Quantitative Polymerase Chain Reaction Detection Research Articles

Discovery and demonstration of the temperature stress response functions of Dermatophagoides farinae proteins 1 and 2

BackgroundDermatophagoides farinae proteins (DFPs) are abundantly expressed in D. farinae; however, their functions remain unknown. Our previous transcriptome sequencing analyses revealed that the basal expression of DFP1 and DFP2 in D. farinae was high and, more importantly, upregulated under temperature stress. Therefore, DFPs were speculated to exert a temperature stress response function.ResultsReal-time quantitative polymerase chain reaction detection revealed that both DFP1 and DFP2 were significantly upregulated under temperature stress. Particularly, DFP1 was upregulated under cold stress. Electrophoresis of D. farinae total proteins revealed an increased abundance of DFP1 and DFP2 (40–55 kDa bands) under temperature stress, which was corroborated by the mass spectrometry results. After silencing DFP1 and DFP2 further, temperature stress led to decreases in gene expression and survival rates. Moreover, DFP1 was identified as the upstream regulator of DFP2.ConclusionThis study highlights the temperature stress response functions of DFP1 and DFP2 at the mRNA and protein levels. These results provide important insights for applying DFP1 and DFP2 as potential target genes for the molecular prevention and control of D. farinae to prevent allergic diseases. The newly established methods provide methodological guidance for the study of genes with unknown functions in mites.

Simple and rapid identification of beef within 30 min using a new food nucleic acid release agent combined with direct-fast qPCR

Simple and rapid molecular detection technologies for authenticating animal species are urgently needed for food safety and authenticity. This study established a new direct-fast quantitative polymerase chain reaction (qPCR) detection technology for beef to achieve rapid and on-site nucleic acid detection in food. This technology can complete nucleic acid extraction in 4 min using a new type of food nucleic acid-releasing agent, followed by direct amplification of the DNA sample by fast qPCR in 25 min. The results indicated that direct-fast qPCR can specifically identify beef and can also identify 0.00001% of beef components in artificially simulated meat mixtures, with a detection precision variation coefficient of <4%. This method can be used to effectively identify beef in different food samples. As a simple, fast, and accurate molecular detection technology for beef, this method may provide a new tool for the on-site detection of beef components in food.

Distribution of extended-spectrum β-lactamase producing Escherichia coli genes in an integrated poultry-fish farming system in Bogor, Indonesia.

The excessive use of antimicrobials in livestock farming leads to the emergence and dissemination of antimicrobial-resistant organisms. This study aimed to detect extended-spectrum β-lactamase (ESBL)-producing Escherichia coli genes in integrated poultry-fish farms in Bogor, Indonesia. A total of 256 samples were collected from six poultry-fish farms. One hundred and seventy-five chicken cloaca swabs, 60 fish skin swabs, six pond water samples, and 15 farmer's hand swabs. ESBL-producing E. coli was confirmed through double-disk diffusion. The specific primers and probe genes for quantitative polymerase chain reaction detection of ESBL-producing E. coli targeted blaTEM, blaCTX-M, blaSHV, and blaOXA-48 genes. Among the 256 samples tested, 145 (56.6%) were positive for E. coli, and 67.6% (98/145) were identified as ESBL-producing E. coli. The most ESBL-producing E. coli isolates were obtained from chicken cloaca (78.3%, 72/92), followed by pond water (66.7%, 4/6), fish skin (47.6%, 20/42), and farmer's hand swabs (40%, 2/5). About 100% of the isolates carried the genes blaTEM and blaCTX-M, whereas 17.3% and 24.5% carried blaSHV and blaOXA-48, respectively. ESBL-producing E. coli genes were investigated in chicken cloaca, fish, pond water, and farmers' hands within an interconnected poultry-fish farming operation. The ESBL-producing E. coli in chickens can transfer resistant genes to aquatic environments. The transfer could harm other aquatic species and food chains, potentially threatening human health.

Screening and Mechanism Study of Three Antagonistic Drugs, Oxysophoridine, Rutin, and Phellodendrine, against Zearalenone-Induced Reproductive Toxicity in Ovine Oocytes.

Zearalenone (ZEN) is a common fungal toxin with reproductive toxicity in various grains. It poses a serious threat to ovine and other animal husbandry industries, as well as human reproductive health. Therefore, investigating the mechanism of toxicity and screening antagonistic drugs are of great importance. In this study, based on the natural compound library and previous Smart-seq2 results, antioxidant and anti-apoptotic drugs were selected for screening as potential antagonistic drugs. Three natural plant compounds (oxysophoridine, rutin, and phellodendrine) were screened for their ability to counteract the reproductive toxicity of ZEN on ovine oocytes in vitro using quantitative polymerase chain reaction (qPCR) and reactive oxygen species detection. The compounds exhibited varying pharmacological effects, notably impacting the expression of antioxidant (GPX, SOD1, and SOD2), autophagic (ATG3, ULK2, and LC3), and apoptotic (CAS3, CAS8, and CAS9) genes. Oxysophoridine promoted GPX, SOD1, ULK2, and LC3 expression, while inhibiting CAS3 and CAS8 expression. Rutin promoted SOD2 and ATG3 expression, and inhibited CAS3 and CAS9 expression. Phellodendrine promoted SOD2 and ATG3 expression, and inhibited CAS9 expression. However, all compounds promoted the expression of genes related to cell cycle, spindle checkpoint, oocyte maturation, and cumulus expansion factors. Although the three drugs had different regulatory mechanisms in enhancing antioxidant capacity, enhancing autophagy, and inhibiting cell apoptosis, they all maintained a stable intracellular environment and a normal cell cycle, promoted oocyte maturation and release of cumulus expansion factors, and, ultimately, counteracted ZEN reproductive toxicity to promote the in vitro maturation of ovine oocytes. This study identified three drugs that antagonize the reproductive toxicity of ZEN on ovine oocytes, and compared their mechanisms of action, providing data support and a theoretical basis for their subsequent application in the ovine breeding industry, reducing losses in the breeding industry, screening of ZEN reproductive toxicity antagonists and various toxin antagonists, improving the study of ZEN reproductive toxicity mechanisms, and even protection of human reproductive health.

Metabolic response and clade variation in Accumulibacter under phosphorus deprivation stress

Two enhanced biological phosphorus removal systems (R1 and R2) fed with different carbon sources (acetic acid and propionic acid) were used to investigate the metabolic response of nitrite-type denitrifying phosphorus removal functional bacteria Accumulibacter to different phosphorus concentrations and the changes of its clade community structure. The results indicated that the systems almost lost the ability to release phosphorus after 15 d phosphorus deprivation, the contents of poly-P in the sludge of R1 and R2 decreased from 0.63 to 0.02 mgP/mgVSS and from 1.23 to 0.05 mgP/mgVSS, respectively. According to the analysis of stoichiometric parameters, the functional bacteria in both systems performed glycogen accumulating organism metabolism to survive under phosphorus-poor condition. Besides, the two systems exhibited a stronger EBPR ability even after 42 d phosphorus deprivation, and the maximum anoxic phosphorus uptake rate was 1.92 times and 2.29 times of those under phosphorus-rich conditions. Real-time quantitative Polymerase Chain Reaction detection indicated that the proportion of Accumulibacter in the two systems decreased from 16.45 % to 8.78 % and from 31.00 % to 8.43 %, respectively, as phosphorus was deprived. Clade IIC maintained its dominance in both systems, while clade IID was gradually washed out from propionic acid system, suggesting that clade IIC had a more flexible metabolic response to phosphorus deprivation stress.

Systematic proteomics analysis revealed different expression of laminin interaction proteins in breast cancer: lower in luminal subtype and higher in claudin-low subtype.

Breast cancer is a major public health concern. Proteomics enables identification of proteins with aberrant properties. Here, we identified proteins with abnormal expression levels in breast cancer tissues and systematically analyzed and validated the data to locate potential diagnostic and therapeutic targets. Protein expression level in breast cancer tissues and para-carcinoma tissues were detected by Isobaric Tags for Relative and Absolute Quantification (iTRAQ) technology and further screened through Gene Expression Profiling Interactive Analysis (GEPIA) database. Cellular components, protein domain and Reactome pathway analysis were performed to screen functional targets. Abnormal expression levels of functional targets were validated by Oncomine database, quantitative real time polymerase chain reaction (qRT-PCR) and proteomics detection. Protein correlation analysis was performed to explain the abnormal expression levels of potential targets in breast cancer. Overall, 207 and 207 proteins were up- and down-regulated, respectively, in breast cancer tissues, and approximately 50% were also detected in the GEPIA database. The overlapping proteins were mainly extracellular proteins containing epidermal growth factor-like domain in leukocyte adhesion molecule (EGF-Lam) domain and enriched in laminin interaction pathway. Moreover, the downregulated laminin interaction proteins could be functional targets, which were also validated through Oncomine-Richardson and Oncomine-Curtis database. However, the lower expression level of laminin interaction proteins only fit for luminal breast cancer cells with no or low metastasis ability because the proteins achieved higher expression level in more invasive claudin-low breast cancer cells. In addition, when compared with corresponding in situ carcinoma tissues, above-mentioned proteins also showed higher expression levels in invasive carcinoma tissues. Finally, we have revealed the negative correlation between the laminin interaction proteins and the claudins. The laminin interaction protein, especially for laminins with β1 and γ1 subunits and their integrin receptors with α1 and α6 subunits, showed lower expression levels in luminal breast cancer with no or lower metastatic ability, but showed higher expression levels in claudin-low breast cancer with higher metastatic ability; and their higher expression could be related to the low claudin expression.

Regulatory roles of long non-coding RNAs in short-term heat stress in adult worker bees

Long non-coding RNAs (lncRNAs) are crucial modulators of post-transcriptional gene expression regulation, cell fate determination, and disease development. However, lncRNA functions during short-term heat stress in adult worker bees are poorly understood. Here, we performed deep sequencing and bioinformatic analyses of honeybee lncRNAs. RNA interference was performed by using siRNA targeting the most highly expressed lncRNA. The silencing effect on lncRNA and the relative expression levels of seven heat shock protein (HSP) genes, were subsequently examined. Overall, 7,842 lncRNAs and 115 differentially expressed lncRNAs (DELs) were identified in adult worker bees following heat stress exposure. Structural analysis revealed that the overall expression abundance, length of transcripts, exon number, and open reading frames of lncRNAs were lower than those of mRNAs. GO analysis revealed that the target genes were mainly involved in “metabolism,” “protein folding,” “response to stress,” and “signal transduction” pathways. KEGG analysis indicated that the “protein processing in endoplasmic reticulum” and “longevity regulating pathway-multiple species” pathways were most enriched. Quantitative real-time polymerase chain reaction (qRT-PCR) detection of the selected DELs confirmed the reliability of the sequencing data. Moreover, the siRNA experiment indicated that feeding siRNA yielded a silencing efficiency of 77.51% for lncRNA MSTRG.9645.5. Upon silencing this lncRNA, the expression levels of three HSP genes were significantly downregulated (p < 0.05), whereas those of three other HSP genes were significantly upregulated (p < 0.05). Our results provide a new perspective for understanding the regulatory mechanisms of lncRNAs in adult worker bees under short-term heat stress.

Porous Agarose Layered Magnetic Graphene Oxide Nanocomposite for Virus RNA Monitoring in Wastewater.

The detection of virus RNA in wastewater has been established as a valuable method for monitoring Coronavirus disease 2019. Carbon nanomaterials hold potential application in separating virus RNA owing to their effective adsorption and extraction capabilities. However, carbon nanomaterials have limited separability under homogeneous aqueous conditions. Due to the stabilities in their nanostructure, it is a challenge to efficiently immobilize them onto magnetic beads for separation. Here, we develop a porous agarose layered magnetic graphene oxide (GO) nanocomposite that is prepared by agglutinating ferroferric oxide (Fe3O4) beads and GO with agarose into a cohesive whole. With an average porous size of approximately 500 nm, the porous structure enables the unhindered entry of virus RNA, facilitating its interaction with the surface of GO. Upon the application of a magnetic field, the nucleic acid can be separated from the solution within a few minutes, achieving adsorption efficiency and recovery rate exceeding 90% under optimized conditions. The adsorbed nucleic acid can then be preserved against complex sample matrix for 3 days, and quantitatively released for subsequent quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection. The developed method was successfully utilized to analyze wastewater samples obtained from a wastewater treatment plant, detecting as few as 10 copies of RNA molecules per sample. The developed aMGO-RT-qPCR provides an efficient approach for monitoring viruses and will contribute to wastewater-based surveillance of community infections.

Aerobic Exercise Attenuates Pressure Overload-Induced Myocardial Remodeling and Myocardial Inflammation via Upregulating miR-574-3p in Mice.

Exercise training can promote cardiac rehabilitation, thereby reducing cardiovascular disease mortality and hospitalization rates. MicroRNAs (miRs) are closely related to heart disease, among which miR-574-3p plays an important role in myocardial remodeling, but its role in exercise-mediated cardioprotection is still unclear. A mouse myocardial hypertrophy model was established by transverse aortic coarctation, and a 4-week swimming exercise training was performed 1 week after the operation. After swimming training, echocardiography was used to evaluate cardiac function in mice, and histopathologic staining was used to detect cardiac hypertrophy, myocardial fibrosis, and cardiac inflammation. Quantitative real-time polymerase chain reaction was used to detect the expression levels of miR-574-3p and cardiac hypertrophy markers. Western blotting detected the IL-6 (interleukin-6)/JAK/STAT inflammatory signaling pathway. Echocardiography and histochemical staining found that aerobic exercise significantly improved pressure overload-induced myocardial hypertrophy (n=6), myocardial interstitial fibrosis (n=6), and cardiac inflammation (n=6). Quantitative real-time polymerase chain reaction detection showed that aerobic exercise upregulated the expression level of miR-574-3p (n=6). After specific knockdown of miR-574-3p in mouse hearts with adeno-associated virus 9 using cardiac troponin T promoter, we found that the protective effect of exercise training on the heart was significantly reversed. Echocardiography and histopathologic staining showed that inhibiting the expression of miR-574-3p could partially block the effects of aerobic exercise on cardiac function (n=6), cardiomyocyte cross-sectional area (n=6), and myocardial fibrosis (n=6). Western blotting and immunohistochemical staining showed that the inhibitory effects of aerobic exercise on the IL-6/JAK/STAT pathway and cardiac inflammation were partially abolished after miR-574-3p knockdown. Furthermore, we also found that miR-574-3p exerts cardioprotective effects in cardiomyocytes by targeting IL-6 (n=3). Aerobic exercise protects cardiac hypertrophy and inflammation induced by pressure overload by upregulating miR-574-3p and inhibiting the IL-6/JAK/STAT pathway.

Prognostic significance of LINC01132 in lung cancer and its regulatory role in tumor progression

BackgroundThe application of long non-coding RNAs (lncRNAs) in cancer has been the focus of research in recent years. This study aimed to discuss the expression and functional mechanism of lncRNA LINC01132 (LINC01132) in lung cancer and explore its prognostic significance in tumors.MethodsThe expression of LINC01132 in lung cancer patients was verified using GSE98929 screening and real-time quantitative polymerase chain reaction (RT-qPCR) detection. The prognostic potential of LINC01132 was evaluated by performing the chi-square analysis of clinical indicators, Kaplan–Meier analysis, and Cox proportional hazard model. Cell Counting Kit-8 (CCK-8), flow cytometry, and Transwell assay were used to characterize the biological functions of the lung cancer cells. The targeting relationship between LINC01132 and microRNA-125a-3p (miR-125a-3p), miR-125a-3p and SMAD2 was predicted by bioinformatics and verified by luciferase activity assay.ResultsLINC01132 was upregulated in lung cancer tissues and cells, which was an independent risk factor for survival and prognostic outcomes of lung cancer patients. Silencing LINC01132 suppressed the proliferation and migration of lung cancer cells and accelerated cell death. The target of LINC01132 was miR-125a-3p, and miR-125a-3p inhibitor could eliminate the inhibitory effect of LINC01132 knockdown on the cells. Additionally, SMAD2 is a downstream target of miR-125a-3p, and knockdown of SMAD2 reversed the effects of miR-125a-3p inhibitor on cell migration and invasion.ConclusionLINC01132 may regulate the progression of lung cancer by targeting the miR-125a-3p /SMAD2 axis and serve as a prognostic biomarker for lung cancer.

SELEX-discovered aptamer that inhibits cellular interleukin-17/interleukin-17 receptor interaction and antagonizes interleukin-17 signaling.

This research is based on a Systematic Evolution of Ligands by EXponential enrichment, also referred to as in vitro selection against the extracellular domain of human interleukin-17 receptor A (IL-17RA). Pull-down assay via quantitative polymerase chain reaction and chemiluminescence detection showed that the cloned RNA with the enriched sequence bound to human IL-17RA and inhibited the interaction between IL-17RA and human interleukin-17A (IL-17A). We also revealed that the newly discovered IL-17RA-binding RNA aptamer bound to cellular IL-17RA, inhibited the cellular IL-17RA/IL-17A interaction, and antagonized cellular IL-17A signaling.

Anemonin ameliorates human diploid fibroblasts 2BS and IMR90 cell senescence by PARP1-NAD+-SIRT1 signaling pathway

ObjectiveAging becomes the most predominant risk factor for all age‐associated pathological conditions with the increase of life expectancy and the aggravation of social aging. Slowing down the speed of aging is considered an effective way to improve health, but so far, effective anti-aging methods are relatively lacking. MethodsAnemonin (ANE) was screened from eight existing small-molecule compounds by cell viability assay. The function of ANE was determined by the analysis of cell proliferation, β -galactosidase (SA-β -Gal) activity, cell cycle, SASP secretion, NAD+/NADH ratio, and other aging-related indicators. The targets of ANE were predicted by Drug Target Prediction System (DTPS) and Swiss Targe Prediction System. The effect of ANE on PARP-1-NAD+-SIRT1 signaling pathway was assessed by quantitative reverse-transcription polymerase chain reaction (RT-PCR), Western blot, PARP1, NAD+ and SIRT1 activity detection. ResultsANE can delay cell senescence; PARP1 is one of the targets of ANE and plays a crucial role in ANE anti-aging; ANE release more NAD+ by inhibiting PARP1 activity, thereby conversely promoting the function of SIRT1 and delay cell senescence. ConclusionsOur study indicates that ANE can delay cellular senescence through the PARP1-NAD+-SIRT1 signaling pathway, which may be considered as an effective anti-aging strategy.

Traditional Chinese Medicine Shi-Bi-Man regulates lactic acid metabolism and drives hair follicle stem cell activation to promote hair regeneration

BackgroundAs a supplement for promoting hair health, Shi-Bi-Man (SBM) is a prescription comprising various traditional Chinese medicines. Though SBM has been reported to promote hair regeneration, its molecular mechanism remains unclear. Cynomolgus monkeys (Macaca fascicularis) are non-human primates with a gene expression profile similar to that of humans. The purpose of this research is to evaluate the effect of SBM on promoting hair regeneration in cynomolgus monkeys and to reveal the underlying mechanism.MethodsThe effect of SBM on hair regeneration was observed by skin administration on 6 cynomolgus monkeys with artificial back shaving. The molecular mechanism of SBM was studied using single-cell RNA sequencing (scRNA-seq) in combination with quantitative polymerase chain reaction (qPCR) detection for gene transcription level, and immunofluorescence staining verification for protein level.ResultsSBM significantly induced hair regeneration in cynomolgus monkeys, increased hair follicle number and facilitated hair follicle development. ScRNA-seq revealed an increase in the number of hair follicle stem cells (HFSCs) with a higher activation state, as evidenced by the higher expression of activation marker LDHA related to metabolism and the proliferation marker MKI67. Immunofluorescence analysis at the protein level and qPCR at the mRNA level confirmed the sequencing data. Cellchat analysis revealed an enrichment of ligand-receptor pairs involved in intercellular communication in Laminin-related pathways.ConclusionSBM significantly promotes hair regeneration in cynomolgus monkeys. Mechanically, SBM can up-regulate LDHA-mediated lactic acid metabolism and drive HFSC activation, which in turn promotes the proliferation and differentiation of HFSCs.

Real-time quantitative reverse transcription polymerase chain reaction detection of SARS-CoV-2 Delta variant in Canadian wastewater.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern are associated with increased infectivity, severity, and mortality of coronavirus disease 2019 (COVID-19) and have been increasingly detected in clinical and wastewater surveillance in Canada and internationally. In this study, we present a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay for detection of the N gene D377Y mutation associated with the SARS-CoV-2 Delta variant in wastewater. Wastewater samples (n=980) were collected from six cities and 17 rural communities across Canada from July to November 2021 and screened for the D377Y mutation. The Delta variant was detected in all major Canadian cities and northern remote regions, and half of the southern rural communities. The sensitivity and specificity of this assay were sufficient for detection and quantitation of the Delta variant in wastewater to aid in rapid population-level screening and surveillance. This study demonstrates a novel cost-effective RT-qPCR assay for tracking the spread of the SARS-CoV-2 Delta variant. This rapid assay can be easily integrated into current wastewater surveillance programs to aid in population-level variant tracking.

Efficacy evaluation and mechanism of Bacillus subtilis EBS03 against cotton Verticillium wilt

BackgroundIn our previous study, a strain EBS03 with good biocontrol potential was screened out of 48 strains of cotton endophyte Bacillus subtilis by evaluating the controlling effect against cotton Verticillium wilt. However, its mechanism for controlling Verticillium wilt remains unclear. The objective of this study was to further clarify its controlling effect and mechanism against cotton Verticillium wilt.ResultsThe results of confrontation culture test and double buckle culture test showed that the inhibitory effects of EBS03 volatile and nonvolatile metabolite on mycelium growth of Verticillium dahliae were 70.03% and 59.00%, respectively; the inhibitory effects of sporulation and microsclerotia germination were 47.16% and 70.06%, respectively. In the greenhouse test, the EBS03 fermentation broth root irrigation had the highest controlling effect at 87.11% on cotton Verticillium wilt, and significantly promoted the growth of cotton seedlings. In the field experiment, the controlling effect of EBS03 fermentation broth to cotton Verticillium wilt was 42.54% at 60 days after cotton sowing, and the boll number per plant and boll weight in EBS03 fermentation broth seed soaking, root irrigation, and spraying treatments significantly increased by 19.48% and 7.42%, 30.90% and 2.62%, 15.99% and 9.20%, respectively. Furthermore, EBS03 improved the resistance of cotton leaves against the infection of V. dahliae, and induced the outbreak of reactive oxygen species and accumulation of callose. In addition, the results of real time fluorescent quantitative polymerase chain reaction (RT-qPCR) detection showed that EBS03 significantly induced upregulation expression level of defense-related genes PAL, POD, PPO, and PR10 in cotton leaves, enhanced cotton plant resistance to V. dahliae, and inhibited colonization level of this fungal pathogen in cotton.ConclusionBacillus subtilis EBS03 has a good biological defense capability, which can inhibit the growth and colonization level of V. dahliae, and activate the resistance of cotton to Verticillium wilt, thus increase cotton yield.

Salivary microbiome in children with Down syndrome: a case-control study

BackgroundDown syndrome (DS), a most frequently occurring genetic disorder, is associated with oral morphological abnormalities and higher incidence rates of oral diseases. Recent studies have analyzed the oral microbiome to elucidate their relationships with oral diseases and general health; however, reports on the oral microbiome in individuals with DS are scarce. This study aimed to characterize the oral microbiome in children with DS.MethodsA total of 54 children aged 1–13 years were enrolled in this case-control study. Of these children, 27 had DS (Case: DS group) and 27 were age-matched healthy children (Control: ND group). Saliva in the oral cavity was collected with a swab, cultured, and tested for cariogenic and periodontopathic bacteria by quantitative polymerase chain reaction (qPCR) detection, and the salivary microbiome was analyzed using next-generation sequencing. The student’s t-test, Fisher’s exact test, Mann–Whitney U test, and permutational multivariate analysis of variance were used for statistical analysis.ResultsResults of culture and qPCR detection tests for cariogenic and periodontopathic bacteria showed no significant differences in the detected bacteria between the DS and ND groups, with the exception of a significantly higher detection rate of Candida albicans in children with DS with mixed dentition. A comparison of the salivary microbiomes by 16S sequencing showed no significant difference in α diversity; however, it showed a significant difference in β diversity. Children with DS had a higher relative abundance of Corynebacterium and Cardiobacterium, and lower relative abundance of TM7.ConclusionsThis study provided basic data on the salivary microbiome of children with DS and showed the microbiological markers peculiar to children with DS. However, further research to identify the relationship with oral diseases is warranted.

Study on the Mitochondrial Autophagy Pathway of Apoptosis Induced by Deferoxamine in the Leukemia Cell Line HL-60

Background: Acute leukemia is one of the most common malignant tumors in children, and it is mainly treated with a combination of chemotherapy and hematopoietic stem cell transplantation. At present, there are many problems with these treatments, such as significant adverse reactions, quick acquisition of drug resistance, and early recurrence. There are no reports on the treatment of leukemia by the mitophagy pathway. Therefore, exploring new ways to treat leukemia has become an urgent task for the effective treatment of leukemia. Objectives: This study aimed to explore the role of the mitochondrial autophagy-mediated apoptosis pathway in deferoxamine (DFO)-induced apoptosis of HL-60 cells and provide an additional experimental basis for the clinical treatment of leukemia with DFO. Methods: Through cell viability detection, Hoechst 33342 staining, and annexin V-APC/7-AAD double staining, we proved that DFO could induce apoptosis in HL-60 cells. Through the determination of intracellular reactive oxygen species (ROS) levels, western blot detection of the mitochondria-related proteins, heat shock protein 60 (HSP60), nip-like protein X (NIX), translocase of inner mitochondrial membrane 23 (TIMM23), translocase of the outer mitochondrial membrane member 20 (TOMM20), quantitative real-time polymerase chain reaction (PCR) detection of the RNA levels of NIX, microtubule-associated protein 1 light chain 3 (LC3), and TIMM23, we demonstrated that DFO induced mitochondrial autophagy in HL-60 cells. Results: DFO induced apoptosis and mitochondrial autophagy in HL-60 cells. Conclusions: This study demonstrated that DFO further induced apoptosis by inducing autophagy in leukemic cells, which provides another experimental basis for the clinical treatment of leukemia with DFO.

Cascade Filtration With PCR Detection and Field-Flow-Fractionation Online With ICP-MS for the Characterization of DNA Interaction With Suspended Particulate Matter.

The variety of applied antibiotics in animal and human medicine results in the release, development, and spread of relevant numbers of antibiotic resistance genes (ARGs) in the environment. The majority of ARGs are present in intracellular forms (in bacteria). Neglected aspects are extracellular variants of ARGs (eARGs) and their fragments, which have been detected in surface-water samples and sediments. The stability of eARGs is expected to be low; however, binding to particulate matter is likely to improve their stability and also affect their transport and dissemination behavior. Few studies have investigated DNA particle interactions, mostly via indirect characterization of adduct formation in model systems but not in real environmental matrices. Therefore, our study aims at a novel approach for direct characterization of desoxyribonucleic acid (DNA) particle interactions using both cascade filtration and field-flow fractionation. Cascade filtration with quantitative polymerase chain reaction (qPCR) detection indicated retention of ARGs on filters with much larger pore sizes supporting the hypothesis of ARG-particle interactions. However, artifacts from membrane clogging or DNA–membrane interaction cannot be excluded. Consequently, asymmetric flow field-flow fractionation was investigated as an alternative separation technique with the advantage of particle separation in a thin channel, reducing the risk of artifacts. The key method parameters, membrane composition, molecular weight cut off, and carrier composition, were systematically investigated using a calf-thymus DNA-spiked surface-water sample as a model. The results clearly showed a shift in the elution time of clay particles suggesting the presence of DNA–clay adducts. Multi-element detection by inductively coupled plasma mass spectrometry (ICP-MS) enabled monitoring of clay via the Al, Fe, and Si signals and DNA via the P signal. Matching peak profiles for the new fraction in the fractograms of the ARG and DNA-spiked water sample support adduct formation. Further evidence was provided by a novel post-channel filtration approach for the separation of free DNA from DNA–clay adducts.

Flavonoid and chromone-rich extract from Euscaphis Konishii Hayata leaf attenuated alcoholic liver injury in mice

Ethnopharmacological relevanceEuscaphis konishii Hayata is a traditional medicinal plant in China, and its leaves are usually used to make dishes for hepatic or gastrointestinal issues by Chinese She nationality. Pharmacological analysis showed that E. konishii leaves contain high levels of flavonoids and chromones with favorable anti-hepatoma effect. Aim of the studyThe extract from E. konishii leaves was detected to evaluate its chemical composition, and the alcoholic liver injury mice model was adopted to elucidate its hepatoprotective effects. Materials and methodsThe total leaf extract from E. konishii was separated by polyamide column to get the flavonoid and chromone-rich extract (FCE). Single compounds from FCE was purified by gel and Sephadex LH-20 chromatography and analyzed by nuclear magnetic resonance (NMR). The chemical component of FCE was confirmed and quantified by HPLC-MS. The OH·, O2-, DPPH and ABTS + free radical assays were adopted to estimate the antioxidant activity of FCE in vitro. The alcohol-fed model mice were established to assess the hepatoprotective capacity of FCE in vivo, through biochemical determination, histopathological analysis, mitochondrial function measurement, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) detection and Western blot determination. Results8 flavonoids and 2 chromones were recognized in the FCEextract by both NMR and HPLC-MS. FCE represented strong free radicals scavenging activity in vitro. With oral administration, FCE (50, 100 and 200 mg/kg) dose-dependently decreased the serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in alcohol-fed mice. FCE gradually reduced the malondialdehyde (MDA) content, increased the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the alcohol-treated liver tissues. FCE also alleviated the hepatic inflammation, inhibited the hepatocyte apoptosis and lessened the alcohol-induced histological alteration and lipid accumulation in the liver tissues. FCE administration inhibited the overexpression of endoplasmic reticulum (ER) chaperones signaling and unfolded protein response (UPR) pathways to defense the ER-induced apoptosis. Pretreatment with FCE also restored the mitochondrial membrane potentials andadenosine triphosphate (ATP) levels, which in turn suppressed the Cytochrome C release and mitochondria-induced apoptosis. ConclusionsFCE conferred great protection against alcoholic liver injury, which might be associated with its viability through suppressing reactive oxygen species (ROS) stress and hepatocyte apoptosis.

Analysis of relationship between distribution of pathogenic microorganisms in olfactory cleft and olfactory disorders among patients with upper respiratory inflammation during the prevention and control of COVID-19

Objective: To explore the relationship between pathogens in the olfactory cleft area and olfactory disorders in patients with upper respiratory inflammation (URI) during the prevention and control of 2019 novel coronavirus disease (COVID-19). Methods: A total of 234 URI patients including acute upper respiratory infection, chronic rhinosinusitis (CRS), allergic rhinitis (AR) were continuously selected from September 2020 to March 2021 in Beijing Anzhen Hospital and 98 healthy adults were enrolled as controls. The secretions from the olfactory cleft of all subjects were collected with nasal swabs under nasal endoscopy. Multiple real-time fluorescent quantitative polymerase chain reaction detection method was used to detect nucleic acids of 33 types of respiratory pathogenic microorganism. Sniffin' Sticks olfactory test was performed on all patients with URI. URI patients with olfactory dysfunction were followed up for 9 (8, 10) months (M (Q1, Q3)). SPSS 20.0 software was used for statistical analysis. Results: Among the 98 controls, 9 (9.18%) were positive for pathogenic microorganisms, including 1 (1.02%) rhinovirus, 1 (1.02%) parainfluenza virus type 3, 3 (3.06%) enterovirus, 1 (1.02%) staphylococcus aureus and 3 (3.06%) Moraxella catarrhalis. Among the 234 URI patients, 111 (47.44%) had olfactory disorders and 123 (52.56%) had normal sense of smell. In the olfactory disorder group (111 cases), 38 cases (34.23%) were positive for pathogenic microorganisms, and 4 cases (3.60%) were mixed infection, including 11 cases of rhinovirus (9.91%), 5 cases of coronavirus 229E (4.50%), 2 cases of coronavirus OC43/NL63 (1.80%), 3 cases of parainfluenza virus type 1 (2.70%), 2 cases of enterovirus (1.80%), 1 case of influenza B virus type BV (0.90%), 11 cases of Staphylococcus aureus (9.91%), 7 cases of Moraxella catarrhalis (6.31%), and 1 case of Klebsiella pneumoniae (0.90%). In the normal smell group (123 cases), 18 cases (14.63%) were positive for pathogenic microorganisms, and 1 case (0.81%) was mixed infection, including 3 cases of rhinovirus (2.44%), 4 cases of coronavirus 229E (3.25%), 1 case of Influenza virus type 3 (0.81%), 3 cases of enterovirus (2.44%), 3 cases of Staphylococcus aureus (2.44%), 4 cases of Moraxella catarrhalis (3.25%), and 1 case of Klebsiella pneumoniae (0.81%). Univariate analysis between the two groups found that there were significant differences in the detection rate of pathogenic microorganisms, rhinovirus and Staphylococcus aureus between the groups (all P<0.05). The detection rate of parainfluenza virus type 1, Staphylococcus aureus, and rhinovirus were different between the patients with olfactory disorder and normal olfactory function in the three subgroups of acute upper respiratory tract infection, CRS and AR, respectively (χ2 value was 3.88, 4.53 and 4.73, respectively, all P<0.05). During the follow-up period, among the 111 patients with olfactory disorder, 71 (63.96%) patients' olfactory function returned to normal, 32 (28.83%) patients' olfactory function improved but not completely returned to normal, 8 (7.21%) patients' olfactory function did not improve. Conclusions: During the prevention and control of COVID-19, rhinovirus or Staphylococcus aureus infection or colonization of URI patients is closely related to olfactory disorders. Parainfluenza virus type 1 infection can cause relatively persistent olfactory disorders in patients with acute upper respiratory tract infection. Staphylococcus aureus and rhinovirus colonization are related to the occurrence of olfactory dysfunction in CRS and AR patients respectively.