Application of ddPCR in detection of the status and abundance of EGFR T790M mutation in the plasma samples of non-small cell lung cancer patients.

Background/Objective: The third-generation epidermal growth factor receptor (EGFR) -tyrosine kinase inhibitor (TKIs), such as osimertinib, designed for targeting the acquired drug-resistant mutation of EGFR T790M, was approved as the first-line therapy for advanced EGFR-mutated non-small cell lung cancer (NSCLC). Thus, detection of the EGFR T790M mutation for NSCLC is crucial. However, tissue samples are often difficult to obtain, especially in patients at advanced stages. This study assessed the performances of droplet digital polymerase chain reaction (ddPCR) and next-generation sequencing (NGS) in detecting EGFR T790M status and abundance in the plasma ctDNA samples of patients with NSCLC. We also explored the association between T790M status and abundance and the response to third-generation EGFR-TKIs. Methods: A total of 201 plasma samples with matched tissues, 821 plasma samples, and 56 patients who received third-generation EGFR-TKIs with response evaluation were included in this study. ddPCR and NGS were used to detect the mutation status and abundance of T790M in the tissues and/or blood samples. Results: The results showed that the sensitivity and the specificity of EGFR T790M mutation status detected by ddPCR in plasma samples were 81.82% and 91.85%, respectively, compared with the tissue samples, with a consistency coefficient of 0.740. Among the 821 plasma samples, the positive rates of EGFR T790M detected by ddPCR and NGS were 34.2% (281/821) and 22.5% (185/821), respectively. With NGS results as the reference, the sensitivity and the specificity of ddPCR were 100% and 84.91%, respectively, and the consistency coefficient of the two methods was 0.717. In addition, we found that a higher EGFR T790M abundance was linked to a higher treatment response rate to the third-generation EGFR-TKIs regardless of the classification of the median value of 0.43% (P = 0.016) or average value of 3.16% (P = 0.010). Conclusion: Taking these data together, this study reveals that ddPCR is an alternatively potent method for the detection of EGFR T790M in the plasma samples of NSCLC patients.

Detection of IDH1 and TERT promoter mutations with droplet digital PCR in diffuse gliomas.

Mutations in isocitrate dehydrogenase (IDH) and telomerase reverse transcriptase promoter (TERTp) exert a far-reaching influence on clinicopathologic diagnosis and prognosis of glioma. Traditional approaches, such as Sanger sequencing and ARMS, lack sensitivity due to tumor heterogeneity and low tumor purity of glioma samples. Therefore, we propose a highly sensitive detection method for IDH1 and TERTp mutations based on ddPCR technology, named IDH1-TERT-mutation ddPCR (IT-ddPCR). We determined the IDH1 and TERTp mutations of 80 patients by Sanger sequencing, ARMS, and IT-ddPCR in parallel. We detected the TERTp mutations of 8 patients with probes by IT-ddPCR and Bio-Rad. IDH1-positive singles were detected in 56 cases by IT-ddPCR. TERTp-positive singles were detected in 50 cases by IT-ddPCR. There was a slight difference in total events, occupancy events, and C228T/C250T droplets between these two different probes. Regression analysis of the TERTp variant frequencies detected by probes of IT-ddPCR and Bio-Rad produced a slope of 1.0425 and a coefficient (R2) of 0.9231. We found that IT-ddPCR showed a higher sensitivity compared with Sanger sequencing and ARMS in the detection of IDH1 and TERTp mutations. There were no significant differences in variant frequencies of TERTp mutations between the two probes of IT-ddPCR and Bio-Rad. Thus, IT-ddPCR can be used to detect low-frequency mutation of IDH1 and TERTp in glioma.

Blocking ß-1,6-glucan synthesis by deleting KRE6 and SKN1 attenuates the virulence of Candida albicans.

ß-1,6-glucan is an important component of the fungal cell wall. The ß-1,6-glucan synthase gene KRE6 was thought to be essential in the fungal pathogen Candida albicans because it could not be deleted in previous efforts. Also, the role of its homolog SKN1 was unclear because its deletion caused no defects. Here, we report the construction and characterization of kre6Δ/Δ, skn1Δ/Δ and kre6Δ/Δ skn1Δ/Δ mutants in C. albicans. While deleting KRE6 or SKN1 had no obvious phenotypic consequence, deleting both caused slow growth, cell separation failure, cell wall abnormalities, diminished hyphal growth, poor biofilm formation and loss of virulence in mice. Furthermore, the GPI-linked cell surface proteins Hwp1 and the invasin Als3 or Ssa1 were not detected in kre6Δ/Δ skn1Δ/Δ mutant. In GMM medium, RT-qPCR and western blotting revealed a constitutive expression of KRE6 and growth conditions-associated activation of SKN1. Like many hypha-specific genes, SKN1 is repressed by Nrg1, but its activation does not involve the transcription factor Efg1. Dysregulation of SKN1 reduces C. albicans ability to damage epithelial and endothelial cells and attenuates its virulence. Given the vital role of ß-1,6-glucan synthesis in C. albicans physiology and virulence, Kre6 and Skn1 are worthy targets for developing antifungal agents.

Characterization of Pph3-mediated dephosphorylation of Rad53 during methyl methanesulfonate-induced DNA damage repair in Candida albicans.

Genotoxic stress causes DNA damage or stalled DNA replication and filamentous growth in the pathogenic fungus Candida albicans The DNA checkpoint kinase Rad53 critically regulates by phosphorylation effectors that execute the stress response. Rad53 itself is activated by phosphorylation and inactivated by dephosphorylation. Previous studies have suggested that the phosphatase Pph3 dephosphorylates Rad53. Here, we used mass spectrometry and mutagenesis to identify Pph3 dephosphorylation sites on Rad53 in C. albicans We found that serine residues 351, 461 and 477, which were dephosphorylated in wild-type cells during the recovery from DNA damage caused by methyl methanesulfonate (MMS), remained phosphorylated in pph3Δ/Δ cells. Phosphomimetic mutation of the three residues (rad53-3D) impaired Rad53 dephosphorylation, exit from cell cycle arrest, dephosphorylation of two Rad53 effectors Dun1 and Dbf4, and the filament-to-yeast growth transition during the recovery from MMS-induced DNA damage. The phenotypes observed in the rad53-3D mutant also occurred in the pph3Δ/Δ mutant. Together, our findings reveal a molecular mechanism by which Pph3 controls DNA damage response in C. albicans.

Curcumin Acetate Nanocrystals for sustained Pulmonary Delivery: Preparation, Characterization and In Vivo Evaluation.

The main objective of this study was to test the hypothesis that inhaled nanocrystals of a highly lipophilic drug could be used as a novel approach for producing sustained pulmonary delivery. Curcumin acetate, an ester prodrug of curcumin, was utilized as a highly lipophilic model drug. Curcumin acetate was subjected to wet ball milling to produce different particle sizes of nanocrystals and microparticles, and the milled curcumin acetate was spray-dried to yield similar inhalable microparticles. Following intrapulmonary administration in rats, pharmacokinetic experiments indicated that curcumin acetate significantly extended the pulmonary absorption time by 7.2-fold compared to curcumin, possibly due to the high lipophilicity of the former. The biodistribution data showed that aerosolized curcumin acetate nanocrystals 123.7 nm in size not only prolonged pulmonary retention, with the AUC value of curcumin acetate being 7.62-fold higher than that of the microparticles 1120 nm in size, but also increased the local in vivo release rate by 3.3-fold and the local availability of converted curcumin by 25.1-fold. In addition, the improved local availability resulted in better pharmacological efficacy in a monocrotaline-induced rat model of pulmonary arterial hypertension. This study was the first to demonstrate that inhalable nanocrystals are a feasible means for the sustained pulmonary delivery of highly lipophilic drugs.

Sds22 participates in Glc7 mediated Rad53 dephosphorylation in MMS-induced DNA damage in Candida albicans.

The protein kinase Rad53 and its orthologs play a fundamental role in regulating the DNA damage checkpoint in eukaryotes. Rad53 is activated by phosphorylation in response to DNA damage and deactivated by dephosphorylation after the damage is repaired. However, the phosphatases involved in Rad53 deactivation are not entirely understood. In this study, by investigating the consequences of overexpressing SDS22, a gene encoding a regulatory subunit of the PP1 phosphatase Glc7, in the human fungal pathogen Candida albicans, we discovered that Sds22 plays an important role in Rad53 dephosphorylation and thus the deactivation of the DNA damage checkpoint. Sds22 cellular levels increase when cells are exposed to DNA damaging agents and decrease after removing the genotoxins. Depletion of Glc7 has similar phenotypes. We provide evidence that Sds2 acts through inhibitory physical association with Glc7. Our findings provide novel insights into the mechanisms for the control of DNA damage checkpoint. Furthermore, SDS22 overexpression reduces C. albicans virulence in a mouse model of systemic infection, suggesting potential targets for developing antifungal drugs.

Tpd3-Pph21 phosphatase plays a direct role in Sep7 dephosphorylation in Candida albicans.

Septins are a component of the cytoskeleton and play important roles in diverse cellular processes including cell cycle control, cytokinesis and polarized growth. In fungi, septin organization, dynamics and function are regulated by phosphorylation, and several kinases responsible for the phosphorylation of several septins have been identified. However, little is known about the phosphatases that dephosphorylate septins. Here, we report the characterization of Tpd3, a structural subunit of the PP2A family of phosphatases, in the pathogenic fungus Candida albicans. We found that tpd3Δ/Δ cells are defective in hyphal growth and grow as pseudohyphae under yeast growth conditions with aberrant septin organization. Western blotting detected hyperphosphorylation of the septin Sep7 in cells lacking Tpd3. Tpd3 and Sep7 colocalize at the bud neck and can coimmunoprecipitate. Furthermore, we discovered similar defects in cells lacking Pph21, a catalytic subunit of the PP2A family, and its physical association with Tpd3. Importantly, purified Tpd3-Pph21 complexes can dephosphorylate Sep7 in vitro. Together, our findings strongly support the idea that the Tpd3-Pph21 complex dephosphorylates Sep7 and regulates morphogenesis and cytokinesis. The tpd3Δ/Δ mutant is greatly reduced in virulence in mice, providing a potential antifungal target.

[Soluble high-expression, purification and bioassay of IGFBP-3].

cDNA for Insulin-like growth factor binding protein 3 was cloned and constructed a prokaryotic expression vector--pET-DsBA-IGFBP3. The construct was transformed into E. coli BL21 (DE3)plysS. The induced fusion protein (D-IGFBP3) was expressed successfully in soluble form. We obtained D-IGFBP3 the purify of which is over 95% after purification by His affinity chromatography. The product was identified by Western-blot. The cell assay showed that the obtained fusion protein can inhibit the growth of MCF-7 and bind with IGF-I in vitro.

Prokaryotic expression, purification, and production of polyclonal antibody against human polypeptide N-acetylgalactosaminyltransferase 14.

Polypeptide N-acetylgalactosaminyltransferase 14 (GalNAc-T14, EC 2.4.1.41) belongs to a large subfamily of glycosyltransferases residing in the Golgi apparatus. N-Acetylgalactosaminyltransferases (GalNAc-Tases) catalyze the first step in the O-glycosylation of mammalian proteins by transferring N-acetyl-D-galactosamine (GalNAc) to peptide substrates. Here, the cloning, expression, purification, and polyclonal antibody preparation of GalNAc-T14 were described. A full-length GalNAc-T14 cDNA was inserted in a prokaryotic expression plasmid pGEX-4T-1 at the EcoRI and XhoI restriction sites. pGEX-4T-T14 was highly expressed in Escherichia coli (E. coli) BL21(DE3) cells after induced by isopropyl-beta-D-thiogalactoside (IPTG). The expressed GST-GalNAc-T14 fusion protein was purified by GSTrap FF chromatography and then used as antigen to immunize rabbits. The obtained antiserum was precipitated by 50% saturated ammonium sulfate and then purified by DEAE-Sepharose FF chromatography. To confirm the activity and specificity of the GalNAc-T14 antibody, we constructed the plasmid pFLAG-GalNAc-T14 to transfect transiently HEK 293T cells. Transiently expressed FLAG-GalNAc-T14 was identified by Western blot analysis with GalNAc-T14 antibody and FLAG monoclonal antibody, respectively. The production of the polyclonal antibody against GalNAc-T14 provides a good tool for studying the biofunctions of GalNAc-T14.

N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner.

Insulin-like growth factor binding protein-3 (IGFBP-3) is known to inhibit cell proliferation and induce apoptosis in IGF-dependent and IGF-independent manners, but the mechanism underlying IGF-independent effects is not yet clear. In a yeast two-hybrid assay, IGFBP-3 was used as the bait to screen a human fetal liver cDNA library for it interactors that may potentially mediate IGFBP-3-regulated functions. N-Acetylgalactosaminyltransferase 14 (GalNAc-T14), a member of the GalNAc-Tases family, was identified as a novel IGFBP-3 binding partner. This interaction involved the ricin-type beta-trefoil domain of GalNAc-T14. The interaction between IGFBP-3 and GalNAc-T14 was reconfirmed in vitro and in vivo, using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assays. Our findings may provide new clues for further study on the mechanism behind the IGF-independent effects of IGFBP-3 promoting apoptosis. The role of GalNAc-T14 as an intracellular mediator of the effects of IGFBP-3 need to be verified in future studies.