Research on fatigue identification methods based on low-load wearable ECG monitoring devices.

The identification of fatigue in personal workers in particular environments can be achieved through early warning techniques. In order to prevent excessive fatigue of medical workers staying in infected areas in the early phase of the coronavirus disease pandemic, a system of low-load wearable electrocardiogram (ECG) devices was used as intelligent acquisition terminals to perform a continuous measurement ECG collection. While machine learning (ML) algorithms and heart rate variability (HRV) offer the promise of fatigue detection for many, there is a demand for ever-increasing reliability in this area, especially in real-life activities. This study proposes a random forest-based classification ML model to identify the four categories of fatigue levels in frontline medical workers using HRV. Based on the wavelet transform in ECG signal processing, stationary wavelet transform was applied to eliminate the main perturbation of ECG in the motion state. Feature selection was performed using ReliefF weighting analysis in combination with redundancy analysis to optimize modeling accuracy. The experimental results of the overall fatigue identification achieved an accuracy of 97.9% with an AUC value of 0.99. With the four-category identification model, the accuracy is 85.6%. These results proved that fatigue analysis based on low-load wearable ECG monitoring at low exertion can accurately determine the level of fatigue of caregivers and provide further ideas for researchers working on fatigue identification in special environments.

Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles.

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

Non-invasive continuous blood pressure prediction based on ECG and PPG fusion map.

To achieve real-time blood pressure monitoring, a novel non-invasive method is proposed in this article. Electrocardiographic (ECG) and pulse wave signals (PPG) are fused from a multi-omics signal-level perspective. A physiological signal fusion matrix and fusion map, which can estimate the blood pressure of blood loss(BPBL), are constructed. The results demonstrate the efficacy of the fusion map model, with correlation values of 0.988 and 0.991 between the estimated BPBL and the true systolic blood pressure (SBP) and diastolic blood pressure (DBP), respectively. The root mean square errors for SBP and DBP were 3.21 mmHg and 3.00 mmHg, respectively. The model validation showed that the fusion map method is capable of simultaneous highlighting of the respective characteristics of ECG and PPG and their correlation, improving the utilization of the information and the accuracy of BPBL. This article validates that physiological signal fusion map can effectively improve the accuracy of BPBL estimation and provides a new perspective for the field of physiological information fusion.

ZY-444 inhibits the growth and metastasis of prostate cancer by targeting TNFAIP3 through TNF signaling pathway.

Prostate cancer is one of the most lethal malignancies, and androgen deprivation therapy remains the mainstay of treatment for prostate cancer patients. Although androgen deprivation can initially come to remission, the disease often develops into castration-resistant prostate cancer (CRPC), which is still dependent on androgen receptor (AR) signaling and is related to a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance uninterrupted emerges, and new therapies are urgently needed. In this study, we identified a potent small molecule compound, ZY-444, that suppressed PCa cells proliferation and metastasis, and inhibited tumor growth both in subcutaneous. Transcriptome sequencing analysis showed that TNFAIP3 was significantly elevated in prostate cancer cells after ZY-444 treatment. Further studies through overexpression of TNFAIP3 confirmed that TNFAIP3, as a direct target gene of ZY-444, contributes to the functions of ZY-444. In addition, we demonstrated the effects of TNFAIP3 on prostate cancer cell apoptosis, migration and proliferation to elucidate the mechanism of ZY-444. We found that TNFAIP3 inhibited the TNF signaling pathway, which could inhibit cell migration and proliferation and contribute to apoptosis. Overall, these findings highlighted TNFAIP3 as a tumor suppressor gene in the regulation of the progression and metastatic potential of prostate cancer and that targeting TNFAIP3 by ZY-444 might be a promising strategy for prostate cancer treatment.

miR­151a­5p promotes the proliferation and metastasis of colorectal carcinoma cells by targeting AGMAT.

Colorectal carcinoma (CRC) is one of the most common types of digestive cancer. It has been reported that the ectopic expression of microRNAs (miRs) plays a critical role in the occurrence and progression of CRC. In addition, it has also been suggested that miR­151a­5p may serve as a useful biomarker for the early detection and treatment of different types of cancer and particularly CRC. However, the specific effects and underlying mechanisms of miR­151a­5p in CRC remain elusive. The results of the current study demonstrated that miR­151a­5p was upregulated in CRC cell lines and clinical tissues derived from patients with CRC. Functionally, the results showed that miR­151a­5p significantly promoted CRC cell proliferation, migration and invasion. Additionally, dual luciferase reporter assays verified that agmatinase (AGMAT) was a direct target of miR­151a­5p and it was positively associated with miR­151a­5p expression. Mechanistically, miR­151a­5p could enhance the epithelial­mesenchymal transition of CRC cells. Taken together, the results of the current study revealed a novel molecular mechanism indicating that the miR­151a­5p/AGMAT axis could serve a crucial role in the regulation of CRC and could therefore be considered as a potential therapeutic strategy for CRC.

Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro.

A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Using the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multi-color RNA immunoFISH and visualized their localization patterns within the cell. The 10-nm resolution achieved by our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive imaging framework that will enable future investigations of coronavirus fundamental biology and therapeutic effects.

A Target-Driven Self-Feedback Paper-Based Photoelectrochemical Sensing Platform for Ultrasensitive Detection of Ochratoxin A with an In2S3/WO3 Heterojunction Structure.

Currently, developing versatile, easy-to-operate, and effective signal amplification strategies hold great promise in photoelectrochemical (PEC) biosensing. Herein, an ultrasensitive polyvinylpyrrolidone-treated In2S3/WO3 (In2S3-P/WO3)-functionalized paper-based PEC sensor was established for sensing ochratoxin A (OTA) based on a target-driven self-feedback (TDSF) mechanism enabled by a dual cycling tactic of PEC chemical-chemical (PECCC) redox and exonuclease III (Exo III)-assisted complementary DNA. The In2S3-P/WO3 heterojunction structure with 3D open-structure and regulable topology was initially in situ grown on Au nanoparticle-functionalized cellulose paper, which was served as a universal signal transducer to directly record photocurrent signals without complicated electrode modification, endowing the paper chip with admirable anti-interference ability and unexceptionable photoelectric conversion efficiency. With the assistance of Exo III-assisted cycling process, a trace amount of OTA could trigger substantial signal reporter ascorbic acid (AA) generated by the enzymatic catalysis of alkaline phosphatase, which could effectively provoke the PECCC redox cycling among the tris(2-carboxyethyl)phosphine acid, AA, and ferrocenecarboxylic at the In2S3-P/WO3 photoelectrode, initiating TDSF signal amplification. Based on the TDSF process induced by the Exo III-assisted recycling and PECCC redox cycling strategy, the developed paper-based PEC biosensor realized ultrasensitive determination of OTA with persuasive selectivity, high stability, and excellent reproducibility. It is believed that the proposed paper-based PEC sensing platform exhibited enormous potential for the detection of other targets in bioanalysis and clinical diagnosis.

Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Employing the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multicolor RNA-immunoFISH and visualized their localization patterns within the cell. The exquisite resolution of our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive framework that supports investigations of coronavirus fundamental biology and therapeutic effects.

Interrogation of the dynamic properties of higher-order heterochromatin using CRISPR-dCas9.

Eukaryotic chromosomes feature large regions of compact, repressed heterochromatin hallmarked by Heterochromatin Protein 1 (HP1). HP1 proteins play multi-faceted roles in shaping heterochromatin, and in cells, HP1 tethering to individual gene promoters leads to epigenetic modifications and silencing. However, emergent properties of HP1 at supranucleosomal scales remain difficult to study in cells because of a lack of appropriate tools. Here, we develop CRISPR-engineered chromatin organization (EChO), combining live-cell CRISPR imaging with inducible large-scale recruitment of chromatin proteins to native genomic targets. We demonstrate that human HP1α tiled across kilobase-scale genomic DNA form novel contacts with natural heterochromatin, integrates two distantly targeted regions, and reversibly changes chromatin from a diffuse to compact state. The compact state exhibits delayed disassembly kinetics and represses transcription across over 600 kb. These findings support a polymer model of HP1α-mediated chromatin regulation and highlight the utility of CRISPR-EChO in studying supranucleosomal chromatin organization in living cells.

Engineering 3D genome organization.

Cancers and developmental disorders are associated with alterations in the 3D genome architecture in space and time (the fourth dimension). Mammalian 3D genome organization is complex and dynamic and plays an essential role in regulating gene expression and cellular function. To study the causal relationship between genome function and its spatio-temporal organization in the nucleus, new technologies for engineering and manipulating the 3D organization of the genome have been developed. In particular, CRISPR-Cas technologies allow programmable manipulation at specific genomic loci, enabling unparalleled opportunities in this emerging field of 3D genome engineering. We review advances in mammalian 3D genome engineering with a focus on recent manipulative technologies using CRISPR-Cas and related technologies.

Effect of Artemisia rupestris L. Extract on Gastrointestinal Hormones and Brain-Gut Peptides in Functional Dyspepsia Rats.

Artemisia rupestris L. is the perennial herb of rupestris belonging to Artemisia (Compositae), which is wildly distributed in Xinjiang (China), middle Asia, and Europe. It is known to have anti-inflammatory, hepatoprotective, immune function regulation, and gastrointestinal function regulation effects. AR is used to treat digestive diseases, but the effects of AR on antifunctional dyspepsia (FD) activity have not yet been reported. In this study, we aimed to investigate the therapeutic effects of Artemisia rupestris L. extract (ARE) on gastrointestinal hormones and brain-gut peptide in functional dyspepsia (FD) rats. Sixty Sprague-Dawley rats were randomly divided into 6 groups. An FD rat model was established by irregular tail clamp stimulation for 14 days except the blank group. After FD rat models, the blank group and model group were given menstruum, and the medicated rats were given corresponding medicine for 14 days. The general observations, bodyweight, and food intake were observed, and the content of serum gastrin (GAS), plasma motilin (MTL), plasma vasoactive intestinal peptide (VIP), and plasma somatostatin (SS) by the enzyme-linked immunosorbent assay was observed. The content of plasma VIP and plasma SS in the ARE group was significantly lower than in the model group, and the content of serum GAS and plasma MTL was increased in the ARE group; the GAS expression of antrum and hypothalamus was increased in the ARE group, and SS expression of antrum and hypothalamus was decreased in the ARE group by immunohistochemical detection; the results of semiquantitative reverse transcription polymerase chain reaction (RT-PCR) indicate that ARE inhibits the mRNA expression of VIP. Our results suggest that ARE can recover gastrointestinal hormone levels and regulation of the peripheral and central nervous system and alter gut peptide levels, which confirm the therapeutic effect of ARE on functional dyspepsia.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Transcriptome-wide discovery of coding and noncoding RNA-binding proteins.

Transcriptome-wide identification of RNA-binding proteins (RBPs) is a prerequisite for understanding the posttranscriptional gene regulation networks. However, proteomic profiling of RBPs has been mostly limited to polyadenylated mRNA-binding proteins, leaving RBPs on nonpoly(A) RNAs, including most noncoding RNAs (ncRNAs) and pre-mRNAs, largely undiscovered. Here we present a click chemistry-assisted RNA interactome capture (CARIC) strategy, which enables unbiased identification of RBPs, independent of the polyadenylation state of RNAs. CARIC combines metabolic labeling of RNAs with an alkynyl uridine analog and in vivo RNA-protein photocross-linking, followed by click reaction with azide-biotin, affinity enrichment, and proteomic analysis. Applying CARIC, we identified 597 RBPs in HeLa cells, including 130 previously unknown RBPs. These newly discovered RBPs can likely bind ncRNAs, thus uncovering potential involvement of ncRNAs in processes previously unknown to be ncRNA-related, such as proteasome function and intermediary metabolism. The CARIC strategy should be broadly applicable across various organisms to complete the census of RBPs.

5-chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(2-methoxy-4-(4-((4-methylpiperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)phenyl)pyrimidine-2,4-diamine (WY-135), a novel ALK inhibitor, induces cell cycle arrest and apoptosis through inhibiting ALK and its downstream pathways in Karpas299 and H2228 cells.

Anaplastic lymphoma kinase (ALK)-positive cancers have rising morbidity and mortality in recent years, and novel chemotherapeutic drugs with no drug resistance and high activity for treating ALK-positive cancers are needed urgently. In this study, we investigated the anti-cancer effect of 5-chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(2-methoxy-4-(4-((4-methylpiperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)phenyl)pyrimidine-2,4-diamine (WY-135), a novel ALK inhibitor, on nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) positive cancer cell line Karpas299 and echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) positive cancer cell line H2228. In vitro enzyme assay showed that WY-135 had better enzyme inhibitory activity than ceritinib. MTT assay showed that WY-135 had similar inhibitory activity with ceritinib in Karpas299 and H2228 cells. The cell cycle analysis proved that WY-135 induced cell cycle arrest at G1 phase in a dose-dependent manner and subsequently progressed into apoptosis. Real-time PCR analysis revealed that the mRNA level of ALK was significantly reduced in Karpas299 and H2228 cells treatment with WY-135. Furthermore, western blot analysis showed that WY-135 significantly suppressed ALK and its downstream protein expression. Taken together, WY-135 exhibits significant anti-cancer activity through inhibiting ALK and its downstream protein expression, arresting cell cycle and eventually inducing cell apoptosis in Karpas299 and H2228 cells. WY-135 is a promising ALK inhibitor with novel structure that has tremendous potentials for therapeutic treatment of NPM-ALK or EML4-ALK positive cancers.

Artificial Cysteine S-Glycosylation Induced by Per-O-Acetylated Unnatural Monosaccharides during Metabolic Glycan Labeling.

The unexpected, non-enzymatic S-glycosylation of cysteine residues in various proteins by per-O-acetylated monosaccharides is described. This artificial S-glycosylation greatly compromises the specificity and validity of metabolic glycan labeling in living cells by per-O-acetylated azido and alkynyl sugars, which has been overlooked in the field for decades. It is demonstrated that the use of unacetylated unnatural sugars can avoid the artifact formation and a corrected list of O-GlcNAcylated proteins and O-GlcNAc sites in HeLa cells has been assembled by using N-azidoacetylgalactosamine (GalNAz).

Solid-State 77Se NMR of Organoselenium Compounds through Cross Polarization Magic Angle Spinning (CPMAS) Method.

Characterization of selenium states by 77Se NMR is quite important to provide vital information for mechanism studies in organoselenium-catalyzed reactions. With the development of heterogeneous polymer-supported organoselenium catalysts, the solid state 77Se NMR comes to the spotlight. It is necessary to figure out an advanced protocol that provides good quality spectra within limited time because solid state 77Se NMR measurements are always time consuming due to the long relaxation time and the relatively low sensitivity. Studies on small molecules and several novel polymer-supported organoselenium materials in this article showed that cross polarization (CP) method with the assistance of magic angle spinning (MAS) was more efficient to get high quality spectra than the methods by using single pulse (SP) or high power 1H decoupling (HPHD) combined with MAS. These results lead to a good understanding of the effect of the molecular structure, the heteronuclear coupling, the long-range ordering of the solid (crystal or amorphous), and the symmetry of 77Se on quality of their spectra.

Methyl 5-[(1H-indol-3-yl)selanyl]-1H-benzoimidazol-2-ylcarbamate (M-24), a novel tubulin inhibitor, causes G2/M arrest and cell apoptosis by disrupting tubulin polymerization in human cervical and breast cancer cells.

Methyl 5-[(1H-indol-3-yl)selanyl]-1H-benzoimidazol-2-ylcarbamate (M-24) is a newly synthesized analogue of nocodazole by our group and has been found to be active for some cancer cells. However, its sensitivity to different cell lines and the underlying anticancer mechanism are still unclear. In this study, we proved that M-24 had strong time- and dose-dependent anti-proliferative effects on human cervical cancer HeLa cells and human breast carcinoma MCF-7 cells. We demonstrated that the growth inhibitory effects of M-24 in both cell lines were associated with microtubule depolymerization. Furthermore, M-24 treatment resulted in cell cycle arrest at the G2/M phase in a dose-dependent manner with subsequent apoptosis induction. Western blotting analysis revealed that up-regulation of cyclin B1 and cdc2 was related with G2/M arrest in both cell lines. In addition, M-24-induced HeLa cell apoptosis was mainly associated with mitochondria-dependent intrinsic pathway. However, M-24-induced MCF-7 cell apoptosis was associated with both mitochondrial and death receptor pathway. In conclusion, M-24 caused apoptosis through disrupting microtubule assembly and inducing cell cycle arrest in HeLa and MCF-7 cells. Therefore, the novel compound M-24 is a promising microtubule-destabilizing agent that has great potential for the therapy of various malignancies especially human cervical and breast cancers.

Asialoglycoprotein receptor-targeted liposomes loaded with a norcantharimide derivative for hepatocyte-selective targeting.

In order to overcome the shortcomings associated with the clinical application of norcantharidin (NCTD), including intense irritation and a short half-life, and to obtain a hepatocyte-selective liposome system with high encapsulation efficiency (EE) and low leakage, we synthesized a C14 alkyl chain norcantharimide derivative of NCTD (2-tetradecylhexahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione, N-14NCTDA). Asialoglycoprotein receptor-targeted, galactosylated liposomes loaded with N-14NCTDA (GAL-Lipo) were prepared by the lipid film hydration method. GAL-Lipo with a satisfactory particle size of approximately 120nm has a higher encapsulation efficiency of more than 98.0%, which is markedly increased compared with NCTD loaded liposomes (EE%=47.6%). In addition, GAL-Lipo remained stable for at least 1 month at 4°C. In cytotoxicity assays, GAL-Lipo demonstrated stronger cytotoxicity effects (IC50=24.58µmolL-1) on Hep G2 cells than free N-14NCTDA (100µmol/L) and conventional liposomes (Con-Lipo, 39.49µmol/L) without the GAL modification. GAL-Lipo can continuously accumulate in Hep G2 cells and be internalized into cells via two pathways, namely caveolin-dependent endocytosis and clathrin-dependent asialoglycoprotein receptors (ASGP-R) mediated endocytosis and produces considerably more significant cellular apoptosis. The results of vivo toxicity studies showed that GAL-Lipo dramatically reduced renal toxicity. In addition, GAL-Lipo has a markedly improved pharmacokinetic profile in vivo and a longer circulation time (AUC=6.700±2.964mgL-1h, t1/2z=1.347±0.519h) than Con-Lipo (AUC=2.319±0.121mgL-1h, t1/2z=0.413±0.238h). In conclusion, N-14NCTDA with an ideal logP is a better alternative for the treatment of primary hepatic carcinoma. GAL-Lipo offers an attractive strategy to specifically target hepatocytes via caveolin-dependent and clathrin-dependent asialoglycoprotein receptor-mediated endocytosis resulting in higher anticancer activity and fewer side-effects.