Bidirectional Peptide Translocation through Ultrasmall Solid-State Nanopores.

It is important to obtain the configuration of polypeptides and the sequence information on amino acids for understanding various life processes and many biological applications. Nanopores, as a newly developed single-molecule detection technology, exhibit unique advantages in real-time dynamics detection. Here, we designed a special peptide chain with 10 arginine in the head and achieved successful single-molecule detection by ultrasmall solid-state nanopores (2-3 nm). Unique bidirectional translocation signals were observed and explained under the framework of charge distribution of the peptide and interaction with the nanopore wall. Two natural peptide chains, histatin-5 and angiopep-2, were also explored by nanopore experiments to confirm our conjecture. Our designed peptide chain could realize multiple detections of the same peptide chain, offering possibilities for high-resolution peptide detection and fingerprinting by solid-state nanopores in the future.

Transgenic expression of artificial microRNA targeting soybean mosaic virus P1 gene confers virus resistance in plant.

RNA silencing is an innate immune mechanism of plants against invasion by viral pathogens. Artificial microRNA (amiRNA) can be engineered to specifically induce RNA silencing against viruses in transgenic plants and has great potential for disease control. Here, we describe the development and application of amiRNA-based technology to induce resistance to soybean mosaic virus (SMV), a plant virus with a positive-sense single-stranded RNA genome. We have shown that the amiRNA targeting the SMV P1 coding region has the highest antiviral activity than those targeting other SMV genes in a transient amiRNA expression assay. We transformed the gene encoding the P1-targeting amiRNA and obtained stable transgenic Nicotiana benthamiana lines (amiR-P1-3-1-2-1 and amiR-P1-4-1-2-1). Our results have demonstrated the efficient suppression of SMV infection in the P1-targeting amiRNA transgenic plants in an expression level-dependent manner. In particular, the amiR-P1-3-1-2-1 transgenic plant showed high expression of amiR-P1 and low SMV accumulation after being challenged with SMV. Thus, a transgenic approach utilizing the amiRNA technology appears to be effective in generating resistance to SMV.

A novel SERS-lateral flow assay (LFA) tray for monitoring of miR-155-5p during pyroptosis in breast cancer cells.

In the study, a novel surface-enhanced Raman scattering (SERS)-lateral flow assay (LFA) tray for the real-time detection of pyroptosis-associated miR-155-5p in breast cancer cells was established and validated. The SERS probe modified with monoclonal antibodies and functionalized HP1@5-FAM was first synthesized. When miR-155-5p was present, HP1@5-FAM on the SERS probe specifically recognized target miRNAs and hybridized with them, resulting in HP2 on the T line only capturing some SERS probes that were not bound to miR-155-5p. The T line appeared as a light orange band or there was no color change, and the corresponding Raman detection result showed a weak or insignificant Raman signal. The SERS probe showed high selectivity, satisfactory stability, and excellent reproducibility, and the limit of detection (LOD) for miR-155-5p was 7.26 aM. Finally, the proposed SERS-LFA tray was applied to detect miR-155-5p in MBA-MD-468 cells that underwent varying degrees of pyroptosis, and the detection results of SERS were consistent with those of the conventional real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. The study demonstrated that the SERS-LFA tray was a convenient and ultrasensitive method for miR-155-5p real-time detection, which could provide more detailed information for pyroptosis and be of potential value in guiding the treatment of breast cancer.

RENAL PROTECTIVE EFFECT AND CLINICAL ANALYSIS OF VITAMIN B 6 IN PATIENTS WITH SEPSIS.

ABSTRACT: Objective: To investigate the protective effect and possible mechanisms of vitamin B 6 against renal injury in patients with sepsis. Methods: A total of 128 patients with sepsis who met the entry criteria in multiple centers were randomly divided into experimental (intravenous vitamin B 6 therapy) and control (intravenous 0.9% sodium chloride therapy) groups based on usual care. Clinical data, the inflammatory response indicators interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor (TNF-α), and endothelin-1 (ET-1), the oxidative stress response indicators superoxide dismutase, glutathione and malondialdehyde, and renal function (assessed by blood urea nitrogen, serum creatinine, and renal resistance index monitored by ultrasound) were compared between the two groups. Results: After 7 d of treatment, the IL-6, IL-8, TNF-α, and ET-1 levels in the experimental group were significantly lower than those in the control group, the oxidative stress response indicators were significantly improved in the experimental group and the blood urea nitrogen, serum creatinine, and renal resistance index values in the experimental group were significantly lower than those in the control group ( P < 0.05). There was no statistical difference between the two groups in the rate of renal replacement therapy and 28 d mortality ( P > 0.05). However, the intensive care unit length of stay and the total hospitalization expenses in the experimental group were significantly lower than those in the control group ( P < 0.05). Conclusion: The administration of vitamin B 6 in the treatment of patients with sepsis attenuates renal injury, and the mechanism may be related to pyridoxine decreasing the levels of inflammatory mediators and their regulation by redox stress.

The Impact of New Regulations on Prevention and Control of E-Cigarettes on Adolescents in Middle Schools - A City in China, 2022-2023.

What is already known about this topic?: To protect the health of young people from the harmful impacts of electronic cigarettes (e-cigarettes), China has enacted various policies and regulations since 2018. As of October 1, 2022, the Electronic Cigarette Management Measures were put into action. They prohibited the sale of flavored e-cigarettes, permitting only those of plain tobacco flavor to be sold. What is added by this report?: The illegal market for flavored e-cigarettes, often disguised as milk tea cups, cola cans, and violent bear images, continues to flourish. There is an increased need to bolster support for the prohibition of flavored e-cigarettes and enhance public awareness of associated regulations. What are the implications for public health practice?: To advance the health of China's youth, it is crucial to improve the implementation and understanding of e-cigarette policies and guidelines.

Abnormal Bone Turnover Observed in Obese Children based on Puberty Stage-Specific Bone Turnover Marker Reference.

CONTEXT: Childhood and adolescence are critical periods for lifelong bone health. The impact of obesity on these phases is controversial, which may be due to the lack of standards for age-, sex-, and puberty-specific bone turnover markers (BTMs) that could sensitively reflect bone metabolism. OBJECTIVE: To generate age-, sex, and puberty stage-specific BTM reference curves in children and adolescents and to explore the effect of obesity on bone metabolism in the Chinese population. METHODS: Our study was part of the Evaluation and Monitoring on School-based Nutrition and Growth in Shenzhen study. A total of 800 participants aged 6∼18 years with normal body mass index (BMI) were selected to establish BTM reference curves for boys and girls at different ages under different pubertal development stages. Additionally, 200 participants with obesity (BMI > 95th percentile) were matched with healthy children from the original cohort at a 1:1 ratio. All participants underwent bone mineral density assessment, and serum levels of procollagen type 1 N-propeptide (P1NP) and ß-C-telopeptide of type I collagen (CTX) were measured. RESULTS: The BTM values presented significant age, sex, and puberty stage differences. Analysis of serum BTMs based on the established reference revealed a higher percentage of low-level P1NP in boys with obesity (P = .005); no significant difference was observed in girls. However, the obese group showed a significantly higher proportion of high ß-CTX levels for girls, not boys (P = .022). CONCLUSION: We provide age-, sex-, and puberty stage-specific P1NP and ß-CTX reference curves. According to these, obesity appeared to be a negative factor for bone formation in boys and for bone resorption in girls.

LORF9 of Marek's disease virus is involved in the early cytolytic replication of B lymphocytes and can act as a target for gene deletion vaccine development.

IMPORTANCE: Marek's disease virus (MDV) is a highly infectious and oncogenic virus that can induce severe T cell lymphomas in chickens. MDV encodes more than 100 genes, most of which have unknown functions. This work indicated that the LORF9 gene is necessary for MDV early cytolytic replication in B lymphocytes. In addition, we have found that the LORF9 deletion mutant has a comparative immunological protective effect with CVI988/Rispens vaccine strain against very virulent MDV challenge. This is a significant discovery that LORF9 can be exploited as a possible target for the development of an MDV gene deletion vaccine.

CIRP attenuates acute kidney injury after hypothermic cardiovascular surgery by inhibiting PHD3/HIF-1α-mediated ROS-TGF-ß1/p38 MAPK activation and mitochondrial apoptotic pathways.

BACKGROUND: The ischemia-reperfusion (IR) environment during deep hypothermic circulatory arrest (DHCA) cardiovascular surgery is a major cause of acute kidney injury (AKI), which lacks preventive measure and treatment. It was reported that cold inducible RNA-binding protein (CIRP) can be induced under hypoxic and hypothermic stress and may have a protective effect on multiple organs. The purpose of this study was to investigate whether CIRP could exert renoprotective effect during hypothermic IR and the potential mechanisms. METHODS: Utilizing RNA-sequencing, we compared the differences in gene expression between Cirp knockout rats and wild-type rats after DHCA and screened the possible mechanisms. Then, we established the hypothermic oxygen-glucose deprivation (OGD) model using HK-2 cells transfected with siRNA to verify the downstream pathways and explore potential pharmacological approach. The effects of CIRP and enarodustat (JTZ-951) on renal IR injury (IRI) were investigated in vivo and in vitro using multiple levels of pathological and molecular biological experiments. RESULTS: We discovered that Cirp knockout significantly upregulated rat Phd3 expression, which is the key regulator of HIF-1α, thereby inhibiting HIF-1α after DHCA. In addition, deletion of Cirp in rat model promoted apoptosis and aggravated renal injury by reactive oxygen species (ROS) accumulation and significant activation of the TGF-ß1/p38 MAPK inflammatory pathway. Then, based on the HK-2 cell model of hypothermic OGD, we found that CIRP silencing significantly stimulated the expression of the TGF-ß1/p38 MAPK inflammatory pathway by activating the PHD3/HIF-1α axis, and induced more severe apoptosis through the mitochondrial cytochrome c-Apaf-1-caspase 9 and FADD-caspase 8 death receptor pathways compared with untransfected cells. However, silencing PHD3 remarkably activated the expression of HIF-1α and alleviated the apoptosis of HK-2 cells in hypothermic OGD. On this basis, by pretreating HK-2 and rats with enarodustat, a novel HIF-1α stabilizer, we found that enarodustat significantly mitigated renal cellular apoptosis under hypothermic IR and reversed the aggravated IRI induced by CIRP defect, both in vitro and in vivo. CONCLUSION: Our findings indicated that CIRP may confer renoprotection against hypothermic IRI by suppressing PHD3/HIF-1α-mediated apoptosis. PHD3 inhibitors and HIF-1α stabilizers may have clinical value in renal IRI.

Prevalence and associated factors of smoking among chinese adolescents: a school-based cross-sectional study.

BACKGROUND: Shenzhen has made great efforts to address the tobacco epidemic in the past decade. This study aims to evaluate the current status of the tobacco epidemic among adolescent in Shenzhen, China. METHODS: The multi-stage random cluster sampling method was used in the school-based cross-sectional study in 2019 and a total of 7,423 junior and high school (both senior and vocational) students were recruited. Information on cigarette use was collected by the electronic questionnaire. Logistic regression analysis was used to examine the associations between current cigarette use and associated factors. ORs with their 95% CIs were reported. RESULTS: The prevalence of current cigarette use among adolescents was 2.3%, with boys (3.4%) significantly higher than girls (1.0%). Smoking rates in junior high schools, senior high schools, and vocational senior high schools were 1.0%, 2.7%, and 4.1%, respectively. The results of multivariate logistic regression analysis indicated that gender, age, parental smoking, teachers smoking in schools, friends smoking, exposure to tobacco marketing, and misconceptions about cigarette use were associated factors for adolescent smoking behaviour. CONCLUSIONS: The prevalence of current smoking was relatively low among adolescent in Shenzhen, China. Personal characteristics, family, and school were associated with current adolescent smokers.

Infection phase-dependent dynamics of the viral and host N6-methyladenosine epitranscriptome in the lifecycle of an oncogenic virus in vivo.

Dynamic alteration of the epitranscriptome exerts regulatory effects on the lifecycle of oncogenic viruses in vitro. However, little is known about these effects in vivo because of the general lack of suitable animal infection models of these viruses. Using a model of rapid-onset Marek's disease lymphoma in chickens, we investigated changes in viral and host messenger RNA (mRNA) N6-methyladenosine (m6 A) modification during Marek's disease virus (MDV) infection in vivo. We found that the expression of major epitranscriptomic proteins varies among viral infection phases, reprogramming both the viral and the host epitranscriptomes. Specifically, the methyltransferase-like 3 (METTL3)/14 complex was suppressed during the lytic and reactivation phases of the MDV lifecycle, whereas its expression was increased during the latent phase and in MDV-induced tumors. METTL3/14 overexpression inhibits, whereas METTL3/14 knockdown enhances, MDV gene expression and replication. These findings reveal the dynamic features of the mRNA m6 A modification program during viral replication in vivo, especially in relation to key pathways involved in tumorigenesis.

Metal-Organic Cage as Single-Molecule Carrier for Solid-State Nanopore Analysis.

The ability to detect biomolecules at the single-molecule level is at the forefront of biological research, precision medicine, and early diagnosis. Recently, solid-state nanopore sensors have emerged as a promising technique for label-free and precise diagnosis assay. However, insufficient sensitivity and selectivity for small analytes are a great challenge for clinical diagnosis applications via solid-state nanopores. Here, for the first time, a metal-organic cage, PCC-57, is employed as a carrier to increase the sensitivity and selectivity of solid-state nanopores based on the intrinsic interaction of the nanocage with biomolecules. Firstly, it is found that the carrier itself is undetectable unless bound with the target analytes and used oligonucleotides as linkers to attach PCC-57 and target analytes. Secondly, two small analytes, oligonucleotide conjugated angiopep-2 and polyphosphoric acid, are successfully distinguished using the molecular carrier. Finally, selectivity of nanopore detection is achieved by attaching PCC-57 to oligonucleotide-tailed aptamers, and the human alpha-thrombin sample is successfully detected. It is believed that the highly designable metal-organic cage could serve as a rich carrier repository for a variety of biomolecules, facilitating single-molecule screening of clinically relevant biomolecules based on solid-state nanopores in the future.

Oligonucleotide Discrimination Enabled by Tannic Acid-Coordinated Film-Coated Solid-State Nanopores.

Discrimination of nucleotides serves as the basis for DNA sequencing using solid-state nanopores. However, the translocation of DNA is usually too fast to be detected, not to mention nucleotide discrimination. Here, we utilized polyphenolic TA and Fe3+, an attractive metal-organic thin film, and achieved a fast and robust surface coating for silicon nitride nanopores. The hydrophilic coating layer can greatly reduce the low-frequency noise of an original unstable nanopore, and the nanopore size can be finely tuned in situ at the nanoscale by simply adjusting the relative ratio of Fe3+ and TA monomers. Moreover, the hydrogen bonding interaction formed between the hydroxyl groups provided by TA and the phosphate groups of DNAs significantly increases the residence time of a short double-strand (100 bp) DNA. More importantly, we take advantage of the different strengths of hydrogen bonding interactions between the hydroxyl groups provided by TA and the analytes to discriminate between two oligonucleotide samples (oligodeoxycytidine and oligodeoxyadenosine) with similar sizes and lengths, of which the current signal patterns are significantly different using the coated nanopore. The results shed light on expanding the biochemical functionality of surface coatings on solid-state nanopores for future biomedical applications.

Deletion of miR-M8 and miR-M13 eliminates the bursa atrophy induced by Marek's disease virus infection.

Marek's disease (MD) is a neoplastic disease of chickens caused by an avian alphaherpesvirus, Marek's disease virus (MDV, also known as Gallid alphaherpesvirus 2 [GaHV2]). A total of 14 microRNA (miRNA) precursors and 26 mature miRNAs have been identified in MDV genome, which were grouped in three distinct clusters. In recent years, our studies revealed the role of MDV encoded cluster 3 miRNAs (or miR-M8-M10) and the specific function of its three members, miR-M6, miR-M7 and miR-M10, in regulating MDV replication and pathogenesis. In this study, we characterized the unique function of the other two members, miR-M8 and miR-M13, in cluster 3 miRNAs. Our results show that miR-M8 and miR-M13 are not important for MDV plaque formation and genome replication in vitro. Animal experiment results show that deletion of miR-M8-5p and miR-M13-5p eliminates the bursa atrophy, but not thymus atrophy, of MDV inoculated chickens. In addition, we found that the survival curve and MD incidences were not affected by disruption of miR-M8 and miR-M13. Taken together, this study uncovers the unique role of miR-M8 and miR-M13 in MDV replication and pathogenesis, which filled the gap in the research of MDV encoded miRNAs.

Marek's disease virus encoded miR-M6 and miR-M10 are dispensable for virus replication and pathogenesis in chickens.

MicroRNAs (miRNAs) are a class of approximately 22 nucleotides long non-coding RNAs, and virus-encoded miRNAs play an important role in pathogenesis. Marek's disease virus (MDV) is an oncogenic avian alphaherpesvirus that causes immunosuppression and tumors in its natural host, chicken. In the MDV genome, 14 miRNA precursors and 26 mature miRNAs were identified, thus MDV has been used as a model to study the function of viral miRNAs in vivo. Recently, a cluster of miRNAs encoded by MDV, Cluster 3 miRNAs (miR-M8-M10), has been shown to restrict early cytolytic replication and pathogenesis of MDV. In this study, we further analyzed the role of miR-M6 and miR-M10, members of cluster miR-M8-M10, in MDV replication and pathogenicity. We found that, compared to parental MDV, deletion of miR-M6-5p significantly enhanced the replication of MDV in cell culture, but not in chickens. The replication of miR-M6-5p deletion MDV was restored once the deleted sequences were re-inserted. Our results also showed that deletion of miR-M10-5p did not affect the replication of MDV in vitro and in vivo. In addition, our animal study results showed that deletion of miR-M6-5p or miR-M10-5p did not alter the pathogenesis of MDV. In conclusion, our study shows that both miR-M6 and miR-M10 are dispensable for MDV replication and pathogenesis in chickens, while also suggests a repressive role of miR-M6 in MDV replication in cell culture.

The effects of histone crotonylation and bromodomain protein 4 on prostate cancer cell lines.

BACKGROUND: The aims of this study were to detect the level of histone crotonylation in prostate cancer (PCa) tissues, analyze the correlations between its level and clinical stage and grade, and explore the effects of bromodomain-containing protein 4 (BRD4) inhibitors and sodium crotonate on the histone crotonylation in PCa cell lines and on the functions of PCa cells. METHODS: PCa tissues from 72 patients and adjacent tissues from 7 patients were collected, and immunohistochemistry was used to measure the level of histone crotonylation. Three human PCa cell lines, PC-3, LNCaP, and C42B, were selected and treated with IC50 value of I-BET762, I-BET726, and CPI-203, respectively. Next, short hairpin RNA (shRNA) transient knockdown was used to inhibit BRD4 expression. Histone crotonylation level and the expression of acetylase were determined by Western blotting. Finally, cell proliferation, migration, and invasion were measured with Cell Counting Kit-8 assay, scratch test, and Transwell test respectively. RESULTS: The level of histone crotonylation in PCa tissue was higher than that in adjacent tissues, and histone lysine crotonylation (Kcr) increased with the increasing malignancy of PCa. Treatments with I-BET762, I-BET726, and CPI-203 could inhibit the proliferation, migration, and invasion of PCa cell lines including PC-3, LNCaP, and C42B, and could also regulate the histone crotonylation and androgen receptor signaling pathways via the regulation of BRD4 expression. CONCLUSIONS: PCa is closely related to histone crotonylation. Inhibition of BRD4 expression can inhibit the proliferation, migration, and invasion of PCa cells.

Real-time Jones phase microscopy for studying transparent and birefringent specimens.

Tissue birefringence is an intrinsic marker of potential value for cancer diagnosis. Traditionally, birefringence properties have been studied by using intensity-based formalisms, through the Mueller matrix algebra. On the other hand, the Jones matrix description allows for a direct assessment of the sample's anisotropic response. However, because Jones algebra is based on complex fields, requiring measurements of both phase and amplitude, it is less commonly used. Here we propose a real-time imaging method for measuring Jones matrices by quantitative phase imaging. We combine a broadband phase imaging system with a polarization-sensitive detector to obtain Jones matrices at each point in a megapixel scale image, with near video rate capture speeds. To validate the utility of our approach, we measured standard targets, partially birefringent samples, dynamic specimens, and thinly sliced histopathological tissue.

Cell-to-cell influence on growth in large populations.

Recent studies have revealed the importance of outlier cells in complex cellular systems. Quantifying heterogeneity in such systems may lead to a better understanding of organ engineering, microtumor growth, and disease models, as well as more precise drug design. We used the ability of quantitative phase imaging to perform long-term imaging of cell growth to estimate the "influence" of cellular clusters on their neighbors. We validated our approach by analyzing epithelial and fibroblast cultures imaged over the course of several days. Interestingly, we found that there is a significant number of cells characterized by a medium correlation between their growth rate and distance (modulus of the Pearson coefficient between 0.25-.5). Furthermore, we found a small percentage of cells exhibiting strong such correlations, which we label as "influencer" cellular clusters. Our approach might find important applications in studying dynamic phenomena, such as organogenesis and metastasis.

An aptamer-Fe3+ modified nanoparticle for lactate oxidation and tumor photodynamic therapy.

To develop a cancer targeting lactate attenuator in vivo for cancer phototherapy and inhibition of HIF-1, we report an aptamer modified photo-responsive nanoparticle (labeled as Mn-D@BPFe-A) for lactate oxidation and cancer phototherapy. Mn-D@BPFe-A was constructed by the assembly of functional complex with BSA, followed by surface metal coordination and the recognition of Fe3+ with GAG containing sequence. Upon irradiation, Mn-D@BPFe-A NPs can oxidize water with the generation of OH, which convert lactate into pyruvate both in vitro and in vivo. Obviously, the Mn-D@BPFe-A exhibits a significant tumor ablation owing to the light driven oxidation of lactic acid and dysfunction of mitochondria. Importantly, it can decrease both the level of lactate in cancer tissues and the expression of HIF-1α and Glut-1 in HepG-2 cells. These results demonstrated that oxidation of lactate with dysfunction of mitochondria by nucleic acid-Fe3+ modified nanoparticle is an effective strategy for the development of non-oxygen dependent photodynamic effect agents.

Mitochondrial dysfunction is responsible for fatty acid synthase inhibition-induced apoptosis in breast cancer cells by PdpaMn.

Targeting cellular metabolism is becoming a hallmark to overcome drug resistance in breast cancer treatment. Activation of fatty acid synthase (FASN) has been shown to promote breast cancer cell growth. However, there is no concrete report underlying the mechanism associated with mitochondrial dysfunction in relation to fatty acid synthase inhibition-induced apoptosis in breast cancer cells. The current study is aimed at exploring the effect of the novel manganese (Mn) complex, labeled as PdpaMn, on lipid metabolism and mitochondrial function in breast cancer cells. Herein, we observed that PdpaMn displayed strong cytotoxicity on breast cancer cell lines and selectively targeted the tumor without affecting the normal organs or cells in vivo. We also observed that PdpaMn could bind to TE domain of FASN and decrease the activity and the level of expression of FASN, which is an indication that FASN could serve as a target of PdpaMn. In addition, we demonstrated that PdpaMn increased intrinsic apoptosis in breast cancer cells relayed by a suppressed the level of expression of FASN, followed by the release of mitochondrial cytochrome c and the activation of caspases-9. Instigated by the above observations, we hypothesized that PdpaMn-induced apoptosis events are dependent on mitochondrial dysfunction. Indeed, we found that mitochondrial membrane potential (MMP) collapse, mitochondrial oxygen consumption reduction and adenosine triphosphate (ATP) release were deeply repressed. Furthermore, our results showed that PdpaMn significantly increased the reactive oxygen species (ROS) production, and the protection conferred by the free radical scavenger N-acetyl-cysteine (NAC) indicates that PdpaMn-induced apoptosis through an oxidative stress-associated mechanism. More so, the above results have demonstrated that mitochondrial dysfunction participated in FASN inhibition-induce apoptosis in breast cancer cells by PdpaMn. Therefore, PdpaMn may be considered as a good candidate for anti-breast cancer therapeutic option.