Double Allele Research Articles

A MSTNDel73C mutation with FGF5 knockout sheep by CRISPR/Cas9 promotes skeletal muscle myofiber hyperplasia.

Mutations in the well-known Myostatin (MSTN) produce a 'double-muscle' phenotype, which makes it commercially invaluable for improving livestock meat production and providing high-quality protein for humans. However, mutations at different loci of the MSTN often produce a variety of different phenotypes. In the current study, we increased the delivery ratio of Cas9 mRNA to sgRNA from the traditional 1:2 to 1:10, which improves the efficiency of the homozygous mutation of biallelic gene. Here, a MSTNDel73C mutation with FGF5 knockout sheep, in which the MSTN and FGF5 dual-gene biallelic homozygous mutations were produced via the deletion of 3-base pairs of AGC in the third exon of MSTN, resulting in cysteine-depleted at amino acid position 73, and the FGF5 double allele mutation led to inactivation of FGF5 gene. The MSTNDel73C mutation with FGF5 knockout sheep highlights a dominant 'double-muscle' phenotype, which can be stably inherited. Both F0 and F1 generation mutants highlight the excellent trait of high-yield meat with a smaller cross-sectional area and higher number of muscle fibers per unit area. Mechanistically, the MSTNDel73C mutation with FGF5 knockout mediated the activation of FOSL1 via the MEK-ERK-FOSL1 axis. The activated FOSL1 promotes skeletal muscle satellite cell proliferation and inhibits myogenic differentiation by inhibiting the expression of MyoD1, and resulting in smaller myotubes. In addition, activated ERK1/2 may inhibit the secondary fusion of myotubes by Ca2+-dependent CaMKII activation pathway, leading to myoblasts fusion to form smaller myotubes.

Relation of rs846910, rs4844880 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) polymorphisms with the risk of preeclampsia: A case-control study

BackgroundThe 11β-hydroxysteroid-dehydrogenase 1 (11β-HSD1) enzyme catalyzes the interconversion of cortisone and cortisol, with mainly oxoreductive activity in intact cells due to co-expression with hexose-6-phosphate dehydrogenase (H6PD). The uterine localization of 11β-HSD1 and its reduced placental expression in women with preeclampsia (PE) suggest a role for 11β-HSD1 in PE pathogenesis. We investigated the association of rs4844880 and rs846910 variants in the HSD11B1 gene with PE in Tunisian women. MethodsThe study cases comprised 334 women who presented with PE and 314 age-matched normotensive women who served as controls. The rs4844880 and rs846910 HSD11B1 gene variants were genotyped by real-time PCR. ResultsThe rs4844880 T > A and rs846910 G > A minor allele frequencies were not different between PE cases and control women, which persisted after adjusting for age, BMI, gestational age, premature delivery, and baby weight. An association was noted for rs4844880 A/A genotype with a heightened risk of PE, which persisted after controlling key covariates. The (minor) A allele of rs4844880 was linked with elevated serum ALT and higher serum AST. In contrast, carriage of the rs846910 minor (A) allele was connected with higher baby weight on delivery and serum AST levels. Setting the major allele homozygotes (T-G) as a reference, a higher prevalence of double minor allele (A-A) haplotype was seen in PE cases than in corresponding controls, which persisted after controlling for age and BMI. Controlling for gestational age and baby weight identified the T-A haplotype and confirmed the association of the A-A haplotype with a heightened risk of PE. ConclusionOur results support an association between HSD11B1 polymorphisms and increased risk of PE and PE-associated clinical features.

First report of whole CFTR gene duplication in a healthy newborn carrying R74W and V855I variants on the same allele

Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder among Caucasians. The improvement of genetic techniques has allowed the identification of an increasing number of genetic variants, including large rearrangements such as duplications. We report the first case of a whole CFTR gene duplication in a healthy newborn, who had normal sweat test, also carrying R74W and V855I variants on the same allele. Familial segregation analysis and the observed frequencies of all the CFTR gene variants, revealed that R74W and V855I were probably both present in a cis arrangement on the allele also containing the duplication (i.e., in a double complex allele). Since R74W is a "variant of varying clinical consequence" its arrangement in trans with one pathogenic variant may not be sufficient to cause a classic CF disease phenotype. Moreover, its duplication could even be an advantage that could compensate for the effect of the alteration.

Large Region of Homozygous (ROH) Identified in Indian Patients with Autosomal Recessive Limb-Girdle Muscular Dystrophy with p.Thr182Pro Variant in SGCB Gene

The sarcoglycanopathies are autosomal recessive limb-girdle muscular dystrophies (LGMDs) caused by the mutations in genes encoding the α, β, γ, and δ proteins which stabilizes the sarcolemma of muscle cells. The clinical phenotype is characterized by progressive proximal muscle weakness with childhood onset. Muscle biopsy findings are diagnostic in confirming dystrophic changes and deficiency of one or more sarcoglycan proteins. In this study, we summarized 1,046 LGMD patients for which a precise diagnosis was identified using targeted sequencing. The most frequent phenotypes identified in the patients are LGMDR1 (19.7%), LGMDR4 (19.0%), LGMDR2 (17.5%), and MMD1 (14.5%). Among the reported genes, each of CAPN3, SGCB, and DYSF variants was reported in more than 10% of our study cohort. The most common variant SGCB p.Thr182Pro was identified in 146 (12.5%) of the LGMD patients, and in 97.9% of these patients, the variant was found to be homozygous. To understand the genetic structure of the patients carrying SGCB p.Thr182Pro, we genotyped 68 LGMD patients using a whole genome microarray. Analysis of the array data identified a large ~1 Mb region of homozygosity (ROH) (chr4:51817441-528499552) suggestive of a shared genomic region overlapping the recurrent missense variant and shared across all 68 patients. Haplotype analysis identified 133 marker haplotypes that were present in ~85.3% of the probands as a double allele and absent in all random controls. We also identified 5 markers (rs1910739, rs6852236, rs13122418, rs13353646, and rs6554360) which were present in a significantly higher proportion in the patients compared to random control set ( n = 128 ) and the population database. Of note, admixture analysis was suggestive of greater proportion of West Eurasian/European ancestry as compared to random controls. Haplotype analysis and frequency in the population database indicate a probable event of founder effect. Further systematic study is needed to identify the communities and regions where the SGCB p.Thr182Pro variant is observed in higher proportions. After identifying these communities and//or region, a screening program is needed to identify carriers and provide them counselling.

Accurate Prediction of Transcriptional Activity of Single Missense Variants in HIV Tat with Deep Learning

Tat is an essential gene for increasing the transcription of all HIV genes, and affects HIV replication, HIV exit from latency, and AIDS progression. The Tat gene frequently mutates in vivo and produces variants with diverse activities, contributing to HIV viral heterogeneity as well as drug-resistant clones. Thus, identifying the transcriptional activities of Tat variants will help to better understand AIDS pathology and treatment. We recently reported the missense mutation landscape of all single amino acid Tat variants. In these experiments, a fraction of double missense alleles exhibited intragenic epistasis. However, it is too time-consuming and costly to determine the effect of the variants for all double mutant alleles through experiments. Therefore, we propose a combined GigaAssay/deep learning approach. As a first step to determine activity landscapes for complex variants, we evaluated a deep learning framework using previously reported GigaAssay experiments to predict how transcription activity is affected by Tat variants with single missense substitutions. Our approach achieved a 0.94 Pearson correlation coefficient when comparing the predicted to experimental activities. This hybrid approach can be extensible to more complex Tat alleles for a better understanding of the genetic control of HIV genome transcription.

Genome-edited TaTFL1-5 mutation decreases tiller and spikelet numbers in common wheat.

Tillering is a critical agronomic trait of wheat (Triticum aestivum L.) that determines the shoot architecture and affects grain yield. TERMINAL FLOWER 1 (TFL1), encoding a phosphatidylethanolamine-binding protein, is implicated in the transition to flowering and shoot architecture in plant development. However, the roles of TFL1 homologs is little known in wheat development. CRISPR/Cas9-mediated targeted mutagenesis was used in this study to generate a set of wheat (Fielder) mutants with single, double or triple-null tatfl1-5 alleles. The wheat tatfl1-5 mutations decreased the tiller number per plant in the vegetative growth stage and the effective tiller number per plant and spikelet number per spike at maturity in the field. RNA-seq analysis showed that the expression of the auxin signaling-related and cytokinin signaling-related genes was significantly changed in the axillary buds of tatfl1-5 mutant seedlings. The results suggested that wheat TaTFL1-5s were implicated in tiller regulation by auxin and cytokinin signaling.

Genetic mutation profiles for children with congenital hypothyroidism in Fujian province

Objective: To explore the mutation characteristics of pathogenic genes in children with congenital hypothyroidism (CH) in Fujian. Methods: The clinical data of 116 unrelated CH children diagnosed in Fujian Provincial Maternal and Child Health Hospital from January 2019 to September 2020 were retrospectively analyzed, including 50 females and 66 males, with an average age of (20±10) days at diagnosis. Targeted exome sequencing technology was used to detect the mutation frequency, type and distribution characteristics of 29 genes related to thyroxine synthesis or thyroid development. Results: Three hundred and fifty-one potential functional mutations were detected in 105 of 116 CH patients, with a detection rate of 90.5% (105/116). DUOX2 (66.4%, 77/116) was the most frequent mutated gene, followed by TG (23.3%, 27/116), DUOXA1 (23.3%, 27/116), and TPO (12.1%, 14/116), which were all involved in thyroid hormone synthesis. Among the 105 children with CH, 70 cases carried double allele mutation. Except for 3 cases of thyroid dysplasia related genes (2 cases of TSHR and 1 case of GLIS3), the rest were also related to thyroid hormone synthesis. The gene with the highest carrier rate was DUOX2 (68.8%, 59/70), followed by TG (8.6%, 6/70), TPO (4.3%, 3/70), DUOXA2 (1.4%, 1/70) and DUOXA1 (1.4%, 1/70). Conclusion: The main mutated genes in CH children in Fujian are the key genes involved in thyroid hormone synthesis, such as DUOX2, TG and TPO.

Interaction of novel proteins, centrin4 and protein of centriole in Leishmania parasite and their effects on the parasite growth

Centrins are cytoskeletal proteins associated with the centrosomes or basal bodies in the eukaryotes. We previously reported the involvement of Centrin 1–3 proteins in cell division in the protozoan parasites Leishmania donovani and Trypanosoma brucei. Centrin4 and 5, unique to such parasites, had never been characterized in Leishmania parasite. In the current study, we addressed the function of centrin4 (LdCen4) in Leishmania. By dominant-negative study, the episomal expression of C-terminal truncated LdCen4 in the parasite reduced the parasite growth. LdCen4 double allele gene deletion by either homologous recombination or CRISPR-Cas9 was not successful in L. donovani. However, CRISPR-Cas9-based deletion of the homologous gene was possible in L. mexicana, which attenuated the parasite growth in vitro, but not ex vivo in the macrophages. LdCen4 also interacts with endogenous and overexpressed LdPOC protein, a homolog of centrin reacting human POC (protein of centriole) in a calcium sensitive manner. LdCen4 and LdPOC binding has also been confirmed through in silico analysis by protein structural docking and validated by co-immunoprecipitation. By immunofluorescence studies, we found that both the proteins share a common localization at the basal bodies. Thus, for the first time, this article describes novel centrin4 and its binding protein in the protozoan parasites.

Genetic Polymorphism of 38 Y-chromosome Short Tandem Repeats in Beijing Han Population from China

Abstract Objective: To investigate 38 Y-chromosome short tandem repeat (Y-STR) genetic polymorphisms in Beijing Han and analyze the genetic distance with neighboring or linguistically similar populations. Materials and Methods: In the study, we selected 531 unrelated male individuals of Beijing Han, and the results were statistically analyzed by testing with GSTAR™ 41Y reagents. Results: The allele peak heights were balanced among the Y loci, the amplified fragment ranged from 100 to 500 bps. A total of 531 haplotypes were detected in 531 samples. Eight null genotypes were observed on locus DYS448. One and three double alleles were observed on single-copy locus DYS576 and DYS19, respectively. DYS385 a/b, DYF387S1 a/b, and DYS527 a/b were more common in double copies, but 3, 13, and 11 triple alleles were detected, respectively. The gene diversity values of Y-STRs except DYS391, DYS438, and DYS645 were >0.5. Twenty-seven Y-STRs of Beijing Han population were selected for genetic distance comparison with 17 populations including Changchun Han, with Rst values ranging from 0.0002 to 0.1703. Conclusion: The 38 Y-STRs in this study have strong male lineage identification ability and have great potential for individual identification, kinship identification, Y-STR database construction, and genetic relationship research.

TP53 Gene Deletion or Mutation in Newly Diagnosed Multiple Myeloma Patients By FISH with Cytoscan Arrays and Targeted Panel Sequencing

Background Multiple myeloma (MM) is a plasma cell clonal malignant disease. In the past 20 years, the progression free survival rate and overall survival rate of patients have been significantly improved, however, due to the potential complex cytogenetic abnormalities, the clinical progresses are heterogeneous and the clinical benefits of patients are not consistent, especially for high-risk patients. In Chinese clinical practice, 17p-, t(4;14), t(14;16) are considered high-risk cytological abnormalities for newly diagnosed multiple myeloma (NDMM) patients according to R-ISS stage and 2022 Chinese guidelines for diagnose and treatment for MM. As a genome guardian, TP53 plays an important role in regulating cell cycle, apoptosis, aging, DNA repair and maintaining gene stability. But the threshold of deletion positive proportion with clinical significance by CD138-sorted FISH was unclear. TP53 mutation is also not regularly examined in Chinese patients. For that aim, our center has taken TP53 seriously as a heterogeneity problem for clinical discussion. Methods The clinical data of 248 NDMM patients in the First Affiliated Hospital of Soochow University of a VRD clinical study, with VRD in combination with autologous stem cell transplantation or VRD treatment for 8 cycles, from September 1, 2018 to August 31, 2021, were analyzed retrospectively. The samples for CD138-sorted FISH, CytoScan and targeted panel sequencing are obtained from the Multiple Myeloma Specialized Disease Bank of the National Center for Clinical Medical Research of Hematological Diseases. Results In this study, the deletion rate of TP53 was 13.0% (32/248), the mutation rate was 2.0% (5/248), the incidence of any TP53 event was 14.5% (36/248), and the incidence of double allele event was 0.8% (2/248). The detection rate of TP53 gene mutation was low, which was related to the short clinical application time of targeted panel sequencing with CD138-sorted samples. All patients with TP53 deletion or mutation were DS-III stage, with anemia (72.2%), hypoalbuminemia (58.3%) and renal insufficiency (38.9%). The most common accompanied abnormalities were 13q14- (63.9%), followed by t (4;14), 1q21+, t (11;14). Abnormal karyotypes were detected by FISH and CytoScan, indicating gene complexity and instability, and the clonal evolution, which is the root of disease recurrence and progression. The positive rate of TP53 mutation of one case study increased from 7.87% at the first recurrence to 53.44% at the second recurrence with 3rd line therapy (Figure 1a), indicating that TP53 gene lead to disease progression and the formation of multidrug resistance. In terms of induction effects, patients with TP53 gene abnormalities had ≥VGPR with 77.8%, lower than that of negative group (84.8%, P=0.0627)(Figure 1b). The median follow-up time is 20 months (1-42 months). Compared with negative ones, TP53 positive subgroup had an adverse PFS result (33 months vs not reached, P=0.0403), while the OS difference hasn't been clinical significant (both not reached, P=0.0541) (Figure 1c). The result showed that TP53 deletion with more than 50% was an independent adverse factor associated with poor prognosis of NDMM patients (PFS: HR=2.2441, 95%CI 1.117-5.335, P=0.0025; OS: HR=2.771, 95%CI 1.006-7.629, P=0.049). The prognosis of patients with TP53 deletion more than 50% was significantly worse than that of the negative group (PFS 25months vs not reached, P=0.0032, OS not reached P=0.0198), but TP53 deletion less than 50% had no difference compared with negative group. (Figure 1d).Conclusions Patients with TP53 deletion or mutation have obvious clinical symptoms and are prone to cytogenetic abnormalities such as 1q+, 1p-, -13, IgH translocation, chromosome abnormality and so on. TP53 deletion with more than 50% was an independent adverse factor associated with poor prognosis of NDMM patients, as a result, TP53 deletion more than 50% is the clinical significant threshold for clinical use. This study comprehensively describes the detection of genetic abnormalities and prognostic survival analysis of NDMM patients by using FISH, CytoScan and targeted panel sequencing. Identifying the real high-risk patients, we can earlier take targeted and more active measures, such as intensive induction treatment, tandem transplantation or CART treatment, to better deepen and consolidate the therapeutic effect for long-term survival benefits of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mixed tuberculosis infections in Northwest of Iran.

Primary Mycobacterium tuberculosis (MTB) infection doesn't provide protection against secondary infection and patients can suffer from multiple strains of M. tuberculosis simultaneously. The aim of this study was to use molecular genotyping to identify cases of mixed infection in Northwest of Iran. One hundred and twenty-one positive culture isolates of M. tuberculosis were prepared from patients consecutively in Northwest of Iran from March 2017 to March 2018 and then microevolution and mix infection were assessed using the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method. MIRU-VNTR analysis revealed that nine samples (7.3%) had double alleles in at least one locus, as observed by five cases of microevolution, and four cases (3.3%) of mixed infection. According to this study, mixed infection in Northwest of Iran has significantly decreased compared to 13 years ago (7.1% decreased to 3.3%), and in order to eradicate tuberculosis it is necessary to identify all cases of mixed infection, at least in recurrent cases, in the future.

Prevalence of nine genetic defects in Chinese Holstein cattle.

Worldwide use of elite sires has caused inbreeding accumulation and high frequencies of genetic defects in dairy cattle populations. In recent years, several genetic defect genes or haplotypes have been identified in Holstein cattle. A rapid and reliable microfluidic chip with Kompetitive allele‐specific PCR (KASP) assay was developed in our previous study for the detection of heterozygotes at eight genetic defect loci of bovine leukocyte adhesion deficiency (BLAD), Brachyspina syndrome (BS), complex vertebral malformation (CVM), Holstein haplotype 1 (HH1), Holstein haplotype 3 (HH3), Holstein haplotype 4 (HH4), Holstein haplotype 5 (HH5) and haplotype for cholesterol deficiency (HCD). This study aimed to extend that assay to include a newly identified genetic defect of Holstein haplotype 6 (HH6) and to estimate the frequencies of carriers for each of the nine genetic defects in six Chinese Holstein herds. Of the 1633 cows, carrier frequencies of the genetic defects were 6.92%, 5.76%, 4.46%, 4.30%, 3.62%, 2.94%, 1.86% and 0.37% for HH1, HH3, CVM, HH5, HCD, BS, HH6 and BLAD, respectively. No carrier was found for HH4. Notably, 27.43% of cows carried at least one genetic defect, while 2.27% and 0.12% of cows carried double and triple genetic defect alleles, respectively. The existence of genetic defects calls for routine molecular testing and effective management of genetic defects by avoiding carrier‐to‐carrier mating in production herds and eliminating or at least reducing the frequency of the defective alleles through marker‐assisted selection in breeding herds.

Generation and identification of a conditional knockout allele for the PSMD11 gene in mice

BackgroundOur previous study have shown that the PSMD11 protein was an important survival factor for cancer cells except for its key role in regulation of assembly and activity of the 26S proteasome. To further investigate the role of PSMD11 in carcinogenesis, we constructed a conditional exon 5 floxed allele of PSMD11 (PSMD11flx) in mice.ResultsIt was found that homozygous PSMD11 flx/flx mice showed normal and exhibited a normal life span and fertility, and showed roughly equivalent expression of PSMD11 in various tissues, suggesting that the floxed allele maintained the wild-type function. Cre recombinase could induce efficient knockout of the floxed PSMD11 allele both in vitro and in vivo. Mice with constitutive single allele deletion of PSMD11 derived from intercrossing between PSMD11flx/flx and CMV-Cre mice were all viable and fertile, and showed apparent growth retardation, suggesting that PSMD11 played a significant role in the development of mice pre- or postnatally. No whole-body PSMD11 deficient embryos (PSMD11−/−) were identified in E7.5–8.5 embryos in uteros, indicating that double allele knockout of PSMD11 leads to early embryonic lethality. To avoid embryonic lethality produced by whole-body PSMD11 deletion, we further developed conditional PSMD11 global knockout mice with genotype Flp;FSF-R26CAG − CreERT2/+; PSMD11flx/flx, and demonstrated that PSMD11 could be depleted in a temporal and tissue-specific manner. Meanwhile, it was found that depletion of PSMD11 could induce massive apoptosis in MEFs.ConclusionsIn summary, our data demonstrated that we have successfully generated a conditional knockout allele of PSMD11 in mice, and found that PSMD11 played a key role in early and postnatal development in mice, the PSMD11 flx/flx mice will be an invaluable tool to explore the functions of PSMD11 in development and diseases.

Genome sequence of Hydrangea macrophylla and its application in analysis of the double flower phenotype.

Owing to its high ornamental value, the double flower phenotype of hydrangea (Hydrangea macrophylla) is one of its most important traits. In this study, genome sequence information was obtained to explore effective DNA markers and the causative genes for double flower production in hydrangea. Single-molecule real-time sequencing data followed by a Hi-C analysis were employed. Two haplotype-phased sequences were obtained from the heterozygous genome of hydrangea. One assembly consisted of 3,779 scaffolds (2.256 Gb in length and N50 of 1.5 Mb), the other also contained 3,779 scaffolds (2.227 Gb in length, and N50 of 1.4 Mb). A total of 36,930 genes were predicted in the sequences, of which 32,205 and 32,222 were found in each haplotype. A pair of 18 pseudomolecules was constructed along with a high-density single-nucleotide polymorphism (SNP) genetic linkage map. Using the genome sequence data, and two F2 populations, the SNPs linked to double flower loci (djo and dsu) were discovered. DNA markers linked to djo and dsu were developed, and these could distinguish the recessive double flower allele for each locus, respectively. The LEAFY gene is a very likely candidate as the causative gene for dsu, since frameshift was specifically observed in the double flower accession with dsu.

Mutational analysis of the phosphorylation sites of the Leishmania mexicana kinesin homologue LmxKin29

Kinesins are motor proteins, have been identified in a wide diversity of eukaryotes, from protists to mammals. The kinetoplastids’ genome sequences have also shown the presence of a large number of kinesins. The current study presents characterise a novel L.mexicana kinesin LmxM29.3.050, thought to be associated with flagellum formation. It was found express in both the amastigote and promastigote life stages. In this study we assigned this protein as a kinesin homologue to the unknown or orphan kinesins superfamily (1). The biochemical analysis showed the MAP kinase homologue LmxMPK3 can phosphorylate full length of LmxKin29 at serine 554. Localisation studies using GFP-tagged LmxKin29 revealed that it is predominantly found in between the nucleus and the flagellar pocket, while in dividing cells LmxKin29 was found at the anterior and posterior ends of the cells. Hence, LmxKin29 might play a role in cytokinesis. Double allele deletion was successfully generated. Morphological analysis of promastigotes with LmxKin29 tagged with GFP displayed no obvious phenotypic differences comparing the mutants with wild type cells.

Dissection of novel candidate genes for grain texture in Russian wheat varieties.

Wheat grain texture is a complex trait. The crucial role of puroindoline genes in grain texture determination was confirmed. New loci, highly associated with the trait, were identified and dissected. Grain texture is an important milling characteristic of common wheat. Previously, it was shown that the Ha locus on 5DS, containing the puroindoline-a, puroindoline-b and GSP-1 genes, has a crucial role in endosperm texture. However, some other loci were detected on different chromosomes, but no specific genes were proposed to be involved in grain hardness organization. In this study, we used a panel of Russian spring wheat varieties. The grain texture of varieties ranged from soft to hard. Analysis of the flour particle size and flour particle specific surface of wheat varieties cultivated in different environments demonstrated high heritability (0.79–0.8) and strong repeatability of the traits. Genotyping of wheat varieties with allele-specific markers for the puroindoline-a and puroindoline-b genes demonstrated that 25% of the studied varieties carried rare allele Pin-D1k, which lacked both Pina and Pinb (double null allele). Our results confirmed the strong impact of this allele on grain texture. Association analysis with the use of SNP genotyping (Illumina 15 K Wheat) and 2-year phenotyping confirmed the key role played by puroindoline genes (5D) in determining the grain texture of Russian wheat varieties. The analysis also detected significant SNPs on the 1B, 3A, 5B, 6A, 6D, 7B, 7D chromosomes. The best candidate loci for the grain texture were located on chromosomes 5B and 7B (FDR ≤ 0.05). We dissected a number of candidate genes and proposed a possible mechanism for their contribution to endosperm texture determination. These genes are involved in the metabolism of galactolipids (DGDG) and carbohydrates (1,3-β-glucan).

Efficient generation of GHR knockout Bama minipig fibroblast cells using CRISPR/Cas9-mediated gene editing.

Dwarfism, also known as growth hormone deficiency (GHD), is a disease caused by genetic mutations that result in either a lack of growth hormone or insufficient secretion of growth hormone, resulting in a person's inability to grow normally. In the past, many studies focusing on GHD have made use of models of other diseases such as metabolic or infectious diseases. A viable GHD specific model system has not been used previously, thus limiting the interpretation of GHD results. The Bama minipig is unique to Guangxi province and has strong adaptability and disease resistance, and an incredibly short stature, which is especially important for the study of GHD. In addition, studies of GHR knockout Bama minipigs and GHR knockout Bama minipig fibroblast cells generated using CRISPR/Cas9 have not been previously reported. Therefore, the Bama minipig was selected as an animal model and as a tool for the study of GHD in this work. In this study, a Cas9 plasmid with sgRNA targeting the first exon of the GHR gene was transfected into Bama minipig kidney fibroblast cells to generate 22 GHR knockout Bama minipig kidney fibroblast cell lines (12 male monoclonal cells and 10 female monoclonal cells). After culture and identification, 11 of the 12 male clone cell lines showed double allele mutations, and the rate of positive alteration of GHR was 91.67%. Diallelic mutation of the target sequence occurred in 10 female clonal cell lines, with an effective positive mutation rate of 100%. Our experimental results not only showed that CRISPR/Cas9 could efficiently be used for gene editing in Bama minipig cells but also identified a highly efficient target site for the generation of a GHR knockout in other porcine models. Thus, the generation of GHR knockout male and female Bama fibroblast cells could lay a foundation for the birth of a future dwarfism model pig. We anticipate that the "mini" Bama minipig will be of improved use for biomedical and agricultural scientific research and for furthering our understanding of the genetic underpinnings of GHD.

Enzymatic and molecular characterization of insecticide resistance mechanisms in field populations of Aedes aegypti from Selangor, Malaysia

BackgroundDengue is a serious public health problem worldwide, including in Selangor, Malaysia. Being an important vector of dengue virus, Aedes aegypti are subjected to control measures which rely heavily on the usage of insecticides. Evidently, insecticide resistance in Ae. aegypti, which arise from several different point mutations within the voltage-gated sodium channel genes, has been documented in many countries. Thus, this robust study was conducted in all nine districts of Selangor to understand the mechanisms of resistance to various insecticides in Ae. aegypti. Mosquitoes were collected from dengue epidemic and non-dengue outbreak areas in Selangor.MethodsUsing the Center for Disease Control and Prevention (CDC) bottle assays, the insecticide resistance status of nine different Ae. aegypti strains from Selangor was accessed. Synergism tests and biochemical assays were conducted to further understand the metabolic mechanisms of insecticide resistance. Polymerase chain reaction (PCR) amplification and sequencing of the IIP-IIS6 as well as IIIS4-IIIS6 regions of the sodium channel gene were performed to enable comparisons between susceptible and resistant mosquito strains. Additionally, genomic DNA was used for allele-specific PCR (AS-PCR) genotyping of the gene to detect the presence of F1534C, V1016G and S989P mutations.ResultsAdult female Ae. aegypti from various locations were susceptible to malathion and propoxur. However, they exhibited different levels of resistance against dichlorodiphenyltrichloroethane (DDT) and pyrethroids. The results of synergism tests and biochemical assays indicated that the mixed functions of oxidases and glutathione S-transferases contributed to the DDT and pyrethroid resistance observed in the present study. Besides detecting three single kdr mutations, namely F1534C, V1016G and S989P, co-occurrence of homozygous V1016G/S989P (double allele) and F1534C/V1016G/S989P (triple allele) mutations were also found in Ae. aegypti. As per the results, the three kdr mutations had positive correlations with the expressions of resistance to DDT and pyrethroids.ConclusionsIn view of the above outcomes, it is important to seek new tools for vector management instead of merely relying on insecticides. If the latter must be used, regular monitoring of insecticide resistance should also be carried out at all dengue epidemic areas. Since the eggs of Ae. aegypti can be easily transferred from one location to another, it is probable that insecticide-resistant Ae. aegypti can be found at non-dengue outbreak sites as well.

Low expression of ACLY associates with favorable prognosis in acute myeloid leukemia

BackgroundAberrant metabolism is a hallmark of cancer cells. Recently, ATP citrate-lyase (ACLY) expression was demonstrated as an independent predictor of clinical outcome in solid tumors. However, no systematic study was conducted to explore the prognostic impact of ACLY on acute myeloid leukemia (AML).MethodsTo assess the prognostic value of ACLY transcript, we conducted a study with a discovery and validation design. We measured ACLY transcript by real-time quantitative PCR in 274 AML patients, and validated the prognostic value in the two independent cohorts using published data. Meta-analysis of gene-expression profile and inhibition ACLY expression in leukemia cell lines were conducted to help us understand the biological insight of low ACLY expression.ResultsLow ACLY expression is less common amongst AMLs with DNMT3A mutations, but coexisted in double allele CEBPA mutations. Moreover, low ACLY expression is associated with favorable overall survival in AML patients and is involved in multiple pathways. These results are also validated in two independent cohorts of AML patients. Moreover, ACLY silencing induces proliferation arrest in THP-1 and MOLM-13 leukemia cell lines.ConclusionWe found low ACLY expression is associated with favorable overall survival in AML patients.

Diversity of Puroindoline genes and their association with kernel hardness in Chinese wheat cultivars and landraces

Kernel hardness is a key trait that greatly affects the milling and baking qualities of common wheat. Kernel hardness is largely controlled by Pina and Pinb genes. Here, we investigate the allelic diversity of Pina and Pinb genes in a collection of 107 Chinese wheat cultivars and landraces. The kernel hardness values of these accessions were determined in two field seasons (2016/2017 and 2017/2018 seasons), and they were strongly correlated between the 2 years, with hardness indexes ranging from 5.4 to 91.8. Five known Pina-D1 alleles and four known Pinb-D1 alleles were detected. In particular, we identified a novel Pina-null allele (designated Pina-D1z) with a 3382-bp deletion that can be detected by the new molecular marker Pina-N5. We also identified a Pina/Pinb double null allele containing a ~ 29-kb deletion. They are useful genotypes for kernel texture improvement. The most frequent genotypes in these accessions were Pina-D1a/Pinb-D1a and Pina-D1a/Pinb-D1b (39.3% and 34.6%, respectively), followed by Pina-D1a/Pinb-D1p (13.2%). A comparison of genotypes between the present study and previous studies suggests that Chinese wheat landraces have more allelic variations in the Pina gene than do cultivars. An association analysis of Puroindolines with kernel hardness shows that 15 Pina-D1 alleles and 6 Pinb-D1 alleles could be associated with hard-kernel phenotypes in common wheat. Notably, this study suggests that Chinese landraces are a valuable resource of genetic variability in Puroindoline genes and provides a deeper understanding of the genotype-to-phenotype association of Puroindoline genes.