Lysis-Free Isolation and Direct Amplification of Pathogenic Bacterial DNA Using Diatom Frustules.

DNA has been implicated as an important biomarker for the diagnosis of bacterial infections. Herein, we developed a streamlined methodology that uses diatom frustules (DFs) to liberate and capture bacterial DNA and allows direct downstream amplification tests without any lysis, washing, or elution steps. Unlike most conventional DNA isolation methods that rely on cell lysis to release bacterial DNA, DFs can trigger the oxidative stress response of bacterial cells to promote bacterial membrane vesicle formation and DNA release by generating reactive oxygen species in aqueous solutions. Due to the hierarchical porous structure, DFs provided high DNA capture efficiency exceeding 80% over a wide range of DNA amounts from 10 pg to 10 ng, making only 10 µg DFs sufficient for each test. Since laborious liquid handling steps are not required, the entire DNA sample preparation process using DFs can be completed within 3 min. The diagnostic use of this DF-based methodology was illustrated, which showed that the DNA of the pathogenic bacteria in serum samples was isolated by DFs and directly detected using polymerase chain reaction (PCR) at concentrations as low as 102 CFU/mL, outperforming the most used approaches based on solid-phase DNA extraction. Furthermore, most of the bacterial cells were still alive after DNA isolation using DFs, providing the possibility of recycling samples for storage and further diagnosis. The proposed DF-based methodology is anticipated to simplify bacterial infection diagnosis and be broadly applied to various medical diagnoses and biological research.

Colony and bacterial LAMP: Simple alternative methods for bacterial determination.

In this study, we developed colony and bacterial LAMP, which directly use bacterial colony and bacterial culture as the templates without DNA extraction for rapid and simple detection of bacteria. The end-point readouts were determined by naked eye under ultraviolet light, and real-time fluorescence curve was also used to confirm that the sensitivity of this method to Salmonella typhimurium and Bacillus cereus was 102 and 103 CFU/reaction, respectively. Results presented here provide alternative methods for colony and bacterial PCR that can greatly contribute to reliable and cost-effective diagnosis in resource-poor settings.

Azacitidine combined with venetoclax alleviates AML-MR with TP53 mutation in SDS: a case report and literature review.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive genetic disease, which is prone to transform into myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). TP53 mutation is a driving factor involved in the transformation of SDS into MDS/AML, and in the evolution of MDS to AML. Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is the only curable approach, however, challenge remains regarding the balance between efficacy and the high risk from treatment-related toxicity and mortality to achieve temporary disease control before transplantation to gain time and opportunities for transplantation. At present, pre-transplant bridging therapy has emerged as one of the important options with improved efficacy, reduced tumor burden, and less treatment-related toxicity. Here we reported azacitidine combined with venetoclax was used as pre-transplant bridging regimen in a TP53-mutant AML-MR case developed from SDS. He achieved complete remission with incomplete recovery and proceeded to Allo-HSCT. We hope to provide some evidence and insight for in-depth research and clinical treatment by presenting this case.

Three-way junction structure-mediated reverse transcription-free exponential amplification reaction for pathogen RNA detection.

Since RNA is an important biomarker of many infectious pathogens, RNA detection of pathogenic organisms is crucial for disease diagnosis and environmental and food safety. By simulating the base mismatch during DNA replication, this study presents a novel three-way junction structure-mediated reverse transcription-free exponential amplification reaction (3WJ-RTF-EXPAR) for the rapid and sensitive detection of pathogen RNA. The target RNA served as a switch to initiate the reaction by forming a three-way junction (3WJ) structure with the ex-trigger strand and the ex-primer strand. The generated trigger strand could be significantly amplified through EXPAR to open the stem-loop structure of the molecular beacon to emit fluorescence signal. The proofreading activity of Vent DNA polymerase, in combination with the unique structure of 2+1 bases at the 3'-end of the ex-primer strand, could enhance the role of target RNA as a reaction switch to reduce non-specific amplification and ensure excellent specificity to differentiate target pathogen from those causing similar symptoms. Furthermore, detection of target RNA showed a detection limit of 1.0×104 copies/mL, while the time consumption was only 20 min, outperforming qRT-LAMP and qRT-PCR, the most commonly used RNA detection methods in clinical practice. All those indicates the great application prospects of this method in clinical diagnostic.

One-pot electrochemical detection of foodborne pathogen based on in situ nucleic acid amplification and wash-free assay.

A signal amplification electrochemical biosensor chip was developed to integrate loop-mediated isothermal amplification (LAMP) based on in situ nucleic acid amplification and methyl blue (MB) serving as the hybridization redox indicator for sensitive and selective foodborne pathogen detection without a washing step. The electrochemical biosensor chip was designed by a screen-printed carbon electrode modified with gold nanoparticles (Au NPs) and covered with polydimethylsiloxane membrane to form a microcell. The primers of the target were immobilized on the Au NPs by covalent attachment for in situ amplification. The electroactive MB was used as the electrochemical signal reporter and embedded into the double-stranded DNA (dsDNA) amplicons generated by LAMP. Differential pulse voltammetry was introduced to survey the dsDNA hybridization with MB, which differentiates the specifically electrode-unbound and -bound labels without a washing step. Pyrene as the back-filling agent can further improve response signaling by reducing non-specific adsorption. This method is operationally simple, specific, and effective. The biosensor showed a detection linear range of 102-107 CFU mL-1 with the limit of detection of 17.7 CFU mL-1 within 40 min. This method showed promise for on-site testing of foodborne pathogens and could be integrated into an all-in-one device.

Engineering of a Chimeric Template Triggers RNase H-Based Isothermal Amplification Approach for Sensitive Detection of Pathogen RNA.

RNA-based detection of pathogenic organisms is an emerging field of research that is crucial for disease diagnosis and environmental and food safety. By rationally engineering an RNA-DNA tandem (RDT) structural template, we proposed a novel RNase H-based isothermal exponential amplification (RH-IEA) reaction to rapidly identify long-stranded RNA. In this strategy, the rigid and compact RDT template selectively recognized the target RNA and formed a stable hybrid with it. Upon site-specific cleavage of RNase H, the 3' overhang of the target RNA was cut off, and a free hydroxyl end at the hydrolysis site was generated to trigger an exponential amplification reaction (EXPAR). This method maintained the high efficiency and rapid amplification kinetics of EXPAR. As a result, the RH-IEA strategy was able to sensitively and specifically detect the characteristic sequence of Escherichia coli O157:H7 RNA, with a detection sensitivity of 1 fg/µL. Besides, the RDT template can be used as an RNA protector to prevent specific segments of the target RNA from being degraded by RNase enzymes, allowing the sample to be stored at room temperature for a long time. With this advantage, the practicality of RH-IEA will be more flexible than the reverse transcription polymerase chain reaction. It was successfully applied in the identification of E. coli O157:H7 in milk with a minimum detection concentration of 1.0 × 102 CFU/mL. Therefore, the RH-IEA method will serve as a powerful tool for detecting long-stranded RNA and will also shed light on the pathogen detection in food safety and molecular diagnosis.

Rapid and sensitive detection of Staphylococcus aureus via an all-in-one staggered strand exchange amplification platform.

Staphylococcus aureus is a common foodborne pathogen that causes food poisoning and infectious diseases in humans and animals. Rapid detection of S. aureus with high sensitivity is of great significance to prevent the spread of this pathogen. In this study, we developed a staggered strand exchange amplification (SSEA) method by refining denaturation bubble-mediated strand exchange amplification (SEA) to detect S. aureus at a constant temperature with high specificity and efficiency. This method employs a DNA polymerase and two sets of forward and reverse primers arranged in tandem that invade denaturation bubbles of double-stranded DNA. In comparison, the sensitivity of SSEA was 20 times that of SEA. Subsequently, magnetic bead (MB)-based DNA extraction was introduced into SSEA to establish an all-in-one SSEA platform that incorporated sample processing, amplification and detection in a single tube. The use of MBs further enhanced the sensitivity of SSEA by two orders of magnitude. Specificity tests showed that the all-in-one SSEA could specifically identify S. aureus and had no cross-reaction with other common foodborne pathogens. For artificially spiked meat samples, the method could detect 1.0 × 102 CFU g-1S. aureus in pork and 1.0 × 103 CFU g-1 in either duck or scallop samples without a bacterial enrichment step. The entire assay can be completed sample-to-answer within 1 h. Thus, we believe that this easy-to-operate diagnostic platform enables sensitive and accurate detection of S. aureus and holds great promise for the food safety industry.

Direct capture and amplification of nucleic acids using a universal, elution-free magnetic bead-based method for rapid pathogen detection in multiple types of biological samples.

Nucleic acid amplification tests (NAATs) have become an attractive approach for pathogen detection, and obtaining high-quality nucleic acid extracts from biological samples plays a critical role in ensuring accurate NAATs. In this work, we established an elution-free magnetic bead (MB)-based method by introducing polyethylene-polypropylene glycol (PEPPG) F68 in lysis buffer and using NaOH solution instead of alcohols as the washing buffer for rapid nucleic acid extraction from multiple types of biological samples, including nasopharyngeal swabs, serum, milk, and pork, which bypassed the nucleic acid elution step and allowed the nucleic acid/MB composite to be directly used as the template for amplification reactions. The entire extraction process was able to be completed in approximately 7 min. Even though the nucleic acid/MB composite could not be used for quantitative real-time PCR (qPCR) assays, this elution-free MB-based method significantly improved the sensitivity of the loop-mediated isothermal amplification (LAMP) assay. The sensitivity of the quantitative real-time LAMP (qLAMP) assays combined with this elution-free MB-based method showed an improvement of one to three orders of magnitude compared with qLAMP or qPCR assays combined with the traditional MB-based method. In addition to manual operation, like the traditional MB-based method, this universal, rapid, and facile nucleic acid extraction method also has potential for integration into automated robotic processing, making it particularly suitable for the establishment of an analysis platform for ultrafast and sensitive pathogen detection in various biological samples both in centralized laboratories and at remote sites.

Identification of pivotal genes with prognostic evaluation value in lung adenocarcinoma by bioinformatics analysis.

Lung cancer remains the leading cause of cancer morbidity and mortality worldwide, and over-diagnosis causes various unnecessary losses in patients' lives and health. How to more effectively screen lung cancer patients and their potential prognostic risk become the focus of our current study. By analyzing the LUAD expression profile in The Cancer Genome Atlas (TCGA), we constructed a weighted gene co-expression network using differentially expressed genes (DEGs) to find the key modules and pivotal genes. A COX proportional risk regression model based on the least absolute shrinkage and selection operator (LASSO) was used to assess the predictive value of the model for the prognosis of LUAD patients. A total of 4107 up-regulated DEGs and 2022 down-regulated DEGs were identified in this study, and enrichment analysis showed that these analyzes were associated with the extracellular matrix of cells and adhesion. Ten gene markers consisting of LDHA, TOP2A, UBE2C, TYMS, TRIP13, EXO1, TTK, TPX2, ZWINT, and UHRF1 were established by extracting the central genes in the key modules, and the upregulation of these genes was accompanied by an increased prognostic risk of patients. Among them, high expression of LDHA, TRIP13, and TTK in LUAD was associated with shorter overall survival and could be used as independent prognostic factors to participate in metabolic processes such as tumor NAD. The present study provides a powerful molecular target for the study of LUAD prognosis and provides a theoretical basis for the diagnosis and treatment of LUAD and the development of targeted inhibitors.

The role of neutrophils in corneal nerve regeneration.

BACKGROUND: To investigate the role of neutrophils in corneal nerve regeneration. METHODS: A mouse model simulating corneal nerve injury was established and samples from corneal scraping with and without neutrophil closure were collected. These samples were used for corneal nerve staining, ribonucleic acid sequencing, and bioinformatics. Differential expression analysis was used to perform enrichment analysis to identify any significant differences between these two groups. The differential genes were then intersected with neutrophil-associated genes and a protein-protein interaction network was constructed using the intersected genes. The immune infiltration between the two groups was examined along with the immune cell variation between the high and low gene expression groups. RESULTS: Neutrophil removal delays corneal epithelial and nerve regeneration. A total of 546 differential genes and 980 neutrophil-associated genes, with 27 genes common to both sets were obtained. Molecular Complex Detection analysis yielded five key genes, namely integrin subunit beta 2 (ITGB2), matrix metallopeptidase 9 (MMP9), epidermal growth factor (EGF), serpin family E member 1 (SERPINE1), and plasminogen activator urokinase receptor (PLAUR). Among these genes, ITGB2, SERPINE1, and PLAUR exhibited increased expression in the neutrophil-confined group, while MMP9 and EGF showed decreased expression, with MMP9 and EGF displaying a more significant difference. Immune infiltration was also observed between the two groups, revealing significant differences in the infiltration of M0 macrophages, activated mast cells, and neutrophils. Moreover, the neutrophil levels were lower in the groups with low MMP9 and EGF expressions and higher in the high-expression group. CONCLUSION: Neutrophil confinement might significantly affect the MMP9 and EGF expression levels. Strategies to inhibit MMP9 could potentially yield therapeutic benefits.

Combination of Cetylpyridinium Chloride and Chlorhexidine Acetate: A Promising Candidate for Rapid Killing of Gram-Positive/Gram-Negative Bacteria and Fungi.

Combined use of the present antimicrobial drugs has been proved to be an alternative approach for antimicrobial agents' development since the co-existed of the drugs working in different mechanism have been demonstrated potentially enhance their antimicrobial activity. In this work, antibacterial and antifungal activity of the cetylpyridinium chloride (CPC)/chlorhexidine acetate (CHA) combination was evaluated for the first time, while a universal concentration for the rapid killing of gram-positive/gram-negative bacteria and fungi was also proposed. The minimum inhibitory concentrations (MIC) of CPC and CHA used alone or in combination were first measured, showing that the combined treatment decreased the MIC against tested gram-positive/gram-negative bacteria and fungi to 1/8-1/2. Growth curve assays demonstrated CPC and CHA had dynamic combined effects against the tested microorganisms at the concentration equal to MIC. Besides, combined use of these two drugs could also enhance their biocidal activity, which was illustrated by fluorescence microscopy and SEM images, as well as soluble protein measurement. More importantly, in vitro acute eye and skin irritation tests showed short-term contact with CPC/CHA combination would not cause any damage to mammalian mucosa and skin. In a word, CPC/CHA combination exhibited broad-spectrum antibacterial and antifungal activity against tested gram-positive/gram-negative bacteria and fungi while without any acute irritation to mammalian mucosa and skin, providing a new perspective on the selection of personal disinfectants.

Mach-Zehnder interferometer based integrated-photonic acetone sensor approaching the sub-ppm level detection limit.

The detection of acetone in the gaseous form in exhaled breath using an integrated sensor can provide an effective tool for disease diagnostics as acetone is a marker for monitoring human metabolism. An on-chip acetone gas sensor based on the principle of Mach-Zehnder interferometer is proposed and demonstrated. The sensing arm of the device is activated with a composite film of polyethyleneimine and amido-graphene oxide as the gas-sensitive adsorption layer. The composite film demonstrates good selectivity to acetone gas, can be used repeatedly, and is stable in long-term use. Room temperature operation has been demonstrated for the sensor with high sensitivity under a 20 ppm acetone environment. The detection limit can reach 0.76 ppm, making it feasible to be used for the clinical diagnosis of diabetes and the prognosis of heart failure.

Electrical potential-assisted DNA-RNA hybridization for rapid microRNA extraction.

Analysis of microRNAs (miRNAs) is important in cancer diagnostics and therapy. Conventional methods used to extract miRNA for analysis are generally time-consuming. A novel approach for rapid and sensitive extraction of miRNAs is urgently need for clinical applications. Herein, a novel strategy based on electrical potential-assisted DNA-RNA hybridization was designed for miRNA extraction. The entire extraction process was accomplished in approximately 3 min, which is much shorter than the commercial adsorption column method, at more than 60 min, or the TRIzol method, at more than 90 min. Additionally, the method offered the advantages of simplicity and specificity during the extraction process by electrical potential-assisted hybridization of single-stranded DNA and RNA. Taking let-7a as an example, satisfactory results were achieved for miRNA extraction in serum, demonstrating the applicability in miRNA nucleic acid amplification.

Single-tube analysis for ultra-fast and visual detection of Salmonella.

Herein, we developed an ultra-fast and visual single-tube nucleic acid detection approach, which combined the advantages of self-settling characteristics of chitosan-functionalized diatomaceous earth (CDE) and accelerated PCR (AC-PCR). DNA was rapidly extracted by CDE within 3 min for the next nucleic acid amplification based on the nucleic acid attached on the chitosan in pH = 5.0. Under the action of gravity, the DNA-enriched CDE self-sediments to the bottom of the tube could be directly used for AC-PCR to achieve single-tube extraction and amplification. Our method detected Salmonella culture fluids with a detection limit of 1 CFU/mL, which was 100-fold more sensitive than conventional method that have not undergone nucleic acid enrichment. Furthermore, it also displayed high specificity and sensitivity for a variety of spiked samples. The entire process could be completed within 17 min in a single tube, and in particular, the result was visualized by the naked eyes. Overall, it is an all-in-one detection strategy without the requirement of redundant procedure, which greatly improved the detection efficiency, and saved the time and the cost. With these advantages, the approach will supply a promising tool in the field of point-of-care testing for Salmonella and other foodborne pathogens.

Multiplex Accelerated PCR System for One-Step Helicobacter pylori cagA + Genotypes Detection: A Guide for Clinical Testing.

Helicobacter pylori cagA + genotype is a leading risk factor for gastric cancer development making accurate identification and timely eradication of H. pylori critical to deadly gastric cancer prevention. Traditional clinical diagnostic methods, including conventional in vitro culture, histological examination, and (13/14)C-urea breath test methods, could only identify the presence of H. pylori, but these means are not capable of identification of cagA + strains. Herein, we firstly built a multiplex detection system based on novel accelerated PCR that could realize one-step detection of as low as 20 copies of H. pylori 16S rDNA and cagA genes within 30 min. In addition, this novel system performed strong anti-jamming capacity, and exhibited that it could specifically differentiate H. pylori cagA- and cagA + genotypes co-existence with other 4 kinds of gastrointestinal pathogens. Furthermore, this one-step system showed remarkable performance on rapid H. pylori infection diagnosis and cagA + genotypes identification in clinical gastric mucosa samples. Specifically, it outperformed histological examination in terms of accuracy and was superior to conventional PCR and DNA sequencing in terms of efficiency. This rapid, sensitive, and reliable H. pylori detection and identification system would break the limitation of traditional methods and realize H. pylori infection diagnosis and cagA + genotypes identification.

Abusive supervision and employee creativity: the mediating role of passion for inventing and the moderating role of financial incentives and innovative culture.

Based on work passion model and the substitutes for leadership perspectives, this study examines the process linking abusive supervision to employee creativity by focusing on the mediating influence of employees' passion for inventing and the moderating influence of financial incentives and innovative culture. Data were obtained from 191 subordinates and their direct supervisor in China. We tested hypotheses using hierarchical multiple regression analyses. The results revealed that abusive supervision was negatively related to employee creativity, and employees' passion for inventing mediated the relationship between abusive supervision and employee creativity. Furthermore, financial incentives weakened the negative relationship between abusive supervision and employees' passion for inventing, while innovative culture could not change the above relationship. This study enriches the understanding of how abusive supervision is related to employee creativity by introducing the emotional mechanism and provides practical implications for reducing the harm of abusive supervision.

A Fluorescence Assay for Exosome Detection Based on Bivalent Cholesterol Anchor Triggered Target Conversion and Enzyme-Free Signal Amplification.

Exosomes are emerging as one of the most promising biomarkers for early disease diagnosis and prognosis. The significant challenges facing the available methods include improving the detection specificity and sensitivity in complex biological samples. Herein, a fluorescence assay was established based on a combination of immunomagnetic separation and a two-step signal amplification strategy for direct isolation and subsequent detection of exosomes. First, immunomagnetic beads capture and enrich the exosomes via antibody-antigen reactions. Second, bivalent cholesterol (BC) anchors spontaneously insert into the lipid bilayer of bead-captured exosomes, forming a "one to many" amplification effect. The simultaneous recognition of the surface protein and the lipid bilayer structure of the exosome significantly eliminates the interference risk from free proteins. The detection of exosomes converts to the detection of BC-anchors. Finally, the sticky end of the BC-anchor acts as the initiator to trigger the enzyme-free DNA circuits for secondary signal amplification. Under the optimal conditions, highly sensitive and selective detection of exosomes was achieved ranging from 5.5 × 103 to 1.1 × 107 particles/µL with a limit of detection of 1.29 × 103 particles/µL. Moreover, this method allows the isolation and quantitative analysis of exosomes in several biological fluids with satisfactory recovery rates (92.25-106.8%). Thus, this approach provides a sensitive, anti-interference platform for isolating and detecting exosomes.

Nucleic acid extraction without electrical equipment via magnetic nanoparticles in Pasteur pipettes for pathogen detection.

The outbreak of COVID-19 makes epidemic prevention and control become a growing global concern. Nucleic acid amplification testing (NAAT) can realize early and rapid detection of targets, thus it is considered as an ideal approach for detecting pathogens of severe acute infectious diseases. Rapid acquisition of high-quality target nucleic acid is the prerequisite to ensure the efficiency and accuracy of NAAT. Herein, we proposed a simple system in which magnetic nanoparticles (MNPs) based nucleic acid extraction was carried out in a plastic Pasteur pipette. Different from traditional approaches, this proposed system could be finished in 15 min without the supports of any electrical instruments. Furthermore, this system was superior to traditional MNPs based extraction methods in the aspects of rapid extraction and enhancing the sensitivity of a NAAT method, accelerated denaturation bubbles mediated strand exchange amplification (ASEA), to the pathogens from various artificial samples. Finally, this Pasteur pipette system was utilized for pathogen detection in actual samples of throat swabs, cervical swabs and gastric mucosa, the diagnosis results of which were identical with that provided by hospital. This rapid, easy-performing and efficiency extraction method ensures the applications of the NAAT in pathogen detection in regions with restricted resources.

A simple methodology for RNA isolation from bacteria by integration of formamide extraction and chitosan-modified silica purification.

RNA isolation from bacteria is technically difficult due to the RNA characteristic of labile and vulnerable degradation. Many reagents were explored for cellular lysis and complete inhibition of RNase. However, the available methods for RNA isolation are either of low efficiency or time-consuming. Here, we developed a rapid and accessible protocol for RNA isolation that combined a simplified cell lysis and RNA release by formamide-based solution and RNA purification by chitosan-modified silica membrane for the first time. With this method, we obtained about ~ 28 µg of total RNA from 108 Escherichia coli cells. The entire procedure can be done within 15 min without redundant pipetting steps. The purity of extracted RNA was comparable to that of commercial kits, but the cost was much lower. Furthermore, the yielded RNA was successfully used in downstream enzymatic reactions, such as reverse transcription and quantitative real-time PCR. This new method would be of benefit for an extensive range of gene expression analyses in bacterial organisms.

Primer design strategy for denaturation bubble-mediated strand exchange amplification.

Denaturation bubble-mediated strand exchange amplification (SEA) is a novel, rapid isothermal nucleic acid amplification has been applied for point-of-care molecular diagnostic in food safety, meat adulteration, forest disease and animal disease. Nevertheless, the absence of specialized strategy for SEA primers design led to long-time of primer screening progress before SEA reaction execution, which would largely increase the time consuming when SEA is utilized for detecting other new targets. In this present work, we investigated the impact of the following primers' attributes on SEA efficiency, including Tm value, 3' end G/C content, self-complementary and 3' complementary, according to which we demonstrated that optimal Tm value and reaction temperature were all 61 °C, while 3'-terminal nucleotide should be G/C, as the SEA reaction induced by the primers possessing these attributes exhibited significantly lower threshold time (Tt) value. Moreover, self-complementary and 3' complementary of primers should be avoided. Besides, we also discussed the consideration priority order of these factors, which was self-complementary and 3' complementary, Tm value and 3' end G/C content in turn. Because the SEA primer design strategy is first presented, our work will greatly promote the application of SEA in point-of-care test.