Genome-Wide CRISPR Screening Identifies DCK and CCNL1 as Genes That Contribute to Gemcitabine Resistance in Pancreatic Cancer.

Pancreatic cancer is one of the most lethal cancers. Due to the difficulty of early diagnosis, most patients are diagnosed with metastasis or advanced-stage cancer, limiting the possibility of surgical treatment. Therefore, chemotherapy is applied to improve patient outcomes, and gemcitabine has been the primary chemotherapy drug for pancreatic cancer for over a decade. However, drug resistance poses a significant challenge to the efficacy of chemotherapy. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) gene-editing system is a powerful tool, and researchers have developed CRISPR/Cas9 library screening as a means to identify the genes associated with specific phenotype changes. We performed genome-wide CRISPR/Cas9 knockout screening in the mouse pancreatic cancer cell line TB32047 with gemcitabine treatment and identified deoxycytidine kinase (DCK) and cyclin L1 (CCNL1) as the top hits. We knocked out DCK and CCNL1 in the TB32047 and PANC1 cell lines and confirmed that the loss of DCK or CCNL1 enhanced gemcitabine resistance in pancreatic cells. Many researchers have addressed the mechanism of DCK-related gemcitabine resistance; however, no study has focused on CCNL1 and gemcitabine resistance. Therefore, we explored the mechanism of CCNL1-related gemcitabine resistance and found that the loss of CCNL1 activates the ERK/AKT/STAT3 survival pathway, causing cell resistance to gemcitabine treatment.

CRISPR-Cas9 Screen Identifies DYRK1A as a Target for Radiotherapy Sensitization in Pancreatic Cancer.

Although radiation therapy has recently made great advances in cancer treatment, the majority of patients diagnosed with pancreatic cancer (PC) cannot achieve satisfactory outcomes due to intrinsic and acquired radioresistance. Identifying the molecular mechanisms that impair the efficacy of radiotherapy and targeting these pathways are essential to improve the radiation response of PC patients. Our goal is to identify sensitive targets for pancreatic cancer radiotherapy (RT) using the kinome-wide CRISPR-Cas9 loss-of-function screen and enhance the therapeutic effect through the development and application of targeted inhibitors combined with radiotherapy. We transduced pancreatic cancer cells with a protein kinase library; 2D and 3D library cells were irradiated daily with a single dose of up to 2 Gy for 4 weeks for a total of 40 Gy using an X-ray generator. Sufficient DNA was collected for next-generation deep sequencing to identify candidate genes. In this study, we identified several cell cycle checkpoint kinases and DNA damage related kinases in 2D- and 3D-cultivated cells, including DYRK1A, whose loss of function sensitizes cells to radiotherapy. Additionally, we demonstrated that the harmine-targeted suppression of DYRK1A used in conjunction with radiotherapy increases DNA double-strand breaks (DSBs) and impairs homologous repair (HR), resulting in more cancer cell death. Our results support the use of CRISPR-Cas9 screening to identify new therapeutic targets, develop radiosensitizers, and provide novel strategies for overcoming the tolerance of pancreatic cancer to radiotherapy.

Rapid Screening of Trace Volatile and Nonvolatile Illegal Drugs by Miniature Ion Trap Mass Spectrometry: Synchronized Flash-Thermal-Desorption Purging and Ion Injection.

The increasing demand for rapid and sensitive roadside identification of trace illegal drugs drives the development of high-performance miniature mass spectrometric instrumentation and methodology. Here, we report a synchronized flash-thermal-desorption purging and ion injection (SFTDPI) method to increase the sensitive and rapid screening of volatile and nonvolatile illegal drugs for miniature ion trap mass spectrometry (ITMS). The flash-thermal desorption could reach 290 °C in 2.5 s, which could achieve efficient vaporization of nonvolatile noscapine with boiling point at 565 °C. ITMS using discontinuous atmospheric pressure interface (DAPI) has an ion utilization ratio of less than 1%, the synchronized purging for flash-thermal desorption and ion injection with DAPI could accurately control the time interval along with the desorption, gas purging, ionization and ion injection, and the sample utilization ratio increases more than five times. The miniature SFTDPI-ITMS presents good performance: (1) more than 60 times improvement in sensitivity was achieved compared to the previously reported thermal-desorption acetone-assisted photoionization ion trap mass spectrometer for nonvolatile drugs, and the minimum detectable quantity reaches 50 pg for fentanyl. (2) Ten kinds of mixing drugs with boiling point difference of 300 °C can be simultaneously identified within 3 s under a single analysis. SFTDPI-ITMS was deployed at the roadside checkpoint of Sino-Burmese border; fentanyl in the captured encapsulated powder and the suspected opiate had been successfully identified.

Rapid On-Site Detection of Illegal Drugs in Complex Matrix by Thermal Desorption Acetone-Assisted Photoionization Miniature Ion Trap Mass Spectrometer.

Illegal drug smugglings and crimes have long been a global concern, and an apparatus which can identify drugs on-the-spot is urgently demanded by law enforcement. A thermal desorption acetone-assisted photoionization miniature ion trap mass spectrometer was developed for on-site and rapid identification of illegal drugs at checkpoints. Acetone was chosen for dopant-assisted photoionization, and the sensitivity of selected drugs was further enhanced with protonated analyte molecular ions [M + H]+. For example, the sensitivity of ephedrine was improved by as high as 22-fold. The mass discrimination effect, which was usually considered as a shortcoming of ion trap mass analyzer, was ingeniously utilized to eliminate the protonated acetone reagent ions and maximize the trapping efficiency of analyte ions in mass analyzer. Twenty-seven drugs were analyzed, and the limits of detection (LODs) of selected illegal drugs were at the nanogram level with analysis time of 2 s. Analyte/dopant ion peak intensity ratios in mass spectra could be used for quantitation to improve the quantitative analysis performance of miniature ion trap mass spectrometer equipped with a discontinuous atmospheric pressure interface (DAPI) with the prerequisite that dopant ions and analyte ions could be simultaneously and effectively trapped by the ion trap. The RSD of signal intensity was reduced from 25.3% to 8.5%, and the linear range was extended from 0.5-25 to 0.5-100 ng/µL for methamphetamine. A temperature-resolved thermal desorption sampling strategy was developed and used to distinguish illegal drug components in plant-based drug samples and drinks containing illegal drugs.

The obstacle problem for conformal metrics on compact Riemannian manifolds.

We prove a priori estimates up to their second order derivatives for solutions to the obstacle problem of curvature equations on Riemannian manifolds ( M n , g ) arising from conformal deformation. With the a priori estimates the existence of a C 1 , 1 solution to the obstacle problem with Dirichlet boundary value is obtained by approximation.

[Association of eight single nucleotide polymorphisms of chromosomes 20 and X with androgenetic alopecia among ethnic Han Chinese from Yunnan].

OBJECTIVE: To assess the association of 8 single nucleotide polymorphisms (SNPs) from chromosomes X and 20 with androgenetic alopecia among ethnic Han population from Yunnan province. METHODS: An eight-SNP co-amplification protocol was developed for the genotyping with a SNaPshot platform. A case-control study was carried out for the 8 SNPs from chromosomes X and 20 in 115 androgenetic alopecia cases and 125 healthy controls. Statistical analysis was conducted with SPSS17.0, Haploview4.2, SHEsis and MDR software. RESULTS: No association was found between the two groups with regard to the 4 SNPs located on the X chromosome. The genotypic frequencies of rs2180439, rs913063 and rs1160312 were significantly different between the two groups (P < 0.05). The frequency of T allele of rs2180439 was significantly higher in the case group (P < 0.05). The frequencies of A alleles of rs913063 and rs1160312 were significantly higher in the case group (P < 0.05). The haplotypes of C-T-C-G, T-C-C-G and T-T-A-A based on rs6137444-rs2180439-rs913063-rs1160312 showed significant difference between the two groups (P <0.05). rs6137444, rs21804393 and rs1160312 have a strong association with androgenetic alopecia. CONCLUSION: The 4 SNPs located on chromosome X were all monomorphic among ethnic Hans from Yunnan. The rs6152, rs16990427, rs1352015, rs1385699 SNPs located on chromosome 20 are associated with androgenetic alopecia in the same population. Individuals with T allele of rs2180439 and A allele of rs913063 and rs1160312 are more likely to develop androgenetic alopecia.

[Association study of CNR1, GAD1 and BDNF polymorphisms with male heroin dependence in the Dai population in Yunnan].

In order to analyze the association of CNR1(Cannabinoid receptor 1), GAD1(Glutamate decarboxylase 1), and BDNF(Brain-derived neurotrophic factor) polymorphisms with male heroin dependence in the Dai population in Yunnan Province, an eight-SNP co-amplification protocol was established to genotype on the SNaPshot platform. A case-control study was performed with 8 SNPs from CNR1, GAD1, and BDNF genes in 165 heroin-dependent males and 170 healthy males of the Dai population. Statistical analyses were conducted with SPSS17.0, Haploview4.2, PHASE2.1, and MDR software. We found that: (1) the genotype frequency of rs13306221 was significant in the case group (P<0.025); (2) the A allelic frequency of rs6265 was significantly higher in the case group; (3) the haplotypes of T-A-C, C-C-C, C-C-T, and T-C-C based on rs1978340-rs3791878-rs11542313 and haplotype A-G based on rs6265-rs13306221 were significant (P<0.05); (4) the haplotype frequencies of T-A-C, C-C-T, and A-G were significantly higher in the case group. These results indicate that the linkage between rs1978340 and rs3791878 in GAD1 has a strong association with heroin dependence. Furthermore, polymorphisms in CNR1 (rs1049353), GAD1 (rs1978340 and rs11542313), and BDNF (rs6265 and rs13306221) were associated with heroin dependence in the Yunnan Dai population, and individuals with the rs6265 A allele were more likely to be heroin dependent.