Comprehensive maturity of nuclear pore complexes regulates zygotic genome activation.

Nuclear pore complexes (NPCs) are channels for nucleocytoplasmic transport of proteins and RNAs. However, it remains unclear whether composition, structure, and permeability of NPCs dynamically change during the cleavage period of vertebrate embryos and affect embryonic development. Here, we report that the comprehensive NPC maturity (CNM) controls the onset of zygotic genome activation (ZGA) during zebrafish early embryogenesis. We show that more nucleoporin proteins are recruited to and assembled into NPCs with development, resulting in progressive increase of NPCs in size and complexity. Maternal transcription factors (TFs) transport into nuclei more efficiently with increasing CNM. Deficiency or dysfunction of Nup133 or Ahctf1/Elys impairs NPC assembly, maternal TFs nuclear transport, and ZGA onset, while nup133 overexpression promotes these processes. Therefore, CNM may act as a molecular timer for ZGA by controlling nuclear transport of maternal TFs that reach nuclear concentration thresholds at a given time to initiate ZGA.

Nicotinamide Mononucleotide Administration Restores Redox Homeostasis via the Sirt3-Nrf2 Axis and Protects Aged Mice from Oxidative Stress-Induced Liver Injury.

Altered adaptive homeostasis contributes to aging and lifespan regulation. In the present study, to characterize the mechanism of aging in mouse liver, we performed quantitative proteomics and found that the most upregulated proteins were related to the oxidation-reduction process. Further analysis revealed that malondialdehyde (MDA) and protein carbonyl (PCO) levels were increased, while nuclear Nrf2 and downstream genes were significantly increased, indicating that oxidative stress induced Nrf2 activation in aged mouse liver. Importantly, nicotinamide mononucleotide (NMN) administration decreased the oxidative stress and the nuclear Nrf2 and Nrf2 downstream gene levels. Indeed, aged mice treated with NMN improved stress resistance against acetaminophen (APAP)-induced liver injury, indicating that NMN restored Nrf2-mediated adaptive homeostasis. Further studies found that NMN increased Sirt3 activities to deacetylate age-associated acetylation at K68 and K122 in Sod2, while its effects on nuclear Nrf2 levels were diminished in Sirt3-deficient mice, suggesting that NMN-enhanced adaptive homeostasis was Sirt3-dependent. Taken together, we demonstrated that Nrf2-regulated adaptive homeostasis was decreased in aged mouse liver and NMN supplementation restored liver redox homeostasis via the Sirt3-Nrf2 axis and protected aged liver from oxidative stress-induced injury.

Decreased NAD Activates STAT3 and Integrin Pathways to Drive Epithelial-Mesenchymal Transition.

Nicotinamide adenine dinucleotide (NAD) plays an essential role in all aspects of human life. NAD levels decrease as humans age, and supplementation with NAD precursors plays a protective role against aging and associated disease. Less is known about the effects of decreased NAD on cellular processes, which is the basis for understanding the relationship between cellular NAD levels and aging-associated disease. In the present study, cellular NAD levels were decreased by overexpression of CD38, a NAD hydrolase, or by treating cells with FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT). Quantitative proteomics revealed that declining NAD levels downregulated proteins associated with primary metabolism and suppressed cell growth in culture and nude mice. Decreased glutathione synthesis caused a 4-fold increase in cellular reactive oxygen species levels, and more importantly upregulated proteins related to movement and adhesion. In turn, this significantly changed cell morphology and caused cells to undergo epithelial to mesenchymal transition (EMT). Secretomic analysis also showed that decreased NAD triggered interleukin-6 and transforming growth factor beta (TGFß) secretion, which activated integrin-ß-catenin, TGFß-MAPK, and inflammation signaling pathways to sustain the signaling required for EMT. We further revealed that decreased NAD inactivated sirtuin 1, resulting in increased signal transducer and activator of transcription 3 (STAT3) acetylation and phosphorylation, and STAT3 activation. Repletion of nicotinamide or nicotinic acid inactivated STAT3 and reversed EMT, as did STAT3 inhibition. Taken together, these results indicate that decreased NAD activates multiple signaling pathways to promote EMT and suggests that age-dependent decreases in NAD may contribute to tumor progression. Consequently, repletion of NAD precursors has potential benefits for inhibiting cancer progression.

Flavonoid 4,4'-dimethoxychalcone selectively eliminates senescent cells via activating ferritinophagy.

Flavonoids are bioactive natural polyphenolic compounds with health benefits, including anti-tumor, anti-inflammatory and anti-aging effects. Our previous studies revealed that a flavonoid 4,4'-dimethoxychalcone (DMC) induced ferroptosis via inhibiting ferrochelatase (FECH). However, the effect of DMC on cellular senescence is unknown. In the present study, we found that DMC treatment selectively eliminated senescent cells, and DMC alone or a combination of DMC and quercetin or dasatinib showed high efficiency in the clearance of senescent cells. We identified FECH was highly expressed in senescent cells compared to non-senescent cells. Mechanistically, we found that DMC inhibited FECH and induced ferritinophagy, which led to an increase of labile iron pool, triggering ferroptosis of senescent cells. Importantly, we found that DMC treatment prevented hair loss, improved motor coordination, and reduced the expression of several senescence-associated secretory phenotype factors (IL-6, IL-1ß, CXCL-10, and MMP12) in the liver of old mice. Collectively, we revealed that, through the induction of ferroptosis, DMC holds the promise as a new senolytics to prevent age-related pathologies.

Development of a new paradigm model for deciphering action mechanism of Danhong injection using a combination of isothermal shift assay and database interrogation.

BACKGROUND: Identification of active components of traditional Chinese Medicine (TCM) and their respective targets is important for understanding the mechanisms underlying TCM efficacy. However, there are still no effective technical methods to achieve this. METHODS: Herein, we have established a method for rapidly identifying targets of a specific TCM and interrogating the targets with their corresponding active components based on Isothermal Shift Assay (iTSA) and database interrogation. RESULTS: We optimized iTSA workflow and identified 110 targets for Danhong injection (DHI) which is used as an effective remedy for cardiovascular and cerebrovascular diseases. Moreover, we identified the targets of the nine major ingredients found in DHI. Database interrogation found that the potential targets for DHI, in which we verified that ADK as the target for salvianolic acid A and ALDH1B1 as the target for protocatechualdehyde in DHI, respectively. CONCLUSION: Overall, we established a novel paradigm model for the identification of targets and their respective ingredients in DHI, which facilitates the discovery of drug candidates and targets for improving disease management and contributes to revealing the underlying mechanisms of TCM and fostering TCM development and modernization.

Dual-pharmacophore artezomibs hijack the Plasmodium ubiquitin-proteasome system to kill malaria parasites while overcoming drug resistance.

Artemisinins (ART) are critical anti-malarials and despite their use in combination therapy, ART-resistant Plasmodium falciparum is spreading globally. To counter ART resistance, we designed artezomibs (ATZs), molecules that link an ART with a proteasome inhibitor (PI) via a non-labile amide bond and hijack parasite's own ubiquitin-proteasome system to create novel anti-malarials in situ. Upon activation of the ART moiety, ATZs covalently attach to and damage multiple parasite proteins, marking them for proteasomal degradation. When damaged proteins enter the proteasome, their attached PIs inhibit protease function, potentiating the parasiticidal action of ART and overcoming ART resistance. Binding of the PI moiety to the proteasome active site is enhanced by distal interactions of the extended attached peptides, providing a mechanism to overcome PI resistance. ATZs have an extra mode of action beyond that of each component, thereby overcoming resistance to both components, while avoiding transient monotherapy seen when individual agents have disparate pharmacokinetic profiles.

Complex roles of nicotinamide N-methyltransferase in cancer progression.

Nicotinamide N-methyltransferase (NNMT) is an intracellular methyltransferase, catalyzing the N-methylation of nicotinamide (NAM) to form 1-methylnicotinamide (1-MNAM), in which S-adenosyl-L-methionine (SAM) is the methyl donor. High expression of NNMT can alter cellular NAM and SAM levels, which in turn, affects nicotinamide adenine dinucleotide (NAD+)-dependent redox reactions and signaling pathways, and remodels cellular epigenetic states. Studies have revealed that NNMT plays critical roles in the occurrence and development of various cancers, and analysis of NNMT expression levels in different cancers from The Cancer Genome Atlas (TCGA) dataset indicated that NNMT might be a potential biomarker and therapeutic target for tumor diagnosis and treatment. This review provides a comprehensive understanding of recent advances on NNMT functions in different tumors and deciphers the complex roles of NNMT in cancer progression.

Flavonoid 4,4'-dimethoxychalcone induced ferroptosis in cancer cells by synergistically activating Keap1/Nrf2/HMOX1 pathway and inhibiting FECH.

Flavonoids are widely distributed in plants as secondary metabolites and have various biological benefits such as anti-tumor, anti-oxidant, anti-inflammatory and anti-aging. We previously reported that 4,4'-dimethoxychalcone (DMC) suppressed cancer cell proliferation by aggravating oxidative stress and inducing G2/M cell cycle arrest. In the present study, we explored the underlying mechanisms by which DMC inhibited cancer cell growth. Given that ferrochelatase (FECH) is a potential target of DMC identified by thermal proteome profiling (TPP) method, herein, we confirmed that DMC inhibited the enzymatic activity of FECH. Furthermore, we proved that DMC induced Keap1 degradation via ubiquitin-proteasome system, which led to the nuclear translocation of Nrf2 and upregulated Nrf2 targeted gene HMOX1. FECH inhibition and HMOX1 upregulation resulted in iron overload and triggered ferroptosis in cancer cells. Collectively, we revealed that DMC induced ferroptosis by synergistically activating Keap1/Nrf2/HMOX1 pathway and inhibiting FECH. Our findings indicate that FECH contributes to the non-canonical ferroptosis induction, shed light on the mechanisms of DMC inhibiting cancer cell growth, and set an example for studying biological functions of flavonoids.

Nicotinamide reprograms adipose cellular metabolism and increases mitochondrial biogenesis to ameliorate obesity.

Obesity poses a global health challenge and is a major risk factor for diabetes mellitus, cardiovascular diseases, hypertension, stroke and certain kinds of cancers. Although the effects of nicotinamide (NAM) on liver metabolism and diseases were well documented, its effects on adipose tissue are yet to be characterized. Herein, we found that NAM supplementation significantly reduced fat mass and improved glucose tolerance in obese mice. Proteomic analysis revealed that NAM supplementation upregulates mitochondrial proteins while quantitative polymerase chain reaction showed that PPARα and PGC1α were both upregulated in adipose tissues, proposing that NAM increased mitochondrial biogenesis in adipose tissue. Indeed, NAM treatment increased proteins related to mitochondrial functions including oxidative phosphorylation, fatty acid oxidation, and TCA cycle. Furthermore, isotope-tracing assisted metabolic profiling revealed that NAM activated NAMPT and increased cellular NAD+ level by 30%. Unexpectedly, we found that NAM also increased glucose derived amino acids to enhance glutathione synthesis for maintaining cellular redox homeostasis. Taken together, our results demonstrated that NAM reprogramed cellular metabolism, enhanced adipose mitochondrial functions to ameliorate symptoms associated with obesity.

Correlation between preoperative psychological personality traits of glioma patients and psychological distress in their primary caregivers.

OBJECTIVE: To explore the relationship between preoperative psychological personality traits of glioma patients and preoperative psychological distress of their primary caregivers. METHODS: A retrospective analysis was conducted on 85 glioma patients (tumor group) who were admitted to our hospital from February 2016 to December 2018 and 85 primary caregivers. Forty-one healthy volunteers were used as controls. RESULTS: The scores of 9 scales of the Minnesota Multiphasic Personality Inventory (MMPI) increased significantly in the tumor group compared to the healthy controls (P < 0.05), including L (lie): [(56.0 ± 11.5 vs. 50.0 ± 10.0)], F (fake): [(53.0 ± 10.6 vs. 41.7 ± 8.8)], Hs (hypochondriasis): [(55.6 ± 11.2 vs. 44.2 ± 8.3)], D (depressive personality): [(49.2 ± 9.6 vs. 42.2 ± 10.3)], Mf (masculinity-femininity): [(49.9 ± 9.4 vs. 42.8 ± 14.0)], Pt (psychasthenia): [(51.5 ± 10.0 vs. 40.9 ± 11.5)], Sc (schizophrenia): [(51.2 ± 10.6 vs. 40.2 ± 10.6)], Ma (hypomania): [(51.8 ± 9.1 vs. 47.7 ± 9.4)] and Si (social introversion): [(46.6 ± 8.7 vs. 36.7 ± 13.0)]. However, the K (defensive responses) scale score in the tumor group decreased significantly (52.1 ± 13.9 vs. 58.8 ± 12.1, P = 0.009). The F (fake): [(rs=0.253, P=0.019 / rs=0.215, P=0.048)], Hs (hypochondriasis): [(rs=0.310, P=0.004 / rs=0.345, P=0.001)], Pt (psychasthenia): [(rs=0.299, P=0.006 / rs=0.258, P=0.017)], and Sc (schizophrenia): [(rs=0.325,P=0.002/rs=0.322, P=0.003)] of preoperative glioma patients were positively correlated with their preoperative psychological distress (depression/anxiety); the D (depressive personality): [(rs=0.229, P=0.035)] of preoperative glioma patients was positively correlated with the preoperative depression symptoms of their primary caregivers, and the Hy (hysteria): [(rs=0.233, P=0.002)] and Pd (psychopathic deviate): [(rs=0.215, P=0.006)] of preoperative glioma patients were positively correlated with the preoperative anxiety symptoms of their primary caregivers. CONCLUSIONS: The preoperative psychological personality traits of glioma patients were positively correlated with preoperative symptoms of depression and anxiety in primary caregivers.

Caregiver burden and influencing factors among family caregivers of patients with glioma: A cross-sectional survey.

OBJECTIVE: The main responsibility of caring for patients with glioma is assumed by family caregivers who experience a considerable burden during the care process. This study aimed to investigate the level of caregiver burden and explore its associated factors among family caregivers of patients with glioma. METHODS: We conducted a cross-sectional study among 131 family caregivers of glioma patients from October 2017 to November 2019. We used the following measurement tools: a demographic questionnaire, the Zarit Burden interview (ZBI), the Hamilton anxiety and depression scale, and the family APGAR index. We used multiple linear regression analysis to determine the factors related to caregiver burden. RESULTS: The ZBI score for the family caregivers of glioma patients was 31.29 (SD = 13.54), and most caregivers (71.7%) reported moderate and severe caregiver burdens. Caregivers' daily sleep time and anxiety symptoms and patients' depressive symptoms independently predicted caregiver burden. CONCLUSIONS: Family caregivers of glioma patients experienced a moderate burden. Personalised psychological intervention and sleep health guidance for patients and caregivers should be considered to reduce family caregiver burden and enhance the quality of life and mental health of both patients and their caregivers.

Nicotinamide N-Methyltransferase Remodeled Cell Metabolism and Aggravated Proinflammatory Responses by Activating STAT3/IL1ß/PGE2 Pathway.

Nicotinamide N-methyltransferase (NNMT) is a cytosolic methyltransferase, catalyzing N-methylation of nicotinamide (NAM) to form 1-methylnicotinamide (1-MNAM), in which S-adenosyl-l-methionine (SAM) is the methyl donor. It has been well documented that NNMT is elevated in multiple cancers and promotes tumor aggressiveness. In the present study, we investigated the effects of NNMT overexpression on cellular metabolism and proinflammatory responses. We found that NNMT overexpression reduced NAD+ and SAM levels, and activated the STAT3 signaling pathway. Consequently, STAT3 activation upregulated interleukin 1ß (IL1ß) and cyclooxygenase-2 (COX2), leading to prostaglandin E2 (PGE2) accumulation. On the other hand, NNMT downregulated 15-hydroxyprostaglandin dehydrogenase (15-PGDH) which catalyzes PGE2 into inactive molecules. Moreover, secretomic data indicated that NNMT promoted secretion of collagens, pro-inflammatory cytokines, and extracellular matrix proteins, confirming NNMT aggravated inflammatory responses to promote cell growth, migration, epithelial-mesenchymal transition (EMT), and chemoresistance. Taken together, we showed that NNMT played a pro-inflammatory role in cancer cells by activating the STAT3/IL1ß/PGE2 axis and proposed that NNMT was a potential therapeutic target for cancer treatment.

Overexpression of 15-Hydroxyprostaglandin Dehydrogenase Inhibits A549 Lung Adenocarcinoma Cell Growth via Inducing Cell Cycle Arrest and Inhibiting Epithelial-Mesenchymal Transition.

PURPOSE: Lung cancer is one of the most commonly diagnosed cancer as well as the leading cause of cancer-related mortality worldwide, among which lung adenocarcinoma (LUAD) is the most frequent form of lung cancer. Previous studies have shown that 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of prostaglandins to reduce their biological activities and behaves as a tumor suppressor in various cancers. Thus, we aimed to systematically examine the effects of 15-PGDH overexpression on cellular processes in lung adenocarcinoma cells. METHODS: The stable 15-PGDH-overexpressing A549 cell line was constructed using lentivirus particles. CCK-8 assay was used to determine the cell proliferation rate and sensitivity to cisplatin. Tandem mass tag (TMT)-based quantitative proteomic analysis was used to identify differentially expressed proteins between control and 15-PGDH-overexpression cells. The cell cycle was determined by a flow cytometer. The expression levels of mesenchymal and epithelial markers were measured using Western blotting. Wound healing and transwell assays were used to detect the cell migration and cell invasion ability, respectively. RESULTS: Analysis of datasets in The Cancer Genome Atlas revealed that the PGDH gene expression level in the lung adenocarcinoma tissues was significantly lower than that in the pericarcinous tissues. 15-PGDH overexpression in A549 cells reduced cell proliferation rate. Quantitative proteomics revealed that 15-PGDH overexpression inhibited PI3K/AKT/mTOR signaling pathway, which is a signaling pathway driving tumor cell growth and epithelial-mesenchymal transition (EMT) process. In addition, both cell cycle and DNA repair-related proteins were down-regulated in 15-PGDH overexpressed cells. 15-PGDH overexpression induced G1/S cell cycle arrest and increased susceptibility to DNA damaging reagent cisplatin. Importantly, overexpression of 15-PGDH inhibited EMT process with the downregulation of ß-catenin and Snail-1 as well as upregulation of E-cadherin and ZO-1. CONCLUSION: 15-PGDH is a tumor suppressor in lung cancer and may serve as a potential therapeutic target to prevent lung adenocarcinoma.

Nicotinamide Mononucleotide Alleviates LPS-Induced Inflammation and Oxidative Stress via Decreasing COX-2 Expression in Macrophages.

Macrophage activation is an important process in controlling infection, but persistent macrophage activation leads to chronic inflammation and diseases, such as tumor progression, insulin resistance and atherosclerosis. Characterizing metabolic signatures of macrophage activation is important for developing new approaches for macrophage inactivation. Herein, we performed metabolomic analysis on lipopolysaccharide (LPS)-activated macrophages and identified the associated changes in metabolites. Notably, the cellular Nicotinamide adenine dinucleotide+ levels were decreased while NADPH was increased, proposing that NAD+ restoration can inhibit macrophage activation. Indeed, supplementation of nicotinamide mononucleotide (NMN) increased cellular NAD+ levels and decreased cytokine productions in LPS-activated cells. Quantitative proteomics identified that nicotinamide mononucleotide downregulated the expressions of LPS-responsive proteins, in which cyclooxygenase-2 (COX-2) expression was significantly decreased in NMN-treated cells. Consequently, the cellular levels of prostaglandin E2 (PGE2) was also decreased, indicating that NMN inactivated macrophages via COX-2-PGE2 pathway, which was validated in activated THP-1 cells and mouse peritoneal macrophages. In conclusion, the present study identified the metabolic characteristics of activated macrophages and revealed that NMN replenishment is an efficient approach for controlling macrophage activation.

Flavonoid 4,4'-dimethoxychalcone suppresses cell proliferation via dehydrogenase inhibition and oxidative stress aggravation.

Flavonoids are natural polyphenolic compounds with a diverse array of biological activities and health-promoting effects. Recent studies have found that 4,4'-dimethoxychalcone (DMC) promoted longevity via autophagy; however, its targets are currently unknown. Herein, we employed an unbiased thermal proteome profiling (TPP) method and identified multiple targets of DMC, including ALDH1A3, ALDH2, and PTGES2. We further determined the dissociation constant (Kd) of DMC and ALDH1A3 to be 2.8 µM using microscale thermophoresis (MST) analysis, which indicated that DMC inhibited ALDH1A3 activity and aggravated cellular oxidative stress. DMC treatment significantly increased cellular reactive oxygen species (ROS) production and inhibited cancer cell growth. Quantitative proteomic analysis showed that DMC upregulated proteins associated with stress-responses and downregulated proteins associated with cell cycle progression, and this was confirmed using cell cycle analysis. Taken together, we showed that TPP is an effective tool with which to identify flavonoid targets and set a precedent for deciphering flavonoid function in the future. We have demonstrated that DMC inhibited cell proliferation via ROS-induced cell cycle arrest and is an anti-proliferative agent in cancer treatment.

Clinical study of preoperative psychological distress and its related factors in the primary caregivers of patients with glioma.

OBJECTIVE: To investigate the preoperative psychological distress in the primary caregivers of glioma patients and its influencing factors and to determine the relationship between preoperative psychological states of glioma patients and their caregivers. METHODS: Using a mixed methods design, the caregivers of patients with malignant glioma were interviewed and completed questionnaires about anxiety/depression and family function during preoperative period. RESULTS: Caregivers of glioma patients had different types of psychological distress before surgery. A total of 46 caregivers (36.5 %) had preoperative psychological depression or anxiety symptom; 30 caregivers (23.8 %) had depression symptoms, 39 caregivers (31.0 %) had anxiety symptoms, and 23 (18.3 %) had both preoperative anxiety and depression symptoms. Female gender was an influential factor for both preoperative depression and anxiety symptoms in caregivers, surgical information was a risk factor for caregivers' preoperative depression symptoms and residence was a risk factor for caregivers' preoperative anxiety symptoms. The preoperative psychological states of patients was positively correlated with the preoperative anxiety and depression symptoms of caregivers. CONCLUSION: The rural female caregivers are more likely to experience psychological distress before surgery, and the preoperative psychological states of patients was positively correlated with the psychological distress in caregivers.

Nicotinamide mononucleotide inhibits hepatic stellate cell activation to prevent liver fibrosis via promoting PGE2 degradation.

Liver fibrosis is a reversible wound-healing response to acute or chronic liver injury that can progress to cirrhosis and liver cancer. Finding new strategies for prevention and management of liver fibrosis is urgently needed. It is known that hepatic stellate cell (HSC) is the primary source of extracellular matrix that drives liver fibrosis progression. Herein, we carried out a comprehensive secretome profiling to identify NMN-induced changes in secretory proteins and found that NMN suppressed the secretion of profibrotic protein and oxidoreductase in activated HSC (LX-2) cells, while real-time quantitative PCR analysis revealed that NMN downregulated profibrotic gene expression, resulting in HSC inactivation. Next, we demonstrated that nicotinamide mononucleotide (NMN) reduced the accumulation of liver extracellular matrix in thioacetamide (TAA) and carbon tetrachloride (CCl4) induced mouse models for liver fibrosis. Furthermore, we determined that NMN inhibited oxidation-mediated 15-PGDH degradation to promote prostaglandin E2 degradation and suppress HSC activation. In summary, our results propose that NMN supplementation is a new therapeutic approach for liver fibrosis prevention.

Effect of backward walking training on knee osteoarthritis: protocol of a systematic review and meta-analysis.

INTRODUCTION: Backward walking (BW) is otherwise known as retrowalking. As opposed to forward walking, BW is a countersequential exercise and is a common method of rehabilitation training and disease-assisted treatment. Studies have shown that BW has a helpful effect on improving lower limb proprioception, gait synergy and improving limb balance. Many studies have concluded that BW can improve the symptoms of patients with knee osteoarthritis (KOA) and can be used for rehabilitation and adjunctive treatment of KOA, but there is a lack of evidence-based medical evidence.This research aims to provide an update to the most recent available evidence on the effect of BW on patients with KOA . METHODS AND ANALYSES: Electronic databases, such as Ovid/MEDLINE, EMBASE, CINAHL, Scopus, Web of Science and PubMed, will be searched by us. We will include studies identified from citation until 12 May 2020 and will not be restricted by geographical setting. The search will not be limited to the language of the publication, but the study of human subjects. Randomised controlled trials (RCTs) on the BW training of KOA will be included, with outcome measures including pain, knee function or balance function. The quality of included RCTs will be evaluated according to the Cochrane Collaboration's risk of bias tool. A meta-analysis or systematic review will be performed to summarise the effects of BW training. We will perform sensitivity analysis on the sample size of RCTs, meta-regression analysis of the follow-up periods, dosages and baselines of outcome measures, and publication bias analysis. ETHICS AND DISSEMINATION: Ethical approval is not required as this study will not involve confidential personal data. The results of this study will be disseminated through a peer-reviewed journal. PROSPERO REGISTRATION NUMBER: CRD42020185694.

RNase-resistant virus-like particles containing long chimeric RNA sequences produced by two-plasmid coexpression system.

RNase-resistant, noninfectious virus-like particles containing exogenous RNA sequences (armored RNA) are good candidates as RNA controls and standards in RNA virus detection. However, the length of RNA packaged in the virus-like particles with high efficiency is usually less than 500 bases. In this study, we describe a method for producing armored L-RNA. Armored L-RNA is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of a two-plasmid coexpression system in which the coat protein and maturase are expressed from one plasmid and the target RNA sequence with modified MS2 stem-loop (pac site) is transcribed from another plasmid. A 3V armored L-RNA of 2,248 bases containing six gene fragments-hepatitis C virus, severe acute respiratory syndrome coronavirus (SARS-CoV1, SARS-CoV2, and SARS-CoV3), avian influenza virus matrix gene (M300), and H5N1 avian influenza virus (HA300)-was successfully expressed by the two-plasmid coexpression system and was demonstrated to have all of the characteristics of armored RNA. We evaluated the 3V armored L-RNA as a calibrator for multiple virus assays. We used the WHO International Standard for HCV RNA (NIBSC 96/790) to calibrate the chimeric armored L-RNA, which was diluted by 10-fold serial dilutions to obtain samples containing 10(6) to 10(2) copies. In conclusion, the approach we used for armored L-RNA preparation is practical and could reduce the labor and cost of quality control in multiplex RNA virus assays. Furthermore, we can assign the chimeric armored RNA with an international unit for quantitative detection.

A novel real-time multiplex reverse transcriptase-polymerase chain reaction for the detection of HIV-1 RNA by using dual-specific armored RNA as internal control.

OBJECTIVE: Transmission of HIV-1 and diagnosis of infection in hospitals and public health settings remains a worldwide concern. HIV-1 detection is sometimes not possible using current commercial assays, probably due to mismatches between the selected primers and probes. METHODS: By screening primers and probes, we developed a dual-specificity probe real-time reverse transcriptase-polymerase chain reaction (DSPrtRT-PCR) assay using dual-specific armored RNA as the internal control. The specificity and sensitivity were compared between the monospecificity probe real-time and DSPrtRT-PCR techniques. RESULTS: The sensitivity of DSPrtRT-PCR improved significantly, with no effect on its specificity. The detection limit was 173 IU/ml. All the HIV-1 group M and group O could be detected. In clinical assays, 1,000 copies/ml of armored RNA was required as internal control. When applied to negative samples, 100% specificity was achieved. Among 60 samples from the tested patients, DSPrtRT-PCR demonstrated high sensitivity, accurately detecting 50 positives and 10 negatives that were confirmed by the COBAS AmpliScreen assay. CONCLUSION: DSPrtRT-PCR is a more efficient and effective viral assay with high sensitivity and specificity as compared to monospecificity probe PCR. It can be widely applied in blood donor screening and qualitative individual detection of HIV-1 RNA.