Biofilm formation and antioxidation were responsible for the increased resistance of N. eutropha to chloramination for drinking water treatment.

Chloramination is an effective strategy for eliminating pathogens from drinking water and repressing their regrowth in water distribution systems. However, the inevitable release of NH4+ potentially promotes nitrification and associated ammonia-oxidizing bacteria (AOB) contamination. In this study, AOB (Nitrosomona eutropha) were isolated from environmental water and treated with two disinfection stages (chloramine disinfection and chloramine residues) to investigate the occurrence mechanisms of AOB in chloramination. The results showed that N. eutropha had considerable resistance to monochloramine compared to Escherichia coli, whose inactivation rate constant was 19.4-fold lower. The higher resistance was attributed to high levels of extracellular polymer substances (EPS) in AOB, which contribute to AOB surviving disinfection and entering the distribution system. In AOB response to the chloramine residues stage, the respiratory activity of N. eutropha remained at a high level after three days of continuous exposure to high chloramine residue concentrations (0.5-1.5 mg/L). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) suggested that the mechanism of N. eutropha tolerance involved a significantly high expression of the intracellular oxidative stress-regulating (sodB, txrA) and protein-related (NE1545, NE1546) genes. Additionally, this process enhanced EPS secretion and promoted biofilm formation. Adhesion predictions based on the XDLVO theory corroborated the trend of biofilm formation. Overall, the naturally higher resistance contributed to the survival of AOB in primary disinfection; the enhanced antioxidant response of surviving N. eutropha accompanied by biofilm formation was responsible for their increased resistance to the residual chloramines.

Development of Fluorescence-Based Assays for Key Viral Proteins in the SARS-CoV-2 Infection Process and Lifecycle.

Since the appearance of SARS-CoV-2 in 2019, the ensuing COVID-19 (Corona Virus Disease 2019) pandemic has posed a significant threat to the global public health system, human health, life, and economic well-being. Researchers worldwide have devoted considerable efforts to curb its spread and development. The latest studies have identified five viral proteins, spike protein (Spike), viral main protease (3CLpro), papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp), and viral helicase (Helicase), which play crucial roles in the invasion of SARS-CoV-2 into the human body and its lifecycle. The development of novel anti-SARS-CoV-2 drugs targeting these five viral proteins holds immense promise. Therefore, the development of efficient, high-throughput screening methodologies specifically designed for these viral proteins is of utmost importance. Currently, a plethora of screening techniques exists, with fluorescence-based assays emerging as predominant contenders. In this review, we elucidate the foundational principles and methodologies underpinning fluorescence-based screening approaches directed at these pivotal viral targets, hoping to guide researchers in the judicious selection and refinement of screening strategies, thereby facilitating the discovery and development of lead compounds for anti-SARS-CoV-2 pharmaceuticals.

Spatio-temporal distribution characteristics and influencing factors of protoporphyrin IX in the estuarine-coastal ecosystems.

Protoporphyrin IX (PPIX), a key precursor for the synthesis of chlorophyll and heme, is fundamental to photosynthetic eukaryotic cells and participates in light absorption, energy transduction, and numerous other cellular metabolic activities. Along with the application of genetic and biochemical techniques over the past few years, our understanding of the formation of PPIX has been largely advanced, especially regarding possible metabolic pathways. However, the ecological role and function of PPIX in natural ecosystems remains unclear. We have previously established a method for quantifying PPIX in marine ecosystems. Here, our results provide evidence that PPIX is not only subtly linked to nutrient uptake but also triggers phytoplankton productivity. PPIX and its derivatives are dynamic spatiotemporally in direct response to increased nutrient availability. Using 16 S rRNA gene amplicon sequencing, PPIX was revealed to interact strongly with many microorganisms, indicating that PPIX serves as a critical metabolite in maintaining microbial metabolism and community development. In summary, we observed that PPIX is linearly related to nutrient availability and microbial diversity. The levels of microbial PPIX reflect ecological health, and the availability of PPIX and nutrients jointly affect microbial community composition.

Anti-Neuroinflammatory Potential of Natural Products in the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is an age-related chronic progressive neurodegenerative disease, which is the main cause of dementia in the elderly. Much evidence shows that the onset and late symptoms of AD are caused by multiple factors. Among them, aging is the main factor in the pathogenesis of AD, and the most important risk factor for AD is neuroinflammation. So far, there is no cure for AD, but the relationship between neuroinflammation and AD may provide a new strategy for the treatment of AD. We herein discussed the main etiology hypothesis of AD and the role of neuroinflammation in AD, as well as anti-inflammatory natural products with the potential to prevent and alleviate AD symptoms, including alkaloids, steroids, terpenoids, flavonoids and polyphenols, which are available with great potential for the development of anti-AD drugs.

Enhanced Water Solubility and Anti-Tumor Activity of Oleanolic Acid through Chemical Structure Modification.

Cancer has been a major health problem in the world in the past decades. It is urgent to develop new, effective and safe drugs for the treatment of cancer. There are many pentacyclic triterpenoids with positive anti-tumor activity and safety in nature. Oleanolic acid (OA), as one of the pentacyclic triterpenoids, also has broad biological activities including liver protection, anti-inflammatory, hypoglycemic, antiviral and anti-tumor. Therefore, to investigate its anti-tumor activity and mechanism, many OA derivatives have been developed. Some derivatives are less toxic to normal hepatocytes, which may be due to the strong liver protection ability of OA. However, the poor water solubility of OA is one of the main reasons for the weak anti-tumor activity. It is reported that some OA derivatives could enhance solubility by chemically linking some hydrophilic groups to improve anti-tumor activity. This review not only summarizes the highly water-soluble OA derivatives that can improve anti-tumor activity reported in recent years, but also introduces their possible anti-tumor mechanisms.

Rapid detection of Pseudomonas aeruginosa using a DNAzyme-based sensor.

In the present study, a DNAzyme was screened in vitro through the use of a DNA library and crude extracellular mixture (CEM) of Pseudomonas aeruginosa. Following eight rounds of selection, a DNAzyme termed PAE-1 was obtained, which displayed high rates of cleavage with strong specificity. A fluorescent biosensor was designed for the detection of P. aeruginosa in combination with the DNAzyme. A detection limit as low as 1.2 cfu/ml was observed. Using proteases and filtration, it was determined that the target was a protein with a molecular weight of 10 kDa-50 kDa. The DNAzyme was combined with a polystyrene board to construct a simple indicator plate sensor which produced a color that identified the target within 10 min. The results were reliable when tap water and food samples were tested. The present study provides a novel experimental strategy for the development of sensors based on a DNAzyme to rapidly detect P. aeruginosa in the field.

Risk factors for disease severity in COVID-19 patients: A single-center retrospective study.

Background: The outbreak of coronavirus disease 2019 (COVID-19) has posed a huge threat to human health. However, little is known regarding the risk factors associated with COVID-19 severity. We aimed to explore early-stage disease risk factors associated with eventual disease severity. Methods: This study enrolled 486 hospitalized, non-intensive care unit (ICU)-admitted adult patients with COVID-19 (age ≥ 18 years) treated at Wuhan Jinyintan Hospital, who were divided into three groups according to disease severity. The demographic, clinical, and laboratory data at admission and clinical outcomes were compared among severity groups, and the risk factors for disease severity were identified by multiple regression analysis. Results: Of 486 patients with COVID-19, 405 (83.33%) were discharged, 33 (6.71%) died outside of the ICU, and 48 (7.20%) were still being treated in the ICU by the time the study period ended. Significant differences in age, lymphocyte counts, and the levels of procalcitonin, aspartate aminotransferase, and D-dimer (P < 0.001 for all) among the three groups. Further analysis showed that older age, decreased lymphocyte counts, and increased procalcitonin, aspartate aminotransferase, and D-dimer levels were significantly associated with disease progression. Conclusion: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may impair the immune system, the blood coagulation system, and hepatic and cardiac function. Some clinical characteristics and laboratory findings can help identify patients with a high risk of disease severity, which can be significant for appropriate resource allocation during the COVID-19 pandemic.

[Study of multimodal monitoring in neurocritical care patients].

OBJECTIVE: To explore the significance of multimodal monitoring in the monitoring and treatment of neurocritical care patients. METHODS: 104 neurocritical care patients admitted to the department of Critical Care Medicine of Fujian Provincial Hospital from March 2019 to January 2020 were enrolled. Patients were randomly assigned into two groups, with 52 in each group. In the routine monitoring treatment group, heart rate, blood pressure, respiratory rate and the changes in consciousness and pupils were monitored after operation. The patients were treated with routine medicine to reduce intracranial pressure (ICP), maintain proper cerebral perfusion pressure (CPP), balance fluid intake and output, and maintain the airway clear. Patients in the multimodal monitoring treatment group were treated with invasive ICP monitoring, ultrasound to assess brain structure, ultrasound to measure optic nerve sheath diameter (ONSD), transcranial color doppler (TCCD), internal jugular venous blood oxygen saturation monitoring, near-infrared spectroscopy (NIRS), non-invasive cerebral blood oxygen saturation monitoring and quantitative electroencephalogram monitoring. According to the monitoring results, the patients were given targeted treatment with the goal of controlling ICP and improving brain metabolism. The length of intensive care unit (ICU) stay, the incidences of neurological complications (secondary cerebral infarction, cerebral hemorrhage, high intracranial pressure, etc.), and the incidences of poor prognosis [6 months after the onset of Glasgow outcome score (GOS) 1 to 3] were compared between the two groups. Spearman rank correlation analysis of the correlation between invasive ICP and the ICP value which was calculated by TCCD. The receiver operating characteristic (ROC) curve of invasive ICP and pulsatility index of middle cerebral artery (PIMCA) were used to predict poor prognosis. RESULTS: The length of ICU stay in the multimodal monitoring treatment group was significantly shorter than that of the routine monitoring treatment group (days: 6.27±3.81 vs. 9.61±5.09, P < 0.01), and the incidence of neurological complications was significantly lower than that in the routine monitoring treatment group (9.62% vs. 25.00%, P < 0.05). In the multimodal monitoring treatment group, 37 cases had a good prognosis and 15 cases had a poor prognosis, while the routine monitoring treatment group had a good prognosis in 27 cases and a poor prognosis in 25 cases. The incidence of poor prognosis in the multimodal monitoring treatment group was lower than that of the routine monitoring treatment group (28.85% vs. 48.08%, P < 0.05). In the multimodal monitoring treatment group, the invasive ICP and PIMCA of patients with good prognosis were significantly lower than those of patients with poor prognosis [invasive ICP (mmHg, 1 mmHg = 0.133 kPa): 16 (12, 17) vs. 22 (20, 24), PIMCA: 0.90±0.33 vs. 1.39±0.58, both P < 0.01]. There was no significant difference in resistance index of the middle cerebral artery (RIMCA) between the good prognosis group and the poor prognosis group (0.63±0.12 vs. 0.66±0.15, P > 0.05). There was a positive correlation between the invasive ICP and the ICP value which was calculated by TCCD (r = 0.767, P < 0.001). ROC curve analysis showed that the area under ROC curve (AUC) of invasive ICP for poor prognosis prediction was 0.906, the best cut-off value was ≥ 18 mmHg, the sensitivity was 86.49%, and the specificity was 86.67%. The AUC of PIMCA for poor prognosis prediction was 0.759, the best cut-off value was ≥ 1.12, the sensitivity was 81.08%, and the specificity was 60.00%. The AUC of invasive ICP was greater than PIMCA (Z = 2.279, P = 0.023). CONCLUSIONS: Comprehensive analysis of multimodal monitoring indicators for neurocritical care patients to guide clinical treatment can reduce the length of hospital stay, and reduce the risk of neurosurgery complications and disability; invasive ICP can predict poor prognosis of neurocritical care patients.

Analgesic action of Rubimaillin in vitro and in vivo.

Pain, a common symptom in clinics, is a serious impediment to quality of life. The analgesic drugs presently in use have poor efficacy, and are associated with undesirable side effects. Rubimaillin (Rub) is a naphthoquinone compound extracted from Chinese herbal medicine, and it has various biological activities. In this study, the analgesic effect of Rub, and its mechanism of action were investigated using glacial acetic acid-induced mice writhing model and a mice model of neurogenic and inflammatory bipolar pain. Analgesic effects were measured in different experimental groups. In vitro, RAW 264.7 cells were used to investigate the release of nitric oxide (NO), iNOS and COX-2 protein in RAW 264.7 cells stimulated with lipopolysaccharide (LPS). The results revealed that Rub reduced the number of acetic acid-induced writhing in mice, inhibited formalin-induced biphasic pain response, and suppressed the production of NO in RAW 264.7 cells. The mechanisms involved in the analgesic and anti-inflammatory effects of rub may be related to the inhibition of cyclooxygenase-2 (COX-2), endogenous inflammatory mediators, and reduction in the content of pain-induced mediators.

APOE polymorphism is associated with blood lipid and serum uric acid metabolism in hypertension or coronary heart disease in a Chinese population.

Aim: To explore the association of APOE polymorphism (rs7412:526C>T and rs429358:388T>C) with glucose, lipid and serum uric acid (UA) metabolism in patients with hypertension or coronary heart disease (CHD). Methods: A total of 544 patients with hypertension or CHD were selected for this study from March 2017 to January 2018. According to the APOE genotypes (excluding the E2/E4 genotype), the subjects were divided into three groups (E2/E2+E2/E3 genotypes, E3/E3 genotype [the wild-type] and E3/E4+E4/E4 genotypes) and the difference of metabolism among the three groups was compared. Results: There were significant differences in total cholesterol (TC), triglycerides, low-density lipoprotein (LDL), high-density lipoprotein and serum UA levels among the three groups. Compared with APOE E3 homozygote, APOE E4 carriers possessed higher TC, triglycerides and LDL levels, whereas APOE E2 carriers had higher high-density lipoprotein level, lower TC and LDL levels. Furthermore, multivariate logistic regression analysis found that setting E3/E3 genotype as the reference group, the carriers of APOE E4 allele (E3/E4+E4/E4 genotypes) were significantly related to hypertriglyceridemia, and APOE E2 allele (E2/E2+E2/E3 genotypes) was significantly correlated with hyperuricemia. Conclusion:APOE polymorphism was associated with blood lipid and serum UA metabolism in patients with hypertension or CHD. Compared with APOE E3 homozygote, APOE E4 allele was related to elevated triglycerides, and APOE E2 allele was correlated with increased serum UA level.

Characterization and mechanism of the effects of Mg-Fe layered double hydroxide nanoparticles on a marine bacterium: new insights from genomic and transcriptional analyses.

BACKGROUND: Layered double hydroxides (LDHs) have received widespread attention for their potential applications in catalysis, polymer nanocomposites, pharmaceuticals, and sensors. Here, the mechanism underlying the physiological effects of Mg-Fe layered double hydroxide nanoparticles on the marine bacterial species Arthrobacter oxidans KQ11 was investigated. RESULTS: Increased yields of marine dextranase (Aodex) were obtained by exposing A. oxidans KQ11 to Mg-Fe layered double hydroxide nanoparticles (Mg-Fe-LDH NPs). Furthermore, the potential effects of Mg-Fe-LDH NPs on bacterial growth and Aodex production were preliminarily investigated. A. oxidans KQ11 growth was not affected by exposure to the Mg-Fe-LDH NPs. In contrast, a U-shaped trend of Aodex production was observed after exposure to NPs at a concentration of 10 µg/L-100 mg/L, which was due to competition between Mg-Fe-LDH NP adsorption on Aodex and the promotion of Aodex expression by the NPs. The mechanism underling the effects of Mg-Fe-LDH NPs on A. oxidans KQ11 was investigated using a combination of physiological characterization, genomics, and transcriptomics. Exposure to 100 mg/L of Mg-Fe-LDH NPs led to NP adsorption onto Aodex, increased expression of Aodex, and generation of a new Shine-Dalgarno sequence (GGGAG) and sRNAs that both influenced the expression of Aodex. Moreover, the expressions of transcripts related to ferric iron metabolic functions were significantly influenced by treatment. CONCLUSIONS: These results provide valuable information for further investigation of the A. oxidans KQ11 response to Mg-Fe-LDH NPs and will aid in achieving improved marine dextranase production, and even improve such activities in other marine microorganisms.

Selection of DNAzymes for Sensing Aquatic Bacteria: Vibrio Anguillarum.

Vibrio anguillarum is a bacterial pathogen that causes serious damage to aquatic fish, and its rapid detection and prevention are critical. DNAzymes are DNA-based catalysts with excellent stability. In this study, in vitro selection of DNAzymes was performed using the crude extracellular matrix (CEM) of V. Anguillarum as the target. Different from previous selections targeting bacterial CEM, this work used an unmodified DNA library, allowing easier adoption of the technology. After seven rounds of selection, a DNAzyme named VAE-2 with high activity and specificity was obtained. It showed the highest activity toward V. Anguillarum among eight types of tested bacterial strains. Polyvalent metal ions are needed for its activity. Protease treatment of the CEM and filtration studies indicated that the target is a protein with a molecular weight between 50 k and 100 k Da. A fluorescent biosensor was designed for V. anguillarum with a detection limit down to 4000 cfu/mL, and detection was demonstrated for real fish tissue and feeding water samples. Being the first work of DNAzyme-based sensing of aquatic bacteria, this study indicates that unmodified DNA can be used for targeting bacterial CEM, and it provides a new framework for developing other RNA-cleaving DNAzymes for rapid detection of pathogenic bacteria and water pollution.

Recognition of Helicobacter pylori by protein-targeting aptamers.

BACKGROUND: Helicobacter pylori (H pylori) is a disease-causing pathogen capable of surviving under acidic conditions of the human stomach. Almost half of the world's population is infected with H pylori, with gastric cancer being the most unsatisfactory prognosis. Although H pylori has been discovered 30 years ago, the effective treatment and elimination of H pylori continue to be problematic. MATERIALS AND METHODS: In our study, we screened nucleic acid aptamers using H pylori surface recombinant antigens as targets. Trypsin was used for separating aptamers that were bound to proteins. Following nine rounds of screening, we performed sequence similarity analyses to assess whether the aptamers can recognize the target protein. Two sequences with desirable recognition ability were selected for affinity detection. Aptamer Hp4 with the strongest binding ability to the H pylori surface recombinant antigen was chosen. After optimization of the binding conditions, we conducted specificity tests for Hp4 using Escherichia coli, Staphylococcus aureus, Vibrioanguillarum, and H pylori. RESULTS: The data indicated that the aptamer Hp4 had an equilibrium dissociation constant (Kd ) of 26.48 ± 5.72 nmol/L to the target protein. This aptamer was capable of exclusively detecting H pylori cells, without displaying any specificity for other bacteria. CONCLUSIONS: We obtained a high-affinity aptamer for H pylori, which is expected to serve as a new molecular probe for detection of H pylori.

Crystal Structure of GH49 Dextranase from Arthrobacter oxidans KQ11: Identification of Catalytic Base and Improvement of Thermostability Using Semirational Design Based on B-Factors.

The crystal structure of Dextranase from the marine bacterium Arthrobacter oxidans KQ11 (Aodex) was determined at a resolution of 1.4 Å. The crystal structure of the conserved Aodex fragment (Ala52-Thr638) consisted of an N-terminal domain N and a C-terminal domain C. The N-terminal domain N was identified as a ß-sandwich, connected to a right-handed parallel ß-helix at the C-terminus. Sequence comparisons, cavity regions, and key residues of the catalytic domain analysis all suggested that the Aodex was an inverting enzyme, and the catalytic acid and base were Asp439 and Asp420, respectively. Asp440 was not a general base in the Aodex catalytic domain, and Asp396 in Dex49A may not be a general base in the catalytic domain. The thermostability of the S357F mutant using semirational design based on B-factors was clearly better than that of wild-type Aodex. This process may promote the aromatic-aromatic interactions that increase the thermostability of mutant Phe357.

Identification of a highly specific DNA aptamer for Vibrio vulnificus using systematic evolution of ligands by exponential enrichment coupled with asymmetric PCR.

Vibrio vulnificus is an important bacterial pathogen that causes serious infections in fish and is also highly pathogenic to humans. Many effective detection methods targeting this pathogen have previously been designed, but many of these methods are time-consuming, complicated and expensive. Thus, these approaches cannot be widely used by small aqacultural concerns. Although DNA aptamers have been used to detect pathogenic bacteria, these have not been applied to marine bacteria, including V. vulnificus. Therefore, we developed a highly specific DNA aptamer for V. vulnificus detection using systematic evolution of ligands by exponential enrichment (SELEX), coupled with asymmetric PCR. After 13 rounds of cross-selection, we identified a novel DNA aptamer (Vapt2). We evaluated the affinity, specificity and limit of detection (LOD) of this aptamer for V. vulnificus. We found that Vapt2 had a high affinity for V. vulnificus (Kd  = 26.8 ± 5.3 nM) and detected this pathogen at a wide range of concentrations (8-2.0 × 108  cfu/ml). Vapt2 bound to V. vulnificus with high selectivity in the presence of other pathogenic bacteria. Our study increases our knowledge of the possible applications of aptamers with respect to marine bacteria. Moreover, our work might provide a framework for the rapid detection of pathogenic bacteria and water pollution.

Trypsin enhances aptamer screening: A novel method for targeting proteins.

A novel screening method for protein aptamer selection was developed in this study. Aptamers with high affinity and specificity to the surface recombinant antigen of Helicobacter pylori (HP-Ag) and to tumor markers carcinoembryonic antigen (CEA), cancer antigen 125 (CA125) and cancer antigen 19-9(CA19-9) were screened using trypsin enhanced screening method. Briefly, the target proteins above were immobilized onto 96-well polystyrene plates and incubated with a single-stranded DNA (ssDNA) library for aptamer selection. Then, trypsin was introduced to digest the proteins and obtain ssDNA that bound to the target proteins with high specificity. The concentration of ssDNA that shed from protein-ssDNA complexes was detected. After sequencing, the enrichment of target-specific aptamers was monitored and the affinity of each aptamer was analyzed. Urea, which has been reported in other article, was used to compare with trypsin. The results revealed that trypsin was more effective than urea for protein aptamer selection. The protocol used in this study provided a novel method for generating aptamers.

Research Progress on Rolling Circle Amplification (RCA)-Based Biomedical Sensing.

Enhancing the limit of detection (LOD) is significant for crucial diseases. Cancer development could take more than 10 years, from one mutant cell to a visible tumor. Early diagnosis facilitates more effective treatment and leads to higher survival rate for cancer patients. Rolling circle amplification (RCA) is a simple and efficient isothermal enzymatic process that utilizes nuclease to generate long single stranded DNA (ssDNA) or RNA. The functional nucleic acid unit (aptamer, DNAzyme) could be replicated hundreds of times in a short period, and a lower LOD could be achieved if those units are combined with an enzymatic reaction, Surface Plasmon Resonance, electrochemical, or fluorescence detection, and other different kinds of biosensor. Multifarious RCA-based platforms have been developed to detect a variety of targets including DNA, RNA, SNP, proteins, pathogens, cytokines, micromolecules, and diseased cells. In this review, improvements in using the RCA technique for medical biosensors and biomedical applications were summarized and future trends in related research fields described.

Purification and Characterization of a Biofilm-Degradable Dextranase from a Marine Bacterium.

This study evaluated the ability of a dextranase from a marine bacterium Catenovulum sp. (Cadex) to impede formation of Streptococcus mutans biofilms, a primary pathogen of dental caries, one of the most common human infectious diseases. Cadex was purified 29.6-fold and had a specific activity of 2309 U/mg protein and molecular weight of 75 kDa. Cadex showed maximum activity at pH 8.0 and 40 °C and was stable at temperatures under 30 °C and at pH ranging from 5.0 to 11.0. A metal ion and chemical dependency study showed that Mn2+ and Sr2+ exerted positive effects on Cadex, whereas Cu2+, Fe3+, Zn2+, Cd2+, Ni2+, and Co2+ functioned as inhibitors. Several teeth rinsing product reagents, including carboxybenzene, ethanol, sodium fluoride, and xylitol were found to have no effects on Cadex activity. A substrate specificity study showed that Cadex specifically cleaved the α-1,6 glycosidic bond. Thin layer chromatogram and high-performance liquid chromatography indicated that the main hydrolysis products were isomaltoogligosaccharides. Crystal violet staining and scanning electron microscopy showed that Cadex impeded the formation of S. mutans biofilm to some extent. In conclusion, Cadex from a marine bacterium was shown to be an alkaline and cold-adapted endo-type dextranase suitable for development of a novel marine agent for the treatment of dental caries.

Reselection Yielding a Smaller and More Active Silver-Specific DNAzyme.

Ag10c is a recently reported RNA-cleaving DNAzyme obtained from in vitro selection. Its cleavage activity selectively requires Ag+ ions, and thus it has been used as a sensor for Ag+ detection. However, the previous selection yielded very limited information regarding its sequence requirement, since only ∼0.1% of the population in the final library were related to Ag10c and most other sequences were inactive. In this work, we performed a reselection by randomizing the 19 important nucleotides in Ag10c in such a way that a purine has an equal chance of being A or G, whereas a pyrimidine has an equal chance of being T or C. The round 3 library of the reselection was carefully analyzed and a statistic understanding of the relative importance of each nucleotide was obtained. At the same time, a more active mutant was identified, containing two mutated nucleotides. Further analysis indicated new base pairs leading to an enzyme with smaller catalytic loops but with ∼200% activity of the original Ag10c, and also excellent selectivity for Ag+. Therefore, a more active mutant of Ag10c was obtained and further truncations were successfully performed, which might be better candidates for developing new biosensors for silver. A deeper biochemical understanding was also obtained using this reselection method.