Pushing the Limits of Capacitively Coupled Contactless Conductivity Detection for Capillary Electrophoresis.

Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) has proven to be an efficient technique for the separation and detection of charged inorganic, organic, and biochemical analytes. It offers several advantages, including cost-effectiveness, nanoliter injection volume, short analysis time, good separation efficiency, suitability for miniaturization, and portability. However, the routine determination of common inorganic cations (NH4+, K+, Na+, Ca2+, Mg2+, and Li+) and inorganic anions (F-, Cl-, Br-, NO2-, NO3-, PO43-, and SO42-) in water quality monitoring typically exhibits limits of detection of about 0.3-1 µM without preconcentration. This sensitivity often proves insufficient for the applications of CE-C4D in trace analysis situations. Here, we explore methods to push the detection limits of CE-C4D through a comprehensive consideration of signal and noise sources. In particular, we (i) studied the model of C4D and its guiding roles in C4D and CE-C4D, (ii) optimized the bandwidth and noise performance of the current-to-voltage (I-V) converter, and (iii) reduced the noise level due to the strong background signal of the background electrolyte by adaptive differential detection. We characterized the system with Li+; the 3-fold signal-to-noise (S/N) detection limit for Li+ was determined at 20 nM, with a linear range spanning from 60 nM to 1.6 mM. Moreover, the optimized CE-C4D method was applied to the analysis of common mixed inorganic cations (K+, Na+, Ca2+, Mg2+, and Li+), anions (F-, Cl-, Br-, NO2-, NO3-, PO43-, and SO42-), toxic halides (BrO3-) and heavy metal ions (Pb2+, Cd2+, Cr3+, Co2+, Ni2+, Zn2+, and Cu2+) at trace concentrations of 200 nM. All electropherograms showed good S/N ratios, thus proving its applicability and accuracy. Our results have shown that the developed CE-C4D method is feasible for trace ion analysis in water quality control.

A systematic mutation analysis of 13 major SARS-CoV-2 variants.

SARS-CoV-2 evolves constantly with various novel mutations. Due to their enhanced infectivity, transmissibility and immune evasion, a comprehensive understanding of the association between these mutations and the respective functional changes is crucial. However, previous mutation studies of major SARS-CoV-2 variants remain limited. Here, we performed systematic analyses of full-length amino acids mutation, phylogenetic features, protein physicochemical properties, molecular dynamics and immune escape as well as pseudotype virus infection assays among thirteen major SARS-CoV-2 variants. We found that Omicron exhibited the most abundant and complex mutation sites, higher indices of hydrophobicity and flexibility than other variants. The results of molecular dynamics simulation suggest that Omicron has the highest number of hydrogen bonds and strongest binding free energy between the S protein and ACE2 receptor. Furthermore, we revealed 10 immune escape sites in 13 major variants, some of them were reported previously, but four of which (i.e. 339/373/477/496) are first reported to be specific to Omicron, whereas 462 is specific to Epslion. The infectivity of these variants was confirmed by the pseudotype virus infection assays. Our findings may help us understand the functional consequences of the mutations within various variants and the underlying mechanisms of the immune escapes conferred by the S proteins.

Correction: A compact and high-performance setup of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D).

Correction for 'A compact and high-performance setup of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D)' by Lin Li et al., Analyst, 2024, https://doi.org/10.1039/d4an00354c.

A compact and high-performance setup of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D).

Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) has the advantages of high throughput (simultaneous detection of multiple ions), high separation efficiency (higher than 105 theoretical plates) and rapid analysis capability (less than 5 min for common inorganic ions). A compact CE-C4D system is ideal for water quality control and on-site analysis. It is suitable not only for common cations (e.g. Na+, K+, Li+, NH4+, Ca2+, etc.) and anions (e.g. Cl-, SO42-, BrO3-, etc.) but also for some ions (e.g. lanthanide ions, Pb2+, Cd2+, etc.) that require complex derivatization procedures to be detected by ion chromatography (IC). However, an obvious limitation of the CE-C4D method is that its sensitivity (e.g. 0.3-1 µM for common inorganic ions) is often insufficient for trace analysis (e.g. 1 ppb or 20 nM level for common inorganic ions) without preconcentration. For this technology to become a powerful and routine analytical technique, the system should be made compact while maintaining trace analysis sensitivity. In this study, we developed an all-in-one version of the CE-C4D instrument with custom-made modular components to make it a convenient, compact and high-performance system. The system was designed using direct digital synthesis (DDS) technology to generate programmable sinusoidal waveforms with any frequency for excitation, a kilovolt high-voltage power supply for capillary electrophoresis separation, and an "effective" differential C4D cell with a low-noise circuitry for high-sensitivity detection. We characterized the system with different concentrations of Cs+, and even a low concentration of 20 nM was detectable without preconcentration. Moreover, the optimized CE-C4D setup was applied to analyse mixed ions at a trace concentration of 200 nM with excellent signal-to-noise ratios. In typical applications, the limits of detection based on the 3σ criterion (without baseline filtering) were 9, 10, 24, 5, and 12 nM for K+, Cs+, Li+, Ca2+, and Mg2+, respectively, and about 7, 6, 6 and 6 nM for Br-, ClO4-, BrO3- and SO42-, respectively. Finally, the setup was also applied for the analysis of all 14 lanthanide ions and rare-earth minerals, and it showed an improvement in sensitivity by more than 25 times.

USP29 activation mediated by FUBP1 promotes AURKB stability and oncogenic functions in gastric cancer.

BACKGROUND: Gastric cancer is a highly prevalent cancer type and the underlying molecular mechanisms are not fully understood. Ubiquitin-specific peptidase (USP) 29 has been suggested to regulate cell fate in several types of cancer, but its potential role in gastric carcinogenesis remains unclear. METHODS: The expression of USP29 in normal and gastric cancer tissues was analyzed by bioinformatics analysis, immunohistochemistry and immunoblot. Gene overexpression, CRISPR-Cas9 technology, RNAi, and Usp29 knockout mice were used to investigate the roles of USP29 in cell culture, xenograft, and benzo[a]pyrene (BaP)-induced gastric carcinogenesis models. We then delineated the underlying mechanisms using mass spectrometry, co-immunoprecipitation (Co-IP), immunoblot, ubiquitination assay, chromatin immunoprecipitation (ChIP), quantitative real-time PCR (qRT-PCR), and luciferase assays. RESULTS: In this study, we found that USP29 expression was significantly upregulated in gastric cancers and associated with poor patient survival. Ectopic expression of USP29 promoted, while depletion suppressed the tumor growth in vitro and in vivo mouse model. Mechanistically, transcription factor far upstream element binding protein 1 (FUBP1) directly activates USP29 gene transcription, which then interacts with and stabilizes aurora kinase B (AURKB) by suppressing K48-linked polyubiquitination, constituting a FUBP1-USP29-AURKB regulatory axis that medicates the oncogenic role of USP29. Importantly, systemic knockout of Usp29 in mice not only significantly decreased the BaP-induced carcinogenesis but also suppressed the Aurkb level in forestomach tissues. CONCLUSIONS: These findings uncovered a novel FUBP1-USP29-AURKB regulatory axis that may play important roles in gastric carcinogenesis and tumor progression, and suggested that USP29 may become a promising drug target for cancer therapy.

The emerging role of USP29 in cancer and other diseases.

Reversible protein ubiquitination is a key process for maintaining cellular homeostasis. Deubiquitinases, which can cleave ubiquitin from substrate proteins, have been reported to be deeply involved in disease progression ranging from oncology to neurological diseases. The human genome encodes approximately 100 deubiquitinases, most of which are poorly characterized. One of the well-characterized deubiquitases is ubiquitin-specific protease 29 (USP29), which is often upregulated in pathological tissues and plays important roles in the progression of different diseases. Moreover, several studies have shown that deletion of Usp29 in mice does not cause visible growth and developmental defects, indicating that USP29 may be an ideal therapeutic target. In this review, we provide a comprehensive summary of the important roles and regulatory mechanisms of USP29 in cancer and other diseases, which may help us better understand its biological functions and improve future studies to construct suitable USP29-targeted therapy systems.

Calibration of the oscillation amplitude of quartz tuning fork-based force sensors with astigmatic displacement microscopy.

Quartz tuning forks and qPlus-based force sensors offer an alternative approach to silicon cantilevers for investigating tip-sample interactions in scanning probe microscopy. The high-quality factor (Q) and stiffness of these sensors prevent the tip from jumping to the contact, even at sub-nanometer amplitude. The qPlus configuration enables simultaneous scanning tunneling microscopy and atomic force microscopy, achieving spatial resolution and spectroscopy at the subatomic level. However, to enable precise measurement of tip-sample interaction forces, confidence in these measurements is contingent upon the accurate calibration of the spring constant and oscillation amplitude of the sensor. Here, we have developed a method called astigmatic displacement microscopy with picometer sensitivity.

Integrin αvß1 facilitates ACE2-mediated entry of SARS-CoV-2.

Integrins have been suggested to be involved in SARS-CoV-2 infection, but the underlying mechanisms remain largely unclear. This study aimed to investigate how integrins facilitate the ACE2-mediated cellular entry of SARS-CoV-2. We first tested the susceptibility of a panel of human cell lines to SARS-CoV-2 infection using the spike protein pseudotyped virus assay and examined the expression levels of integrins in these cell lines by qPCR, western blot and flow cytometry. We found that integrin αvß1 was highly enriched in the SARS-CoV-2 susceptible cell lines. Additional studies demonstrated that RGD (403-405)→AAA mutant was defective in binding to integrin αvß1 compared to its wild type counterpart, and anti-αvß1 integrin antibodies significantly inhibited the entry of SARS-CoV-2 into the cells. Further studies using mouse NIH3T3 cells expressing human ACE2, integrin αv, integrin ß1, and/or integrin αvß1 suggest that integrin αvß1 was unable to function as an independent receptor but could significantly facilitate the cellular entry of SASR-CoV-2. Finally, we observed that the Omicron exhibited a significant increase in the ACE2-mediated viral entry. Our findings may enhance our understanding of the pathogenesis of SARS-CoV-2 infection and offer potential therapeutic target for COVID-19.

Thymosin α1 modulated the immune landscape of COVID-19 patients revealed by single-cell RNA and TCR sequencing.

BACKGROUND: The Coronavirus disease-19 (COVID-19) pandemic has posed a serious threat to global health. Thymosin α1 (Tα1) was considered to be applied in COVID-19 therapy. However, the data remains limited. METHODS: Participants with or without Tα1 treatment were recruited. Single cell RNA-sequencing (scRNA-seq) and T cell receptor-sequencing (TCR-seq) of the peripheral blood mononuclear cell (PBMC) samples were done to analyze immune features. The differential expression analysis and functional enrichment analysis were performed to explore the mechanism of Tα1 therapy. RESULTS: 33 symptomatic SARS-CoV-2-infected individuals (COV) and 11 healthy controls (HC) were enrolled in this study. The proportion of CD3+ KLRD1+ NKT, TBX21+ CD8+ NKT was observed to increase in COVID-19 patients with Tα1 treatment (COVT) than those without Tα1 (COV) (p = 0.024; p = 0.010). These two clusters were also significantly higher in Health controls with Tα1 treatment (HCT) than those without Tα1 (HC) (p = 0.016; p = 0.031). Besides, a series of genes and pathways related to immune responses were significantly higher enriched in Tα1 groups TBX21+ CD8+ NKT, such as KLRB1, PRF1, natural killer cell-mediated cytotoxicity pathway, chemokine signaling pathway, JAK-STAT signaling pathway. The increased TRBV9-TRBJ1-1 pair existed in both HCs and COVID-19 patients after Tα1 treatment. 1389 common complementarity determining region 3 nucleotides (CDR 3 nt) were found in COV and HC, while 0 CDR 3 nt was common in COVT and HCT. CONCLUSIONS: Tα1 increased CD3+ KLRD1+ NKT, TBX21+ CD8+ NKT cell proportion and stimulated the diversity of TCR clones in COVT and HCT. And Tα1 could regulate the expression of genes associated with NKT activation or cytotoxicity to promote NKT cells. These data support the use of Tα1 in COVID-19 patients.

Identification of USP29 as a key regulator of nucleotide biosynthesis in neuroblastoma through integrative analysis of multi-omics data.

Cancer cells show enhanced nucleotide biosynthesis, which is essential for their unlimited proliferation, but the underlying mechanisms are not entirely clear. Ubiquitin specific peptidase 29 (USP29) was reported to sustain neuroblastoma progression by promoting glycolysis and glutamine catabolism; however, its potential role in regulating nucleotide biosynthesis in tumor cells remains unknown. In this study, we depleted endogenous USP29 in MYCN-amplified neuroblastoma SK-N-BE2 cells by sgRNAs and conducted metabolomic analysis in cells with or without USP29 depletion, we found that USP29 deficiency caused a disorder of intermediates involved in glycolysis and nucleotide biosynthesis. De novo nucleotide biosynthesis was analyzed using 13C6 glucose as a tracer under normoxia and hypoxia. The results indicated that USP29-depleted cells showed inhibition of nucleotide anabolic intermediates derived from glucose, and this inhibition was more significant under hypoxic conditions. Analysis of RNA sequencing data in SK-N-BE2 cells demonstrated that USP29 promoted the gene expression of metabolic enzymes involved in nucleotide anabolism, probably by regulating MYC and E2F downstream pathways. These findings indicated that USP29 is a key regulator of nucleotide biosynthesis in tumor cells.

Distinct miRNAs associated with various clinical presentations of SARS-CoV-2 infection.

MicroRNAs (miRNAs) have been shown to play important roles in viral infections, but their associations with SARS-CoV-2 infection remain poorly understood. Here, we detected 85 differentially expressed miRNAs (DE-miRNAs) from 2,336 known and 361 novel miRNAs that were identified in 233 plasma samples from 61 healthy controls and 116 patients with COVID-19 using the high-throughput sequencing and computational analysis. These DE-miRNAs were associated with SASR-CoV-2 infection, disease severity, and viral persistence in the patients with COVID-19, respectively. Gene ontology and KEGG pathway analyses of the DE-miRNAs revealed their connections to viral infections, immune responses, and lung diseases. Finally, we established a machine learning model using the DE-miRNAs between various groups for classification of COVID-19 cases with different clinical presentations. Our findings may help understand the contribution of miRNAs to the pathogenesis of COVID-19 and identify potential biomarkers and molecular targets for diagnosis and treatment of SARS-CoV-2 infection.

[Guilingji Capsules reduce 900 MHz collphone electromagnetic radiation-induced testicular oxidative damage and downregulate Prdx2 protein expression in the rat testis].

OBJECTIVE: To investigate the relationship of electromagnetic radiation (EMR) from 900 MHz cellphone frequency with testicular oxidative damage and its influence on the Prdx2 protein expression in the rat testis, and to explore the mechanism of Guilingji Capsules (GC) alleviating oxidative damage to the testis tissue. METHODS: Fifty healthy SD male rats were randomly divided into five groups of equal number, sham-EMR, 4-h EMR, 8-h EMR, 4-h EMR+GC and 8-h EMR+GC and exposed to 900 MHz EMR (370 µW/cm2) for 0, 4 or 8 hours daily for 15 successive days. The rats of the latter two groups were treated intragastrically with GC suspension and those of the first three groups with pure water after exposure to EMR each day. After 15 days of exposure and treatment, all the rats were sacrificed and their testis tissue collected for observation of the histomorphological and ultrastructural changes by HE staining and transmission electron microscopy, measurement of the levels of serum glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) with thiobarbiuric acid and determination of the Prdx2 protein expression by immunohistochemistry and Western blot. RESULTS: Compared with the rats in the sham-EMR group, those in the 4-h and 8-h EMR groups showed different degrees of histomorphological and ultrastructural changes in the testis tissue, significantly decreased levels of GSH (ï¼»80.62 ± 10.99ï¼½ vs ï¼»69.58 ± 4.18ï¼½ and ï¼»66.17 ± 8.45ï¼½ mg/L, P < 0.05) and SOD (ï¼»172.29 ± 10.98ï¼½ vs ï¼»158.92 ± 6.46ï¼½ and ï¼»148.91 ± 8.60ï¼½ U/ml, P < 0.05) and increased level of MDA (ï¼»7.51 ± 1.73ï¼½ vs ï¼»9.84 ± 1.03ï¼½ and ï¼»11.22 ± 2.13ï¼½ umol/ml, P < 0.05), even more significantly in the 8-h than in the 4-h EMR group (P < 0.05). In comparison with the sham-EMR group, the expression of the Prdx2 protein was markedly downregulated in the 4-h and 8-h EMR groups (0.56 ± 0.03 vs 0.49 ± 0.03, 0.21 ± 0.01, P < 0.05), but again upregulated in the 4-h and 8-h EMR+GC groups (0.55±0.03 and 0.37±0.04) (P < 0.05). CONCLUSIONS: Electromagnetic radiation from cellphones can cause ultrastructural damage to the testis tissue of male rats, while Guilingji Capsules can alleviate it, presumably by upregulating the Prdx2 protein expression in the testis tissue and reducing testicular oxidative damage.

Evaluation of HBV serological markers in treatment-naïve HBV mono-infected patients and HBV-HIV co-infected patients.

OBJECTIVES: Many studies have investigated the utility of hepatitis B virus (HBV) serological markers in HBV-infected patients. However, only a few studies have examined HBV serological markers in HBV-human immunodeficiency virus (HIV) co-infected patients. Here, we conducted a cross-sectional study to evaluate correlations of HBV serological markers in treatment-naïve HBV mono-infected patients and HBV-HIV co-infected patients. METHODS: HBsAg, HBV DNA, HBV RNA, and HBcrAg were quantified in 51 HBV mono-infected patients and 33 HBV-HIV co-infected patients recruited at Tianjin Second People's Hospital from 2016 to 2019. RESULTS: There was no significant difference in serum levels of HBV DNA (P = 0.056), HBV RNA (P = 0.387), HBcrAg (P = 0.714) and HBsAg (P = 0.165) between the patient groups. In HBV mono-infected patients, strong positive correlations were confirmed between HBV RNA and HBV DNA (r=0.620, P < 0.01), HBcrAg and HBV DNA (r=0.802, P < 0.001), and HBcrAg and HBV RNA (r=0.727, P < 0.01). In HBV-HIV co-infected patients, serum HBsAg was very strongly correlated with HBcrAg (r=0.838, P < 0.001). In HBeAg-positive HBV mono-infected patients, all HBV serological markers correlated with each other, whereas only HBV RNA correlated with HBcrAg in HBeAg-negative HBV mono-infected patients (r=0.688, P = 0.007). In HBeAg-positive HBV-HIV co-infected patients, only HBsAg correlated with HBcrAg (r=0.725, P<0.001), whereas HBcrAg and HBV RNA correlated with each other in HBeAg-negative patients (r = 0.683, P=0.010). Moreover, CD4 T-cell counts were not significantly associated with HBsAg, HBV DNA, HBV RNA, and HBcrAg levels. CONCLUSION: Compared with HBsAg and HBV DNA, which are widely used in clinical settings, our study confirmed that new HBV serological markers, such as HBV RNA and HBcrAg, have some utility in HBV mono-infected patients and HBV-HIV co-infected patients for monitoring the progression of liver disease.

HIV-1 Genetic Diversity and High Prevalence of Pretreatment Drug Resistance in Tianjin, China.

Diversity of genotypes and prevalence of pretreatment drug resistance (PDR) are challenges for the epidemic control and vaccine development of HIV-1. However, little is known about the situation in Tianjin. Blood samples were collected from newly diagnosed, antiretroviral treatment (ART)-naive HIV/AIDS patients from January 2016 to November 2019. The target fragment in the pol gene was sequenced after RNA extraction and gene amplification. The HIV-1 genotype was identified by phylogenetic analysis. Drug resistance was carried out using the Stanford University HIVdb algorithm. A total of 305 pol sequences from 279 non-PDR individuals and 35 PDR individuals were successfully amplified. The most prevalent genotype was CRF01_AE (65.6%, 200/305), followed by CRF07_BC (22.0%, 67/305) and B (3.0%, 9/305). A variety of circulating recombinant forms (CRFs) and unique recombinant forms were found. The overall incidence of PDR was 11.5% (35/305), with 9.8% (30/305) to non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs). The most frequent mutation pattern against NNRTIs was V179D/E/T (6.9%, 21/305), with M184V (1.0%, 3/305) and K65R (1.0%, 3/305) against nucleoside RT inhibitors (NRTIs). M64L (0.1%, 1/305) was the sole mutation found against protease inhibitors (PIs). Eight variants generated at least low-level resistance to NNRTIs (2.6%, 8/305), which was much higher than that to NRTIs (1.6%, 5/305) and PIs (0/305) (p < .05). Genotypic drug resistance testing before initiating ART in newly diagnosed HIV/AIDS patients may be necessary in Tianjin, China. The non-NNRTI-based regimen may be preferred as initial therapy in Tianjin.