Intracellular generation of single-strand template increases the knock-in efficiency by combining CRISPR/Cas9 with AAV.

Targeted integration of transgenes facilitates functional genomic research and holds prospect for gene therapy. The established microhomology-mediated end-joining (MMEJ)-based strategy leads to the precise gene knock-in with easily constructed donor, yet the limited efficiency remains to be further improved. Here, we show that single-strand DNA (ssDNA) donor contributes to efficient increase of knock-in efficiency and establishes a method to achieve the intracellular linearization of long ssDNA donor. We identified that the CRISPR/Cas9 system is responsible for breaking double-strand DNA (dsDNA) of palindromic structure in inverted terminal repeats (ITRs) region of recombinant adeno-associated virus (AAV), leading to the inhibition of viral second-strand DNA synthesis. Combing Cas9 plasmids targeting genome and ITR with AAV donor delivery, the precise knock-in of gene cassette was achieved, with 13-14% of the donor insertion events being mediated by MMEJ in HEK 293T cells. This study describes a novel method to integrate large single-strand transgene cassettes into the genomes, increasing knock-in efficiency by 13.6-19.5-fold relative to conventional AAV-mediated method. It also provides a comprehensive solution to the challenges of complicated production and difficult delivery with large exogenous fragments.

Effects of Hinge-region Natural Polymorphisms on Human Immunodeficiency Virus-Type 1 Protease Structure, Dynamics, and Drug Pressure Evolution.

Multidrug resistance to current Food and Drug Administration-approved HIV-1 protease (PR) inhibitors drives the need to understand the fundamental mechanisms of how drug pressure-selected mutations, which are oftentimes natural polymorphisms, elicit their effect on enzyme function and resistance. Here, the impacts of the hinge-region natural polymorphism at residue 35, glutamate to aspartate (E35D), alone and in conjunction with residue 57, arginine to lysine (R57K), are characterized with the goal of understanding how altered salt bridge interactions between the hinge and flap regions are associated with changes in structure, motional dynamics, conformational sampling, kinetic parameters, and inhibitor affinity. The combined results reveal that the single E35D substitution leads to diminished salt bridge interactions between residues 35 and 57 and gives rise to the stabilization of open-like conformational states with overall increased backbone dynamics. In HIV-1 PR constructs where sites 35 and 57 are both mutated (e.g. E35D and R57K), x-ray structures reveal an altered network of interactions that replace the salt bridge thus stabilizing the structural integrity between the flap and hinge regions. Despite the altered conformational sampling and dynamics when the salt bridge is disrupted, enzyme kinetic parameters and inhibition constants are similar to those obtained for subtype B PR. Results demonstrate that these hinge-region natural polymorphisms, which may arise as drug pressure secondary mutations, alter protein dynamics and the conformational landscape, which are important thermodynamic parameters to consider for development of inhibitors that target for non-subtype B PR.

Erlotinib promotes endoplasmic reticulum stress-mediated injury in the intestinal epithelium.

Erlotinib, a popular drug for treating non-small cell lung cancer (NSCLC), causes diarrhea in approximately 55% of patients receiving this drug. In the present study, we found that erlotinib induced barrier dysfunction in rat small intestine epithelial cells (IEC-6) by increasing epithelial permeability and down-regulating E-cadherin. The mRNA levels of various pro-inflammatory cytokines (Il-6, Il-25 and Il-17f) were increased after erlotinib treatment in IEC-6 cells. Erlotinib concentration- and time-dependently induced apoptosis and endoplasmic reticulum (ER) stress in both IEC-6 and human colon epithelial cells (CCD 841 CoN). Intestinal epithelial injury was also observed in male C57BL/6J mice administrated with erlotinib. Knockdown of C/EBP homologous protein (CHOP) with small interference RNA partially reversed erlotinib-induced apoptosis, production of IL-6 and down-regulation of E-cadherin in cultured intestinal epithelial cells. In conclusion, erlotinib caused ER stress-mediated injury in the intestinal epithelium, contributing to its side effects of diarrhea in patients.

Emergence of blaOXA-181-bearing tigecycline-resistant Klebsiella aerogenes in China.

We report the isolation of a blaOXA-181-positive, tigecycline-resistant Klebsiella aerogenes strain KA04 from a Chinese inpatient's fecal sample. Species identification was performed using MALDI-TOF MS. The antibiotic susceptibilities were assessed via the broth microdilution method. To elucidate the transmission and genetic structure of the blaOXA-181 gene, conjugation assays and whole-genome sequencing (WGS) were performed. KA04 displayed resistance to carbapenems, quinolones, piperacillin/tazobactam and tigecycline. Through WGS and conjugation experiments, it was possible to confirm blaOXA-181 and qnrS1 genes causing antibiotic resistance were located on a 51-kb IncX3 type mobile plasmid, blaOXA-181 gene could be successfully transferred into E. coli EC600 at a conjugation frequency of 1.1 × 10- 4. tet(A) gene was located on both the chromosome and non-transmissible IncFIB(K) plasmid. This is a tigecycline-resistant K. aerogenes harboring blaOXA-181 isolate from human fecal sample, highlighting a significant public health concern. Further comprehensive surveillance is needed.

Ripening of avocado fruits studied by spectroscopic techniques.

Avocados are considered very healthy due to the high content mono-unsaturated lipid, essential vitamins and minerals, minimal sugar and no cholesterol and are therefore sometimes referred to as "the perfect fruits". Avocados, mainly grown in Latin-America, are harvested unripe and sent overseas. However, the ripening process is very difficult to assess visually and tactilely. A tool for precise noninvasive judgment of the status would be valuable as the fruit is too expensive to be cut open unripe or overdue. A white-light source and a light-emitting diode unit with four excitation wavelengths (365, 385, 395, and 405 nm) were used for reflectance and fluorescence spectroscopy in a fiber-coupled set-up for noninvasive monitoring. Twelve non-ripe avocados, with approximately the same size and appearance, were studied and divided into three groups and kept at three different storage conditions; at room temperature, in a refrigerator and a combination of the two. We showed that fluorescence was useful for following the ripening process. A method, which compensates for the spatial variations in spectral properties around a fruit, is described. Remote fluorescence monitoring, intended for orchard use, was also demonstrated. A low-cost device based on fluorescence for avocado ripeness assessment is proposed.

Darunavir-Resistant HIV-1 Protease Constructs Uphold a Conformational Selection Hypothesis for Drug Resistance.

Multidrug resistance continues to be a barrier to the effectiveness of highly active antiretroviral therapy in the treatment of human immunodeficiency virus 1 (HIV-1) infection. Darunavir (DRV) is a highly potent protease inhibitor (PI) that is oftentimes effective when drug resistance has emerged against first-generation inhibitors. Resistance to darunavir does evolve and requires 10-20 amino acid substitutions. The conformational landscapes of six highly characterized HIV-1 protease (PR) constructs that harbor up to 19 DRV-associated mutations were characterized by distance measurements with pulsed electron double resonance (PELDOR) paramagnetic resonance spectroscopy, namely double electron-electron resonance (DEER). The results show that the accumulated substitutions alter the conformational landscape compared to PI-naïve protease where the semi-open conformation is destabilized as the dominant population with open-like states becoming prevalent in many cases. A linear correlation is found between values of the DRV inhibition parameter Ki and the open-like to closed-state population ratio determined from DEER. The nearly 50% decrease in occupancy of the semi-open conformation is associated with reduced enzymatic activity, characterized previously in the literature.

Non-intrusive studies of gas contents and gas diffusion in hen eggs.

A detailed study of the condition of eggs was performed using tunable diode lasers to monitor free gas in hen eggs. We detected oxygen and water vapor signals from 13 unfertilized eggs and studied the growth of the egg air cell over a time period of 3 weeks. We also studied the gas exchange through the egg shell, which is of particular interest for fertilized eggs. Four fertilized and five unfertilized eggs were followed over 3 weeks, the hatching period for hen eggs, and significant variations were found both in time and for the two types of eggs. Our results indicate that the techniques could be developed for automatic control of egg freshness, as well as for monitoring the hatching progress of fertilized eggs.

Towards an optical diagnostic system for otitis media using a combination of otoscopy and spectroscopy.

An improved method, where conventional otoscope investigation of human suspicious otitis media is combined with diffuse reflectance spectroscopy and gas in scattering media absorption spectroscopy (GASMAS) is being developed. Otitis media is one of the most common infectious diseases in children, whose Eustachian tube connecting the middle ear with the nasal cavity is more horizontal than for adults, which leads to impaired fluid drainage. At present, the use of an otoscope to visually observe possible changes in the tympanic membrane appearance is the main diagnostics method for otitis media. Inaccurate diagnosis related to similar symptoms, and the difficulty for small children to describe the condition experienced, frequently leads to over-prescription of antibiotics and alarming increase in bacterial resistance development. More accurate diagnostic methods are highly desirable. Diffuse reflectance spectroscopy is a non-invasive quantitative spectroscopic technique that enables to objectively quantify changes in the hemoglobin content of the tympanic membrane related to inflammation. If an infection is present, the ventilatory function of the Eustachian tube is frequently impaired and the middle-ear cavity will be filled with fluid. GASMAS, a non-invasive detection method, can non-invasively determine if gas is replaced by fluid in the middle-ear cavity.

LGALS3 Promotes Treatment Resistance in Glioblastoma and Is Associated with Tumor Risk and Prognosis.

BACKGROUND: LGALS3 promotes tumor progression in diverse cancers. However, the involvement of LGALS3 in glioblastoma has not yet been broadly illuminated. METHODS: Microarray was performed to detect the gene expression profiles of radioresistance in T98G cells and identified a universally upregulated gene, LGALS3. The impact of LGALS3 on the survival of glioblastoma cells facing ionizing irradiation or temozolomide was investigated by the Cell Counting Kit-8 (CCK-8). A total of 120 glioblastoma cases were collected to analyze the relationship between LGALS3 expression and patient prognosis. Another 961 patients with glioma and 1,351 healthy controls were recruited to study the association of SNPs across the LGALS3 gene with glioblastoma susceptibility. The functional SNP sites were also studied in cellular experiments. RESULTS: An effective protection of LGALS3 from ionizing irradiation or temozolomide-induced cell death in T98G and U251 cells was found. In addition, high expression of LGALS3 could work as an independent risk factor for survival of patients with glioblastoma. Two SNP sites (rs4644 and rs4652) across the LGALS3 gene were associated with increased risk for glioblastoma, and the C allele of rs4652 and the A allele of rs4644 could enhance glioblastoma resistance to radio-chemotherapy, but not cell proliferation. CONCLUSIONS: Our results suggest that LGALS3 is an important biomarker influencing glioblastoma risk and prognosis and a potential target for treating the malignancy, especially ones with resistance against the standard therapy. IMPACT: LGALS3 promotes glioblastoma cells' resistance to ionizing irradiation and temozolomide and predicts poor prognosis. Targeting LGALS3 may limit the therapeutic resistance in glioblastoma and increase patient survival.

New applications of CRISPR/Cas9 system on mutant DNA detection.

The detection of mutant DNA is critical for precision medicine, but low-frequency DNA mutation is very hard to be determined. CRISPR/Cas9 is a robust tool for in vivo gene editing, and shows the potential for precise in vitro DNA cleavage. Here we developed a DNA mutation detection system based on CRISPR/Cas9 that can detect gene mutation efficiently even in a low-frequency condition. The system of CRISPR/Cas9 cleavage in vitro showed a high accuracy similar to traditional T7 endonuclease I (T7E1) assay in estimating mutant DNA proportion in the condition of normal frequency. The technology was further used for low-frequency mutant DNA detection of EGFR and HBB somatic mutations. To the end, Cas9 was employed to cleave the wild-type (WT) DNA and to enrich the mutant DNA. Using amplified fragment length polymorphism analysis (AFLPA) and Sanger sequencing, we assessed the sensitivity of CRISPR/Cas9 cleavage-based PCR, in which mutations at 1%-10% could be enriched and detected. When combined with blocker PCR, its sensitivity reached up to 0.1%. Our results suggested that this new application of CRISPR/Cas9 system is a robust and potential method for heterogeneous specimens in the clinical diagnosis and treatment management.

Development of risk prediction models for glioma based on genome-wide association study findings and comprehensive evaluation of predictive performances.

Over 14 common single nucleotide polymorphisms (SNP) have been consistently identified from genome-wide association studies (GWAS) as associated with glioma risk in European background. The extent to which and how these genetic variants can improve the prediction of glioma risk has was not been investigated. In this study, we employed three independent case-control datasets in Chinese populations, tested GWAS signals in dataset1, validated association results in dataset2, developed prediction models in dataset2 for the consistently replicated SNPs, refined the consistently replicated SNPs in dataset3 and developed tailored models for Chinese populations. For model construction, we aggregated the contribution of multiple SNPs into genetic risk scores (count GRS and weighed GRS) or predicted risks from logistic regression analyses (PRFLR). In dataset2, the area under receiver operating characteristic curves (AUC) of the 5 consistently replicated SNPs by PRFLR(SNPs) was 0.615, higher than those of all GRSs(ranging from 0.607 to 0.611, all P>0.05). The AUC of genetic profile significantly exceeded that of family history (fmc) alone (AUC=0.535, all P<0.001). The best model in our study comprised "PRURA +fmc" (AUC=0.646) in dataset3. Further model assessment analyses provided additional evidence. This study indicates that genetic markers have potential value for risk prediction of glioma.

Whole exome-wide association study identifies a missense variant in SLC2A4RG associated with glioblastoma risk.

In this study, we conducted a genome-wide scan of single nucleotide polymorphisms (SNPs) to identify coding variants that is associated with the risk of glioblastoma (GBM), the most common and most malignant subtype of glioma. We genotyped 1038 GBM cases and 1008 controls in a Chinese Han population using Illumina HumanExome Beadchip v1.0. A missense variant, rs8957 (E[GAG]233D[GAU], SLC2A4RG, 20q13.33), was found being associated with GBM risk, with an odd ratio (OR) of 1.43 (95% confidence interval (CI) = 1.25-1.64, P = 1.72E-07). The G>T transversion at rs8957 leading to changes of subcellular localization of SLC2A4RG, possibly due to the impairment of its nuclear export signal or protein folding. Moreover, the amino acid substitution compromised the function of SLC2A4RG as a cancer suppressor by promoting cell growth through de-inhibition of CDK1 in U87 and U251 cell lines. These results suggest SLC2A4RG plays an important role in the etiology of GBM and may be a potential therapeutic target.

Development of background-free tame fluorescent probes for intracellular live cell imaging.

Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule's roles and functions. Besides being nontoxic and specific, desirable labelling probes should be highly cell permeable without nonspecific interactions with other cellular components to warrant high signal-to-noise ratio. While it is critical, rational design for such probes is tricky. Here we report the first predictive model for cell permeable background-free probe development through optimized lipophilicity, water solubility and charged van der Waals surface area. The model was developed by utilizing high-throughput screening in combination with cheminformatics. We demonstrate its reliability by developing CO-1 and AzG-1, a cyclooctyne- and azide-containing BODIPY probe, respectively, which specifically label intracellular target organelles and engineered proteins with minimum background. The results provide an efficient strategy for development of background-free probes, referred to as 'tame' probes, and novel tools for live cell intracellular imaging.

VAMP8 facilitates cellular proliferation and temozolomide resistance in human glioma cells.

BACKGROUND: Malignant glioma is a common and lethal primary brain tumor in adults. Here we identified a novel oncoprotein, vesicle-associated membrane protein 8 (VAMP8), and investigated its roles in tumorigenisis and chemoresistance in glioma. METHODS: The expression of gene and protein were determined by quantitative PCR and Western blot, respectively. Histological analysis of 282 glioma samples and 12 normal controls was performed by Pearson's chi-squared test. Survival analysis was performed using the log-rank test and Cox proportional hazards regression. Cell proliferation and cytotoxicity assay were conducted using Cell Counting Kit-8. Autophagy was detected by confocal microscopy and Western blot. RESULTS: VAMP8 was significantly overexpressed in human glioma specimens and could become a potential novel prognostic and treatment-predictive marker for glioma patients. Overexpression of VAMP8 promoted cell proliferation in vitro and in vivo, whereas knockdown of VAMP8 attenuated glioma growth by arresting cell cycle in the G0/G1 phase. Moreover, VAMP8 contributed to temozolomide (TMZ) resistance by elevating the expression levels of autophagy proteins and the number of autophagosomes. Further inhibition of autophagy via siRNA-mediated knockdown of autophagy-related gene 5 (ATG5) or syntaxin 17 (STX17) reversed TMZ resistance in VAMP8-overexpressing cells, while silencing of VAMP8 impaired the autophagic flux and alleviated TMZ resistance in glioma cells. CONCLUSION: Our findings identified VAMP8 as a novel oncogene by promoting cell proliferation and therapeutic resistance in glioma. Targeting VAMP8 may serve as a potential therapeutic regimen for the treatment of glioma.

High expression of N-myc (and STAT) interactor predicts poor prognosis and promotes tumor growth in human glioblastoma.

Glioma is the most malignant brain tumor and glioblastoma (GBM) is the most aggressive type. The involvement of N-myc (and STAT) interactor (NMI) in tumorigenesis was sporadically reported but far from elucidation. This study aims to investigate roles of NMI in human glioma. Three independent cohorts, the Chinese tissue microarray (TMA) cohort (N = 209), the Repository for Molecular Brain Neoplasia Data (Rembrandt) cohort (N = 371) and The Cancer Genome Atlas (TCGA) cohort (N = 528 or 396) were employed. Transcriptional or protein levels of NMI expression were significantly increased according to tumor grade in all three cohorts. High expression of NMI predicted significantly unfavorable clinical outcome for GBM patients, which was further determined as an independent prognostic factor. Additionally, expression and prognostic value of NMI were associated with molecular features of GBM including PTEN deletion and EGFR amplification in TCGA cohort. Furthermore, overexpression or depletion of NMI revealed its regulation on G1/S progression and cell proliferation (both in vitro and in vivo), and this effect was partially dependent on STAT1, which interacted with and was regulated by NMI. These data demonstrate that NMI may serve as a novel prognostic biomarker and a potential therapeutic target for glioblastoma.

Membrane assisted micro-solid phase extraction of pharmaceuticals with amino and urea-grafted silica gel.

Individual polar sorbents with surface-displayed amino groups (APS) and non-nucleophilic urea-groups (UPS), were prepared by chemical modification of granular silica gel with bifunctional silane coupling reagents. In this preliminary study, they were separately employed for micro-solid phase extraction (µ-SPE) of the quarternary salt of Amitriptyline (Ami), Carbamazepine (Cbz), Ketoprofen (Ket) and Diclofenac (Dfn) from aqueous samples in conjunction with high performance liquid chromatography. The resulting enrichment factors for both APS and UPS are comparable and exceeded those of µ-SPE involving commercial C18-silica gel sorbents. The presence of highly polar, but non-basic and non-nucleophilic surfaces on UPS prompted the development of a UPS-based µ-SPE method. Good linear correlation was found over a concentration range of 0-50 µg L(-1) with limits of detection ranging from 0.66 to 2.36 µg L(-1)). Limits of quantification between 1.61 and 7.88 µg L(-1) were obtained. HPLC analyses indicated that relative recoveries of 123% for Ami, 65.6% for Cbz, 71.2% for Ket and 66.5% for Dfn were obtained during µ-SPE of spiked (10 µg L(-1)) environmental water samples with percentage relative standard deviations (%RSD) of between 2.1% and 12.6%.