One-step genotyping of α-thalassaemia by multiplex symmetric PCR melting curve.

AIMS: Alpha-thalassaemia is one of the most common monogenic disorders worldwide. Due to high guanine-cytosine (GC) content and high mutation diversity in α-globin gene cluster, deletional and non-deletional mutations were usually separately detected with different methods. The aim of this study was to develop a novel one-step method for α-thalassaemia genotyping. METHODS: A multiplex symmetric PCR melting curve strategy was designed for one-step α-thalassaemia genotyping. Based on this strategy, a novel method was developed to simultaneously detect four common deletional (-α3.7 , -α4.2 , _ _SEA , --THAI ) and five common non-deletional (αCD30(-GAG)α, αCD31(G>A)α, αWSα, αQSα, αCSα) α-thalassaemia mutations in a closed-tube reaction. This method was also evaluated by double-blind detection of 235 genotype-known samples and 1630 clinical samples. RESULTS: All nine α-thalassaemia mutations could be accurately identified by this novel method within 3 hours. The evaluation results also showed a 100% concordance with comparison methods. CONCLUSIONS: This method is rapid, accurate, low-cost and easy to operate, which can be used for molecular screening and genetic diagnosis of α-thalassaemia in clinical practice. The multiplex symmetric PCR melting curve strategy designed in this study can also provide an effective approach to the method development for high GC content templates and multiple mutations.

Application of an optimized interpretation model in capillary hemoglobin electrophoresis for newborn thalassemia screening.

INTRODUCTION: Newborn screening is an important supplement to thalassemia control and prevention. Capillary electrophoresis (CE) technology has several advantages for thalassemia screening but with low sensitivity, especially for thalassemia carriers. This study aims to illustrate the application of an optimized interpretation model in newborn thalassemia screening by capillary hemoglobin electrophoresis. METHODS: Two thousand, two hundred fifty-eight neonates selected from four regions in China were enrolled and were screened for α-thalassemia and ß-thalassemia by capillary electrophoresis. Results were interpreted based on an optimized model integrated with multiple parameters. Molecular analysis was carried out in synchrony and used as the gold standard for the screening performance assessment. The consistency among different regions and thalassemia genotypes were also investigated. RESULTS: Among the 2258 neonates, 485 were identified to have a likely diagnosis of thalassemia, and 422 α-thalassemia, 80 ß-thalassemia, and 21 α/ß-thalassemia cases were confirmed by molecular analysis, including 277 α-thalassemia silent carriers, 135 α-thalassemia trait carriers, 10 Hemoglobin H disease, and 80 ß-thalassemia trait carriers. The screening sensitivity, specificity, positive, and negative predictive value for α-thalassemia and ß-thalassemia were 84.83%, 99.14%, 95.98%, 96.41%, and 88.75%, 98.73%, 76.34%, and 99.48%, respectively. The optimized interpretation model showed higher performance for thalassemia carriers, though some neonates with silent α-thalassemia genotypes (-α3.7 /αα, -α4.2 /αα, and αWS α/αα) and ß-28 /ßN genotype were still missed. The screening performance among different regions was comparable. CONCLUSIONS: Capillary hemoglobin electrophoresis with the optimized interpretation model shows reliable performance for newborn thalassemia screening. It is applicable to large-scale population screening.

Rapid detection of SARS-CoV-2 with CRISPR-Cas12a.

The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.

Chiglitazar Preferentially Regulates Gene Expression via Configuration-Restricted Binding and Phosphorylation Inhibition of PPARγ.

Type 2 diabetes mellitus is often treated with insulin-sensitizing drugs called thiazolidinediones (TZD), which improve insulin resistance and glycemic control. Despite their effectiveness in treating diabetes, these drugs provide little protection from eminent cardiovascular disease associated with diabetes. Here we demonstrate how chiglitazar, a configuration-restricted non-TZD peroxisome proliferator-activated receptor (PPAR) pan agonist with moderate transcription activity, preferentially regulates ANGPTL4 and PDK4, which are involved in glucose and lipid metabolism. CDK5-mediated phosphorylation at serine 273 (S273) is a unique regulatory mechanism reserved for PPARγ, and this event is linked to insulin resistance in type 2 diabetes mellitus. Our data demonstrates that chiglitazar modulates gene expression differently from two TZDs, rosiglitazone and pioglitazone, via its configuration-restricted binding and phosphorylation inhibition of PPARγ. Chiglitazar induced significantly greater expression of ANGPTL4 and PDK4 than rosiglitazone and pioglitazone in different cell models. These increased expressions were dependent on the phosphorylation status of PPARγ at S273. Furthermore, ChIP and AlphaScreen assays showed that phosphorylation at S273 inhibited promoter binding and cofactor recruitment by PPARγ. Based on these results, activities from pan agonist chiglitazar can be an effective part of a long-term therapeutic strategy for treating type 2 diabetes in a more balanced action among its targeted organs.

ING5 is a Tip60 cofactor that acetylates p53 in response to DNA damage.

Posttranslational modification of p53 is a critical event in regulating the expression of its target genes. p53 is acetylated at lysine 120 (K120) by acetyltranferases Tip60 (KAT5) and hMOF (KAT8) in response to DNA damage. Identification of cofactors for these two enzymes will shed light on the mechanism by which cells make a choice between cell-cycle arrest and apoptosis. It has been reported that ING5, a member of the inhibitor of growth (ING) family, is involved in p53-dependent pathways, but its exact role is unknown. In this study, we found that ING5 expression was significantly increased and that ING5 assisted Tip60, but not hMOF, in acetylating p53 at K120 in response to DNA damage. ING5 had no effect on acetylation of p53 at K373/382, but it formed a complex with p53 and Tip60. ING5 was required for acetylation of p53 at K120, and p53 acetylated at K120 subsequently bound to the promoters of its target apoptotic genes, BAX and GADD45, to promote their expression and lead to apoptosis. Mutation of K120 to K120R abolished the effects of ING5 on p53-induced gene expression. Thus, we conclude that ING5 functions as a cofactor of Tip60 in the acetylation of p53 at K120 in response to DNA damage.

Acetyl-Coenzyme A acyltransferase 2 attenuates the apoptotic effects of BNIP3 in two human cell lines.

BNIP3 is a unique pro-apoptotic protein which belongs to the BH3-only subset of the Bcl-2 family and localizes on mitochondrial membrane. Despite the inherent difficulty of identifying binding partners for membrane proteins, several binding partners for BNIP3 have been identified. In this study, a modified split-ubiquitin membrane yeast two-hybrid system was constructed and used to identify acetyl-Coenzyme A acyltransferase 2 (ACAA2) as a new BNIP3 binding partner. The interaction between BNIP3 and ACAA2 was confirmed by pull-down and co-immunoprecipitation assays. ACAA2 was also found to co-localize with BNIP3 in mitochondria. Furthermore, the apoptosis induced by over-expressed BNIP3 via transfection or hypoxia treatment was abolished by ACAA2 in human hepatocellular carcinoma HepG2 cells and osteosarcoma U-2 OS cells. These results strongly suggest that ACAA2 be a functional BNIP3 binding partner and provide a possible linkage between fatty acid metabolism and apoptosis of cells.

The orphan nuclear receptor Rev-erbbeta recruits Tip60 and HDAC1 to regulate apolipoprotein CIII promoter.

Nuclear hormone receptors function as ligand activated transcription factors. Ligand binding and modification such as acetylation have been reported to regulate nuclear hormone receptors. The orphan receptors, Rev-erbalpha and Rev-erbbeta, are members of the nuclear receptor superfamily and act as transcriptional repressors. In this study, the role of recruitment of co-factors by Rev-erbbeta and acetylation of Rev-erbbeta in modulating apolipoprotein CIII (apoCIII) transcription were investigated. Rev-erbbeta was found to transcriptionally repress apoCIII after binding to the apoCIII promoter. Tip60, a histone acetyl-transferase (HAT), was a novel binding partner for Rev-erbbeta and recruited to the apoCIII promoter by Rev-erbbeta. Tip60 was able to acetylate Rev-erbbeta and relieve the apoCIII repression mediated by Rev-erbbeta. This de-repression effect depended on acetylation of Rev-erbbeta at its RXKK motif by Tip60. In addition, histone deacetylase 1 (HDAC1) interacted with Rev-erbbeta and was recruited to the apoCIII promoter by Rev-erbbeta to antagonize Tip60's activity. Taken together, we have provided evidence that Rev-erbbeta modulates the apoCIII gene expression by recruiting different transcription co-activator or co-repressor.

Human homologue of SETA binding protein 1 interacts with cathepsin B and participates in TNF-Induced apoptosis in ovarian cancer cells.

Lysosomal cysteine protease cathepsin B has been reported to play an important role in apoptosis of many different cancer cells, but the regulation of cathepsin B in apoptosis is poorly understood. Human homologue of SETA binding protein 1 (hSB1) was identified to interact with cathepsin B by yeast-two hybrid method, and the interaction was confirmed in vitro GST pull-down assay and in vivo coimmunoprecipitation experiment. hSB1 was co-localized with cathepsin B in cellular lysosomes. Our previous study has shown that TNF can induce ovarian cancer cells OV-90 apoptosis and the apoptosis process is cathepsin B-depended. Here we provide evidence that overexpression of cathepsin B-interacting protein hSB1 could suppress TNF-triggered apoptosis in OV-90 cells, but has no effect on cellular cathepsin B activity. hSB1 may function as a regulator of cathepsin B-mediated apoptosis.

Construction, expression and characterization of human interferon alpha2b-(G4S)n-thymosin alpha1 fusion proteins in Pichia pastoris.

AIM: Interferon alpha2b (IFNalpha2b) and thymosin alpha1 (Talpha1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNalpha2b and Talpha1 linked by different lengths of (G4S)n (n = 1-3) were constructed and expressed in Pichia pastoris. METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex 75 gel filtration and analyzed by SDS-PAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins. RESULTS: DNA sequencing confirmed that the fusion genes of IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNalpha2b monoclonal antibody and Talpha1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay. CONCLUSION: The recombinant IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNalpha2b and immunomodulatory activity of Talpha1 in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.