmic strategies. In heart failure and atrial fibrillation, a persistent Na V) trigger the fast upstroke of the action potential (AP), making them important for the physiological conduction of electrical impulses in the heart. Under physiological conditions, NaV channels (predominantly NaV1.5) quickly become inactive after activation. However, in some cardiac pathologies such as ischemia and heart failure (HF), NaV channels were described to remain persistently open or reopen, thus creating the late sodium current (INaL) as a persistent inward current  levels and an increased occurrence of Ca2+ sparks in the human atrium [NaL through specific targeting of NaV1.8 has the capability to reduce the reverse mode NCX, thereby also mitigating diastolic proarrhythmogenic SR-Ca2+ leak [NaL and an increased risk of arrhythmias is indeed complex. This complexity arises from the fact that the increased leak of Ca2+ from the SR can induce a transient inward current, which, in turn, leads to arrhythmogenic delayed afterdepolarizations. Additionally, it can also result in significant spontaneous proarrhythmic Ca2+ release from the SR [In our previous studies, we demonstrated that the In atrium ,28. Furta2+ leak ,21,31. T2+ spark frequency as well as a decreased frequency of spontaneous Ca2+ waves in human atrial SCN10A KO CMs and in control CMs after pharmacological inhibition. As Ca2+ waves represent a major proarrhythmic trigger, we hereby establish the principle of NaV1.8-induced INaL and its triggering role in cellular arrhythmogenesis that is independent of neuronal influence in isolated human atrial CMs. Interestingly, we found no effects on intracellular Ca2+ transients in either SCN10A KO or following NaV1.8 inhibition in control CMs. Thus, we propose that the intracellular Ca2+ handling and likely contractile function of CMs remain mostly unaffected by NaV1.8. In summary, our results and current evidence indicate that the discussed NaV1.8-induced INaL mainly influences arrhythmogenesis on a subcellular level while leaving cellular Ca2+ release and contractile function unaffected.In the present study, we show a reduction in spontaneous SR CaV1.8 was detected in atria, and human hypertrophied and failing ventricles [V1.8 using SCN10A KO iPSC-CMs or pharmacological inhibition can reverse cellular proarrhythmic effects in the atria. Both inhibition and KO of NaV1.8 potently suppressed proarrhythmogenic triggers  while leaving the peak Na+ current unaffected. These findings suggest targeting NaV1.8-dependent INaL constitutes a novel therapeutic anti-arhythmic strategy for the treatment of atrial rhythm disorders.The currently available anti-arrhythmic drugs, particularly for patients with structural heart disease, are limited in their effectiveness. Drugs like flecainide or amiodarone, which are commonly used, demonstrate suboptimal efficacy and are associated with significant adverse side effects, including life-threatening ventricular arrhythmias and organ toxicity. Therefore, new, safer, and more precise compounds for the treatment of atrial arrhythmias are highly desirable. Nantricles ,22,31. TSCN10A KO iPSC line was generated from a control iPSC line by CRISPR/Cas9 genome editing as described in detail in previous studies [SCN10A KO iPSCs were differentiated into functionally beating, atrial iPSC-derived cardiomyocytes as described in [All procedures conducted in this study adhered to the principles outlined in the Declaration of Helsinki and received approval from the local ethics committee of the University Medicine of Göttingen (Az-10/9/15). Informed consent was signed by all tissue donors. A homozygous  studies ,23. The ribed in .V1.8 knockout lines and their corresponding healthy isogenic control line.In order to achieve directed atrial cardiac differentiation of the induced pluripotent stem cells (iPSCs), manipulation of the Wnt signaling pathway was employed, as previously described . The celV1.8-induced sodium currents, a specific NaV1.8 blocker PF-01247324 ) was used. Cellular electrophysiological measurements were performed under slight beta-adrenergic stimulation (isoproterenol (Iso), 50 nmol/L, Sigma-Aldrich, Taufkirchen, Germany)) [For selective inhibition of Naermany)) . Prior tThe patch-clamp experiments was performed as previously described ,22. BrieAction potential recordings were performed using the whole-cell patch-clamp technique. To elicit action potentials, square current pulses with amplitudes of 0.5–1 nA and durations of 1–5 ms were applied. The stimulation frequency was increased gradually from 0.5 to 2 Hz.NaL) was measured using the ruptured-patch whole-cell patch-clamp technique. The pipette used had a resistance ranging from 2 to 3 mega-ohms (MΩ). INaL recordings were performed exclusively in CMs where a seal with a resistance of over 1 giga-ohm (GΩ) was achieved, and the access resistance remained below 7 MΩ. After a stabilization period of 3 min, the iPSC-derived CMs were held at a holding potential of −120 mV and then depolarized to −35 mV for 1000 ms with 10 pulses and a basic cycle length of 2 s. The INaL was quantified as the integral current amplitude between 100 and 500 ms and was normalized to the membrane capacitance.The late sodium current  for 15 min at RT for de-esterification of the dye. The solution was substituted with Tyrode’s solution . Cells exhibiting major Ca2+ release events (Ca2+ wavelets or waves) were excluded from the calculation of Ca2+ spark frequency and separately classified as proarrhythmic cells as a proportion of all cells.A total of 35.000 atrial iPSC-CMs plated on glass-bottom FluoroDishes were incubated with the Caribed in ) and the2+ indicator Fura 2-AM  for 15 min at RT. Subsequently, the cells were washed with Tyrode’s solution for de-esterification and incubated with pharmacological agents as described above. The measurements were performed using a fluorescence detection system  connected to an inverted microscope with oil immersion lens (40×). Cardiomyocytes were subjected to electrical field stimulation at 1 Hz for the duration of the experiment to ensure steady intracellular Ca2+ concentrations. Recording of Ca2+ transients for analysis was performed at 1 Hz at steady state. For each cell, the stimulation was paused for 30 s to detect spontaneous Ca2+ release events and evaluate the spontaneous beating frequency of the iPSC-CMs. Ca2+ transients were analyzed using the software IonWizard (IonOptix).A total of 35.000 atrial CMs were dissociated and plated as described above and loaded with the radiometric Cap values are two-sided and considered statistically significant if p < 0.05.The data are reported as mean ± SEM, unless otherwise stated. Analysis was carried out with Prism 9 software . For comparisons of two groups, unpaired Student’s t test was used in the case of parametric distribution of the data. Three or more groups including more than one differentiation experiment were compared using nested one-way ANOVA. The results were corrected for multiple comparisons by Sidak’s correction. Fisher’s exact test was used to statistically compare proportions. V1.8, which contributes to the INaL in the heart, is down-regulated in atrial SCN10A-KO iPSC-CMs and, importantly, contributes to INaL formation in human atrial CMs. NaV1.8 KO or the inhibition of NaV1.8 modulates proarrhythmogenic triggers such as INaL and diastolic SR-Ca2+ leak in human atrial CMs. Therefore, NaV1.8 might represent a novel treatment target for antiarrhythmic strategies.In conclusion, we showed that the neuronal sodium channel Na


The inhibition of those channels in different experimental models was effective to ameliorate the post-ischemic damage. However, at a clinical level, the results were contradictory. Recent results of our group obtained in an ¨ex vivo¨ heart model demonstrated that a chemical derived from acetazolamide, the N-methylacetazolamide (NMA) protected the heart against ischemia-reperfusion injury, diminishing the infarct size and improving the post-ischemic recovery of myocardial function and mitochondrial dynamic. A significant inhibitory action on L-type Ca2+ channels was also detected after NMA treatment, suggesting this action as responsible for the beneficial effects on myocardium exerted by this compound. Although these results were promising, the effectiveness of NMA in the treatment of ischemic heart disease in humans as well as the advantages or disadvantages in comparison to the classic calcium antagonists needs to be investigated.The increase of intracellular Ca IHD is ial role .2+ through the cardiac α1 subunit of voltage-gated L-type Ca2+ channel (CaC) which activates ryanodine receptor 2 (RyR2). Ca2+ release triggers shortening of the contractile unit, the sarcomere, resulting in the force generation. Muscle relaxation occurs when Ca2+ is removed from the contractile unit through the combined action of Ca2+ pumps and Na+/Ca2+ exchangers  belongs to a group of chemicals derived from the carbonic anhydrase (CA) inhibitor acetazolamide. In this case, a methyl group substituted one Hzolamide .+ efflux or the intracellular pH recovery  Ca2+ load, which is released at the onset of reperfusion and produces an abrupt rise in cytosolic Ca2+ and the consequent decrease in SR Ca2+ content and Ca2+ transient  and the activation of mitochondrial fragmentation -assessed by dephosphorylation at Ser637Drp1 (a dynamin-related protein-1)- both events occurring during ischemia-reperfusion  compounds, and NMA was the administration time; while the classic calcium blockers were given prior to ischemia, NMA was administered at the beginning of reperfusion. This fact is very important, making ¨a priori¨ the NMA a superior tool for the treatment of ischemic heart disease in patients. Although this fact is very important, clinical trials will be mandatory to demonstrate the effectiveness of NMA in humans and its advantages or disadvantages in comparison to other calcium antagonists.Taking into account previous data , we cons