Sensitive Detection of KRAS Mutations by Clustered Regularly Interspaced Short Palindromic Repeats

Kirsten rat sarcoma viral oncogene (KRAS) is the isoform most frequently mutated in human tumors. Testing for activating KRAS mutations has important implications for diagnosis and the personalized medicine of cancers. The current techniques for detecting KRAS mutations have moderate sensitivity. The emerging clustered regularly interspaced short palindromic repeats (CRISPR) system shows great promise in the detection of nucleic acids and is revolutionizing medical diagnostics. This study aimed to develop CRISPR–Cas12a as a sensitive test to detect KRAS mutations. Serially diluted DNA samples containing KRAS mutations are subjected to CRISPR–Cas12a and polymerase chain reaction (PCR). CRISPR–Cas12a and PCR can specifically detect 0.01% and 0.1% mutant KRAS DNA in the presence of wild-type KRAS DNA, respectively. Twenty pairs of lung tumor and noncancerous lung tissues are tested by CRISPR–Cas12a, PCR, and direct sequencing. CRISPR–Cas12a could identify the G12C mutation in five of 20 tumor tissues, while both PCR and direct sequencing discovered the KRAS mutation in three of the five tumor tissues. Furthermore, the results of CRISPR–Cas12a for testing the mutation could be directly and immediately visualized by a UV light illuminator. Altogether, CRISPR–Cas12a has a higher sensitivity for the detection of KRAS mutations compared with PCR and sequencing analysis, and thus has diagnostic and therapeutic implications. Nevertheless, the technique needs to be validated for its clinical significance in a large and prospective study

Rapid and Sensitive Detection of SARS-CoV-2 Using Clustered Regularly Interspaced Short Palindromic Repeats

Rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for controlling the pandemic of coronavirus disease 2019. Polymerase chain reaction (PCR)-based technique is the standard test for detection of SARS-CoV-2, which, however, requires complicated sample manipulation (e.g., RNA extraction) and is time-consuming. We previously demonstrated that clustered regularly interspaced short palindromic repeats (CRISPR) could precisely detect Human papillomavirus and somatic mutations of Epidermal growth factor receptor gene and Kirsten rat sarcoma viral oncogene homolog gene in plasma. The objective of this study was to develop CRISPR as a rapid test for sensitive detection of SARS-CoV-2. We first combined reverse transcription-isothermal recombinase polymerase amplification and CRSIPR to detect SARS-CoV-2 in genomic RNA of cells infected with the virus. The CRISPR assay with guide RNA against the M gene of SARS-CoV-2 had a sensitivity of 0.1 copies per µL for detection of the virus. We then used the CRSIPR assay to directly analyze raw SARS-CoV-2 samples. The CRISPR assay could sensitively detect SARS-CoV-2 in one hour without RNA extraction. This assay can be performed at a single temperature and with minimal equipment. The results were immediately visualized either by a UV light illuminator or paper strips. The diagnostic value of the test was confirmed in nasopharyngeal swab specimens. Altogether, we have developed a rapid CRISPR test for sensitive detection of SARS-CoV-2

Utilizing MiSeq Sequencing to Detect Circulating microRNAs in Plasma for Improved Lung Cancer Diagnosis

Non-small cell lung cancer (NSCLC) is a major contributor to cancer-related deaths, but early detection can reduce mortality. NSCLC comprises mainly adenocarcinoma (AC) and squamous cell carcinoma (SCC). Circulating microRNAs (miRNAs) in plasma have emerged as promising biomarkers for NSCLC. However, existing techniques for analyzing miRNAs have limitations, such as restricted target detection and time-consuming procedures. The MiSeqDx System has been shown to overcome these limitations, making it a promising tool for routine clinical settings. We investigated whether the MiSeqDx could profile cell-free circulating miRNAs in plasma and diagnose NSCLC. We sequenced RNA from the plasma of patients with AC and SCC and from cancer-free smokers using the MiSeqDx to profile and compare miRNA expressions. The MiSeqDx exhibits high speed and accuracy when globally analyzing plasma miRNAs. The entire workflow, encompassing RNA to data analysis, was completed in under three days. We also identified panels of plasma miRNA biomarkers that can diagnose NSCLC with 67% sensitivity and 68% specificity, and detect SCC with 90% sensitivity and 94% specificity, respectively. This study is the first to demonstrate that rapid profiling of plasma miRNAs using the MiSeqDx has the potential to offer a straightforward and effective method for the early detection and classification of NSCLC

Rapid and Sensitive Detection of SARS-CoV-2 Using Clustered Regularly Interspaced Short Palindromic Repeats

Rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for controlling the pandemic of coronavirus disease 2019. Polymerase chain reaction (PCR)-based technique is the standard test for detection of SARS-CoV-2, which, however, requires complicated sample manipulation (e.g., RNA extraction) and is time-consuming. We previously demonstrated that clustered regularly interspaced short palindromic repeats (CRISPR) could precisely detect Human papillomavirus and somatic mutations of Epidermal growth factor receptor gene and Kirsten rat sarcoma viral oncogene homolog gene in plasma. The objective of this study was to develop CRISPR as a rapid test for sensitive detection of SARS-CoV-2. We first combined reverse transcription-isothermal recombinase polymerase amplification and CRSIPR to detect SARS-CoV-2 in genomic RNA of cells infected with the virus. The CRISPR assay with guide RNA against the M gene of SARS-CoV-2 had a sensitivity of 0.1 copies per µL for detection of the virus. We then used the CRSIPR assay to directly analyze raw SARS-CoV-2 samples. The CRISPR assay could sensitively detect SARS-CoV-2 in one hour without RNA extraction. This assay can be performed at a single temperature and with minimal equipment. The results were immediately visualized either by a UV light illuminator or paper strips. The diagnostic value of the test was confirmed in nasopharyngeal swab specimens. Altogether, we have developed a rapid CRISPR test for sensitive detection of SARS-CoV-2

In Vivo Osseointegration Performance of Titanium Dioxide Coating Modified Polyetheretherketone Using Arc Ion Plating for Spinal Implant Application

Polyetheretherketone (PEEK), which has biomechanical performance similar to that of human cancellous bone, is used widely as a spinal implant material. However, its bioinertness and hydrophobic surface properties result in poor osseointegration. This study applies a novel modification method, arc ion plating (AIP), that produces a highly osteoblast compatible titanium dioxide (TiO2) coatings on a PEEK substrate. This PEEK with TiO2 coating (TiO2/PEEK) was implanted into the femurs of New Zealand white male rabbits to evaluate its in vivo performance by the push-out test and histological observation. Analytical results show that AIP can prepare TiO2 coatings on bullet-shaped PEEK substrates as implant materials. After prolonged implantation in rabbits, no signs of inflammation existed. Newly regenerated bone formed more prominently with the TiO2/PEEK implant by histological observation. The shear strength of the bone/implant interface increases as implantation period increases. Most importantly, bone bonding performance of the TiO2/PEEK implant was superior to that of bare PEEK. The rutile-TiO2 coatings achieved better osseointegration than the anatase-TiO2 coatings. Therefore, AIP-TiO2 can serve as a novel surface modification method on PEEK for spinal interbody fusion cages

Polyurethane/Nanosilver-Doped Halloysite Nanocomposites: Thermal, Mechanical Properties, and Antibacterial Properties

In this study, the researchers successfully embellished the surface of halloysite (Ag/HNTs) with silver using halloysite, silver nitrate (AgNO3), and polyvinylpyrrolidone (PVP). The researchers then prepared polyurethane that contained pyridine ring by using 4,4′-diphenylmethane diisocyanate (MDI) and polytetramethylene glycol (PTMG) as the hard chain segment and the soft chain segment of polyurethane (PU), as well as 2,6-pyridinedimethanol (2,6-PDM) as the chain extension agent. This was followed by the preparation of Ag/HNTs/PUs nanocomposite thin films, achieved by mixing Ag/HNTs with different ratios into polyurethane that contains pyridine ring. First, the Ag/HNTs powders were analyzed using energy-dispersive X-ray spectroscopy, X-ray diffraction, and transmission electron microscopy. Subsequently, Fourier-transform infrared spectroscopy was used to examine the dispersibility of Ag/HNTs in PU, whereas the thermal stability and the viscoelasticity of Ag/HNTs/PU were examined using thermal gravimetric analysis, differential scanning calorimetry, and dynamic mechanical analysis. When the mechanical properties of Ag/HNTs/PU were tested using a universal strength tester, the results indicated a maximum increase of 109.5% in tensile strength. The researchers then examined the surface roughness and the hydrophobic ability of the Ag/HNTs/PU thin films by using atomic force microscopy and water contact angle. Lastly, antibacterial testing on Escherichia coli revealed that when the additive of Ag/HNTs reached 2.0 wt%, 99.3% of the E. coli were eliminated. These results indicated that the addition of Ag/HNTs into PU could enhance the thermal stability, mechanical properties, and antibacterial properties of PU, implying the potential of Ag/HNTs-02 as biomedicine material

Treating delayed endoscopic sphincterotomy-induced bleeding: Epinephrine injection with or without thermotherapy

AIM: To compare the hemostatic efficacy between epinephrine injection alone and epinephrine injection combined with thermotherapy for delayed post-endoscopic sphincterotomy (ES) bleeding