CARMA3 Drives NF-κB Activation and Promotes Intervertebral Disc Degeneration: Involvement of CARMA3-BCL10-MALT1 Signalosome.

Intervertebral disc degeneration (IDD) diseases are common and frequent diseases in orthopedics. The caspase recruitment domain (CARD) and membrane-associated guanylate kinase-like protein 3 (CARMA3) is crucial in the activation of the NF-κB pathway. However, the biological function of CARMA3 in IDD remains unknown. Here, CARMA3 expression was elevated in nucleus pulposus (NP) tissues of IDD rats and nutrient deprivation (ND)-induced NP cells. The main pathological manifestations observed in IDD rats were shrinkage of the NP, reduction of NP cells, fibrosis of NP tissues, and massive reduction of proteoglycans. These changes were accompanied by a decrease in the expression of collagen II and aggrecan, an increase in the expression of the extracellular matrix (ECM) catabolic proteases MMP-3, MMP-13, and metalloprotease with ADAMTS-5, and an increase in the activity of the pro-apoptotic protease caspase-3. The expression of p-IκBαSer32/36 and p-p65Ser536 was also upregulated. However, these effects were reversed with the knockdown of CARMA3. Mechanistically, CARMA3 bound to BCL10 and MALT1 to form a signalosome. Knockdown of CARMA3 reduced the CARMA3-BCL10-MALT1 signalosome-mediated NF-κB activation. CARMA3 activated the NF-κB signaling pathway in a manner that bound to BCL10 and MALT1 to form a signalosome, which affects NP cell damage and is involved in the development of IDD. This supports CARMA3-BCL10-MALT1-NF-κB as a promising targeting axis for the treatment of IDD.

Is Ancient Medical Treatment an Option for Curating Osteosarcoma Combined with Chemotherapy? A Basic Analysis of Clinic Pharmacy.

BACKGROUND: As a malignant tumor, osteosarcoma (OS) ranks first place among adolescent cancers and is susceptible to developing resistance to chemotherapeutic agents. Differently, traditional Chinese medicine (TCM) has multiple pharmacodynamic targets and complex biological components, which can inhibit tumor survival and drug resistance and gradually play an important role in the treatment of sarcoma. METHODS: This study is to systematically evaluate the safety and efficacy of TCM combined with chemotherapy performed in the clinical treatment of OS. Based on multiple mainstream databases, eleven articles on the relationship between natural products and chemotherapy involving 656 patients were selected from all the literature published as of June 2022. Revman 5.4 software was used for a comprehensive search analysis, supplemented by established exclusion criteria, the Jadad scale, and the evaluation methods provided by Cochrane. RESULTS: The efficiency of TCM combined with chemotherapy was significantly increased compared with chemical drugs alone [OR=2.56, 95% CI (1.36,4.79), Z=2.92, P=0.003]. Meanwhile, the adverse reactions such as nausea and vomiting, hepatotoxicity, and hematological changes caused by chemical drugs were alleviated correspondingly. CONCLUSION: This study indicates that the mode of TCM combined with chemotherapy sheds light on the clinical treatment of OS, which is much better than the one-way mode.

Label-free detection of biomolecules using inductively coupled plasma mass spectrometry (ICP-MS).

Bioassays using inductively coupled plasma mass spectrometry (ICP-MS) have gained increasing attention because of the high sensitivity of ICP-MS and the various strategies of labeling biomolecules with detectable metal tags. The classic strategy to tag the target biomolecules is through direct antibody-antigen interaction and DNA hybridization, and requires the separation of the bound from the unbound tags. Label-free ICP-MS techniques for biomolecular assays do not require direct labeling: they generate detectable metal ions indirectly from specific biomolecular reactions, such as enzymatic cleavage. Here, we highlight the development of three main strategies of label-free ICP-MS assays for biomolecules: (1) enzymatic cleavage of metal-labeled substrates, (2) release of immobilized metal ions from the DNA backbone, and (3) nucleic acid amplification-assisted aggregation and release of metal tags to achieve amplified detection. We briefly describe the fundamental basis of these label-free ICP-MS assays and discuss the benefits and drawbacks of various designs. Future research is needed to reduce non-specific adsorption and minimize background and interference. Analytical innovations are also required to confront challenges faced by in vivo applications.

Highly sensitive detection of aflatoxin B1 byCRISPR/Cas12a-assisted single nanoparticle counting.

CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated protein (Cas) have gained extensive applications in bioassays. However, CRISPR-based detection platforms are often hampered by limited analytical sensitivity, while nucleic acid-based amplification strategies are usually indispensable for additional signal enhancement with potential risks of amplification leakages. To address these challenges, an amplification-free CRISPR-based bioassay of aflatoxin B1 (AFB1) was proposed by applying single nanoparticle counting. Single-particle mode inductively coupled plasma mass spectrometry (Sp-ICPMS) has been regarded as a sensitive tool for nanoparticle counting since one nanoparticle can generate considerable signals above backgrounds. With AFB1, activator strands were introduced to initiate the trans-cleavage of CRISPR/Cas12a for cutting the nanoparticles-tagged-magnetic beads, which were transduced to nanoparticle count signals after separation. Finally, a pico-mole level limit-of-detections (LODs) with moderate selectivity was achieved. Certified reference materials (CRMs) analysis and recovery tests were conducted with promising results. To our best knowledge, this is the first report of the single particle counting-based CRISPR/Cas12a biosensing study.

Rapid and sensitive point-of-care PTS-CRISPR assay for food safety monitoring of aflatoxin B1.

Food safety is crucial for human health, but its effective on-site monitoring remains a challenge. Pregnancy test strip (PTS) is the successful point-of-care testing (POCT) product of the highest market share in the world, with the cost as low as $0.10 per test. Herein, combined with the CRISPR (clustered regularly interspaced short palindromic repeats) system, PTS-CRISPR was for the first time introduced into food safety monitoring, for rapid and sensitive POCT of aflatoxin B1. The low-cost, easy-to-operate PTS-CRISPR is expected to bring security to the grassroots food market.

Recent advances in RNA sample preparation techniques for the detection of SARS-CoV-2 in saliva and gargle.

Molecular detection of SARS-CoV-2 in gargle and saliva complements the standard analysis of nasopharyngeal swabs (NPS) specimens. Although gargle and saliva specimens can be readily obtained non-invasively, appropriate collection and processing of gargle and saliva specimens are critical to the accuracy and sensitivity of the overall analytical method. This review highlights challenges and recent advances in the treatment of gargle and saliva samples for subsequent analysis using reverse transcription polymerase chain reaction (RT-PCR) and isothermal amplification techniques. Important considerations include appropriate collection of gargle and saliva samples, on-site inactivation of viruses in the sample, preservation of viral RNA, extraction and concentration of viral RNA, removal of substances that inhibit nucleic acid amplification reactions, and the compatibility of sample treatment protocols with the subsequent nucleic acid amplification and detection techniques. The principles and approaches discussed in this review are applicable to molecular detection of other microbial pathogens.

Isotope-encoded tetrahedral DNA for multiple SARS-CoV-2 variant diagnosis.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed an unprecedented demand for accurate and cost-effective diagnostic assays to discriminate between different variants. Whilst many bioassays have been successfully demonstrated for SARS-CoV-2 detection, diagnosis of its variants remains challenging and mainly relies on time-consuming and costly sequencing techniques. Herein, we proposed a triplevalent tetrahedral DNA nanostructure (tTDN) with three overhang isotope probes capable of multiplex simultaneous analysis. HV69/70 del (alpha-specific), K417N (beta-specific) and T478K (delta-specific) and omicron with common mutations above of the SARS-CoV-2 S gene were detected selectively with the aid of the TDN scaffold and MNAzyme system, and a sensitive strategy enabling the screening of four kinds of variants of concern (VOC) was achieved.

A Photopatternable Conjugated Polymer with Thermal-Annealing-Promoted Interchain Stacking for Highly Stable Anti-Counterfeiting Materials.

Photoresponsive polymers can be conveniently used to fabricate anti-counterfeiting materials through photopatterning. However, an unsolved problem is that ambient light and heat can damage anti-counterfeiting patterns on photoresponsive polymers. Herein, photo- and thermostable anti-counterfeiting materials are developed by photopatterning and thermal annealing of a photoresponsive conjugated polymer (MC-Azo). MC-Azo contains alternating azobenzene and fluorene units in the polymer backbone. To prepare an anti-counterfeiting material, an MC-Azo film is irradiated with polarized blue light through a photomask, and then thermally annealed under the pressure of a photonic stamp. This strategy generates a highly secure anti-counterfeiting material with dual patterns, which is stable to sunlight and heat over 200 °C. A key for the stability is that thermal annealing promotes interchain stacking, which converts photoresponsive MC-Azo to a photostable material. Another key for the stability is that the conjugated structure endows MC-Azo with desirable thermal properties. This study shows that the design of photopatternable conjugated polymers with thermal-annealing-promoted interchain stacking provides a new strategy for the development of highly stable and secure anti-counterfeiting materials.

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern.

The continuing evolution of the SARS-CoV-2 virus has led to the emergence of many variants, including variants of concern (VOCs). CRISPR-Cas systems have been used to develop techniques for the detection of variants. These techniques have focused on the detection of variant-specific mutations in the spike protein gene of SARS-CoV-2. These sequences mostly carry single-nucleotide mutations and are difficult to differentiate using a single CRISPR-based assay. Here we discuss the specificity of the Cas9, Cas12, and Cas13 systems, important considerations of mutation sites, design of guide RNA, and recent progress in CRISPR-based assays for SARS-CoV-2 variants. Strategies for discriminating single-nucleotide mutations include optimizing the position of mismatches, modifying nucleotides in the guide RNA, and using two guide RNAs to recognize the specific mutation sequence and a conservative sequence. Further research is needed to confront challenges in the detection and differentiation of variants and sublineages of SARS-CoV-2 in clinical diagnostic and point-of-care applications.

A data-driven approach for discovering the most probable transition pathway for a stochastic carbon cycle system.

Many natural systems exhibit tipping points where changing environmental conditions spark a sudden shift to a new and sometimes quite different state. Global climate change is often associated with the stability of marine carbon stocks. We consider a stochastic carbonate system of the upper ocean to capture such transition phenomena. Based on the Onsager-Machlup action functional theory, we calculate the most probable transition pathway between the metastable and oscillatory states via a neural shooting method. Furthermore, we explore the effects of external random carbon input rates on the most probable transition pathway, which provides a basis to recognize naturally occurring tipping points. Particularly, we investigate the transition pathway's dependence on the transition time and further compute the optimal transition time using a physics-informed neural network, toward the maximum carbonate concentration state in the oscillatory regimes. This work may offer some insights into the effects of noise-affected carbon input rates on transition phenomena in stochastic models.

The prognostic value of Epstein-Barr virus infection in Hodgkin lymphoma: A systematic review and meta-analysis.

Introduction: Epstein-Barr virus (EBV) contributes significantly to the development and occurrence of B-cell lymphomas. However, the association between EBV infection status and clinical outcomes in Hodgkin lymphoma (HL) patients has long been controversial. Therefore, we aimed to estimate the prognostic significance of EBV infection in HL survival. Methods: We searched PubMed, Embase, Web of Science, and the Cochrane Library for relevant cohort studies from the date of their inception to February 20, 2022. Hazard ratios (HRs) and 95% confidence intervals (CIs) for overall survival (OS), Failure-free survival (FFS), Progression-free survival (PFS), Event-free survival (EFS) and disease-specific survival (DSS) were extracted from the studies or calculated. Subgroup analyses were conducted independently on the five survival outcomes to investigate the source of heterogeneity. Results: A total of 42 qualified studies involving 9570 patients were identified in our meta-analysis. There was an association between EBV positivity and significantly poorer OS (HR=1.443, 95% CI: 1.250-1.666) and DSS (HR=2.312, 95% CI: 1.799-2.972). However, the presence of EBV in HL showed no effect on FFS, PFS or EFS. In subgroup analyses of OS, DSS and FFS stratified by age groups, EBV positivity was associated with poorer prognosis in elderly patients. Meanwhile, in children and adolescents with EBV-positive HL, we also observed a trend toward a better prognosis, though the results were not statistically significant. Conclusions: EBV-positive status is associated with poor OS and DSS in HL patients. EBV infection should therefore be considered a valuable prognostic marker and risk-stratifying factor in HL, especially in older patients. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42022328708.

Single-Nanoparticle Differential Immunoassay for Multiplexed Gastric Cancer Biomarker Monitoring.

Precision medicine demands the best application of multiple unambiguous biomarkers to bring uniform decisions in disease prognosis. The remarkable development of heterogeneous immunoassay greatly promotes precision medicine when combined with the biomarker combination strategy. Nevertheless, the cumbersome washing steps in heterogeneous immunoassay have inevitably compromised the accuracy because of the sample losses and nature change of the matrix, challenging the further exploration of a more facile and lower limit-of-detection analysis. The new methodologies with high throughputs and specificity are never out of date to provide simultaneous evaluations and uniform decisions on multiple analytes through a simple process. Herein, we propose a new wash-free immunoassay, named differential assay, for multiplexed biomarker monitoring. The method is based on counting the number difference of unbound nanoparticle tags before and after immunoreactions from a solid support (i.e., magnetic microsphere) by single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS), discarding the tedious washing steps. We primarily explore the proof-of-concept proposal within two types (sandwich and competitive assay), demonstrating the good feasibility for further facile clinical practice. To provide efficient multiplexed evaluations, we synthesized PtNPs with four diameters and screened the most suitable size for efficient differential immunoassay. The wash-free strategy was successfully utilized in simultaneous serological biomarker (CA724, CA199, and CEA) evaluation, with results in good accordance with those measured by the clinical routine method. Potentially, the proposed differential bioassay can be regarded as a more facile and valuable tool in malignancy prognosis and cancer recurrence monitoring.

Effects of different water management and fertilizer methods on soil temperature, radiation and rice growth.

In recent decades, the application of organic fertilizer to agricultural soils has attracted wide attention. However, few studies have carefully explored the effects of humic acid fertilizer on soil temperature, radiation, and the physiology of plant leaves, especially when coupled with different irrigation methods. To provide a better growing environment for crops and explore the best regulation method of humic acid fertilizer and irrigation in the farmland soil environment on the Songnen Plain, China, through field experiments, we selected rice as the test crop and applied humic acid fertilizer to the soil with different irrigation methods. The effects of different humic acid fertilizers and irrigation methods on the soil temperature and radiation changes during different growth stages were examined, and the subtle differences in agronomic and fluorescence characteristics in different growth stages of rice plants were compared. The results showed that the soil temperature was not significantly different among all the treatments. However, radiation interception was obviously different, and the best value was observed in the CT5 treatment. The fluorescence indices and leaf chlorophyll relative content (SPAD) differed with the change in humic acid fertilizer application and irrigation methods. At the jointing and heading stages, the Fv /Fm values of the CT5, FT5 and WT5 treatments were larger than those of the other treatments, and the best value was recorded in the CT5 treatment. The differences in NPQ at these two stages were significant, and the NPQ in the CT5 treatment was significantly higher than that in the other treatments (P < 0.05). In general, the QP under control irrigation was greater than that under flood and wet irrigation (P < 0.05). Moreover, there were no significant differences among the gradients under the different humic acid fertilizer application methods in terms of QP (P > 0.05). Additionally, SPAD values were higher under the CT5 and FT5 treatments.

Variational inference of the drift function for stochastic differential equations driven by Lévy processes.

In this work, we consider the nonparametric estimation problem of the drift function of stochastic differential equations driven by the α-stable Lévy process. We first optimize the Kullback-Leibler divergence between the path probabilities of two stochastic differential equations with different drift functions. We then construct the variational formula based on the stationary Fokker-Planck equation using the Lagrangian multiplier. Moreover, we apply the empirical distribution to replace the stationary density, combining it with the data information, and we present the estimator of the drift function from the perspective of the process. In the numerical experiment, we investigate the effect of the different amounts of data and different α values. The experimental results demonstrate that the estimation result of the drift function is related to both and that the exact drift function agrees well with the estimated result. The estimation result will be better when the amount of data increases, and the estimation result is also better when the α value increases.

Standard-free single magnetic bead evaluation: a stable nanoplatform for prostate disease differentiation.

Explicit interpretation of heterogeneity between prostate-specific antigen (PSA) subtypes is essential for prostate cancer differentiation during different disease courses, whereas a universal protocol with uniform criteria is still lacking across the globe. In this work, a standard-free single magnetic bead (SMB) nanoplatform utilizing metal nanoparticles with optimal diameters was proposed for prostate disease differentiation in a 134-donor model. The inaccuracy of detection in absolute quantification was diminished via evaluations of metal intensities on the single magnetic bead. The intrinsic proportion of fPSA in tPSA was successfully evaluated by direct use of the Pt to Au intensity ratio (Pt/Au ratio), exhibiting better differentiation between healthy and unhealthy, benign prostatic hyperplasia (BPH) and cancer individuals compared with solo fPSA or tPSA. We generated thresholds respectively for prostate disease differentiation, envisioning that this standard-free SMB nanoplatform would establish a standardized methodology with uniform criteria worldwide in cancer diagnosis, staging, and postoperative assessments.

An Onsager-Machlup approach to the most probable transition pathway for a genetic regulatory network.

We investigate a quantitative network of gene expression dynamics describing the competence development in Bacillus subtilis. First, we introduce an Onsager-Machlup approach to quantify the most probable transition pathway for both excitable and bistable dynamics. Then, we apply a machine learning method to calculate the most probable transition pathway via the Euler-Lagrangian equation. Finally, we analyze how the noise intensity affects the transition phenomena.

Dual-amplified CRISPR-Cas12a bioassay for HIV-related nucleic acids.

Nucleic acid amplification strategies have successfully dominated ultrasensitive bioassays, but they sometimes bring high time-consumption, multi-step operation, increased contamination risk, and mismatch-related inaccuracy. We proposed a nucleic acid amplification-free method called the AuNPs-tagging based CRISPR-Cas12a bioassay platform. The signal amplification was realized by integrating the self-amplification effect of CRISPR-Cas12a with the enhancement effect of the large number of detectable atoms inside each gold nanoparticle. The proposed method achieved a low LOD of 1.05 amol in 40 min for HIV-related DNA.

HOGG1-assisted DNA methylation analysis via a sensitive lanthanide labelling strategy.

The assessment of DNA methylation level is an important indicator for the diagnosis and treatment of some diseases. DNA methylation assays are usually based on nucleic acid amplification strategies, which are time-consuming and complicated in operation procedures. Herein, we proposed a sensitive lanthanide-labelled ICP-MS method for DNA methylation analysis that exploited the feature of Human 8-oxoGuanine DNA Glycosylase (hOGG1), which specifically recognizes 8-oxo-G/5mC base pairs to effectively distinguish methylated DNA. A low limit of detection of 84 pM was achieved, and a 0.1% methylation level can be discriminated in the mixture, without any amplification procedure. Compared with commonly used nucleic acid amplification strategies, this proposed method is time-saving and low probability of false positive. Moreover, this work has been successfully validated in human serum samples, the recovery rates is between 96.7% and 105%, and the relative standard deviation (RSD) is in the range of 3.0%-3.5%, indicating that this method has the potential to be applied in clinical and biological samples quantitative analysis.

Element coding based accurate evaluation of CRISPR/Cas9 initial cleavage.

As a powerful gene editing tool, the kinetic mechanism of CRISPR/Cas9 has been the focus for its further application. Initial cleavage events as the first domino followed by nuclease end trimming significantly affect the final on-target rate. Here we propose EC-CRISPR, element coding CRISPR, an accurate evaluation platform for initial cleavage that directly characterizes the cleavage efficiency and breaking sites. We benchmarked the influence of 19 single mismatch and 3 multiple mismatch positions of DNA-sgRNA on initial cleavage, as well as various reaction conditions. Results from EC-CRISPR demonstrate that the PAM-distal single mismatch is relatively acceptable compared to the proximal one. And multiple mismatches will not only affect the cleavage efficiency, but also generate more non-site #3 cleavage. Through in-depth research of kinetic behavior, we uncovered an abnormally higher non-#3 proportion at the initial stage of cleavage by using EC-CRISPR. Together, our results provided insights into cleavage efficiency and breaking sites, demonstrating that EC-CRISPR as a novel quantitative platform for initial cleavage enables accurate comparison of efficiencies and specificities among multiple CRISPR/Cas enzymes.