Mutation Characteristics of Phenylalanine Hydroxylase Gene in Children with Phenylketonuria in Yinchuan City.

BACKGROUND: Phenylalanine hydroxylase deficiency (PAHD) is an autosomal recessive disorder affecting phenylalanine (Phe) metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene. It has a complex phenotype with many variants and genotypes in various populations. This study sets out to analyze the screening results of children with phenylketonuria (PKU) in Yinchuan City and characterize the mutation variants of the PAH gene. METHODS: Phenylketonuria screening results were retrospectively analyzed in 398,605 neonates (207,361 males and 191,244 females) born in different maternity hospitals in Yinchuan City between January 2017 and December 2021. Screening for genetic metabolic diseases was performed with parental consent at their own expense. A comprehensive diagnosis was performed by integrating tandem mass spectrometry (MS/MS) findings with clinical presentations. High-throughput sequencing (HTS) was used to detect genetic and metabolic disease-associated genes in children with PKU who were clinically diagnosed and voluntarily tested. The identified loci were validated through Sanger sequencing and parental verification. RESULTS: Among the screened newborns, 45 (11.3/100,000) PKU cases were diagnosed. In the 38 cases that underwent self-financed PAH sequencing, 56 mutations were detected in 76 chromosomes, with an overall detection rate of 73.7%. All patients harbored mutant genes, and the 56 mutations detected identified represented 14 variants, including 8 missense mutations, 2 splicing mutations, 2 nonsense mutations, and 2 silent mutations. The mutations were primarily distributed in exons 2, 3, 6, 7, 9, 11, and intron 4, with the highest frequency observed in exon 7 (25 [44.7%]), followed by exon 11 (15 [26.7%]). The most prevalent mutations were exon 7-p.R252W (10 [17.9%]) and exon 7-p.R261Q (8 [14.3%]). CONCLUSIONS: The PAH gene mutations in children with PKU in Yinchuan City are predominantly concentrated in exons 6, 7, and 11, with the highest detection rates observed for p.R252W and p.R261Q mutations.

Diagnostic Value and High-Risk Factors of Two-Dimensional Ultrasonography Combined with Four-Dimensional Ultrasonography in Prenatal Ultrasound Screening of Fetal Congenital Malformations.

Objective: This study mainly analyzes the diagnostic value of two-dimensional ultrasonography (2D-US) combined with four-dimensional ultrasonography (4D-US) in prenatal ultrasound screening of fetal congenital malformations (CMs) and explores the high-risk factors affecting fetal malformations. Methods: The clinical and imaging data of 2247 pregnant women who underwent prenatal fetal malformation screening in the General Hospital of Ningxia Medical University between February 2020 and October 2021 were collected and analyzed, retrospectively. All pregnant women underwent 2D-US, and those with suspected fetal malformations were further inspected by 4D-US. The accuracy of ultrasound examination results relative to actual pregnancy outcomes was analyzed, taking the neonatal malformation after induced labor or actual delivery as the gold standard, and the risk factors influencing the occurrence of fetal malformations were discussed. Results: A total of 87 cases (3.87%) of fetal malformations were detected out of the 2247 parturients examined. The accuracy, sensitivity, and specificity of 2D-US diagnosis were 81.40%, 43.68%, and 82.92%, respectively, while the data were 83.67%, 51.72%, and 84.95% for 4D-US, respectively, and 93.59%, 90.80%, and 93.70%, respectively, for 2D-US +4D-US. The combined diagnosis of 2D-US +4D-US achieved statistically higher accuracy, sensitivity, and specificity than either of them alone. One-way analysis of variance and multivariate logistic regression analysis identified that the independent risk factors affecting fetal malformation were age ≥ 35, history of adverse pregnancy and childbirth, medication during pregnancy, toxic exposure during pregnancy, and history of seropositive for TORCH-IgM. Folic acid supplementation was a protective factor. Conclusions: Prenatal US is an effective approach for screening fetal malformations. 2D-US +4D-US can effectively improve the diagnostic rate of fetal malformations. For pregnant women with high-risk factors, prevention should be given priority, and prenatal screening and prenatal diagnosis should be standardized to reduce the occurrence of fetal malformations.

Identification of novel deep intronic PAH gene variants in patients diagnosed with phenylketonuria.

Phenylketonuria (PKU) is caused by phenylalanine hydroxylase (PAH) gene variants. Previously, 94.21% of variants were identified using Sanger sequencing and multiplex ligation-dependent probe amplification. To investigate the remaining variants, we performed whole-genome sequencing for four patients with PKU and unknown genotypes to identify deep intronic or structural variants. We identified three novel heterozygous variants (c.706+368T>C, c.1065+241C>A, and c.1199+502A>T) in a deep PAH gene intron. We detected a c.1199+502A>T variant in 60% (6/10) of PKU patients with genetically undetermined PKU. In silico predictions indicated that the three deep variants may impact splice site selection and result in the inclusion of a pseudo-exon. A c.1199+502A>T PAH minigene and reverse transcription PCR (RT-PCR) on blood RNA from a PKU patient with biallelic variants c.1199+502A>T and c.1199G>A confirmed that the c.1199+502A>T variant may strengthen the predicted branch point and leads to the inclusion of a 25-nt pseudo-exon in the PAH mRNA. Reverse transcription polymerase chain reaction (RT-PCR) on the minigene revealed that c.706+368T>C may create an SRSF2 (SC35) binding site via a 313-nt pseudo-exon, whereas c.1065+241C>A may produce an 81-nt pseudo-exon that strengthens the predicted SRSF1 (SF2/ASF) binding site. These results augment current knowledge of PAH genotypes and show that deep intronic analysis of PAH can genetically diagnose PKU.

[Gene variant analysis of a fetus with autosomal recessive polycystic kidney disease].

OBJECTIVE: To explore the genetic basis for a fetus with autosomal recessive polycystic kidney disease (ARPKD). METHODS: Fetal tissue and peripheral blood samples were respectively obtained from the abortus and the couple. Following extraction of genomic DNA, genetic testing was carried out. RESULTS: The fetus was found to carry compound heterozygous variants of the PKHD1 gene, namely c.5336A>T (p.N1779I) and c.9455delA (p.N3152Tfs*10), which were respectively inherited from the husband and wife. CONCLUSION: The c.5336A>T and c.9455delA variants of the PKHD1 gene probably account for the ARPKD in the fetus. Above results have enabled genetic counseling and prenatal diagnosis for the couple.

Role of bioengineering and laborers in integration of farmland resources toward to improve dimension of sustainable agriculture in China.

Farmland transfer is one of the essential approaches for achieving large-scale farming and its management affects productive efficiency, environment pollution and food sustainable security supply. Present study was carried out investigation based representative agricultural development area Guanzhong Plain of Shaanxi, aimed at explore the role of biotechnology and laborers in integration of farmland toward to improve sustainable agriculture in rural China by employed the profit and Tobit models evaluation. The conclusion demonstrated that labor's and agricultural management model as main stay, intensive farming has positive effect-based economic and environmental benefits than fragmentation management, female laborers have weaker effect on farmland renting-out behavior among smallholders while male laborers were superior promoters in increasing the area of rented-in farmland and farm scale. Finally, bioengineering development and agricultural intensification management as a rational choice that has great potential value for large-scale cultivation that contributing a promising future for achieving cleaner production, environment and human health further providing huge economic and social and environmental benefits in sustainability agriculture. Additionally, government policies require intensive intervention to accelerate large-scale management and biotechnology implementation.Abbreviation: Aaflf: Average age of female labor force; Incom(log): Log of annual household income; Noflf: Number of women in the labor force; Nooaf: Number of old adults in family; NTFs: non-transfer families; OLS: ordinary least square; Palff: Proportion of agricultural laborers in the female labor force; Palmf: Proportion of agricultural laborers in the male labor force; RIFs: rented-in families; ROFs: rented-out families; Whhf: Whether the household head is female.

Formononetin inhibits tumor growth by suppression of EGFR-Akt-Mcl-1 axis in non-small cell lung cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) activating mutations play crucial roles in the tumorigenesis of human non-small cell lung cancer (NSCLC). The mechanism regarding how EGFR signaling regulates myeloid cell leukemia sequence 1 (Mcl-1) protein stability and ubiquitination remains undefined. METHODS: MTS assay was used for natural product library screening. The effect of formononetin (Formo) on NSCLC cells was determined by MTS assay and soft agar assay. Molecular modeling was performed to analyze the potential different binding modes between Formo and EGFR WT or mutants. Mcl-1 protein level and the inhibitory effect of Formo on EGFR signaling were examined by immunoblot, in vitro kinase assay, in vitro pulldown and ATP competition assays, co-immunoprecipitation assay, ubiquitination analysis, in vivo xenograft model, and immunohistochemical staining. RESULTS: Formo was identified as an EGFR inhibitor by a 98 commercially available natural product screening. Formo suppresses WT and mutant EGFR kinases activity in vitro, ex vivo, and in vivo. Molecular modeling indicates that Formo docks into the ATP-binding pocket of both WT and mutant EGFR. Formo inhibits EGFR-Akt signaling, which in turn activates GSK3ß and promotes Mcl-1 phosphorylation in NSCLC cells. Treatment with Formo enhances the interaction between Mcl-1 and SCFFbw7, which eventually promotes Mcl-1 ubiquitination and degradation. Depletion of either GSK3ß or SCFFbw7 compromised Formo-induced Mcl-1 downregulation. Finally, Formo inhibits the in vivo tumor growth in a xenograft mouse model. CONCLUSION: This study highlights the importance of promoting ubiquitination-dependent Mcl-1 turnover might be an alternative strategy to enhance the anti-tumor efficacy of EGFR-TKI.

Repression of Hexokinases II-Mediated Glycolysis Contributes to Piperlongumine-Induced Tumor Suppression in Non-Small Cell Lung Cancer Cells.

Deregulation of glycolysis is a common phenomenon in human non-small cell lung cancer (NSCLC). In the present study, we reported the natural compound, piperlongumine, has a profound anti-tumor effect on NSCLC via regulation of glycolysis. Piperlongumine suppressed the proliferation, colony formation and HK2-mediated glycolysis in NSCLC cells. We demonstrated that exposure to piperlongumine disrupted the interaction between HK2 and VDAC1, induced the activation of the intrinsic apoptosis signaling pathway. Moreover, our results revealed that piperlongumine down-regulated the Akt signaling, exogenous overexpression of constitutively activated Akt1 in HCC827 and H1975 cells significantly rescued piperlongumine-induced glycolysis suppression and apoptosis. The xenograft mouse model data demonstrated the pivotal role of suppression of Akt activation and HK2-mediated glycolysis in mediating the in vivo antitumor effects of piperlongumine. The expression of HK2 was higher in malignant NSCLC tissues than that of the paired adjacent tissues, and was positively correlated with poor survival time. Our results suggest that HK2 could be used as a potential predictor of survival and targeting HK2 appears to be a new approach for clinical NSCLC prevention or treatment.

Butein suppresses hepatocellular carcinoma growth via modulating Aurora B kinase activity.

Aurora B is aberrantly expressed in various tumors and shown to be a promising target for cancer therapy. Butein, a chalcone isolated from Rhus cerniciflua, has demonstrated antitumor activities in different cancers. In this study, we aimed to validate whether Aurora B kinase was the direct target of butein to exhibit its potency in hepatocellular carcinoma (HCC). Comparing with the normal cell line and tissue, Aurora B was overexpressed in all tested HCC cells and the majority of tumor tissue. Knocking down of Aurora B with shRNA substantially inhibited HCC cell proliferation, colony formation and delayed tumor growth in nude mice. Except computer docking, a series of kinase assays revealed butein directly interacted with Aurora B and inhibited its kinase activity. Along with the decrease of Aurora B and histone H3 phosphorylation, HCC cells were induced G2/M cell cycle arrest and subjected to cell apoptosis. Butein-mediated antitumor activities were substantially impaired in Aurora B knockdown cells, suggesting Aurora B was an important target of butein in HCC. Oral administration of butein substantially restrained HCC xenograft growth and the expressions of Ki67 and phosphor-histone H3 were significantly decreased in butein-treated tissue. To the best of our knowledge, our studies revealed that Aurora B was the direct target of butein in HCC.

Repression of Noxa by Bmi1 contributes to deguelin-induced apoptosis in non-small cell lung cancer cells.

Deguelin, a natural rotenoid isolated from several plants, has been reported to exert anti-tumour effects in various cancers. However, the molecular mechanism of this regulation remains to be fully elucidated. Here, we found that deguelin inhibited the growth of non-small cell lung cancer (NSCLC) cells both in vitro and in vivo by downregulation of Bmi1 expression. Our data showed that Bmi1 is highly expressed in human NSCLC tissues and cell lines. Knockdown of Bmi1 significantly suppressed NSCLC cell proliferation and colony formation. Deguelin treatment attenuated the binding activity of Bmi1 to the Noxa promoter, thus resulting in Noxa transcription and apoptosis activation. Knockdown of Bmi1 promoted Noxa expression and enhanced deguelin-induced apoptosis, whereas overexpression of Bmi1 down-regulated Noxa protein level and deguelin-induced apoptosis. Overall, our study demonstrated a novel apoptotic mechanism for deguelin to exert its anti-tumour activity in NSCLC cells.

[Clinical and genetic features of ring chromosome 13 syndrome: an analysis of one case].

A girl aged 5 months was admitted due to developmental delay. Physical examination showed delayed physical development, unusual facies (microcephalus, hypertelorism, low-set ears, wide nasal bridge, and short philtrum), and an absence of the labium minus at one side. The peripheral blood karyotype was 46,XX,r(13)(p11q33)[82]/45,XX,-13[10]/46,XX,r(13;13)(p11q33;p11q33)[8], and array-based comparative genomic hybridization showed an 87.5 Mb duplication in 13q11q33.2 region and an 8.2 Mb deletion in 13q33.2q34 region. Fluorescence in situ hybridization showed terminal depletion of the long arm of the ring chromosome 13. The girl was diagnosed with ring 13 syndrome. This syndrome has various clinical phenotypes and is closely associated with the amount and site of the loss of genetic material in chromosomal band and different rates of chimerism.

Deguelin attenuates non-small cell lung cancer cell metastasis through inhibiting the CtsZ/FAK signaling pathway.

Lung cancer is the leading cause of cancer-related death among both men and women every year, mainly due to metastasis. Although natural compound deguelin has been reported to inhibited cell migration and invasion in various cancer cells, the details of this regulation progress remain to be fully elucidated. In this study, we investigated the underlying mechanism of deguelin-suppressed metastasis of non-small cell lung cancer (NSCLC) cells. Our results demonstrate that deguelin inhibits NSCLC cell migration, invasion, and metastasis both in vitro and in vivo. These inhibitory effects of deguelin were mediated by suppressing of Cathepsin Z (CtsZ) expression and interrupting the interaction of CtsZ with integrin ß3. Moreover, deguelin inhibits the activation of CtsZ downstream FAK/Src/Paxillin signaling. Knockdown of CtsZ mimicked the effect of deguelin on NSCLC cells migration and invasion. Our study reveals that deguelin exerts its anti-metastatic effect both in vitro and in vivo is partly dependent on the suppression of CtsZ signaling. Deguelin would be a potential anti-metastasis agent against NSCLC.

Genome sequence of Anopheles sinensis provides insight into genetics basis of mosquito competence for malaria parasites.

BACKGROUND: Anopheles sinensis is an important mosquito vector of Plasmodium vivax, which is the most frequent and widely distributed cause of recurring malaria throughout Asia, and particularly in China, Korea, and Japan. RESULTS: We performed 454 next-generation sequencing and obtained a draft sequence of A. sinensis assembled into scaffolds spanning 220.8 million base pairs. Analysis of this genome sequence, we observed expansion and contraction of several immune-related gene families in anopheline relative to culicine mosquito species. These differences suggest that species-specific immune responses to Plasmodium invasion underpin the biological differences in susceptibility to Plasmodium infection that characterize these two mosquito subfamilies. CONCLUSIONS: The A. sinensis genome produced in this study, provides an important resource for analyzing the genetic basis of susceptibility and resistance of mosquitoes to Plasmodium parasites research which will ultimately facilitate the design of urgently needed interventions against this debilitating mosquito-borne disease.

Functional characterization of an arrestin gene on insecticide resistance of Culex pipiens pallens.

BACKGROUND: Continuous and excessive application of insecticides has resulted in the rapid development of insecticide resistance in several mosquito species, including Culex pipiens pallens. Previous studies in our laboratory found that arrestin gene expression was higher in the deltamethrin-resistant (DR) strain than in the deltamethrin-susceptible (DS) strain of Cx. pipiens pallens. Similarly, other studies reported that arrestin was highly expressed in permethrin-resistant Cx. quinquefasciatus and in dichlorodiphenyltrichloroethane (DDT)-resistant Drosophila melanogaster. METHODS: Full-length cDNAs of an arrestin gene were cloned from Cx. pipiens pallens via polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE). The mRNA levels of the arrestin gene in the whole life cycle of DR and DS strains of Cx. pipiens pallens were investigated via quantitative real-time PCR. In addition, the relationship between arrestin and deltamethrin (DM) resistance were identified using genetic overexpression strategies and arrestin RNAi in mosquito cells. Cell viability was analyzed with cholecystokinin octapeptide after DM treatment. Moreover, the mRNA levels of cytochrome P450 6A1 (CYP6A1) and opsin in the transfected cells and controls were analyzed. RESULTS: Complete arrestin gene sequence was cloned and expressed throughout the life cycle of Cx. pipiens pallens. Moreover, arrestin was significantly upregulated in the DR strain, compared with that in the DS strain at the egg, pupae, and adult stages. Arrestin overexpression comparably increased the mosquito cell viability, whereas arrestin knockdown by siRNA decreased mosquito cell viability with deltamethrin (DM) treatment. Meanwhile, the mRNA levels of CYP6A1 and opsin were upregulated in mosquito cells transfected with arrestin and downregulated in mosquito cells with arrestin knockdown. CONCLUSION: This study presented the first evidence that arrestin might be associated with insecticide resistance in Cx. pipiens pallens.

Cloning and characterization of prophenoloxidase A3 (proPOA3) from Culex pipiens pallens.

The prophenoloxidase subunit A3 (proPOA3) gene was cloned from Culex pipiens pallens, which had an open reading frame of 2061 bp encoding a putative 686 amino acid protein. The deduced amino acid sequence shares 98% with proPOA3 from Culex quinquefasciatus. ProPOA3 is expressed at all developmental stages of C. pipiens pallens. Significant negative correlation was observed between proPOA3 expression and deltamethrin resistance in resistant C. pipiens pallens. Furthermore, proPOA3 expression levels were significantly lower in deltamethrin-resistant mosquitoes than in susceptible mosquitoes collected at four locations in Eastern China. However, we did not find any substantial change in proPOA3 expression in field-collected resistant Anopheles mosquitoes. Moreover, overexpressing proPOA3 in C6/36 cells led to more sensitivity to deltamethrin treatment. In laboratory and field-collected resistant C. pipiens pallens, a valine to isoleucine mutation (769G>A) and two synonymous mutations (1116G>C and 1116G>A) were identified in proPOA3. In addition, the mutation frequency of 769G>A and 1116G>C increased gradually, which corresponded with raised deltamethrin resistance levels. Taken together, our study provides the first evidence that proPOA3 may play a role in the regulation of deltamethrin-resistance in C. pipiens pallens.

Transient transfection of Cryptosporidium parvum using green fluorescent protein (GFP) as a marker.

Cryptosporidium parvum is a protozoan parasite that infects a variety of mammals. The parasite has been shown to harbor a dsRNA virus (CPV) and in the present study, we have developed a CPV transient transfection system for this parasite by using green fluorescent protein (GFP) to replace the partial gene encoding region of the larger dsRNA (CPV-L) and the smaller dsRNA (CPV-S) virus. Two viral RNA-mediated transfection vectors: pCPVL-GFP and pCPVS-GFP were successfully constructed and both in vitro transcripts were electroporated into oocysts and sporozoites. Transient expression of GFP was detected in C. parvum oocysts and excysted sporozoites by fluorescence microscopy and by RT-PCR detection of GFP mRNA and antisense RNA in transfected C. parvum oocysts. Our study provides a new approach for studying gene expression and regulation in C. parvum and will hopefully lead to the construction of a stable CPV transfection system in the future.