High-efficiency genetic engineering toolkit for virus based on lambda red-mediated recombination.

PURPOSE: Viruses, such as Ebola virus (EBOV), evolve rapidly and threaten the human health. There is a great demand to exploit efficient gene-editing techniques for the identification of virus to probe virulence mechanism for drug development. METHODS: Based on lambda Red recombination in Escherichia coli (E. coli), counter-selection, and in vitro annealing, a high-efficiency genetic method was utilized here for precisely engineering viruses. EBOV trVLPs assay and dual luciferase reporter assay were used to further test the effect of mutations on virus replication. RESULTS: Considering the significance of matrix protein VP24 in EBOV replication, the types of mutations within vp24, including several single-base substitutions, one double-base substitution, two seamless deletions, and one targeted insertion, were generated on the multi-copy plasmid of E. coli. Further, the length of the homology arms for recombination and in vitro annealing, and the amount of DNA cassettes and linear plasmids were optimized to create a more elaborate and cost-efficient protocol than original approach. The effects of VP24 mutations on the expression of a reporter gene (luciferase) from the EBOV minigenome were determined, and results indicated that mutations of key sites within VP24 have significant impacts on EBOV replication. CONCLUSION: This precise mutagenesis method will facilitate effective and simple editing of viral genes in E. coli.

A Dual Load-Modulated Doherty Power Amplifier Design Method for Improving Power Back-Off Efficiency.

In this paper, the load modulation process of a Doherty power amplifier (DPA) is analyzed to address the issue of why designed DPAs have a very low efficiency in the back-off state in some cases. A general formula of the real load modulation process is also given for analyzing the load modulation of a peak PA matching network. This provides a new perspective for improving the back-off efficiency of a DPA. To improve the power back-off efficiency of a DPA, a dual load-modulated DPA (D-DPA) design method is proposed. The core principle of the proposed design method is to control the load modulation process from the carrier PA to the peaking PA based on the design method of the traditional two-way DPA. The efficiency of the peaking PA in the back-off region is enhanced, thereby improving the efficiency in the entire back-off region of the DPA. Based on the proposed design method, a D-DPA operating at 2 GHz is designed and fabricated. The test results show that the saturated output power and gain are 43.7 dBm and 9.7 dB, respectively, while the efficiency at 6 dB output power back-off is 59.2%. The designed D-DPA eliminates the efficiency pit of the traditional two-way DPA in the output power back-off region.

Inhibitory Effects of 3-Methylcholanthrene Exposure on Porcine Oocyte Maturation.

3-methylcholanthrene (3-MC) is a highly toxic environmental pollutant that impairs animal health. 3-MC exposure can cause abnormal spermatogenesis and ovarian dysfunction. However, the effects of 3-MC exposure on oocyte maturation and embryo development remain unclear. This study revealed the toxic effects of 3-MC exposure on oocyte maturation and embryo development. 3-MC with different concentrations of 0, 25, 50, and 100 µM was applied for in vitro maturation of porcine oocytes. The results showed that 100 µM 3-MC significantly inhibited cumulus expansion and the first polar body extrusion. The rates of cleavage and blastocyst of embryos derived from 3-MC-exposed oocytes were significantly lower than those in the control group. Additionally, the rates of spindle abnormalities and chromosomal misalignments were higher than those in the control group. Furthermore, 3-MC exposure not only decreased the levels of mitochondria, cortical granules (CGs), and acetylated α-Tubulin, but also increased the levels of reactive oxygen species (ROS), DNA damage, and apoptosis. The expression of cumulus expansion and apoptosis-related genes was abnormal in 3-MC-exposed oocytes. In conclusion, 3-MC exposure disrupted the nuclear and cytoplasmic maturation of porcine oocytes through oxidative stress.

Does Hepatitis B Virus Affect the Coagulation Parameters for HBV-Related Decompensated Cirrhosis?

BACKGROUND: To evaluate the effect of the hepatitis B virus (HBV) on the coagulation parameters in patients with hepatitis B virus-related decompensated cirrhosis (HBV-DeCi). METHODS: A retrospective analysis was conducted on the medical records of 112 patients with HBV-DeCi. Baseline clinical and laboratory characteristics were retrieved. Subjects were subdivided into 3 groups. Group I: 22 cases of hepatitis B were HBsAg, HBeAg, and HbcAb positive; Group II: 67 patients were HBsAg, HBeAb, and HbcAb positive; Group III: 23 patients were HBsAb, HBeAb and HbcAb positive. The coagulation indicators, such as prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (FIB), and international normalized ratio (INR, a method to standardize reporting of the PT, using the formula, INR = (PTpatient/PTcontrol)ISI) of each groups were analyzed, The correlation between the characteristics of coagulation function and the type of hepatitis infection were studied. RESULTS: The FIB values of Group I and II were lower than those of Group III, and Group I had significantly longer TT compared to Group III. CONCLUSIONS: In patients with HBV-DeCi, hepatitis B virus has an effect on coagulation parameters; therefore, antiviral treatment must be carried out as soon as possible.

Virology and Serological Characteristics of Chronic Hepatitis B Patients with the Co-existence of HBsAg and Anti-HBs Antibodies.

BACKGROUND: To investigate the virological and serological characteristics of chronic hepatitis B patients concurrently positive for HBsAg and anti-HBs. METHODS: Chronic hepatitis B patients with coexistence of HBsAg and anti-HBs were screened by electrochemiluminescence immunoassay (ECLIA). The serum biomarkers such as HBV markers, HBV DNA load, AFP, and liver function were detected, and the virological and serological features were analyzed. RESULTS: The simultaneous seropositivity for hepatitis-B surface antigen and anti-HBS antibodies was 3.867%. There was no significant difference in the detection rate between men and women (p > 0.05). The HBV DNA detection rate, HBV DNA load, and liver function index of the high HBsAg titer group were significantly higher than those of the low titer group. There was no significant difference in HBV DNA load, AFP, and liver function between different levels of anti-HBs (p > 0.05). CONCLUSIONS: In chronic hepatitis B patients, there is a certain proportion of patients with coexistence of HBsAg and anti-HBs. The emergence of anti-HBs does not mean that HBsAg can be completely and effectively eliminated. HBV DNA load can be replicated continuously with the presence of anti-HBs, and its follow-up is worthy of clinical attention.

Comprehensive study of altered proteomic landscape in proximal renal tubular epithelial cells in response to calcium oxalate monohydrate crystals.

BACKGROUND: Calcium oxalate monohydrate (COM), the major crystalline composition of most kidney stones, induces inflammatory infiltration and injures in renal tubular cells. However, the mechanism of COM-induced toxic effects in renal tubular cells remain ambiguous. The present study aimed to investigate the potential changes in proteomic landscape of proximal renal tubular cells in response to the stimulation of COM crystals. METHODS: Clinical kidney stone samples were collected and characterized by a stone component analyzer. Three COM-enriched samples were applied to treat human proximal tubular epithelial cells HK-2. The proteomic landscape of COM-crystal treated HK-2 cells was screened by TMT-labeled quantitative proteomics analysis. The differentially expressed proteins (DEPs) were identified by pair-wise analysis. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEPs were performed. Protein interaction networks were identified by STRING database. RESULTS: The data of TMT-labeled quantitative proteomic analysis showed that a total of 1141 proteins were differentially expressed in HK-2 cells, of which 699 were up-regulated and 442 were down-regulated. Functional characterization by KEGG, along with GO enrichments, suggests that the DEPs are mainly involved in cellular components and cellular processes, including regulation of actin cytoskeleton, tight junction and focal adhesion. 3 high-degree hub nodes, CFL1, ACTN and MYH9 were identified by STRING analysis. CONCLUSION: These results suggested that calcium oxalate crystal has a significant effect on protein expression profile in human proximal renal tubular epithelial cells.

Rapid and visual detection of dengue virus using recombinase polymerase amplification method combined with lateral flow dipstick.

Dengue virus (DENV), a member of the genus Flavivirus within the family Flaviviridae, is one of the most significant mosquito-borne viruses that causing dengue fever in human. A rapid diagnostic would be helpful to detect DENV infection in a timely manner. In the last decade, recombinase polymerase amplification (RPA) technique has been experiencing rapid development and widely employed to detect various other pathogens. In present study, a reverse transcription RPA (RT-RPA) assay combined with lateral flow dipstick (LFD) was established for rapid detection of DENV. The assay could detect DENV-1, -2, -3 and -4. The minimal detection limit of the RT-RPA-LFD assay was 10 copies RNA molecules. The assay was DENV-specific since it had no non-specific reactions with other common human pathogens. The clinical performance of the RT-RPA assay was validated using 120 clinical samples. The coincidence rate between RT-RPA-LFD and qRT-PCR for the clinical samples was 100%, indicating the RT-RPA-LFD assay had good diagnostic performance on clinical samples. The RT-RPA-LFD assay required no sophisticated instrument, providing a possible solution for DENV diagnosis in recourse-limited settings where DENV infection is epidemic.

Development of a reverse transcription recombinase polymerase amplification assay for rapid detection of human respiratory syncytial virus.

Respiratory syncytial virus (RSV) is one of the most important causative agents that causing respiratory tract infection in children and associated with high morbidity and mortality. A diagnostic method would be a robust tool for identification of RSV infection, especially in the resource-limited settings. Recombinase polymerase amplification (RPA) is a novel isothermal amplification technique which has been widely employed to detect human/animal pathogens. In present study, a probe-based reverse transcription RPA (RT-RPA) assay was established for the detection of RSV. The primers and probe were designed based on the sequences of the conserved nucleocapsid (N) gene. The minimal detection limit of the RT-RPA assay for the detection of RSV B was 19 copies of RNA molecules at 95% probability, whereas the detection limit for RSV A was 104 copies molecule. The assay was RSV-specific since it had no non-specific reactions with other common human pathogens. The clinical performance of the RT-RPA assay was validated using 188 nasopharyngeal aspirates (NPAs). The nucleic acid extraction of the samples was performed by use of the magnetic bead-based kit which didn't require the heavy and expensive centrifuge. The coincidence rates between RT-RPA and qRT-PCR for the clinical samples was 96%, indicating the RT-RPA assay had good diagnostic performance on clinical samples. The real-time RT-RPA assay combined with the manual genome extraction method make it potential to detect clinical samples in field, providing a possible solution for RSV diagnosis in remote rural areas in developing countries.

Delivery of cell membrane impermeable peptides into living cells by using head-to-tail cyclized mitochondria-penetrating peptides.

A series of cyclic Arg-rich mitochondria-penetrating peptides were prepared with variation in the macrocycle size and the chirality of Arg residues. A cyclic heptapeptide was demonstrated to be an efficient mitochondria-specific delivery vector for delivering membrane impermeable peptides.

MiR-106b promotes therapeutic antibody expression in CHO cells by targeting deubiquitinase CYLD.

MicroRNAs (miRNAs) function as important regulators of major cellular processes, such as cell cycle, proliferation, development, and apoptosis. Recently, miRNA engineering of Chinese hamster ovary (CHO) cells has emerged as a promising strategy for enhancing therapeutic antibody production. Previously, we have reported that inhibition of deubiquitinase cylindromatosis (CYLD) remarkably enhanced the therapeutic antibody production in CHO cells. However, the mechanisms regulating CYLD in CHO cells remain elusive. Herein, we demonstrated that miR-106b targets CYLD directly, as shown by a series of bioinformatics analyses and experimental assays. Stable overexpression of miR-106b in CHO cells promoted CHO cell viability and subsequent antibody expression in transient transfection assay. Furthermore, the results in fed-batch culture showed that stable overexpression of miR-106b in a CHO-IgG cell line achieved about 0.66-fold promotion in product titer compared to the parental cells. Meanwhile, overexpression of miR-106b did not affect the quality of antibody. Taken together, our findings highlight the effect of miR-106b inhibition in CYLD synthesis and its function in antibody expression as a new target for improving CHO manufacturing cells.

Spurious Low WBC Count from Sysmex XN3000: a Case Report.

BACKGROUND: We present a case of spurious low WBC count in a liver transplant patient. The patient is a 56-year-old man with liver cancer. METHODS: His routine blood test revealed a decrease in WBC count: 0.03 x 109/L compared to 19.30 x 109/L before. The WBCs in the blood smear appeared higher than that reported by the XN without any aggregation. We diluted the blood sample to 1:7 with the DCL of the XN. RESULTS: The diluted result matches the blood smear. CONCLUSIONS: Dilution mode may be a good choice when there is WNR and WDF discordance, and a smear must be reviewed.

Inactivation of deubiquitinase CYLD enhances therapeutic antibody production in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are promising host engineering cells for industry manufacturing of therapeutic antibodies. However, cell death due to apoptosis remains a huge challenge to augment antibody production, and developing CHO cells with enhanced anti-apoptosis and proliferation ability is fundamental for cell line development and high-yielding bioprocesses. Deubiquitinase cylindromatosis (CYLD) has been proved to be a tumor suppressor by negatively regulating NF-κB and Wnt/ß-catenin signaling pathways. Its mutation or deletion is a common chromosome variation in several types of cancers. Here, we engineered CHO CYLD-/- cells by CRISPR-Cas9 editing technology. These cells displayed stronger cell proliferation and anti-apoptosis ability compared to parental cells. Three antibody expression plasmid kits were transiently transfected into these cells. Our data showed that inactivation of CYLD increased the highest titers of rituximab, Herceptin, and one bispecific antibody by 105, 63, and 228%, respectively. Reversely, overexpression of CYLD could promote cell apoptosis, whereas inhibiting cell proliferation and antibody production. Furthermore, inhibition of CYLD in CHO cells stably expressing an IgG antibody (CHO-IgG) achieved about 50% increase in product titer compared to parental cells. Meanwhile, inhibition of CYLD did not affect the quality of antibody. Thus, our data demonstrated that inactivation of CYLD could promote CHO cell proliferation, anti-apoptosis ability, and subsequent antibody production, suggesting that CYLD is a potential functional target for CHO cell engineering.

Elevated expression of TANK-binding kinase 1 enhances tamoxifen resistance in breast cancer.

Resistance to antiestrogens is one of the major challenges in breast cancer treatment. Although phosphorylation of estrogen receptor α (ERα) is an important factor in endocrine resistance, the contributions of specific kinases in endocrine resistance are still not fully understood. Here, we report that an important innate immune response kinase, the IκB kinase-related TANK-binding kinase 1 (TBK1), is a crucial determinant of resistance to tamoxifen therapies. We show that TBK1 increases ERα transcriptional activity through phosphorylation modification of ERα at the Ser-305 site. Ectopic TBK1 expression impairs the responsiveness of breast cancer cells to tamoxifen. By studying the specimens from patients with breast cancer, we find a strong positive correlation of TBK1 with ERα, ERα Ser-305, and cyclin D1. Notably, patients with tumors highly expressing TBK1 respond poorly to tamoxifen treatment and show high potential for relapse. Therefore, our findings suggest that TBK1 contributes to tamoxifen resistance in breast cancer via phosphorylation modification of ERα.

Up-regulation of CYLD enhances Listeria monocytogenes induced apoptosis in THP-1 cells.

Listeria monocytogenes (Lm), a facultative anaerobic gram-positive bacterium, causes listeriosis. Immune cell apoptosis is considered to be one pathogenic factor for listeriosis. As a deubiquitinase, CYLD is an important regulator both in innate immune response and apoptosis by negatively modulating NF-κB pathway. However the role of CYLD in Lm induced apoptosis remains unclear. Here we found that CYLD is significantly up-regulated in macrophages upon its infection. There is a moderate decrease in Lm proliferation and apoptotic cells in siRNA-induced CYLD knockdown THP-1 cells. Thereby CYLD may be involved in cell apoptosis mediated by Lm infection and its proliferation.

Downregulation of microRNA miR-526a by enterovirus inhibits RIG-I-dependent innate immune response.

UNLABELLED: Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that induces type I interferon-mediated host-protective innate immunity against viral infection. Although cylindromatosis (CYLD) has been shown to negatively regulate innate antiviral response by removing K-63-linked polyubiquitin from RIG-I, the regulation of its expression and the underlying regulatory mechanisms are still incompletely understood. Here we show that RIG-I activity is regulated by inhibition of CYLD expression mediated by the microRNA miR-526a. We found that viral infection specifically upregulates miR-526a expression in macrophages via interferon regulatory factor (IRF)-dependent mechanisms. In turn, miR-526a positively regulates virus-triggered type I interferon (IFN-I) production, thus suppressing viral replication, the underlying mechanism of which is the enhancement of RIG-I K63-linked ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic miR-526a expression inhibits the replication of EV71 virus. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and suggest a novel mechanism for the evasion of the innate immune response controlled by EV71. IMPORTANCE: RNA virus infection upregulates the expression of miR-526a in macrophages through IRF-dependent pathways. In turn, miR-526a positively regulates virus-triggered type I IFN production and inhibits viral replication, the underlying mechanism of which is the enhancement of RIG-I K-63 ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein; cells with overexpressed miR-526a were highly resistant to EV71 infection. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and propose a novel mechanism for the evasion of the innate immune response controlled by EV71.

miR-526a regulates apoptotic cell growth in human carcinoma cells.

MicroRNAs (miRNAs) play vital roles in the regulation of cell cycle, cell growth, apoptosis, and tumorigenesis. Our previous studies showed that miR-526a positively regulated innate immune response by suppressing CYLD expression, however, the functional relevance of miR-526a expression and cell growth remains to be evaluated. In this study, miR-526a overexpression was found to promote cancer cell proliferation, migration, and anchor-independent colony formation. The molecular mechanism(s) of miR-526a-mediated growth stimulation is associated with rapid cell cycle progression and inhibition of cell apoptosis by targeting CYLD. Taken together, these results provide evidence to show the stimulatory role of miR-526a in tumor migration and invasion through modulation of the canonical NF-κB signaling pathway.

Effects of different ages on frozen semen quality and in vitro fertilization efficiency in Wannan black pigs.

According to previous studies, the quality and fertilization rate of fresh sperm from boars of different ages were significantly different. However, the difference of freeze-thaw sperm quality and fertility in boars of different ages is unclear. In this study, boars of a Chinese native breed were assigned into two groups. Each group consisted of five boars aged aged either 2-3 years (young boars = YB) or 5-6 years (aging boars = AB) A total of 60 ejaculates for each group were collected and cryopreserved. Semen quality and in vitro fertility of post-thaw sperm was evaluated. The results showed that the concentration and motility of fresh sperm collected from AB were similar to YB, but their semen volume was higher than that in YB (p < 0.05). Frozen-thawed sperm of AB had lower viability than YB, and higher abnormal rate and reactive oxygen species (ROS) levels of YB (p < 0.05). There was no effect of the age on post-thaw sperm motility and time survival. Functional assessments indicated that increasing age markedly compromises the integrity of the sperm plasma membrane and acrosome, as well as mitochondrial functionality post-thaw, albeit without affecting DNA integrity. Furthermore, increasing age of boars reduces the ability of sperm to bind to the oocyte zona pellucida after thawing, delaying the time of the first embryo cleavage after fertilization. Finally, the early developmental efficiency of in vitro fertilized embryos progressing from 4-cell to blastocyst derived from post-thaw sperm in AB significantly decreased compared to those from YB (p < 0.05). Taken together, these results suggest that increasing age in boars impairs the quality and in vitro fertility of frozen thawed sperm.

Enrichment of C17:0-rich saturated fatty acids from sheep tail fat for adjuvant therapy of non-small-cell lung cancer.

Heptadecanoic acid (C17:0), an odd-chain saturated fatty acid (OCSFA) in ruminant lipid, has been demonstrated to be potential for treating cancers. Our results also showed that sheep tail fat (STF) with higher level of C17:0-containing saturated fatty acids (SFAs) whereas lower level of oleic acid (C18:1), performed remarkable inhibition against non-small-cell lung cancer (NSCLC) cells. To enrich the content of C17:0, a C17:0-rich SFA concentrate (HRSC) was prepared from STF by solvent crystallization and urea complexation methods (hexane/STF = 3.5/1, 4 °C for 8 h, and 80% ethanol/urea/free fatty acids = 8/1/1, 4 °C for 6 h). The content of C17:0 was up from 3.02 to 6.34% and the recovery was 4.17%. Biological experiments showed that HRSC exerted better antiproliferative effect against NSCLC cells. Moreover, HRSC performed enhanced inhibitory effect in A549 cell xenograft mouse model. Therefore, HRSC has the potential to be applied in adjuvant therapy for NSCLC. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01504-w.

Emamectin benzoate exposure impaired porcine oocyte maturation.

Emamectin benzoate (EB) is a widely used insecticide that can damage the central nervous and immune systems. EB exposure significantly reduced the number of eggs laid, hatching rate, and developmental rate of lower organisms such as nematodes. However, effects of EB exposure on the maturation of higher animals such as porcine oocytes remains unknown. Here we reported that EB exposure severely impaired porcine oocyte maturation. EB exposure with 200 µM prevented cumulus expansion and reduced the rates of first polar body (pb1) extrusion, cleavage and blastocyst after parthenogenetic activation. Moreover, EB exposure disrupted spindle organization, chromosome alignment, and polymerization of microfilaments, but also apparently decreased the levels of acetylated α-tubulin (Ac-Tub) in oocytes. In addition, EB exposure perturbed mitochondria distribution and increased levels of reactive oxygen species (ROS), but did not affect the distribution of cortical granules (CGs) in oocytes. Excessive ROS caused DNA damage accumulation and induced early apoptosis of oocytes. EB exposure led to the abnormal expression of cumulus expansion and apoptosis-associated genes. Altogether, these results demonstrate that EB exposure impaired nuclear and cytoplasmic maturation of porcine oocytes probably through oxidative stress and early apoptosis.

BRD4 Targets the KEAP1-Nrf2-G6PD Axis and Suppresses Redox Metabolism in Small Cell Lung Cancer.

Accumulating evidence has witnessed the Kelch-like ECH-associated protein 1(KEAP1)- nuclear factor (erythroid-derived 2)-like 2 (Nrf2) axis is the main regulatory factor of cell resistance to endogenous and exogenous oxidative assaults. However, there are few studies addressing the upstream regulatory factors of KEAP1. Herein, bioinformatic analysis suggests bromodomain-containing protein 4 (BRD4) as a potential top transcriptional regulator of KEAP1 in lung cancer. Using molecular and pharmacological approaches, we then discovered that BRD4 can directly bind to the promoter of KEAP1 to activate its transcription and down-regulate the stability of Nrf2 which in turn transcriptionally suppresses glucose-6-phosphate dehydrogenase (G6PD) in small cell lung cancer (SCLC), a highly proliferative and aggressive disease with limited treatment options. In addition, BRD4 could associate with the Nrf2 protein in a non-KEAP1-dependent manner to inhibit Nrf2 activity. Furthermore, simultaneous application of JQ1 and ATRA or RRx-001 yielded synergistic inhibition both in vitro and in vivo. These data suggest metabolic reprogramming by JQ1 treatment improves cell resistance to oxidative stress and might be a resistance mechanism to bromodomain and extra-terminal domain (BET) inhibition therapy. Altogether, our findings provide novel insight into the transcriptional regulatory network of BRD4 and KEAP1 and transcriptional regulation of the pentose phosphate pathway in SCLC.