Further Dimensions for Sensing in Biofluids: Distinguishing Bioorganic Analytes by the Salt-Induced Adaptation of a Cucurbit[7]uril-Based Chemosensor.

Insufficient binding selectivity of chemosensors often renders biorelevant metabolites indistinguishable by the widely used indicator displacement assay. Array-based chemosensing methods are a common workaround but require additional effort for synthesizing a chemosensor library and setting up a sensing array. Moreover, it can be very challenging to tune the inherent binding preference of macrocyclic systems such as cucurbit[n]urils (CBn) by synthetic means. Using a novel cucurbit[7]uril-dye conjugate that undergoes salt-induced adaptation, we now succeeded in distinguishing 14 bioorganic analytes from each other through the facile stepwise addition of salts. The salt-specific concentration-resolved emission provides additional information about the system at a low synthetic effort. We present a data-driven approach to translate the human-visible curve differences into intuitive pairwise difference measures. Ion mobility experiments combined with density functional theory calculations gave further insights into the binding mechanism and uncovered an unprecedented ternary complex geometry for CB7. TThis work introduces the non-selectively binding, salt-adaptive cucurbit[n]uril system for sensing applications in biofluids such as urine, saliva, and blood serum.

[Study on Parametric Release of Ethylene Oxide Sterilization of Medical Devices].

This study briefly introduces the basic theory of sterilization, the characteristics of ethylene oxide sterilization for medical devices and the key factors about sterilization effectiveness, analyzes and compares three methods used in the product release of medical devices sterilized by ethylene oxide: test for sterility, traditional release and parametric release, and focuses on the theoretical basis, feasibility, validation requirements, advantages and disadvantages of parametric release.

Legionella qingyii sp. nov., isolated from water samples in China.

Three Legionella-like strains, designed km488T, km489 and km521, were isolated from freshwater samples in China. Cells were Gram-stain-negative, rod-shaped and non-spore-forming. Growth was observed on BCYEα agar, but not on BCYEα agar without l-cysteine, chocolate agar with PolyViteX or Columbia blood agar. The major fatty acids (>5 %) of strains km488T, km489 and km521 were C16 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The mip gene sequences (574 nt) showed the isolates were almost identical with more than 99.7 % sequence similarities, and closely matched to L. gormanii ATCC 33297T with 95.4-95.6 % sequence similarities. Phylogenetic analyses based on concatenated gene (16S rRNA, mip, rpoB and rnpB) sequences indicated that the isolates formed a distinct cluster along with L. gormanii within the genus Legionella. Matrix-assisted laser desorption ionization time-of-flight analyses also demonstrated a clear separation between the isolates and other closely and distantly related Legionella species. DNA-DNA hybridization studies demonstrated that the isolates were closely related (92.0 -95.0 % DNA-DNA relatedness) but differentiated from their phylogenetic neighbours (<70 % DNA-DNA relatedness). The whole genome of km488T was sequenced, and showed a G+C content of 37.8 mol%. Based on the findings from this polyphasic taxonomic study, the isolates are considered to represent a single novel species, for which the name Legionella qingyii sp. nov. is proposed. The type strain is km488T (KCTC 15636T=CCTCC AB 2018025T=NRBC 113223T).

[Analysis of 10 patients with duplications of 15q11q13 region and autism features].

OBJECTIVE To analyze the clinical and genetic features of 10 unrelated patients with duplications of 15q11q13 region and autism features.METHODS Karyotyping,chromosomal microarray analysis (CMA) and fluorescence in situ hybridization (FISH) were carried out for the patients and their parents.RESULTS Eight patients presented with a supernumerary marker chromosome (SMC) of unknown origin by G-banding analysis and triplication of the 15q11q13 region by high-resolution CMA analysis. Two remaining patients had normal karyotypes but duplications of the 15q11q13 region. All duplications have encompassed the Prader Willi/Angelman syndrome critical region (PWACR). Similar gains in copy number were not detected among the parents of the patients,suggesting a de novo origin for them. Analysis of SNP-array data of the family trios using Chromosome Analysis Suite Software found that the copy number gains have originated from the mothers.The diagnosis of 15q11q13 duplication syndrome was ascertained. For patients with SMC detected by karyotyping analysis,a FISH assay using probes specific for the 15q11q13 region showed that such SMC also derived from chromosome 15q11q13 region and contained two copy numbers, which was consistent with the result of CMA.CONCLUSION Ten patients with autism and 15q11q13 duplications were identified with combined karyotyping, CMA and FISH analysis. A phenotype - genotype correlation was established.

[Development of ultrasonic power meter].

This article describes the design and development of an ultrasonic power meter which is consist of an electronic balance, a practice target, an acoustic enclosures and a blocking. The electronic balance mounted on the blocking is linked with the practice target by connecting rod. By adjusting the blocking makes the practice target suspended above ultrasound probe, and then the ultrasonic power can be measured. After initial tests, the ultrasonic power meter performanced with good stability and high precision.

Efficient optimization parameter calibration method-based DEM simulation for compacted loess slope under dry-wet cycling.

Dry-wet cycles can cause significant deterioration of compacted loess and thus affect the safety of fill slopes. The discrete element method (DEM) can take into account the non-homogeneous, discontinuous, and anisotropic nature of the geotechnical medium, which is more capable of reflecting the mechanism and process of instability in slope stability analysis. Therefore, this paper proposes to use the DEM to analyze the stability of compacted loess slopes under dry-wet cycles. Firstly, to solve the complex calibration problem between macro and mesoscopic parameters in DEM models, an efficient parameter optimization method was proposed by introducing the chaotic particle swarm optimization with sigmoid-based acceleration coefficients algorithm (CPSOS). Secondly, during the parameter calibration, a new indicator, the bonding ratio (BR), was proposed to characterize the development of pores and cracks in compacted loess during dry-wet cycles, to reflect the impact of dry-wet action on the degradation of bonding between loess aggregates. Finally, according to the results of parameter calibration, the stability analysis model of compacted loess slope under dry-wet cycling was established. The results show that the proposed optimization calibration method can accurately reflect the trend of the stress-strain curve and strength of the actual test results under dry-wet cycles, and the BR also reflects the degradation effect of dry-wet cycles on compacted loess. The slope stability analysis shows that the DEM reflects the negative effect of dry-wet cycles on the safety factor of compacted loess slopes, as well as the trend of gradual stabilization with dry-wet cycles. The comparison with the finite element analysis results verified the accuracy of the discrete element slope stability analysis.

Identification method of thyroid nodule ultrasonography based on self-supervised learning dual-branch attention learning framework.

Thyroid ultrasound is a widely used diagnostic technique for thyroid nodules in clinical practice. However, due to the characteristics of ultrasonic imaging, such as low image contrast, high noise levels, and heterogeneous features, detecting and identifying nodules remains challenging. In addition, high-quality labeled medical imaging datasets are rare, and thyroid ultrasound images are no exception, posing a significant challenge for machine learning applications in medical image analysis. In this study, we propose a Dual-branch Attention Learning (DBAL) convolutional neural network framework to enhance thyroid nodule detection by capturing contextual information. Leveraging jigsaw puzzles as a pretext task during network training, we improve the network's generalization ability with limited data. Our framework effectively captures intrinsic features in a global-to-local manner. Experimental results involve self-supervised pre-training on unlabeled ultrasound images and fine-tuning using 1216 clinical ultrasound images from a collaborating hospital. DBAL achieves accurate discrimination of thyroid nodules, with a 88.5% correct diagnosis rate for malignant and benign nodules and a 93.7% area under the ROC curve. This novel approach demonstrates promising potential in clinical applications for its accuracy and efficiency.

Binding affinity-based intracellular drug detection enabled by a unimolecular cucurbit[7]uril-dye conjugate.

Label-free fluorescence-based chemosensing has been increasingly brought into focus due to its simplicity and high sensitivity for intracellular monitoring of molecules. Currently used methods, such as conventional indicator displacement assays (IDAs), pose limitations related to dissociation upon dilution, random diffusion of the released indicators, and high sensitivity to interference by agents from the ambient cellular environment (e.g., salts, enzymes, and proteins). Herein we report a potentially widely applicable strategy to overcome the limitations of conventional IDAs by employing a macrocyclic cucurbit[7]uril (CB7) host covalently coupled to a nitrobenzoxadiazole (NBD) fluorescent dye (CB7-NBD conjugate). As a proof of concept, we demonstrated that the CB7-NBD unimolecular conjugate responded to various target analytes even in the complex live cell system. Moreover, the sensing system was compatible with fluorescence imaging, fluorescence-assisted cell sorting (FACS), and fluorescence spectrometry with a microplate reader. These experiments demonstrated an application of covalently bound unimolecular CB7-NBD conjugate as a sensor for detecting diverse analytes in the intracellular compartment of live cells.

Deep Learning Performance of Ultra-Widefield Fundus Imaging for Screening Retinal Lesions in Rural Locales.

Importance: Retinal diseases are the leading cause of irreversible blindness worldwide, and timely detection contributes to prevention of permanent vision loss, especially for patients in rural areas with limited medical resources. Deep learning systems (DLSs) based on fundus images with a 45° field of view have been extensively applied in population screening, while the feasibility of using ultra-widefield (UWF) fundus image-based DLSs to detect retinal lesions in patients in rural areas warrants exploration. Objective: To explore the performance of a DLS for multiple retinal lesion screening using UWF fundus images from patients in rural areas. Design, Setting, and Participants: In this diagnostic study, a previously developed DLS based on UWF fundus images was used to screen for 5 retinal lesions (retinal exudates or drusen, glaucomatous optic neuropathy, retinal hemorrhage, lattice degeneration or retinal breaks, and retinal detachment) in 24 villages of Yangxi County, China, between November 17, 2020, and March 30, 2021. Interventions: The captured images were analyzed by the DLS and ophthalmologists. Main Outcomes and Measures: The performance of the DLS in rural screening was compared with that of the internal validation in the previous model development stage. The image quality, lesion proportion, and complexity of lesion composition were compared between the model development stage and the rural screening stage. Results: A total of 6222 eyes in 3149 participants (1685 women [53.5%]; mean [SD] age, 70.9 [9.1] years) were screened. The DLS achieved a mean (SD) area under the receiver operating characteristic curve (AUC) of 0.918 (0.021) (95% CI, 0.892-0.944) for detecting 5 retinal lesions in the entire data set when applied for patients in rural areas, which was lower than that reported at the model development stage (AUC, 0.998 [0.002] [95% CI, 0.995-1.000]; P < .001). Compared with the fundus images in the model development stage, the fundus images in this rural screening study had an increased frequency of poor quality (13.8% [860 of 6222] vs 0%), increased variation in lesion proportions (0.1% [6 of 6222]-36.5% [2271 of 6222] vs 14.0% [2793 of 19 891]-21.3% [3433 of 16 138]), and an increased complexity of lesion composition. Conclusions and Relevance: This diagnostic study suggests that the DLS exhibited excellent performance using UWF fundus images as a screening tool for 5 retinal lesions in patients in a rural setting. However, poor image quality, diverse lesion proportions, and a complex set of lesions may have reduced the performance of the DLS; these factors in targeted screening scenarios should be taken into consideration in the model development stage to ensure good performance.

Chemiluminescent Cucurbit[n]uril-Based Chemosensor for the Detection of Drugs in Biofluids.

Chemiluminescence-based detection methods offer a superior signal-to-noise ratio and are commonly adopted for biosensors. This work presents the design and implementation of a supramolecular assay based on a chemiluminescent chemosensor. Specifically, an indicator displacement assay (IDA) with the supramolecular host-guest complex of chemiluminescent phenoxy 1,2-dioxetane and cucurbit[8]uril enables the low-micromolar detection of drugs in human urine and human serum samples. Cucurbit[8]uril thereby acts as a non-surfactant chemiluminescence enhancer and a synthetic receptor. Additionally, we show that adding an equimolar amount of cucurbit[8]uril to a commercially available dioxetane used in standard enzymatic chemiluminescence immunoassays enhances the chemiluminescence by more than 15 times. Finally, we demonstrate that a chemiluminescence resonance energy transfer between a unimolecular macrocyclic cucurbit[7]uril-dye conjugate and a phenoxy 1,2-dioxetane can be utilized to detect the herbicide paraquat at a micromolar concentration in aqueous media.

Mutation Spectrum of EGFR From 21,324 Chinese Patients With Non-Small Cell Lung Cancer (NSCLC) Successfully Tested by Multiple Methods in a CAP-Accredited Laboratory.

Genotyping epidermal growth factor receptor (EGFR) gene in patients with advanced non-small cell lung cancers (NSCLC) is essential for identifying those patients who may benefit from targeted therapies. Systemically evaluating EGFR mutation detection rates of different methods currently used in clinical setting will provide valuable information to clinicians and laboratory scientists who take care of NSCLC patients. This study retrospectively reviewed the EGFR data obtained in our laboratory in last 10 years. A total of 21,324 NSCLC cases successfully underwent EGFR genotyping for clinical therapeutic purpose, including 5,244 cases tested by Sanger sequencing, 13,329 cases tested by real-time PCR, and 2,751 tested by next-generation sequencing (NGS). The average EGFR mutation rate was 45.1%, with 40.3% identified by Sanger sequencing, 46.5% by real-time PCR and 47.5% by NGS. Of these cases with EGFR mutations identified, 93.3% of them harbored a single EGFR mutation (92.1% with 19del or L858R, and 7.9% with uncommon mutations) and 6.7% harbored complex EGFR mutations. Of the 72 distinct EGFR variants identified in this study, 15 of them (single or complex EGFR mutations) were newly identified in NSCLC. For these cases with EGFR mutations tested by NGS, 65.3% of them also carried tumor-related variants in some non-EGFR genes and about one third of them were considered candidates of targeted drugs. NGS method showed advantages over Sanger sequencing and real-time PCR not only by providing the highest mutation detection rate of EGFR but also by identifying actionable non-EGFR mutations with targeted drugs in clinical setting.

Covalent cucurbit[7]uril-dye conjugates for sensing in aqueous saline media and biofluids.

Non-covalent chemosensing ensembles of cucurbit[n]urils (CBn) have been widely used in proof-of-concept sensing applications, but they are prone to disintegrate in saline media, e.g. biological fluids. We show here that covalent cucurbit[7]uril-indicator dye conjugates are buffer- (10× PBS buffer) and saline-stable (up to 1.4 M NaCl) and allow for selective sensing of Parkinson's drug amantadine in human urine and saliva, where the analogous non-covalent CB7⊃dye complex is dysfunctional. The in-depth analysis of the covalent host-dye conjugates in the gas-phase, and deionized versus saline aqueous media revealed interesting structural, thermodynamic and kinetic effects that are of general interest for the design of CBn-based supramolecular chemosensors and systems. This work also introduces a novel high-affinity indicator dye for CB7 through which fundamental limitations of indicator displacement assays (IDA) were exposed, namely an impractical slow equilibration time. Unlike non-covalent CBn⊃dye reporter pairs, the conjugate chemosensors can also operate through a SN2-type guest-dye exchange mechanism, which shortens assay times and opens new avenues for tailoring analyte-selectivity.

Correction: Mutation spectrums of TSC1 and TSC2 in Chinese women with lymphangioleiomyomatosis (LAM).

[This corrects the article DOI: 10.1371/journal.pone.0226400.].

One-step preparation of gold nanovectors using folate modified polyethylenimine and their use in target-specific gene transfection.

Gene transfection, as an effective treatment for inherited and acquired life threatening diseases caused by genetic deficiencies and abnormalities, has evolved as a promising therapeutic strategy for cancer and other intractable diseases. Non-target-specific vectors will affect normal cells as well as pathogenic cells, resulting in a relative decrease in transfection efficiency and unnecessary cytotoxicity. In the present work, gold nanoparticles (AuNPs) functionalized with folate (FA)-modified polyethylenimine (PEI-FA) were prepared by a single step method (without additional reducing agent) for targeted gene transfection in tumor cells. Moreover, an improved compound vector system was developed by mixing PEI-AuNPs and PEI-FA-AuNPs. It was shown that the compound vector system not only greatly increased transfection efficiency in HeLa cells, but also reduced cytotoxicity. By comparison, the transfection efficiency in L929 cells lacking folate receptor, was clearly lower than in tumor cells. The specific gene transfection of HeLa cells using this vector system could be clearly observed by confocal laser scanning microscopy in a co-culture system of HeLa cells and L929 cells. This transfection system with high-efficiency, high-specificity and low-toxicity appears to have potential in targeted cancer treatment and drug delivery.

Mutation spectrums of TSC1 and TSC2 in Chinese women with lymphangioleiomyomatosis (LAM).

The aim of our study was to elucidate the landscapes of genetic alterations of TSC1 and TSC2 as well as other possible non-TSC1/2 in Lymphangioleiomyomatosis (LAM) patients. Sixty-one Chinese LAM patients' clinical information was collected. Tumor biopsies and matched leukocytes from these patients were retrospectively analyzed by next generation sequencing (NGS), chromosomal microarray analysis (CMA), and multiplex ligation-dependent probe amplification (MLPA). Eighty-six TSC1/2 variants were identified in 46 of the 61 LAM patients (75.4%) in which TSC2 and TSC1 variants were 88.37% and 11.63% respectively. The 86 variants are composed of (i) 52 single nucleotide variants (SNVs) (including 30 novel variants), (ii) 23 indels (including 21deletions, and 2 insertions), (iii) a germline duplication of exon 31-42 of TSC2, (iv) a 2.68 Mb somatic duplication containing TSC2, and (v) 9 regions with copy-neutral loss of heterogeneity (CN-LOHs) present only in the LAM patients with single TSC1/2 mutations. Sixty-one non-TSC1/2 variants in 31 genes were identified in 37 LAM patients. Combined applications of different techniques are necessary to achieve maximal detection rate of TSC1/2 variants in LAM patients. Thirty novel TSC1/2 variants expands the spectrum of TSC1/2 in LAM patients. Identification of 61 non-TSC1/2 variants suggests that alternative genes might have contributed to the initiation and progression of LAM.

Parallel Analyses of Somatic Mutations in Plasma Circulating Tumor DNA (ctDNA) and Matched Tumor Tissues in Early-Stage Breast Cancer.

PURPOSE: Early detection and intervention can decrease the mortality of breast cancer significantly. Assessments of genetic/genomic variants in circulating tumor DNA (ctDNA) have generated great enthusiasm for their potential application as clinically actionable biomarkers in the management of early-stage breast cancer.Experimental Design: In this study, 861 serial plasma and matched tissue specimens from 102 patients with early-stage breast cancer who need chemotherapy and 50 individuals with benign breast tumors were deeply sequenced via next-generation sequencing (NGS) techniques using large gene panels. RESULTS: Cancer tissues in this cohort of patients showed profound intratumor heterogeneities (ITHGs) that were properly reflected by ctDNA testing. Integrating the ctDNA detection rate of 74.2% in this cohort with the corresponding predictive results based on Breast Imaging Reporting and Data System classification (BI-RADS) could increase the positive predictive value up to 92% and potentially dramatically reduce surgical overtreatment. Patients with positive ctDNA after surgery showed a higher percentage of lymph node metastasis, indicating potential recurrence and remote metastasis. The ctDNA-positive rates were significantly decreased after chemotherapy in basal-like and Her2+ tumor subtypes, but were persistent despite chemotherapy in luminal type. The tumor mutation burden in blood (bTMB) assessed on the basis of ctDNA testing was positively correlated with the TMB in tumor tissues (tTMB), providing a candidate biomarker warranting further study of its potentials used for precise immunotherapy in cancer. CONCLUSIONS: These data showed that ctDNA evaluation is a feasible, sensitive, and specific biomarker for diagnosis and differential diagnosis of patients with early-stage breast cancer who need chemotherapy.

Sweet Switch: Sugar-Responsive Bioactive Surfaces Based on Dynamic Covalent Bonding.

Smart bioactive surfaces that can modulate interactions with biological systems are of great interest. In this work, a surface with switchable bioactivity in response to sugars has been developed. It is based on dynamic covalent bonding between phenylboronic acid (PBA) and secondary hydroxyls on the "wide" rim of ß-cyclodextrin (ß-CD). The system reported consists of gold surface modified with PBA-containing polymer brushes and a series of functional ß-CD derivatives conjugated to diverse bioactive ligands (CD-X). CD-X molecules are attached to the surface to give specified bioactivity such as capture of a specific protein or killing of attached bacteria. Subsequent treatment with cis-diol containing biomolecules having high affinity for PBA (e.g. fructose) leads to the release of CD-X together with the captured proteins, killed bacteria, and so forth from the surface. The surface bioactivity is thereby "turned off". Effectively, this constitutes an on-off bioactivity switch in a mild and noninvasive way, which has the potential in the design of dynamic bioactive surfaces for biomedical applications.

A supramolecular approach for versatile biofunctionalization of magnetic nanoparticles.

A convenient and versatile approach for biofunctionalization of magnetic nanoparticles (MNPs) was developed based on supramolecular host-guest interaction. Adamantane groups were introduced on the surface of MNPs for further incorporation of specific biofunctional ß-cyclodextrin derivatives, endowing MNPs with the desired bioactivity (e.g. biorecognition capability and biocidal activity).

Using porous magnetic iron oxide nanomaterials as a facile photoporation nanoplatform for macromolecular delivery.

The intracellular delivery of exogenous macromolecules such as functional proteins, antibodies, polysaccharides and nucleic acids into living cells for biomedical applications is of great interest. Even though great efforts have been devoted to this task, universal delivery systems that provide excellent intracellular delivery performance combined with easy cell recovery are urgently needed. Magnetic iron oxide nanoparticles show promising potential for various biomedical applications because of their advantages such as high biocompatibility and cost-effectiveness. Herein, a new facile platform for macromolecular delivery was developed based on the photothermal properties of porous magnetic iron oxide nanoparticles (P-MNPs). The near-infrared radiation (NIR) absorption behavior of P-MNPs remarkably facilitates the delivery of macromolecules into cells while maintaining high cell viability. Furthermore, the assistance of polycationic polyethylenimine improves the efficiency of DNA delivery. Most importantly, the cells could be easily recovered after macromolecular delivery by trypsinization, which is of great significance for further practical application of the delivery system. The facile and cost-effective platform proposed in this work provides a new avenue for the utilization of P-MNPs in macromolecular delivery.

Microfluidic channels with renewable and switchable biological functionalities based on host-guest interactions.

Poly(dimethylsiloxane) (PDMS)-based microfluidic systems are gaining increasing attention due to their ease of fabrication, optical transparency and mechanical properties. However, the inherent hydrophobicity and chemical inertness of PDMS hinder its wider application in microfluidic systems. There is thus a strong need for methods for surface modification of PDMS-based microfluidic channels. In this work, oligo(ethylene glycol)methacrylate (OEGMA) and adamantane-containing OEGMA (OEGMA-Ada) were graft copolymerized on PDMS microchannel surfaces using a simple photochemical process to give PDMS-POA. OEGMA was chosen for its resistance to non-specific protein adsorption, and OEGMA-Ada was chosen for its subsequent attachment of mannose with bacteria binding affinity or biotin with avidin binding affinity. ß-CD decorated with biotin (CD-B) and/or mannose (CD-M) was attached to the PDMS-POA microchannels via host-guest interactions between the adamantane and ß-CD moieties. The data obtained suggest that the functions of the PDMS-POA/CD-B and PDMS-POA/CD-M microchannels with respect to biotin binding and bacterial adhesion were renewable. In addition, the biofunction of the PDMS-POA microchannels could be switched by treatment with SDS to release the CD component followed by treatment with a different ß-CD derivative. Different from previous surface modification strategies for PDMS-based microfluidic channels, the combination of visible light-induced grafting and host-guest chemistry provides modified PDMS microchannels with renewable and switchable biofunctions for the detection and measurement of specific proteins and bacteria.