Circadian disturbances by altering the light-dark cycle negatively affects hematopoietic function of bone marrow in mice.

Circadian rhythms in metabolically active tissues are crucial for maintaining physical health. Circadian disturbance (CD) can cause various health issues, such as metabolic abnormalities and immune and cognitive dysfunctions. However, studies on the role of CD in immune cell development and differentiation, as well as the rhythmic expression of the core clock genes and their altered expression under CD, remain unclear. Therefore, we exposed C57bl/6j mice to repeated reversed light-dark cycles for 90 days to research the effects of CD on bone marrow (BM) hematopoietic function. We also researched the effects of CD on endogenous circadian rhythms, temporally dependent expression in peripheral blood and myeloid leukocytes, environmental homeostasis within BM, and circadian oscillations of hematopoietic-extrinsic cues. Our results confirmed that when the light and dark cycles around mice were frequently reversed, the circadian rhythmic expression of the two main circadian rhythm markers, the hypothalamic clock gene, and serum melatonin, was disturbed, indicating that the body was in a state of endogenous CD. Furthermore, CD altered the temporally dependent expression of peripheral blood and BM leukocytes and destroyed environmental homeostasis within the BM as well as circadian oscillations of hematopoietic-extrinsic cues, which may negatively affect BM hematopoiesis in mice. Collectively, these results demonstrate that circadian rhythms are vital for maintaining health and suggest that the association between CD and hematopoietic dysfunction warrants further investigation.

Elucidating the Role of CRISPR/Cas in Single-Step Isothermal Nucleic Acid Amplification Testing Assays.

Developing assays that combine CRISPR/Cas and isothermal nucleic acid amplification has become a burgeoning research area due to the novelty and simplicity of CRISPR/Cas and the potential for point-of-care uses. Most current research explores various two-step assays by appending different CRISPR/Cas effectors to the end of different isothermal nucleic acid amplification methods. However, efforts in integrating both components into more ideal single-step assays are scarce, and poor-performing single-step assays have been reported. Moreover, lack of investigations into CRISPR/Cas in single-step assays results in incomplete understanding. To fill this knowledge gap, we conducted a systematic investigation by developing and comparing assays that share the identical recombinase polymerase amplification (RPA) but differ in CRISPR/Cas12a. We found that the addition of CRISPR/Cas12a indeed unlocks signal amplification but, at the same time, impedes RPA and that CRISPR/Cas12a concentration is a key parameter for attenuating RPA impediment and ensuring assay performance. Accordingly, we found that our protospacer adjacent motif (PAM)-free CRISPR/Cas12a-assisted RPA assay, which only moderately impeded RPA at its optimal CRISPR/Cas12a concentration, outperformed its counterparts in assay design, signal, sensitivity, and speed. We also discovered that a new commercial Cas12a effector could also drive our PAM-free CRISPR/Cas12a-assisted RPA assay and reduce its cost, though simultaneously lowering its signal. Our study and the new insights can be broadly applied to steer and facilitate further advances in CRISPR/Cas-based assays.

Revealing an initiation inhibition of RCA and its application in nucleic acid detection.

Rolling circle amplification is a widely used biosensing technique. Although various secondary structures have been employed in RCA, their effects on RCA efficiency have seldom been reported. Here, we find that stems in circular templates can strongly inhibit RCA, and the primer-stem distance is responsible for the inhibition. Based on the results, we propose an initiation inhibition mechanism and present a design principle for a general RCA assay. Inspired by this mechanism, we further propose a new nucleic acid detection method. The results verify that this method can increase RCA detection sensitivity according to the target recycling principle. Besides DNA detection, it can also achieve single mismatch discrimination of miRNA detection after optimization. This method also shows convenient visualization detection. The initiation inhibition of RCA could be helpful for RCA applications as promising detection techniques.

Applying biosensor development concepts to improve preamplification-free CRISPR/Cas12a-Dx.

Development of CRISPR/Cas-based in vitro diagnostic devices, or CRISPR/Cas-Dx, has become an intensely researched area. Among the different classes of CRISPR/Cas-Dx, the class based on the Cas12a enzyme (i.e., CRISPR/Cas12a-Dx or simply Cas12a-Dx), is predominantly employed for detecting DNA targets. Current research in Cas12a-Dx has focused on appending Cas12a-Dx to preamplification techniques or coupling Cas12a-Dx to different detection modalities, which has inevitably overshadowed the detection performance of Cas12a-Dx and overlooked its intrinsic detection capability without preamplification. We recognize that Cas12a-Dx, which relies on DNA-activated Cas12a to cleave single-stranded DNA, shares significant similarity with other nuclease-based DNA biosensors, whose performances can be influenced by parameters ranging from the reaction buffer to the reaction temperature. We are thus inspired to probe the limits of preamplification-free Cas12a-Dx by exploring and systematically evaluating several potential parameters that may impact its detection sensitivity and time. Using a previously reported fluorescence-based Cas12a-Dx as the test bed, we have identified that the Cas12a enzyme, the reaction buffer, the substrate label, the substrate concentration, and the reaction temperature can be optimized to significantly improve the signal-to-background ratio and the reaction rate of Cas12a-Dx. Based on these findings, we have improved the limit of detection (LOD) of the Cas12a-Dx to 100 fM, while reduced the time-to-positive to <46 min, representing the most sensitive LOD without preamplification and the fastest time-to-positive for this LOD to date. More broadly, our work provides a roadmap for further advancing Cas12a-Dx and perhaps other classes of CRISPR/Cas-Dx.

Screening substrate-binding positions by rolling circle amplification suggesting a binding model of Nt.BstNBI.

Nicking endonucleases (NEs) become increasingly attractive for their promising applications in isothermal amplification. Unfortunately, in comparison with their applications, their catalytic mechanism studies have relatively lagged behind due to a paucity of crystal structure information. Nt.BstNBI is one of those widely used NEs. However, many aspects of its catalytic mechanism still remained to be explored. Herein, we employed only rolling circle amplification (RCA) assay as a major analytic tool and succeeded in identifying the potential binding positions and regions of the DNA substrate based on locked nucleic acid modification, DNA duplex length of substrate, and substrate mismatch designs. Based on these data, we, for the first time, revealed that Nt.BstNBI was likely to recognize six adjacent positions of the recognition sequence (G1rt, A2rt, G3rt, A2rb, C3rb, and T4rb) in the major groove and hold three positions of the cleavage sequence (N3ct, N4ct, and N7cb) in the minor groove of DNA duplex for nicking. Moreover, this work also demonstrated the unexpected efficiency of RCA to study the macromolecular interaction for certain kind of nucleases in an easy and high-throughput way.

Screening oligonucleotide sequences for silver staining and d-galactose visual detection using RCA silver staining in a tube.

Oligonucleotides were screened for strongly silver-stained repetitive sequences. An 'AG'-clustered purine sequence showed strong staining, and the staining density can be compromised by disrupting the continuity of the 'AG'-clustered sequence. The staining-favored sequence was then employed in rolling circle amplification (RCA) for its product detection. A tube-staining method was developed for convenient and visual RCA assay. Moreover, by introducing GalR into RCA, d-galactose was detected by RCA tube-staining with naked eyes without any equipment. About 10 mM d-galactose can be easily identified, and the detection of d-galactose was specific in comparison with that of several other monosaccharides.

A sensitive colorimetric assay system for nucleic acid detection based on isothermal signal amplification technology.

Rapid and accurate detection of microRNAs in biological systems is of great importance. Here, we report the development of a visual colorimetric assay which possesses the high amplification capabilities and high selectivity of the rolling circle amplification (RCA) method and the simplicity and convenience of gold nanoparticles used as a signal indicator. The designed padlock probe recognizes the target miRNA and is circularized, and then acts as the template to extend the target miRNA into a long single-stranded nucleotide chain of many tandem repeats of nucleotide sequences. Next, the RCA product is hybridized with oligonucleotides tagged onto gold nanoparticles. This interaction leads to the aggregation of gold nanoparticles, and the color of the system changes from wine red to dark blue according to the abundance of miRNA. A linear correlation between fluorescence and target oligonucleotide content was obtained in the range 0.3-300 pM, along with a detection limit of 0.13 pM (n = 7) and a RSD of 3.9% (30 pM, n = 9). The present approach provides a simple, rapid, and accurate visual colorimetric assay that allows sensitive biodetection and bioanalysis of DNA and RNA nucleotides of interest in biologically important samples. Graphical abstract The colorimetric assay system for analyzing target oligonucleotides.

Suppression of rolling circle amplification by nucleotide analogs in circular template for three DNA polymerases.

Among wide applications of nucleotide analogs, their roles in enzyme catalytic reactions are significant in both fundamental and medical researches. By introducing analogs into circular templates, we succeeded in determining effects of four analogs on RCA efficiency for three different DNA polymerases. Results showed an obvious suppression effect for 2'-OMeRNA modification, which might be due to the size of the C2'-modified moieties. 2'-F RNA, LNA and PS had little interference, suggesting good analog candidates for application in RCA. Different polymerases and nucleobases made a little difference according to analogs we used. These results are useful for understanding polymerase catalytic mechanism and analogs applications in RCA reaction.

Effects of 2'-O-methyl nucleotide on ligation capability of T4 DNA ligase.

To further understand the ligation mechanism, effects of 2'-O-methyl nucleotide (2'-OMeN) on the T4 DNA ligation efficiency were investigated. Fluorescence resonance energy transfer assay was used to monitor the nick-joining process by T4 DNA ligase. Results showed that substitutions at 5'- and 3'-ends of the nick decreased the ligation efficiency by 48.7% ± 6.7% and 70.6% ± 4.0%, respectively. Substitutions at both 5'- and 3'-ends decreased the ligation efficiency by 76.6% ± 1.3%. Corresponding kinetic parameters, Vmax, Km, and kcat, have been determined in each case by using the Michaelis-Menten equation. The kinetic data showed that the 2'-OMeN substitutions reduced the maximal initial velocity and increased the Michaelis constant of T4 DNA ligase. Mismatches at 5'- and 3'-ends of the nick have also shown different influences on the ligation. Results here showed that the sugar pucker conformation at 3'-end impairs the ligation efficiency more profoundly than that at 5'-end. Different concentrations of Mg(2+), Ca(2+), K(+), Na(+), and ATP were also demonstrated to affect the T4 DNA ligase activity. These results enriched our knowledge about the effects of 2'-OMeN substitutions on the T4 DNA ligase.

Effects of cations on small fragment of DNA polymerase I using a novel FRET assay.

DNA polymerase I (PolI) digested by protease produces a small fragment (SF) containing 5'-3' exonuclease activity. The 5'-3' exonuclease activity of polI cleaves the downstream RNA primer strands during DNA replication in vivo. Previous in vitro studies suggested its capability of cleaving duplex from 5' terminal and a flap-structure-specific endonuclease activity. From the crystal structures of other nucleases and biochemical data, a two-metal-ion mechanism has been proposed but has not been determined. In this study, we cloned, expressed, and purified the SF protein, and established a novel fluorescence resonance energy transfer (FRET) assay to analyze the catalytic activity of the SF protein. The effects of several metal ions on its catalytic capability were analyzed using this FRET assay. Results showed that Mg2+, Mn2+, and Zn2+ were able to activate the cleavage of SF, while Ca2+, Ni2 +, and Co2+ were not suitable for SF catalysis. The effects of K+, Na+, and dNTP were also determined.

Comparison between better and poorly differentiated locally advanced gastric cancer in preoperative chemotherapy: a retrospective, comparative study at a single tertiary care institute.

BACKGROUND: Gastric cancer is the third leading cause of cancer-related mortality in China, and the long-term survival for locally advanced gastric cancer is very poor. Simple surgery cannot yield an ideal result because of the high recurrence rate after tumor resection. Preoperative chemotherapy could help to reduce tumor volume, improve the R0 resection rate (no residual tumor after surgery), and decrease the risk of local tumor recurrence. The aim of this study was to evaluate the influence of pathological differentiation in the effect of preoperative chemotherapy for patients with locally advanced gastric cancer. METHODS: Patients with locally advanced gastric cancer (n = 32) received preoperative chemotherapy under the XELOX (capecitabine plus oxaliplatin) regimen. According to pathological examination, patients' tumors were classified into better (well and moderate) and poorly differentiated (lower differentiated and undifferentiated) groups, and the clinical response rate, type of gastrectomy, and negative tumor residual rate were compared between the two groups of patients. Morphological changes and toxic reactions were monitored after chemotherapy. RESULTS: The results showed that the clinical response rate in the better differentiated group was significantly higher than that in the poorly differentiated group (100% versus 25%, P = 0.000). The partial gastrectomy rate in the better differentiated group was significantly higher than that in the poorly differentiated group (87.5% versus 25% P = 0.000). A significant shrinking of tumor and necrosis of tumor tissues caused by chemotherapy could be observed. CONCLUSIONS: In conclusion, the better differentiated group with locally advanced gastric cancer is suitable for preoperative chemotherapy under the XELOX regimen, and as a result of effective preoperative chemotherapy, much more gastric tissue can be preserved for the better differentiated group.

Network Pharmacology and Bioinformatics Analysis to Identify the Molecular Targets and its Biological Mechanisms of Sciadopitysin against Glioblastoma.

Glioblastoma multiform (GBM) is categorized as the most malignant subtype of gliomas, which comprise nearly 75% of malignant brain tumors in adults. Increasing evidence suggests that network pharmacology will be a novel method for identifying the systemic mechanism of therapeutic compounds in diseases like cancer. The present study aimed to use a network pharmacology approach to establish the predictive targets of sciadopitysin against GBM and elucidate its biological mechanisms. Firstly, targets of sciadopitysin were obtained from the SwissTargetPrediction database, and genes associated with the pathogenesis of GBM were identified from the DiGeNET database. Sixty-four correlative hits were identified as anti-glioblastoma targets of sciadopitysin. Functional enrichment and pathway analysis revealed significant biological mechanisms of the targets. Interaction of protein network and cluster analysis using STRING resulted in two crucial interacting hub genes, namely, HSP90 and AKT1. Additionally, the in vitro cytotoxic potential of sciadopitysin was assessed on GBM U87 cells. The findings indicate that the pharmacological action of sciadopitysin against GBM might be associated with the regulation of two core targets: HSP90 and AKT1. Thus, the network pharmacology undertaken in the current study established the core active targets of sciadopitysin, which may be extensively applied with further validations for treatment in GBM.

Advances in the detection of ß-lactamase: A review.

ß-lactamase, an enzyme secreted by bacteria, is the main resistant mechanism of Gram-negative bacteria to ß-lactam antibiotics. The resistance of bacteria to ß-lactam antibiotics can be evaluated by testing the activity of ß-lactamase. Traditional phenotypic detection is a golden principle, but it is time-consuming. In recent years, many new methods have emerged, which improve the efficiency by virtue of their sensitivity, low cost, easy operation, and other advantages. In this paper, we systematically review these researches and emphasize their limits of detection, sample operation, and test duration. Noteworthily, some detection systems can identify the ß-lactamase subtype conveniently. We mainly divide these tests into three categories to elaborate their characteristics and application status. Both advantages and disadvantages of these methods are discussed. Additionally, we analyze the recent 5 years published researches to predict the trend of development in this field.

The novel subclusters based on cancer-associated fibroblast for pancreatic adenocarcinoma.

Introduction: Pancreatic adenocarcinoma (PAAD) is a fatal disease characterized by promoting connective tissue proliferation in the stroma. Activated cancer-associated fibroblasts (CAFs) play a key role in fibrogenesis in PAAD. CAF-based tumor typing of PAAD has not been explored. Methods: We extracted single-cell sequence transcriptomic data from GSE154778 and CRA001160 datasets from Gene Expression Omnibus or Tumor Immune Single-cell Hub to collect CAFs in PAAD. On the basis of Seurat packages and new algorithms in machine learning, CAF-related subtypes and their top genes for PAAD were analyzed and visualized. We used CellChat package to perform cell-cell communication analysis. In addition, we carried out functional enrichment analysis based on clusterProfiler package. Finally, we explored the prognostic and immunotherapeutic value of these CAF-related subtypes for PAAD. Results: CAFs were divided into five new subclusters (CAF-C0, CAF-C1, CAF-C2, CAF-C3, and CAF-C4) based on their marker genes. The five CAF subclusters exhibited distinct signaling patterns, immune status, metabolism features, and enrichment pathways and validated in the pan-cancer datasets. In addition, we found that both CAF-C2 and CAF-C4 subgroups were negatively correlated with prognosis. With their top genes of each subclusters, the sub-CAF2 had significantly relations to immunotherapy response in the patients with pan-cancer and immunotherapy. Discussion: We explored the heterogeneity of five subclusters based on CAF in signaling patterns, immune status, metabolism features, enrichment pathways, and prognosis for PAAD.

Stretchable and Healable Conductive Elastomer Based on PEDOT:PSS/Natural Rubber for Self-Powered Temperature and Strain Sensing.

Self-powered elastic Conductors based on thermoelectric materials with the ability to harvest energy from the living environment are considered as important for electronic devices under off-grid, maintenance-free, or unfeasible battery replacement circumstances. Poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) is perhaps the most well-known organic conductor. However, the application of PEDOT:PSS in flexible devices is limited by its brittleness and various unrecoverable properties under strain. Various polymer blends based on water-soluble polymers and PEDOT:PSS have been prepared. Nevertheless, they fail to illustrate good balance between electrical conductivity and mechanical performance due to various issues, including the phase morphology with PEDOT:PSS as the dispersed phase; thus, the formation of a conductive network between PEDOT:PSS is prohibited. In this study, PEDOT:PSS is incorporated into natural rubber (NR), with NR as the dispersed phase. For 10 wt % PEDOT:PSS-NR composite films doped with dimethyl sulfoxide (DMSO), the conductivity was up to 87 S/cm and the elongation at break was maintained at 490%. More importantly, self-powered temperature- and tensile strain-sensing abilities were also realized. Furthermore, it is also demonstrated that most of the unrecoverable strain and conductivity under cyclic tensile strain could be healed by water and phosphate-buffered saline (PBS) post-treatment. This work provides interesting insights for preparing healed and stretchable self-powered electronic sensors.

Polymerization behavior of Klenow fragment and Taq DNA polymerase in short primer extension reactions.

DNA polymerases amplify DNA fragments through primer extension reactions. However, polymerization behavior of short primers in the primer extension process has not been systematically explored. In this study, we examined the minimal primer length required for primer extension, and the effect of primer length, mismatches and other conditions on DNA polymerization using a non-radioactive method. Under the condition we conducted, the shortest primers polymerized by Klenow fragment (KF) and Taq DNA polymerase in our experiments were respectively heptamer and octamer. The extension efficiency was also affected by the up-stream overhanging structure of the primer-template complex. We hypothesized a simple model to interpret these observations based on the polymerase structures. Furthermore, it was found that the longer the primer, the more efficient is the primer extension. These polymerization behavior of short primers lay foundation about DNA polymerization mechanism and development of novel nucleic acid detection assays.

A Strategy Employing a TF-Splinting Duplex Nanoswitch to Achieve Single-Step, Enzyme-Free, Signal-On Detection of l-Tryptophan.

Transcription factor (TF)-based metabolite detection mainly depends on TF-regulated gene expression in cells. From TF activation to gene transcription and translation, the signal travels a relatively long way before it is received. Here, we propose a TF-splinting duplex DNA nanoswitch to detect metabolites. We show its feasibility using tryptophan repressor (TrpR) to detect l-tryptophan as a model. The assay has been optimized and characterized after obtaining a proof of concept, and the detection of l-tryptophan in complex biological samples is feasible. Unlike an equivalent gene expression approach, the whole process is a single-step, enzyme-free, and signal-on method. It can be completed within 20 min. This proposed TF-splinting duplex has the potential to be applied to the quick and convenient detection of other metabolites or even TFs.

Repressor logic modules assembled by rolling circle amplification platform to construct a set of logic gates.

Small molecule metabolites and their allosterically regulated repressors play an important role in many gene expression and metabolic disorder processes. These natural sensors, though valuable as good logic switches, have rarely been employed without transcription machinery in cells. Here, two pairs of repressors, which function in opposite ways, were cloned, purified and used to control DNA replication in rolling circle amplification (RCA) in vitro. By using metabolites and repressors as inputs, RCA signals as outputs, four basic logic modules were constructed successfully. To achieve various logic computations based on these basic modules, we designed series and parallel strategies of circular templates, which can further assemble these repressor modules in an RCA platform to realize twelve two-input Boolean logic gates and a three-input logic gate. The RCA-output and RCA-assembled platform was proved to be easy and flexible for complex logic processes and might have application potential in molecular computing and synthetic biology.

Production of dumbbell probe through hairpin cleavage-ligation and increasing RCA sensitivity and specificity by circle to circle amplification.

Dumbbell probe (DP) attracts increasing interests in rolling circle amplification (RCA). A universal DP production method through cleavage-ligation of hairpin was proposed and optimized. The production is characterized by restriction endonuclease (RE)-induced cleavage ends ligation. It has the advantage of phosphorylation-free, splint-free and purification-free. To optimize designing, we found that the position of RE cleavage sequence in the stem and the primer position in the loop affected the formation and amplification of DP obviously. Both sticky and blunt ends cleaved by RE produce DP efficiently. Moreover, we introduced this DP into circle to circle (C2C) RCA based on the same cleavage-ligation principle, and acquired high sensitivity. By combining a two-ligation design and the C2C strategy, specificity for detecting let-7 family members was increased extremely. Furthermore, coreaction of different steps facilitated convenient formation and amplification process of DP.

Mapping the nicking efficiencies of nickase R.BbvCI for side-specific LNA-substituted substrates using rolling circle amplification.

We used a novel asymmetric cleavage analysis method based on rolling circle amplification (RCA) to determine the effects of LNA modification of substrate on the two subunits of R.BbvCI cleavage. We designed two sets of cleavage circular substrates by using two different ligation strategies and analyzed the single strand cleavage efficiency affected by different modification positions both from the cleaved strands and the uncleaved strands. Results showed that the effects of LNA on cleavage rates of modified strands and unmodified strands were both site-dependent. The Nb.BbvCI and Nt.BbvCI were affected by LNA modification in different way. Most of the modification positions showed strong inhibition of both of these two nickases cleavage. However, the modification in T3 position of bottom strand hardly affected both of the two nickases activities. The results suggested an intimated interaction between the two subunits of R.BbvCI, and the T3 position in bottom strand might be a less tight position which was hard to be disturbed.