Controlling the Crystal Growth of DNA Molecules via Strategic Chemical Modifications.

Intermolecular interactions are critical to the crystallization of biomolecules, yet the precise control of biomolecular crystal growth based on these interactions remains elusive. To understand the connections between the crystallization kinetics and the strength of intermolecular interactions, herein we have employed DNA triangular crystals and modified ones as a versatile tool to investigate how the strength of intermolecular interaction affects crystal growth. Interestingly, we have found that the 2'-O-methylation at sticky ends of the DNA triangle could strengthen its intermolecular interaction, resulting in the accelerated formation of smaller crystals. Conversely, phosphorothioate modification could weaken the sticky-end cohesion and delay the nucleation, resulting in formation of fewer but larger crystals. In addition, these modification effects were consistently observed in the crystallization of a DNA decamer. In one word, our experimental results demonstrate that the strength of intermolecular interaction directly impacts crystal growth. It suggests that 2'-O-methylation and phosphorothioate modification represents a rational strategy for controlling DNA molecules grow into desired crystals and it also facilitates structural determination.

EUS­guided antegrade stenting for malignant jaundice following Roux­en­Y reconstruction: the treatment of multiple biliary strictures.

Multiple malignant biliary strictures are rare, and the application of multiple stents can achieve better drainage. EUS-guided biliary drainage (EUS-BD) can be offered as an alternative technique when ERCP unsuccessful. We applied Endoscopic ultrasonic guided antegrade stenting technique to treat a case of multiple biliary strictures following Roux-en-Y reconstruction.

Tandem Repeat Generation and Novel Isothermal Amplification Based on Nonspecific Tailing and Replication Slippage.

BACKGROUND: Isothermal amplification is considered to be one of the most promising tools for point-of-care testing molecular diagnosis. However, its clinical application is severely hindered by nonspecific amplification. Thus, it is important to investigate the exact mechanism of nonspecific amplification and develop a high-specific isothermal amplification assay. METHODS: Four sets of primer pairs were incubated with Bst DNA polymerase to produce nonspecific amplification. Gel electrophoresis, DNA sequencing, and sequence function analysis were used to investigate the mechanism of nonspecific product generation, which was discovered to be nonspecific tailing and replication slippage mediated tandem repeats generation (NT&RS). Using this knowledge, a novel isothermal amplification technology, bridging primer assisted slippage isothermal amplification (BASIS), was developed. RESULTS: During NT&RS, the Bst DNA polymerase triggers nonspecific tailing on the 3'-ends of DNAs, thereby producing sticky-end DNAs over time. The hybridization and extension between these sticky DNAs generate repetitive DNAs, which can trigger self-extension via replication slippage, thereby leading to nonspecific tandem repeats (TRs) generation and nonspecific amplification. Based on the NT&RS, we developed the BASIS assay. The BASIS is carried out by using a well-designed bridging primer, which can form hybrids with primer-based amplicons, thereby generating specific repetitive DNA and triggering specific amplification. The BASIS can detect 10 copies of target DNA, resist interfering DNA disruption, and provide genotyping ability, thereby offering 100% accuracy for type 16 human papillomavirus detection. CONCLUSION: We discovered the mechanism for Bst-mediated nonspecific TRs generation and developed a novel isothermal amplification assay (BASIS), which can detect nucleic acids with high sensitivity and specificity.

Endoscopic ultrasound-guided fine needle aspiration in the diagnosis of non-Hodgkin's diffuse large B-cell lymphoma with pancreatic metastasis.

Non-Hodgkin lymphoma with pancreatic metastasis is extremely rare, it's easy to be misdiagnose as pancreatic solid tumor and delay treatment. We report a case of Endoscopic ultrasound-guided fine-needle aspiration in the diagnosis of non-Hodgkin's diffuse large B-cell lymphoma with pancreatic metastasis.

Endoscopic management of stent displacement during EUS-guided pancreatic pseudocyst drainage.

The therapeutic effect of EUS-Guided Pancreatic Pseudocyst Drainage (EUS-PPD) is widely recognized, and intraoperative stent displacement is a rare but potentially serious condition. We report a case of the cyst stent displace into the cyst cavity during EUS-PPD, we successfully reduced the stent in time under the guidance of EUS and fluoroscopy in the final.

Signal Extraction, Transformation, and Magnification for Ultrasensitive and Specific Detection of Nucleic Acids.

Nucleic acid noises caused by the background and nonspecificity amplifications can jeopardize accurate polymerization and detection of nucleic acids, especially when they are analyzed in low copies. We hypothesize to reduce the noises by designing a system for specific signal extraction, transformation, and magnification to improve the specificity and sensitivity. Herein, by developing an extractor-trigger complex (ET-Combo) for the system, we have established isothermal and hybridizing combined amplifications: a one-pot detection system with two-step amplification coupled by ET-Combo. To our surprise, the signal extraction is only successful when ET-Combo is included in the first amplification. Our signal extracting, filtering, and relaying system with ET-Combo is rapid and specific, removing the noises generated during the isothermal amplification under elevated temperatures. To match the first amplification, we have designed and established a hybridizing chain reaction at high temperature. This one-pot system can resist disruption of background noises and allow detection of DNA up to five copies (single digit). With the high sensitivity, specificity, and noise resistance, our system has been successfully used to diagnose clinical samples of human papillomavirus (HPV) with the genotyping specificity.

Self-Replication-Assisted Rapid Preparation of DNA Nanowires at Room Temperature and Its Biosensing Application.

A rapid room-temperature DNA nanowires preparation strategy on the basis of self-replicating catalyzed hairpin assembly (SRCHA) was reported. In this system, three hairpin probes (P1, P2, and P3) were well-designed and partially hybridize to each other, and two split trigger DNA sequences were integrated into P1 and P3, respectively. When the SRCHA was initiated by the trigger DNA, a series of DNA assembly steps based on the toehold-mediated DNA strand displacement were activated, and the Y shaped DNA (P1-P2-P3) was formed. In that case, the two split trigger DNA sequences will come into close-enough proximity to form the trigger DNA replicas, which can initiate the additional SRCHA reaction cycles for DNA nanowire preparation, and eventually a rapid room-temperature DNA nanowires preparation strategy without need of fuel strands was successfully developed. Furthermore, the prepared DNA nanowires have been used to develop a rapid and signal amplified sensing platform for sensitive adenosine triphosphate (ATP) detection.

Self-Replicating Catalyzed Hairpin Assembly for Rapid Signal Amplification.

A rapid signal amplification system based on the self-replicating catalyzed hairpin assembly is reported in which two hairpins, H1 and H2, were well-designed in which two split target/trigger DNA and two split G-quadruplex sequences were respectively integrated into H1 and H2. Target/trigger DNA can be cyclically used in this system to form the duplex DNA assemblies (H1-H2), which will bring the two G-quadruplex fragments into close-enough proximity to induce the formation of intact G-quadruplex as a colorimetric signal readout. Similarly, the two split target/trigger DNA sequences will reunite into a DNA sequence that is identical to the target/trigger DNA; then, the obtained replica can also be cyclically used as a new activator unit to trigger the CHA reaction, leading to the rapidly and significantly enhanced formation of target/trigger DNA replicas with the concomitant generation of a higher signal. This self-replication-based autocatalytic signal amplified approach has been successfully used to develop a rapid and visual assay for DNA and small molecule detection.

Rapid identification and prognosis evaluation by dual-phase computed tomography angiography for stroke patients with a large ischemic region in the anterior circulation treated with endovascular thrombectomy.

Purpose: To investigate the value of dual-phase head-and-neck computed tomography angiography (CTA) in assessing advantages and risks associated with mechanical thrombectomy for stroke with a large ischemic region in the anterior circulation within 6 h of onset. Methods: We retrospectively analyzed the data of patients with acute occlusion of the internal carotid artery or middle cerebral artery-M1 segment. Baseline dual-phase CTA was performed for collateral grading using the 4-point visual collateral score (0, 0% filling; 1, >0% and ≤50% filling; 2, >50 and <100% filling; 3, 100% filling). The rates of modified Rankin score (MRS) ≤ 3 at 90 days, any intracranial hemorrhage (ICH) within 48 h, malignant cerebral edema within 24 h, and all-cause 90-day mortality were analyzed. Results: Among the 69 study patients, 15, 26, 17, and 11 patients had collateral grades of 0, 1, 2, and 3, respectively. At 90 days, the MRS was ≤3 in 0, 8.33, 29.41, and 36.36% of patients with grades 0, 1, 2, and 3, respectively. ICH incidence was 73.33, 57.69, 29.41, and 18.18% for grades 0, 1, 2, and 3, respectively, while the incidence of malignant brain edema was 100, 76.92, 35.29, and 0%, respectively. All-cause 90-day mortality was 53.33% for grade 0 and 30.77% for grade 1; no deaths occurred at grades 2 and 3. Conclusion: Collateral grading based on dual-phase CTA enables simple and rapid preoperative evaluation prior to mechanical thrombectomy for acute anterior-circulation stroke with a large ischemic focus, particularly for patients presenting within the 6-h time window.

EUS-guided drainage for pancreatic duct stone combined with main pancreatic duct stenosis after pancreaticoduodenectomy: a case report.

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Isothermal amplification based on specific signal extraction and output for fluorescence and colorimetric detection of nucleic acids.

Due to the complexity of compositions and low abundance of target in clinical sample, nucleic acids detection often suffers from false positives caused by nonspecific amplification. In in vitro diagnosis (IVD), PCR usually employ TaqMan probe to report specific signals and block false positive signals. However, nucleic acid isothermal amplifications, such as loop-mediated isothermal amplification (LAMP), lack of mature specific signal output mechanism, which prevents them from being used for IVD and point-of-care testing (POCT). In this work, we constructed a specific signal extract-to-output isothermal detection system (SSEI). SSEI contains a well-designed DNA probe for specific signal extraction and output in LAMP. This probe is a double-stranded DNA with an overhang sequence and named as extract-to-output probe (ETO probe). ETO probe can recognize the target-specific intermediate products in LAMP and release another signal-output probe (OP) to report the target-specific signals. With these unique properties, SSEI can detect as low as 10 copies of target DNA per reaction either by fluorescence detector or naked eyes. Moreover, due to the excellent performance against background nucleic acids interference, this biosensing platform had been successfully used for hepatitis B virus (HBV) clinical samples detection.

Nucleic acid amplification with specific signal filtration and magnification for ultrasensitive colorimetric detection.

Recently, sensitive, fast and low cost nucleic acid isothermal amplification technologies (such as loop-mediated isothermal amplification, LAMP) have attracted great attention in the urgent needs of point-of-care testing (POCT) and regular epidemic prevention and control. However, unlike PCR which usually employs TaqMan probe to report specific signals, specific-signal-output strategies in isothermal amplification are immature and visual detection even rare, which limits their popularity in POCT. We hypothesize to address this issue by designing a visual-signal-report system to both filtrate and magnify the target information in isothermal amplification. In this work, we developed a specific signal filtration and magnification colorimetric isothermal sensing platform (SFMC for short) for ultrasensitive detection of DNA and RNA. SFMC consists of two processes: an isothermal amplification with specific signal filtration and a self-replication catalyzed hairpin assembly (SRCHA) for rapid target-specific signal magnification and outputting. With these unique properties, this biosensing platform could detect target DNA as low as 5 copies per reaction and target RNA as low as 10 copies per reaction by naked eyes. Benefited from the excellent colorimetric detection performance, this biosensing platform has been successfully used for African swine fever virus (ASFV) and SARS-CoV-2 detection.

Selenium atom on phosphate enhances specificity and sensitivity of DNA polymerization and detection.

DNA polymerization is of high specificity in vivo. However, its specificity is much lower in vitro, which limits advanced applications of DNA polymerization in ultrasensitive nucleic acid detection. Herein, we report a unique mechanism of single selenium-atom modified dNTP (dNTPαSe) to enhance polymerization specificity. We have found that both dNTPαSe (approximately 660 fold) and Se-DNA (approximately 2.8 fold) have lower binding affinity to DNA polymerase than canonical ones, and the Se-DNA duplex has much lower melting-temperature (Tm) than the corresponding canonical DNA duplex. The reduced affinity and Tm can destabilize the substrate-primer-template-enzyme assembly, thereby largely slowing down the mismatch of DNA polymerization and enhancing the amplification specificity and in turn detection sensitivity. Furthermore, the Se-strategy enables us to develop the selenium enhanced specific isothermal amplification (SEA) for nucleic acid detection with high specificity and sensitivity (up to detection of single-digit copies), allowing convenient detection of clinical HPV and COVID-19 viruses in the low-copy number. Clearly, we have discovered the exciting mechanism for enhancing DNA polymerization accuracy, amplification specificity and detection sensitivity by SEA, up to two orders of magnitude higher.

Phosphate modified magnetite@ferrihydrite as an magnetic adsorbent for Cd(II) removal from water, soil, and sediment.

This work successfully fabricated a novel magnetic adsorbent, i.e., phosphate modified magnetite@ferrihydrite (Mag@Fh-P), and explored its potential application for Cd(II) removal from water, soil, and sediment. To synthesize the adsorbent, ferrihydrite-coated magnetite (Mag@Fh) was firstly developed with partially acid-dissolved natural magnetite particles, followed by in-situ synthesis of ferrihydrite on magnetite surface via alkali addition. Selection of natural magnetite as iron source for ferrihydrite synthesis and as magnetic core is believed to save the cost of adsorbent. Then, phosphate was loaded on Mag@Fh by impregnation-heating treatment to produce Mag@Fh-P. Batch adsorption experiments revealed that the Cd(II) adsorption on Mag@Fh-P could reach equilibrium within 60 min, and the calculated adsorption capacity using Langmuir model was 64.1 mg/g, which was significantly higher than that on magnetite (0.44 mg/g) and Mag@Fh (23.9 mg/g). The results from X-ray photoelectron spectroscopy analysis and batch adsorption experiments confirmed that both ligand exchange and electrostatic attraction contributed to Cd(II) adsorption. Besides, Mag@Fh-P can also be an efficient amendment for soil and sediment remediation. The spent Mag@Fh-P could be easily recovered via magnetic separation, accompanied by the significant decrease in total Cd(II) concentration in soil/sediment. At an adsorbent dosage of 2 wt%, 0.82 and 0.74 mg/kg of total Cd(II) in soil and sediment was removed, respectively. In all, the synthesized Mag@Fh-P as adsorbent has the merits of cost effectiveness, fast adsorption rate, high adsorption capacity, and easy separation, and thus it has promising application for the removal of heavy metal cations from water, soil, and sediment.

Facile synthesis of Al/Fe bimetallic (oxyhydr)oxide-coated magnetite for efficient removal of fluoride from water.

In this work, we developed a novel magnetic bimetallic Al/Fe (oxyhydr)oxide adsorbent through a facile and cost-effective method and explored its potential to adsorb fluoride in water. Its synthesis involved corrosion of natural magnetite in aluminium chloride solution, followed by titration with NaOH solution for in-situ synthesis of Al/Fe (oxyhydr)oxide-coated magnetite (Mag@Al2Fe). Characterization data indicated a uniform coating of Al/Fe (oxyhydr)oxide on magnetite, and the resulting composite possessed large specific surface area (∼90 m2/g) and good magnetic property. In batch adsorption experiments, the isotherm and kinetic data fitted well to the Langmuir model and pseudo-second-order model, respectively. The maximum adsorption capacity of Mag@Al2Fe is 26.5 mg/g, which was much higher than natural magnetite (0.44 mg/g). Moreover, this material retained high adsorption capacity toward fluoride within a wide pH range (3.0-8.0) and offered facile magnetic separation from water. Influence of competing ions was also evaluated which showed that the presence of Cl- and NO3 - posed negligible interference, while HCO3 - and SO4 2- had negative effects on fluoride adsorption. Thermodynamic investigations revealed that fluoride adsorption was exothermic and spontaneous. The observed increase in solution pH and formation of Al-F and Fe-F bonds (as indicated by XPS analysis) after fluoride adsorption suggested the major adsorption mechanism of ligand exchange. Besides, the adsorption/desorption cycle studies demonstrated the well-retained performance of Mag@Al2Fe for repeated application after regeneration by 0.5 mol/L NaOH solution. Facile synthesis, high defluoridation, lower cost, and quick separation of Mag@Al2Fe indicates its promising potential for drinking water defluoridation.

Molecular analysis of spoilage-related bacteria in pasteurized milk during refrigeration by PCR and denaturing gradient gel electrophoresis.

Bacterial diversity in fluid milk products has been extensively studied in order to improve milk quality. Here, we illustrate the utility of viable counts and PCR-denaturing gradient gel electrophoresis (DGGE) for monitoring the microbial spoilage of pasteurized milk during shelf life. Five pasteurized milk samples stored at 4 degrees C were examined at 10 and 5 days before expiration and on the expiration day. With bacterial DNA extracted directly from the samples, PCR-DGGE analysis indicated that Pseudomonas became dominant in four samples. Meanwhile, the aerobic plate count of these four samples exceeded the regulatory limit of 20,000 CFU/ml at 5 days before expiration, and the rapid psychrotrophic count markedly surpassed the aerobic plate count on the expiration day. Streptococcus and Buttiauxella spp. were detected in several samples. Sequence analysis of DGGE fragments revealed high diversity among Pseudomonas spp. in the milk samples. P. putida and P. migulae grew to high numbers during refrigerated storage. Further identification of Pseudomonas at the species level was facilitated by PCR and multiplex PCR using species-specific primers; consequently, P. fluorescens and P. fragi were observed. These results highlight an important role of Pseudomonas in the shelf life of pasteurized milk.

Rapid DNA detection based on self-replicating catalyzed hairpin assembly using nucleotide base analog pyrrolo-deoxycytidine as fluorophore.

A rapid signal amplified DNA detection method based on self-replicating catalyzed hairpin assembly (SRCHA) has been proposed. In this SRCHA system, two split target DNA sequences were respectively integrated into hairpin auxiliary probes H1 and H2. H2 was used as fluorescent probe which containing a fluorescent nucleotide base analog pyrrolo-deoxycytidine (P-dC) at the end of the stem. Target DNA can be circularly used in this SRCHA system to form the helix DNA H1-H2 complex, the structure change of H2 will move P-dC from hairpin stem to flexible ssDNA sticky end, leading to fluorescence increase due to the less stacking interaction. Meanwhile, the two spilt target DNA sequence was reunited and the target DNA replicate was obtained, which also can be circularly used as new activator to trigger additional CHA reaction and fluorescence signal was then rapidly and significantly enhanced. This SRCHA system has been successfully employed for DNA detection with picomolar within around 15min, and provides a potential technology for the real-time rapid bioanalysis.