cDNA Sequence Research Articles

Platelet-Restricted Clonal Hematopoiesis

Clonal hematopoiesis (CH) is a common age-related condition in which somatic mutations accumulate in hematopoietic stem cells (HSCs), leading to the clonal expansion of mutated cells. CH has been causatively linked with risk of disease, including atherosclerotic cardiovascular disease (CVD), and reduced overall survival. The current model of hematopoiesis suggests substantial heterogeneity and lineage-priming within the HSC compartment, including the existence of multipotent but platelet (PLT)-biased HSCs. However, all CH studies to date are based on whole blood or granulocyte (GRA) DNA, and therefore they do not assess for PLT-restricted clones, which we reasoned could be particularly important given the association of CH with CVD. We aimed to assess for presence of PLT-biased CH in a cohort of normal individuals, hypothesizing that PLT-biased CH might represent a distinct subgroup of persons with CH. We analysed a cohort of 151 individuals, aged 22.0 to 98.6 years ( x̅ = 74.9; 95% CI, 50.0 to 91.8), with normal blood counts, and no known hematological malignancy. We first established a method for isolation of high-purity cDNA from PLTs alongside isolation of gDNA and cDNA from matched GRAs. Myeloid cell contamination was extremely low in the PLT fraction as assessed by flow cytometry and molecular analysis. We first performed targeted analysis for presence of the JAK2V617F mutation in PLT and GRA fractions due to the known association of JAK2V617F with CVD and myeloid neoplasms, including essential thrombocythemia. We developed a high sensitivity allele specific JAK2V617F droplet digital PCR (ddPCR) assay (variant allele frequency [VAF] limit of detection 0.1%) for parallel assessment of gDNA and cDNA from GRAs and PLTs respectively. When including all cases with a VAF > 0.1%, JAK2V617F CH was detected in 21 (13.9%) individuals, consisting of 2 cases of non-lineage-restricted CH (present in both GRA and PLT fractions), 6 cases of GRA-restricted CH, and 13 cases of PLT-restricted CH. Only 6 cases showed VAF ≥ 2% (consistent with CH of indeterminate potential) and all were PLT restricted. Comparison of median JAK2V617F VAF in PLT cDNA with VAF in GRA gDNA revealed significantly higher VAF in the PLT-derived material (1.00% vs. 0.26%; p = 0.04). We next developed a custom, targeted, hybridisation capture next-generation sequencing (NGS) panel for parallel ultra-deep sequencing of both gDNA and cDNA, covering the major genes and hotspot mutations involved in CH ( DNMT3A, TET2, ASXL1, IDH2, JAK2, CALR, MPL, CBL, KRAS, GNB1, SF3B1, SRSF2, U2AF1, PPM1D and TP53). The panel comprised a total of 250 120-nucleotide-long probes (including 27 cDNA-specific probes) with a combined footprint size 25,083 bp. Performance studies showed acceptable off-target effect and good coverage, alignment rate, uniformity, and complexity. Agreement between the NGS-measured VAF for GRA gDNA and GRA cDNA was high (ρ c = 0.952, alpha = 0.05). In total, we screened GRA gDNA and matched PLT cDNA samples from 42 individuals. At the resolution of VAF ≥ 2%, analysis of PLTs substantially enhanced the number of individuals with detected CH from 12 (28.6%) to 26 (61.9%) (Fig. 1). Amongst the detected variants (n = 44), mutations detected in the GRA fraction (n = 20) accounted for less than half of the detected variants, while the PLT fraction included ~75% of the variants (n = 33), including 55% (n = 24) that were PLT-restricted. Notably, lineage-restriction for the above variants held true when taking into consideration clones with VAF < 2% too. Median VAF for all CH variants was 3.9% (95% CI, 2.2% to 32.3%), with no difference between GRA and PLT CH. The detected variants were distributed across DNMT3A, TET2, ASXL1, JAK2, SF3B1, and PPM1D genes, with evidence of lineage bias towards PLTs for TET2- and JAK2-related CH. PLT-restricted clones included high-VAF (> 5%) variants in DNMT3A (n = 4; x̅VAF = 13.2%), TET2 (n = 2; x̅VAF = 7.7%), JAK2 (n = 2; x̅VAF = 21.6%), and SF3B1 (n = 1), including an individual with DNMT3AQ842R and the SF3B1K666N at VAFs 16.7% and 12.5% respectively. Selected NGS-detected variants were orthogonally validated. We describe PLT-restricted CH as a frequent phenomenon in persons with normal blood parameters. Given the known association between CH and risk of CVD and hematological malignancy, further studies on larger patient cohorts are warranted to determine the importance of PLT vs. GRA CH in relation to clinical endpoints.

Allele-Specific Expression of Leukemia Genes Is Associated with Pathogenicity in Poor Risk AML

Introduction: An imbalance in gene expression of the different alleles of a gene (allele-specific expression or ASE) is an intriguing genome-wide and dynamic phenomenon that could have an important role in tumorigenesis. Little is known about the causes and consequences of the differential cis- and trans-regulation on resultant ASE, aside from the impact of copy number alterations (CNAs) or imprinting. Acute myeloid leukemia (AML) is an aggressive clonal hematopoietic stem cell malignancy, with a low mutational burden in comparison with other cancers, where the impact of ASE may constitute a novel mechanism of pathogenesis. Aim: Application of Whole Genome Sequencing (WGS), RNA-seq and Oxford Nanopore methylation sequencing to study the ASE landscape in a cohort of poor risk AML patients and shed light on the mechanisms triggering this phenomenon as well as its temporal dynamics. Methods: ASE landscape was explored using paired germline and tumour WGS and RNA-seq on diagnostic bone marrow or peripheral blood samples from 33 cytogenetically poor risk AML patients, and additional RNA-seq from 18 samples corresponding to follow-up timepoints (including remission, relapse and refractory samples) from eight of the patients.Heterozygous SNPs were identified in the genomic DNA, and allele frequencies in RNA-seq reads were used to quantify ASE, after filtering for CNAs in the tumor DNA. Cas9-enriched Oxford Nanopore sequencing was then performed on available patient samples at diagnosis and subsequent time points. Allele-specific methylation was determined on the Nanopore sequencing using Guppy v6.3. Results: In the diagnostic cohort, we identified 4,802 protein-coding genes exhibiting ASE in at least one sample, with a median of 169 genes per sample (range 119-468, with one outlier sample showing ASE in 1,705 genes). Overall, 283 protein-coding genes showed ASE in ≥ 20% patients across the 33 samples. These genes included known imprinted genes and the transcription factor GATA2, where ASE was previously associated with AML, validating our ASE pipeline and filtering criteria. We also identified for the first time recurrent ASE in other myeloid genes such as KMT2A (20%), the homeobox genes PBX2 (41%), PBX3 (20%), HOXA6 (20%) and HOXB cluster ( HOXB2 (31%), HOXB3 (27%), HOXB5 (40%), HOXB6 (30%)); and the myeloid differentiation genes CDKN2A (40%), PDGFRB (25%) and PRTN3 (57%). Eighteen primary AML samples from 8/33 patients had paired diagnostic, remission and relapse or refractory material, allowing us to track the dynamic ASE pattern. RNA-seq and Sanger sequencing of cDNA in these samples showed that the ASE observed at diagnosis in HOXB2, HOXB3, HOXB6, PBX3 and GATA2 was lost in the corresponding remission sample (bi-allelic expression), with ASE returning at relapse and maintained in the refractory samples. In order to explore the epigenetic mechanisms underlying these allele-specific changes in expression, Cas9-enriched Oxford Nanopore sequencing was performed in 2 patients with available DNA at diagnosis and sequential time points, to determine the pattern of allele-specific methylation of the promoter region of GATA2 (patient 1) and HOXB2 (patient 2). In patient 1 at diagnosis, 90% of CpG sites were methylated at the GATA2 promoter region on one allele versus 61% in the other allele, with the hypomethylated allele corresponding to the allele that is overexpressed in the ASE sample. However, in the remission sample, the proportion of methylated CpG sites on the two alleles was similar (83% and 73%, respectively), in agreement with the expression of both GATA2 alleles at this stage. Likewise, in the diagnostic sample of patient 2, 6% of CpG sites were methylated in the HOXB2 promoter region of one allele, corresponding to the overexpressed allele in the ASE sample, while the other promoter allele had 17% of CpG sites methylated; with no differences in CpG methylation between alleles in the remission samples (9% versus 9%), again correlating with the bi-allelic expression of HOXB2 at remission. Conclusions: Combining WGS and RNA-seq in AML primary samples, we have identified key leukemia genes showing recurrent dynamic ASE associated with disease evolution: since it is found at diagnosis, lost in remission, but recovered in relapse and persisting in refractory stages. Our results also suggest allele-specific methylation as the potential mechanism orchestrating this phenomenon.

Highly Sensitive Chip-Based Digital PCR Platform for Quantitative Detection of BCR::ABL1 Transcripts throughout CML Treatment

Introduction: With various BCR::ABL1 tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) has become a manageable disease and current therapeutic goal is treatment-free remission (TFR). To achieve a successful TFR in more patients, a precise assessment of minimal residual disease (MRD) is one of the prerequisite requirements. Since undetectable BCR::ABL1 fusion transcripts by conventional Taqman™-based quantitative polymerase chain reaction (qPCR) is not an indicator for complete eradication of the CML clone, it requires the development of more sensitive technologies. Currently, to overcome some limitations of conventional qPCR, such as rigorous standardization process and variations in sensitivity between laboratories, digital quantitative-PCR (dqPCR) methods have been developed for accurate and precise detection and its use in the routine clinical practice is gradually expanding. However, current dqPCR methods require the inclusion of one or more negative partitions to calculate the concentration using Poisson distribution. Consequently, this leads to a more than a 10-fold decrease in the maximum detectable concentration in a single test compared to conventional qPCR. Therefore, we have developed a novel technology, High Dynamic Range Digital Real-time PCR (so called HDR-drPCR) to achieve wider dynamic range, which allows more stable, accurate and sensitive monitoring of BCR::ABL1 in a timely manner. Methods: For linearity assessment, we used BCR::ABL1 positive (e14a2) K562 cell line diluted to 7 points by 10-fold serial dilutions from 25 to 2.5x10 -5 %IS with BCR::ABL1 negative HL60 cells. In HDR-drPCR, the GUSB gene was employed as a control gene to determine %IS values. For this purpose, the 1 st WHO international genetic reference panel was utilized to calculate the correction factor (CF). With the results from cell lines, we also evaluated equivalence between conventional qPCR and HDR-drPCR using 219 clinical specimens. Among them, 100 samples were determined as DMR (<0.01%IS) based on the qPCR test results In detail, conventional qPCR was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche, USA) for cDNA synthesis and Real-Q BCR-ABL Quantification Kit (Biosewoom, South Korea) for qPCR. For HDR-drPCR (1-step reverse transcriptase PCR), 10 6 to 10 7 copies per reaction of GUSB gene were applied to disposable cartridges of Genotizer™ HDR BCR::ABL1 Major IS detection kit (Optolane Technologies, South Korea) with LOAA platform (Optolane Technologies, Korea). Based on the results, correlation analysis between qPCR and HDR-drPCR was performed using correlation graphs and Blend-Altman plots. Results: Linearity was assessed by the dilution of RNA of K562 with RNA of HL60 using 10-fold serial dilution of up to - 7 log. The result showed accurate linearity (R 2 = 0.9972) at the range of BCR::ABL1/GUSB ratio of 10 1 to 10 -5(%). Notably, HDR-drPCR technology allowed to detect the level of BCR::ABL1 transcript of MR 6.34 (0.00005 %IS) (Figure 1A). The correlation analysis for 209 clinical samples showed that measured values from both qPCR and HDR-drPCR were closely correlated with the R 2 of 0.973 1 (Figure 1B) . The Bland-Altman plot showed a mean bias of 0.159, and the limit of agreement at the 95% confidence interval (LOA95) was calculated as -0.387 to 0.705. Furthermore, the concordance rate between the two methods was 96.17%, as measured by the proportion of samples in which the difference between the two methods was within a factor of 5. This indicates a strong concordance between the two methods. HDR-drPCR reliably detected BCR::ABL1 transcripts in all 90 clinical specimens measured below 0.01%IS (DMR) using qPCR. Additionally, HDR-drPCR detected trace amounts of BCR::ABL1 transcripts in 6 out of 10 specimens that were not detected by qPCR, showcasing its improved sensitivity compared to the conventional qPCR method. Conclusion: Taken together, we believe our high dynamic range real-time digital PCR(HDR-drPCR) may minimize false positive results and maximize the analytical sensitivity of detecting BCR::ABL1 fusion transcripts and thus, would be useful for accurately assessing treatment in routine clinical practice and facilitating the application of treatment-free remission (TFR). We will include these results in further intensive studies focusing on DMR clinical specimens Study support: Funded by Korea Research Foundation

Phosphate deficiency responsive TaSPX3 is involved in the regulation of shoot phosphorus in Arabidopsis plants

SPX (SYG/PHO81/XPR1) domain genes have been reported to play vital roles in the Phosphorus (Pi) signaling network in Arabidopsis thaliana and rice. However, the functions of SPX proteins in wheat remain largely unknown. In this study, the full-length cDNA sequence of the TaSPX3 gene was cloned from the common wheat variety Zhengmai9023. The expression of TaSPX3 was up-regulated in eight different genotypes of wheat under low phosphorus (LP) stress, indicating that TaSPX3 responds to Pi limitation in multiple wheat genotypes. The transcription level of TaSPX3 was also detected in the absence of seven different elements, showing certain specificity for Pi deficiency in wheat. Over expressing TaSPX3 in Arabidopsis can alleviate Pi deficiency symptoms at the seedling stage and promote the growth of plant, and advance the flowering period at the adult stage. The expression of 7 genes associated with the Pi starvation signal pathways was analyzed using qRT-PCR. The results showed that TaSPX3, along with AtSPX1, AtRNS1, AtIPS1, AtPAP2, AtPAP17 and AtAT4, were all induced by Pi deficiency. This study reveals that the TaSPX3 gene in wheat is involved in the response to phosphorus stress and may affect shoot phosphorus levels through AT4 or PAPs-related pathways. Overall, our study provides new insights into the regulation of plant response under LP conditions and the molecular mechanism underlying the role of the wheat SPX gene in coping with LP stress.

GjSOX9 Cloning, Expression, and Comparison with gjSOXs Family Members in Gekko japonicus.

SOX9 plays a crucial role in the male reproductive system, brain, and kidneys. In this study, we firstly analyzed the complete cDNA sequence and expression patterns for SOX9 from Gekko japonicus SOX9 (gjSOX9), carried out bioinformatic analyses of physiochemical properties, structure, and phylogenetic evolution, and compared these with other members of the gjSOX family. The results indicate that gjSOX9 cDNA comprises 1895 bp with a 1482 bp ORF encoding 494aa. gjSOX9 was not only expressed in various adult tissues but also exhibited a special spatiotemporal expression pattern in gonad tissues. gjSOX9 was predicted to be a hydrophilic nucleoprotein with a characteristic HMG-Box harboring a newly identified unique sequence, "YKYQPRRR", only present in SOXE members. Among the 20 SOX9 orthologs, gjSOX9 shares the closest genetic relationships with Eublepharis macularius SOX9, Sphacrodactylus townsendi SOX9, and Hemicordylus capensis SOX9. gjSOX9 and gjSOX10 possessed identical physicochemical properties and subcellular locations and were tightly clustered with gjSOX8 in the SOXE group. Sixteen gjSOX family members were divided into six groups: SOXB, C, D, E, F, and H with gjSOX8, 9, and 10 in SOXE among 150 SOX homologs. Collectively, the available data in this study not only facilitate a deep exploration of the functions and molecular regulation mechanisms of the gjSOX9 and gjSOX families in G. japonicus but also contribute to basic research regarding the origin and evolution of SOX9 homologs or even sex-determination mode in reptiles.

Ultrasensitive detection of circulating tumor cells via DNA walker driven by a DNA circuit synergized with MOF-on-MOF nanozyme

Circulating tumor cells (CTCs) monitoring as a means of liquid biopsy can be effective in aiding the diagnosis, treatment monitoring, and prognosis of cancer. Herein, we fabricated a dual-mode cytosensor for ultrasensitive detection of CTCs via DNA walker powered by a DNA circuit and MOF-on-MOF nanozyme. The anti-EpCAM aptamer was hybridized with complementary DNA (cDNA) and modified onto the magnetic nanospheres (MNs). In the presence of CTCs, cDNA strand can be released by cell-aptamer specific recognition and was separated through a magnet to trigger the subsequent DNA walking process. In the DNA walking process, the cDNA strand could trigger cascade toehold-mediated DNA displacement reactions by fuel nanozyme-functionalized DNA strands and recycle to further accelerate the DNA circuit thus amplifying the output signal. What’s more, since the MOF-on-MOF nanozyme had good peroxidase activity, electrochemical and colorimetric dual-mode signal output can be achieved in the presence of 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) for CTCs determination through an ITO chamber. As a proof of concept, by virtue of the outstanding synergistic signal amplification of DNA walker and MOF-on-MOF nanozymes, the designed dual-mode cytosensor exhibited a comprehensive linear range of 5 to 5 × 105 cells/mL and a sensitivity of 5 cells/mL and performed well in complex environment. This work provided important references for further investigations on CTC-related diagnosis and individualized treatment.

Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing

BackgroundElevated TCRαβ+CD4-CD8- double-negative T-cells (DNT) and serum biomarkers help identifying FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified upon standard exon sequencing (ALPS-undetermined: ALPS-U). ObjectiveWe aimed to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. MethodsGenetic analysis included whole FAS gene sequencing, copy number variation analysis and sequencing of FAS cDNA and other FAS pathway related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss-of-heterozygosity (sLOH). ResultsNine of 16 ALPS-U patients lacked FAS expression on CD57+DNT predicting heterozygous “loss of expression” FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7/9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT, one patient showed a FAS exon duplication. Three patients had reduced FAS expression, two of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the four ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. ConclusionA combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing. Clinical implicationDetection of complex FAS pathway gene alterations by extended genetic analysis allows targeted therapy with sirolimus.

The influence of coiled-coil motif of serine recombinase toward the directionality regulation

Serine integrases promote the recombination of two complementary DNA sequences, attP and attB, to create hybrid sequences, attL and attR. The reaction is unidirectional in the absence of an accessory protein called recombination directionality factor. We utilized tethered particle motion (TPM) experiments to investigate the reaction behaviors of two model serine integrases from Listeria innocua phage LI and Streptomyces coelicolor phage C31. Detailed kinetic analyses of wild-type and mutant proteins were carried out to verify the mechanisms of recombination directionality. In particular, we assessed the influence of a coiled-coil motif (CC) that is conserved in the C-terminal domain of serine integrases and is an important prerequisite for efficient recombination. Compared to wild type, we found that CC deletions in both serine integrases reduced the overall abundance of integrase (Int) att-site complexes and favored the formation of nonproductive complexes over recombination-competent complexes. Furthermore, the rate at which CC mutants formed productive synaptic complexes and disassembled aberrant nonproductive complexes was significantly reduced. It is notable that while the φC31 Int CC is essential for recombination, the LI Int CC plays an auxiliary role for recombination to stabilize protein-protein interactions and to control the directionality of the reaction.

Kidney cell culture of Rana dybowskii and study molecules of TRIF in Rana dybowskii cells under the stress of Aeromonas hydrophila

As a model organism, the cell culture method of Rana dybowskii (Rd) has not received widespread attention. We obtained kidney cells from Rana dybowskii by tissue block culture and enzyme digestion, and optimized the culture method by changing different culture conditions. We can more conveniently study the operation of the Rana dybowskii immune system through cell lines. One of the most important adaptor proteins in the Toll-like receptors (TLRs) signaling pathway, TIR-domain-containing adaptor inducing interferon-β (TRIF), plays a role in the apoptosis pathway and participates in the ubiquitin-mediated protein hydrolysis pathway. This rationalizes the significance of TRIF in the immune cascade signaling pathway. To obtain and analyze the full-length sequence of cDNA, sequential structures of amino acids, as well as Rana dybowskii TRIF expression profiles, the present work carried out multiple-sequence alignment, rapid amplification of cDNA ends (RACE), the establishment of a phylogenetic tree, search for the conserved domain, and qRT-PCR. Results revealed 1733-bp full-length cDNA of RdTRIF with an open reading frame (ORF) of 1281 bp, which encoded one protein containing 426 amino acid residues. The amino acid sequence of RdTRIF confirmed the presence of one Toll/interleukin-1 receptor (TIR) domain. As a kind of hydrophilic protein, the pI and molecular weight of RdTRIF were 5.67 and 24.76 kDa, respectively. We predicted 51 candidate phosphorylation sites, including 32 serine residues, 16 threonine residues along with 3 tyrosine residues. The phylogenetic tree and multiple-sequence alignment substantiated the highest homology of RdTRIF protein to Xenopus tropicalis homologs. According to the qRT-PCR results, the expression of RdTRIF was noted under the stress of Ah in the kidney.

A novel homozygous splice-site mutation of JK gene leads to Jk(a-b-) phenotype.

This study aimed to investigate the molecular mechanism of the Jk(a-b-) phenotype in a Chinese transfusion patient. Many different mutation types relating to Jk(a-b-) phenotype have been reported. However, the splice-site mutation is relatively rare and the related functional verification is lacking. In this study, the blood sample was collected from a transfusion patient with the Jk(a-b-) phenotype. Serotyping was performed using routine serological methods. The exons sequences and coding regions of the JK gene were amplified using polymerase chain reaction and directly sequenced. To perform a minigene splicing assay, the intronic mutation sequences were cloned into a pSPL3 splice reporting vector. The splicing reporter minigene assay was performed in HEK 293T cells. The Jk(a-b-) phenotype of the blood sample was identified through serological testing. Sequencing results revealed that the sample had a novel homozygous splice-site mutation JK*02N (NM_015865.7: c.663+3A>C). Further analysis, including cDNA sequencing and minigene splicing assay, confirmed that the novel splice-site mutation resulted in exon skipping. Interestingly, different numbers of exons being skipped were obtained by the two methods. This study revealed a novel homozygous splicing-site mutation associated with the Jk(a-b-) phenotype in Chinese population. Our results emphasise the importance of the in vitro functional method minigene splicing assay, while also acknowledging its potential limitations when compared to cDNA sequencing.

An Adapted GeneSwitch Toolkit for Comparable Cellular and Animal Models: A Proof of Concept in Modeling Charcot-Marie-Tooth Neuropathy.

Investigating the impact of disease-causing mutations, their affected pathways, and/or potential therapeutic strategies using disease modeling often requires the generation of different in vivo and in cellulo models. To date, several approaches have been established to induce transgene expression in a controlled manner in different model systems. Several rounds of subcloning are, however, required, depending on the model organism used, thus bringing labor-intensive experiments into the technical approach and analysis comparison. The GeneSwitch™ technology is an adapted version of the classical UAS-GAL4 inducible system, allowing the spatial and temporal modulation of transgene expression. It consists of three components: a plasmid encoding for the chimeric regulatory pSwitch protein, Mifepristone as an inducer, and an inducible plasmid. While the pSwitch-containing first plasmid can be used both in vivo and in cellulo, the inducible second plasmid can only be used in cellulo. This requires a specific subcloning strategy of the inducible plasmid tailored to the model organism used. To avoid this step and unify gene expression in the transgenic models generated, we replaced the backbone vector with standard pUAS-attB plasmid for both plasmids containing either the chimeric GeneSwitch™ cDNA sequence or the transgene cDNA sequence. We optimized this adapted system to regulate transgene expression in several mammalian cell lines. Moreover, we took advantage of this new system to generate unified cellular and fruit fly models for YARS1-induced Charco-Marie-Tooth neuropathy (CMT). These new models displayed the expected CMT-like phenotypes. In the N2a neuroblastoma cells expressing YARS1 transgenes, we observed the typical "teardrop" distribution of the synthetase that was perturbed when expressing the YARS1CMT mutation. In flies, the ubiquitous expression of YARS1CMT induced dose-dependent developmental lethality and pan-neuronal expression caused locomotor deficit, while expression of the wild-type allele was harmless. Our proof-of-concept disease modeling studies support the efficacy of the adapted transgenesis system as a powerful tool allowing the design of studies with optimal data comparability.

Cloning, computational analysis and expression profiling of steroid 5 alpha-reductase 1 (SRD5A1) gene during reproductive phases and ovatide stimulation in endangered catfish, Clarias magur

The cloning and characterization of the complete coding sequence of the Clarias magur SRD5A1 (CmSRD5A1) gene, which encodes an enzyme responsible for regulating steroid levels by converting testosterone into 5α-dihydrotestosterone (DHT), have been successfully achieved. DHT plays a vital role in enabling the complete expression of testosterone's actions in neuroendocrine tissues. The ORF of the full-length cDNA sequence of SRD5A1 was 795 bp, translating into 265 amino acids, with a total length of 836 bp including UTRs. Like other vertebrates, the signal peptide analysis revealed that SRD5A1 is a non-secretory protein, and hydropathy profiles indicated that it is hydrophobic in nature. The 3D structure of CmSRD5A1 sequence generated above was predicted using highly accurate AlphaFold 2 in Google Colab online platform. CmSRD5A1 contains seven transmembrane helices connected by six loops, with the N-termini located on the periplasmic side and C-termini on the cytosolic side. Structural superimposition with known bacterial and human SRD5As showed very high structural similarity. The electrostatic potential calculation and surface analysis of CmSRD5A1 revealed the presence of a large cavity with two openings one highly electropositive towards the cytosolic side and another relatively neutral towards the transmembrane region. The structural comparison revealed that the electropositive side of the cavity should bind to NADPH and the steroid hormone in the hydrophobic environment. Polar residues binding to NADPH are highly conserved and the same as known strictures. The conserved residues involved in hydrogen bonding with the ketone group at C-3 in the steroids hence fevering Δ4 double-bond reduction are identified as E66 and Y101. Our findings showed that SRD5A1 expression was lower during the spawning phase than the preparatory phase in female fish, while the administration of Ovatide (a GnRH analogue) resulted in up-regulation of expression after 6 h of injection in the ovary. In males, the lowest expression was observed during the preparatory phase and peaked at 16 h post- Ovatide injection in the testis. The expression of SRD5A1 in the brain of female fish was slightly higher during the Ovatide stimulation phase than the spawning phase. This study represents the first report on the cloning and characterization of the full-length cDNA of SRD5A1 in Indian catfish.

Overexpression of cardiac calsequestrin as a novel gene-therapy approach to treat CPVT1: in silico and in vivo proves of principle

Abstract Background More than 200 gain-of-function mutations in the cardiac ryanodine receptor (RyR2) cause catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1). From a molecular point of view, 3 types of mutations exist [1]: Class 1 mutants enhance RyR2 opening in response to sarcoplasmic reticulum (SR) free Ca2+; Class 2 and Class 3 mutants enhance RyR2 opening in response to both cytosolic and SR free Ca2+. Purpose The study's objective was to evaluate in silico and in vivo, in CPVT1 knock-in mice carriers of the Class 1 mutation RyR2(R4496C/+), whether overexpression of cardiac calsequestrin (CASQ2) rescues the arrhythmic phenotype in CPVT1 mice. Rationale: Several studies performed in the 2000’s have collectively demonstrated that an increase in CASQ2 promotes the closure of RyR2 channels [2]: inspired by this evidence, here we test the hypothesis that CASQ2 gene therapy might be a novel treatment for CPVT1. Methods (1) In silico study: Properties of Class 1 and Class 2 RyR2s mutants (Figure, panels A-C) were introduced into an in silico model of the murine ventricular myocyte [3]. Virtual myocytes were paced either at baseline or during simulated delivery of 100 nM Isoproterenol at 1 Hz for 180 seconds. To simulate CASQ2 overexpression, protein levels were progressively increased, and the changes in the arrhythmic burden were documented. (2) In vivo study (performed in compliance with ethical regulations): an adeno-associated viral vector containing the cDNA sequence of CASQ2 was injected into RyR2(R4496C/+) mice (tail iv, 3×10^13 vg/kg) at 4 weeks of age. Eight weeks post-infection, animals were treated with an ip bolus of caffeine (120mg/kg) and epinephrine (2 mg/kg) to elicit arrhythmias (Epi/Caff Test). Results (1) In silico: dose-response curves were simulated to correlate CASQ2 levels with arrhythmia inducibility during virtual Isoproterenol delivery. For the simulated prototypical Class 1 mutation, 1.4-fold CASQ2 expression suppressed arrhythmias. For the prototypical Class 2 mutation, 1.8-fold CASQ2 expression was needed (Figure, panel D). (2) In vivo: At 12 weeks of age, both RyR2(R4496C/+) treated and non-treated littermates received the ip Epi / Caff bolus. Arrhythmias were observed in 17/32 (53%) untreated mice and in 0/12 treated animals (p=0.0012). Conclusion(s) We provide proof-of-principle demonstrating that in mice, the overexpression of CASQ2 may be a powerful and safe therapeutic strategy for CPVT1.

Unclassified glutathione-S-transferase AiGSTu1 confers chlorantraniliprole tolerance in Agrotis ipsilon.

Chlorantraniliprole (CAP) is a diamide insecticide with high efficacy against many pest insects, including the black cutworm, Agrotis ipsilon. Agrotis ipsilon is a serious pest causing significant yield losses in crops. Glutathione-S-transferases (GSTs) belong to a family of metabolic enzymes that can detoxify a wide range of pesticides. However, little is known about the functions of GSTs in CAP tolerance in A. ipsilon. A cDNA sequence (designated AiGSTu1) encoding an unclassified GST was identified from A. ipsilon. AiGSTu1 is highly expressed during the 3rd -instar larval and the pupal stages. Most of the mRNA transcripts were found in larval Malpighian tubules. Exposure to CAP strongly enhanced AiGSTu1 expression, GST activity, hydrogen peroxide (H2 O2 ) and malondialdehyde levels in larvae. H2 O2 treatment upregulated the transcription level of AiGSTu1, suggesting that CAP-induced oxidative stress may activate AiGSTu1 expression. The activity of recombinant AiGSTu1 was inhibited by CAP in a dose-dependent manner. Metabolism assay results demonstrated that AiGSTu1 is capable of depleting CAP. Overexpression of AiGSTu1 enhanced the tolerance of Escherichia coli cells to H2 O2 and the oxidative stress inducer, cumene hydroperoxide. Silencing of AiGSTu1 by RNA interference increased the susceptibility of A. ipsilon larvae to CAP. The findings of this study provide valuable insights into the potential role of AiGSTu1 in CAP detoxification and will improve our understanding of CAP tolerance in A. ipsilon. © 2023 Society of Chemical Industry.

Characterization and pathogenicity analysis of a newly isolated strain of infectious hematopoietic necrosis virus

Rainbow trout is one of the fastest-growing aquaculture species and infectious hematopoietic necrosis virus (IHNV) is endemic throughout almost all rainbow trout farms in China nowadays. In this study, IHNV GS21 was identified as the causative pathogen, which resulted in massive mortality of rainbow trout occurring in northwest China. GS21 isolate was propagated in Chinook salmon embryonic cell line (CHSE-214) and induced apparent cytopathic effects (CPE) at 3 days post-infection (dpi). Phylogenetic analysis revealed that GS21 isolate was clustered with other reported Chinese isolates within the J genogroup. Moreover, the complete cDNA sequence of GS21 isolate was obtained and it possesses more than 98 % of ANI values and 89 % of DDH values with other Chinese IHNV isolates. The detailed sequence analysis of G gene revealed the distinct amino acid substitutions of G230, G252, G270, and I277 in GS21 isolate. Furthermore, the artificially infected rainbow trout exhibited similar clinical disease symptoms as natural infection did. The cumulative mortality infected by GS21 isolate of 104 PFU/mL reached 93 % at approximately 13.5 °C. Additionally, viral loads in tissues increased first and declined then as well as the expression of immune-associated genes. Collectively, our results characterized a novel IHNV GS21 isolate that can lead to massive mortality in juvenile rainbow trout and provided a basis to define the pathogenic characteristics and evolutionary relationship of IHNV and host immune response against IHNV infection.

Identification of Pla a 7 as a novel pollen allergen group in Platanus acerifolia pollen

BackgroundPlatanus acerifolia is recognized as a source of allergenic pollen worldwide. Currently, five Platanus acerifolia pollen allergens belonging to different protein families have been identified, in which profilin and enolase were characterized by our group recently. Besides, we also screened and identified a novel allergen candidate as triosephosphate isomerase, which was different from already known types of pollen allergens. However, the role of this novel allergen group in Platanus acerifolia pollen allergy was unclear. Therefore, we further investigated the allergenicity and clarify its clinical relevance in this study. MethodsThe natural triosephosphate isomerase from Platanus acerifolia pollen was purified by three steps of chromatography and identified by mass spectrometry. The cDNA sequence of this protein was matched from in-house transcripts based on internal peptide sequences, which was further confirmed by PCR cloning. The recombinant triosephosphate isomerase was expressed and purified from E. coli. Allergenicity analysis of this protein was carried out by enzyme linked immunosorbent assay, immunoblot, and basophil activation test. ResultsA novel allergen group belonging to triosephosphate isomerase was firstly identified in Platanus acerifolia pollen and named as Pla a 7. The cDNA of Pla a 7 contained an open reading frame of 762 bp encoding 253 amino acids. The natural Pla a 7 displayed 41.4% IgE reactivity with the patients’ sera by ELISA, in which the absorbance value showed correlation to the serum sIgE against Platanus acerifolia pollen extract. Inhibition of IgE-binding to pollen extracts reached 26%-94% in different Pla a 7-positive sera. The recombinant Pla a 7 exhibited weaker IgE-reactivity in ELISA than its natural form, but showed comparable activity in immunoblot. The allergenicity was further confirmed by basophil activation test. ConclusionsTriosephosphate isomerase (Pla a 7) was first recognized as pollen allergen in Platanus acerifolia pollen, which is a completely different type of pollen allergen from those previously reported. This finding is essential to enrich information on allergen components and pave the way for molecular diagnosis or treatment strategies for Platanus acerifolia pollen allergy.

Characterization and expression analysis of seven lipid metabolism-related genes in yellow catfish Pelteobagrus fulvidraco fed high fat and bile acid diet

SREBPs, such as SREBP1 and SREBP2, were the key transcriptional factors regulating lipid metabolism. The processing of SREBPs involved many genes, such as scap, s1p, s2p, cideb. Here, we deciphered the full-length cDNA sequences of scap, srebp1, srebp2, s1p, s2p, cideb and cidec from yellow catfish Pelteobagrus fulvidraco. Their full-length cDNA sequences ranged from 1587 to 3884 bp, and their ORF length from 1191 to 2979 bp, encoding 396–992 amino acids. Some conservative domains were predicted, including the multiple transmembrane domains in SCAP, the bHLH-ZIP domain in SREBP1 and SREBP2, the ApoB binding region, ER targeting region and LD targeting region in CIDEb, the LD targeting region in the CIDEc, the conserved catalytic site and processing site in S1P, and the transmembrane helix domain in S2P. Their mRNA expression could be observed in the heart, spleen, liver, kidney, brain, muscle, intestine and adipose, but varied with tissues. The changes of their mRNA expression in responses to high-fat (HFD) and bile acid (BA) diets were also investigated in the brain, heart, intestine, kidney and spleen tissues. In the brain, HFD significantly increased the mRNA expression of seven genes (scap, srebp1, srebp2, s1p, s2p, cideb and cidec), and the BA attenuated the increase of scap, srebp1, srebp2, s1p, s2p, cideb and cidec mRNA expression induced by HFD. In the heart, HFD significantly increased the mRNA abundances of six genes (srebp1, srebp2, scap, s2p, cideb and cidec), and BA attenuated the increase of their mRNA abundances induced by HFD. In the intestine, HFD increased the cideb, s1p and s2p mRNA abundances, and BA attenuated the HFD-induced increment of their mRNA abundances. In the kidney, HFD significantly increased the scap, cidec and s1p mRNA expression, and BA diet attenuated the increment of their mRNA expression. In the spleen, HFD treatment increased the scap, srebp2, s1p and s2p mRNA expression, and BA diet attenuated HFD-induced increment of their mRNA expression. Taken together, our study elucidated the characterization, expression profiles and transcriptional response of seven lipid metabolic genes, which would serve as the good basis for the further exploration into their function and regulatory mechanism in fish.

Identification and molecular characterization of Rab7 form Yellow River Carp Cyprinus carpio

Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. In this study, full-length cDNA sequences of Rab7 were cloned from Yellow River Carp Cyprinus carpio, which were designated as CcRab7, and its function during pathogen infection was also investigated. The results showed that the full-length cDNA of CcRab7 was composed of 1511 bp, and open reading frame is 618 bp encoding 205 amino acids with 23.10 kDa. The deduced amino acid sequence of CcRab7 shared high similarity to that of Pimephales promelas (99.02%). In this paper, real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression levels in 11 tissues and organs of healthy Yellow River Carp, and the tissue distribution of CcRab7 as well as its defense mechanism against Spring viremia of carp virus (SVCV) and Aeromonas hydrophila in Yellow River Carp C. carpio was investigated. The results revealed that the mRNA transcripts of CcRab7 were widely detected in various tissues of C. carpio, with the highest expression levels in head kidney and blood, indicating that CcRab7 was involved in the innate immune process. Yellow River Carp was immunized with A. hydrophila and SVCV respectively, and the expression changes of CcRab7 were detected in gill, spleen, liver and skin of Yellow River Carp. The mRNA transcripts of CcRab7 in the main organs (gill, and liver, skin and spleen) were significantly affected by SVCV and A. hydrophila infection. The results showed that the CcRab7 expression levels was obviously up-regulated at different time points. The eukaryotic recombinant plasmids of CcRab7 and pEGFP-N3 transfected into Grass carp ovary cell lines (GCO) cells for subcellular localization. The results provide a basis for further investigation into the immunological role of CcRab7 in Yellow River Carp.

Identification and functional characterization of a novel truncating splicing variant in COL4A5 gene causes X-linked Alport syndrome with astigmatism.

Identification and functional characterization of a novel truncating splicing variant in COL4A5 gene causes X-linked Alport syndrome with astigmatism.

Identification of transcriptionally active transposons in Barley

BackgroundThe genomes of many major crops including barley (Hordeum vulgare) consist of numerous transposons. Despite their important roles in crop genome evolution and morphological variations, most of these elements are silent or truncated and unable to be mobile in host genomes. Thus far, only a very limited number of active transposons were identified in plants.ResultsWe analyzed the barley full-length cDNA (FLcDNA) sequences and detected 71 unique FLcDNAs exhibiting significant sequence similarity to the extant transposase proteins. These FLcDNAs were then used to search against the genome of a malting barley cultivar ‘Morex’, seven new intact transposons were identified. Sequence alignments indicated that six intact transposons contained the entire FLcDNAs whereas another one served as 3’ untranslated region (3’ UTR) of a barley gene. Our reverse transcription-PCR (RT-PCR) experiment further confirmed the expression of these six transposons and revealed their differential expression. We conducted genome-wide transposon comparisons and detected polymorphisms of three transposon families between the genomes of ‘Morex’ and other three genotypes including the wild barley (Hordeum spontaneum, B1K-04-12) and two cultivated barley varieties, ‘Golden Promise’ and ‘Lasa Goumang’. Lastly, we screened the transcripts of all annotated barley genes and found that some transposons may serve as the coding regions (CDSs) or UTRs of barley genes.ConclusionWe identified six newly expressed transposons in the barley genome and revealed the recent mobility of three transposon families. Our efforts provide a valuable resource for understanding the effects of transposons on barley genome evolution and for developing novel molecular tools for barley genetic improvement and other research.