Development of plate-type and tubular chemiluminescence immunoassay against African swine fever virus p72.

African swine fever (ASF) is a highly contagious and fatal viral disease that has caused huge economic losses to the pig and related industries worldwide. At present, rapid, accurate, and sensitive laboratory detection technologies are important means of preventing and controlling ASF. However, because attenuated strains of African swine fever virus (ASFV) are constantly emerging, an ASFV antibody could be used more effectively to investigate the virus and control the disease on pig farms. The isolation of ASFV-specific antibodies is also essential for the diagnosis of ASF. Therefore, in this study, we developed two chemiluminescence immunoassays (CLIAs) to detect antibodies directed against ASFV p72: a traditional plate-type blocking CLIA (p72-CLIA) and an automatic tubular competitive CLIA based on magnetic particles (p72-MPCLIA). We compared the diagnostic performance of these two methods to provide a feasible new method for the effective prevention and control of ASF and the purification of ASFV. The cut-off value, diagnostic sensitivity (Dsn), and diagnostic specificity (Dsp) of p72-CLIA were 40%, 100%, and 99.6%, respectively, in known background serum, whereas those of p72-MPCLIA were 36%, 100%, and 99.6%, respectively. Thus, both methods show good Dsn, Dsp, and repeatability. However, when analytical sensitivity was evaluated, p72-MPCLIA was more sensitive than p72-CLIA or a commercial enzyme-linked immunosorbent assay. More importantly, p72-MPCLIA reduced the detection time to 15 min and allowed fully automated detection. In summary, p72-MPCLIA showed superior diagnostic performance and offered a new tool for detecting ASFV infections in the future. KEY POINTS: • Two chemiluminescence immunoassay (plate-type CLIA and tubular CLIA) methods based on p72 monoclonal antibody (mAb) were developed to detect ASFV antibody. • Both methods show good diagnostic performance (Dsn (100%), Dsp (99.6%), and good repeatability), and p72-MPCLIA detects antibodies against ASFV p72 with high efficiency in just 15 min.

Silver nanoparticles induce endothelial cytotoxicity through ROS-mediated mitochondria-lysosome damage and autophagy perturbation: The protective role of N-acetylcysteine.

Silver nanoparticles (AgNPs) are used increasingly often in the biomedical field, but their potential deleterious effects on the cardiovascular system remain to be elucidated. The primary aim of this study was to evaluate the toxic effects, and the underlying mechanisms of these effects, of AgNPs on human umbilical vein endothelial cells (HUVECs), as well as the protective role of N-acetylcysteine (NAC) against cytotoxicity induced by AgNPs. In this study, we found that exposure to AgNPs affects the morphology and function of endothelial cells which manifests as decreased cell proliferation, migration, and angiogenesis ability. Mechanistically, AgNPs can induce excessive cellular production of reactive oxygen species (ROS), leading to damage to cellular sub-organs such as mitochondria and lysosomes. More importantly, our data suggest that AgNPs causes autophagy defect, inhibits mitophagy, and finally activates the mitochondria-mediated apoptosis signaling pathway and evokes cell death. Interestingly, treatment with ROS scavenger-NAC can effectively suppress AgNP-induced endothelial damage.Our results indicate that ROS-mediated mitochondria-lysosome injury and autophagy dysfunction are potential factors of endothelial toxicity induced by AgNPs. This study may provide new evidence for the cardiovascular toxicity of AgNPs and serve as a reference for the safe use of nanoparticles(NPs) in the future.

Sodium Selenite Ameliorates Silver Nanoparticles Induced Vascular Endothelial Cytotoxic Injury by Antioxidative Properties and Suppressing Inflammation Through Activating the Nrf2 Signaling Pathway.

Silver nanoparticles (AgNP) are the dominant nanomaterials in commercial products and the medical field, but the widespread occurrence of AgNP has become a global threat to human health. Growing studies indicate that AgNP exposure can induce vascular endothelial toxicity by excessive oxidative stress and inflammation, which is closely related to cardiovascular disease (CVD), but the potential intrinsic mechanism remains poorly elucidated. Thus, it has been crucial to control the toxicological effects of AgNP in order to improve their safety and increase the outcome of their applications.Multiple researches have demonstrated that sodium selenite (Se) possesses the capability to counteract the toxicity of AgNP, but the functional role of Se in AgNP-induced CVD is largely unexplored. The aim of this study was to explore the potential protective effect of Se on AgNP-induced vascular endothelial lesion and elucidate the underlying mechanisms. An in vivo model of toxicity in animals was established by the instillation of 200 µL of AgNP into the trachea of rats both with (0.2 mg/kg/day) and without Se treated. In vitro experiments, human umbilical vein endothelial cells (HUVECs) were incubated with AgNP (0.3 µg/mL ) and Se for a duration of 24 h. Utilizing transmission electron microscopy, we observed that the internalization of AgNP-induced endothelial cells was desquamated from the internal elastic lamina, the endoplasmic reticulum was dilated, and the medullary vesicle formed. Se treatment reduced the levels of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), inhibited the release of pro-inflammatory cytokines (specifically tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6), improved endothelial cell permeability, integrity, and dysfunction, and prevented damage to the aortic endothelium caused by AgNP. Importantly, we found that Se showed the capacity against AgNP with biological functions in guiding the intracellular reactive oxygen species (ROS) scavenging and meanwhile exhibiting anti-inflammation effects. Se supplementation decreased the intracellular ROS release and suppressed NOD-like receptor protein 3 (NLRP3) and nuclear factor kappa-B (NF-κB) mediated inflammation within AgNP-intoxicated rats and HUVECs. The anti-oxidant stress and anti-inflammatory effects of Se were at least partly dependent on nuclear factor erythroid 2-related factor 2 (Nrf2). Overall, our results indicated that the protectiveness of Se against AgNP-induced vascular endothelial toxicity injury was at least attributed to the inhibition of oxidative ROS and pro-inflammatory NF-κB/NLRP3 inflammasome by activating the Nrf2 and antioxidant enzyme (HO-1) signal pathway.

Evaluation of humoral and cellular immune responses induced by a cocktail of recombinant African swine fever virus antigens fused with OprI in domestic pigs.

BACKGROUND: African swine fever (ASF) is a highly fatal disease in domestic pigs caused by ASF virus (ASFV), for which there is currently no commercial vaccine available. The genome of ASFV encodes more than 150 proteins, some of which have been included in subunit vaccines but only induce limited protection against ASFV challenge. METHODS: To enhance immune responses induced by ASFV proteins, we expressed and purified three fusion proteins with each consisting of bacterial lipoprotein OprI, 2 different ASFV proteins/epitopes and a universal CD4+ T cell epitope, namely OprI-p30-modified p54-TT, OprI-p72 epitopes-truncated pE248R-TT, and OprI-truncated CD2v-truncated pEP153R-TT. The immunostimulatory activity of these recombinant proteins was first assessed on dendritic cells. Then, humoral and cellular immunity induced by these three OprI-fused proteins cocktail formulated with ISA206 adjuvant (O-Ags-T formulation) were assessed in pigs. RESULTS: The OprI-fused proteins activated dendritic cells with elevated secretion of proinflammatory cytokines. Furthermore, the O-Ags-T formulation elicited a high level of antigen-specific IgG responses and interferon-γ-secreting CD4+ and CD8+ T cells after stimulation in vitro. Importantly, the sera and peripheral blood mononuclear cells from pigs vaccinated with the O-Ags-T formulation respectively reduced ASFV infection in vitro by 82.8% and 92.6%. CONCLUSIONS: Our results suggest that the OprI-fused proteins cocktail formulated with ISA206 adjuvant induces robust ASFV-specific humoral and cellular immune responses in pigs. Our study provides valuable information for the further development of subunit vaccines against ASF.

Identification of p72 epitopes of African swine fever virus and preliminary application.

African swine fever virus (ASFV) causes a highly lethal hemorrhagic viral disease (ASF) of pigs that results in serious losses in China and elsewhere. The development of a vaccine and diagnosis technology for ASFV is essential to prevent and control the spread of ASF. The p72 protein of ASFV is highly immunogenic and reactive, and is a dominant antigen in ASF vaccine and diagnostic research. In this study, 17 p72 monoclonal antibodies (mAbs) were generated. Epitope mapping by a series of overlapping peptides expressed in Escherichia coli showed that these mAbs recognized a total of seven (1-7) linear B cell epitopes. These mAbs did not show significant neutralizing activity. Epitopes 1 (249HKPHQSKPIL258), 2 (69PVGFEYENKV77), 5 (195VNGNSLDEYSS205), and 7 (223GYKHLVGQEV233) are novel. Sequence alignment analysis revealed that the identified epitopes were highly conserved among 27 ASFV strains from nine genotypes. Preliminary screening using known positive and negative sera indicated the diagnostic potential of mAb-2B8D7. The results provide new insights into the antigenic regions of ASFV p72 and will inform the diagnosis of ASFV.

A vesicular stomatitis virus-based African swine fever vaccine prototype effectively induced robust immune responses in mice following a single-dose immunization.

Introduction: African swine fever (ASF) is a highly contagious hemorrhagic fever disease in pigs caused by African swine fever virus (ASFV). It is very difficult to control and prevent ASF outbreaks due to the absence of safe and effective vaccines. Methods: In order to develop a safe and effective ASF vaccine for the control and prevention of ASF, two ASFV recombinant vesicular stomatitis virus (VSV) live vector vaccine prototypes, containing the gene of p72, and a chimera of p30 and p54, were developed based on the replication-competent VSV, and named VSV-p72 and VSV-p35. The immune potency of VSV-p72 or VSV-p35 alone and in combination was evaluated in BALB/c mice via intramuscular and intranasal vaccination. Results: The results indicated that whether administered alone or in combination, the two vaccine prototypes showed acceptable safety in mice and, more importantly, induced high-level specific antibodies against p72, p30, and p54 of ASFV and a strong cellular immune response 28 days after vaccination. The sera from mice vaccinated with the vaccine prototypes significantly inhibited ASFV from infecting porcine alveolar macrophages (PAMs) in vitro. Most notably, the immunized sera from a mixture of VSV-p35 and VSV-p72 inhibited ASFV from infecting PAMs, with an inhibition rate of up to 78.58%. Conclusion: Overall, our findings suggest that ASFV recombinant VSV live vector vaccine prototypes may become a promising candidate vaccine for the control and prevention of ASF.

A comprehensive review of pancreatic cancer and its therapeutic challenges.

Pancreatic cancer is a devastating and lethal human malignancy with no curable chemo-treatments available thus far. More than 90% of pancreatic tumors are formed from ductal epithelium as pancreatic ductal adenocarcinoma (PDAC), which often accompany with the expression of mutant K-ras. The incidences of pancreatic cancer are expected to increase rapidly worldwide in the near future, due to environmental pollution, obesity epidemics and etc. The dismal prognosis of this malignancy is contributed to its susceptibility to tumor micro-metastasis from inception and the lack of methods to detect precursor lesions at very early stages of the onset until clinical symptoms occur. In recent years, basic and clinical studies have been making promising progresses for discovering markers to determine the subtypes or stages of this malignancy, which allow effectively implementing personalized therapeutic interventions. The purpose of this review is to discuss the existing knowledge of the molecular mechanisms of pancreatic cancer and the current state of treatment options with the emphasis on targeting therapeutic approaches. The specific focuses are on the molecular mechanisms of the disease, identifications of drug resistance, establishment of immune escaping mechanisms as well as potential of targeting identified pathways in combinations with existing chemo-drugs.

Isolation, identification and pathogenic characteristics of tick-derived parainfluenza virus 5 in northeast China.

The number of parainfluenza virus 5 (PIV5) infection cases has increased worldwide over the past six decades; however, factors underlying this increase remain unclear. PIV5 has been emerging or re-emerging in humans and animal species. To date, no information is yet available regarding PIV5 infection in arthropod ticks. Here, we successfully isolated tick-derived PIV5 from the Ixodes persulcatus species designated as HLJ/Tick/2019 in Heilongjiang, China. Phylogenetic analysis revealed that the tick-derived PIV5 is closely related to subclade 2.2.6, which has become the dominant subtype prevalent in dogs, pigs and wildlife across China. Further experiments to understand the importance of this virus as an infectious vector revealed that a ferret animal model experimentally infected with Tick/HLJ/2019 via the oronasal and ocular inoculation routes developed moderate respiratory distress with pneumonia and neurologic tissue damage from inflammation for the first time. Further surveillance of PIV5 in vectors of viral transmission is necessary to enhance our knowledge of its ecology in reservoirs and facilitate the control of re-emerging diseases.

Antigenicity and immunogenicity of recombinant proteins comprising African swine fever virus proteins p30 and p54 fused to a cell-penetrating peptide.

African swine fever (ASF) is a highly fatal swine disease threatening the global pig industry. Currently, vaccine is not commercially available for ASF. Hence, it is desirable to develop effective subunit vaccines against ASF. Here, we expressed and purified two recombinant fusion proteins comprising ASFV proteins p30 and p54 fused to a novel cell-penetrating peptide Z12, which were labeled as ZPM (Z12-p30-modified p54) and ZPMT (Z12-p30-modified p54-T cell epitope). Purified recombinant p30 and modified p54 expressed alone or fused served as controls. The transduction capacity of these recombinant proteins was assessed in RAW264.7 cells. Both ZPM and ZPMT exhibited higher transduction efficiency than the other proteins. Subsequently, humoral and cellular immune responses elicited by these proteins were evaluated in mice. ZPMT elicited the highest levels of antigen-specific IgG responses, cytokines (interleukin-2, interferon-γ, and tumor necrosis factor-α) and lymphocyte proliferation. Importantly, sera from mice immunized with ZPM or ZPMT neutralized greater than 85% of ASFV in vitro. Our results indicate that ZPMT induces potent neutralizing antibody responses and cellular immunity in mice. Therefore, ZPMT may be a suitable candidate to elicit immune responses in swine, providing valuable information for the development of subunit vaccines against ASF.

LINC00665 promotes the progression of acute myeloid leukemia by regulating the miR-4458/DOCK1 pathway.

This study aimed to explore the role of LINC00665, miR-4458 and DOCK1 and their interactions in the development of acute myeloid leukemia (AML). The relative expression of LINC00665, miR-4458 and DOCK1 in AML samples was measured using qRT-PCR, and the protein level of DOCK1 in AML cell lines was examined using western blot. CCK8, BrdU, transwell, cell adhesion, and caspase-3 activity assays were carried out to evaluate the viability, proliferation, migration, adhesion, and apoptosis of AML cells, respectively. Luciferase reporter, RIP, and RNA pull-down assays were also performed to confirm the target relationship among LINC00665, miR-4458 and DOCK1. Findings revealed that LINC00665 and DOCK1 were aberrantly overexpressed in AML tissues and that the expression of miR-4458 was low in AML tissues. Silencing LINC00665 or DOCK1 presented significant restriction to the proliferation, migration and adhesion of AML cells. Apart from that, it was found that inhibiting miR-4458 could enhance the proliferation, migration and adhesion of AML cells but suppress the apoptosis of AML cells. Experimental results also indicated that LINC00665 exerted its positive function on AML cells by sponging miR-4458 and that miR-4458 influenced the progression of AML cells by targeting DOCK1 directly. Overall, this finding not only provided a novel molecular pathway for the diagnosis and treatment of AML but also showed that LINC00665 could enhance the progression of AML by regulating the miR-4458/DOCK1 pathway.

LncRNA TRHDE-AS1 inhibit the scar fibroblasts proliferation via miR-181a-5p/PTEN axis.

Hypertrophic scar (HS), a fibroproliferative disorder caused by abnormal wound healing after skin injury, which is characterized by excessive deposition of extracellular matrix and invasive growth of fibroblasts. Recent studies have shown that some non-coding RNA implicated the formation of HS, but the mechanism remains unclear. In this study, we found that lncRNA TRHDE-AS1 was downregulated in HS tissues and HSFs, and the level of lncRNA TRHDE-AS1 negatively correlated with the level of miR-181a-5p in HS tissue and HSFs. Overexpressed lncRNA TRHDE-AS1 significantly suppressed miR-181a-5p level, while promoted HSFs apoptosis and inhibited HSFs proliferation. Further study shown that PTEN was a direct target of miR-181a-5p, and lncRNA TRHDE-AS1 served as a molecular sponge for miR-181a-5p to regulate the expression of PTEN. Overexpression of PTEN could eliminate lncRNA TRHDE-AS1-mediated proliferation suppression of HSFs. In conclusion, our study suggested that lncRNA TRHDE-AS1/miR-181a-5p/PTEN axis plays an important role in promoting hypertrophic scar formation, which may be effectively used as a therapeutic target for hypertrophic scar treatment.

Identification and biochemical characterisation of a novel methionine aminopeptidase from the taiga tick Ixodes persulcatus.

Methionine aminopeptidases (MetAPs), which remove the initiator methionine from nascent peptides, are essential in all organisms and considered to be a valuable targets for the treatment of various diseases, including cancer, malaria, and bacterial infections. However, MetAPs have not been reported in hard ticks (family Ixodidae), and their bioinformatics characterisation in tick's genome sequences is limited. In this study, we cloned, identified, and characterised a novel MetAP from Ixodes persulcatus, a vector for pathogens causing Lyme borreliosis and tick-borne encephalitis. The sequence analysis showed that I. persulcatus MetAP was a type 1 enzyme carrying C-terminal motifs conserved in the M24A family of metallopeptidases. Protein-protein docking simulations using human MetAP revealed conservation of substrate and metal-binding residues in the catalytic site cleft of the novel enzyme, which was designated IpMetAP. Recombinant IpMetAP expressed in Escherichia coli revealed its significant enzymatic activity with the synthetic substrate H-Met-4-methyl-coumaryl-7-amide at pH 7.5 with Km of 0.014 mM, kcat of 0.25 s-1, and overall catalytic efficiency (kcat/Km) of 18.36 mM-1 s-1. The activity of IpMetAP was enhanced by the addition of divalent cations Mn2+ and Co2+ and significantly inhibited by EDTA and bestatin. Site-directed mutagenesis of conserved amino acids indicated that the substitution of metal-binding residues D226 and H288 completely abolished the IpMetAP enzymatic activity, whereas that of the other sites had only moderate effects on substrate hydrolysis. The catalytic properties of IpMetAP suggest that the enzyme behaves similar to other MetAPs and such characterization expands our knowledge of aminopeptidases and protein metabolism of tick.

MOB1 Inhibits Malignant Progression of Colorectal Cancer by Targeting PAK2.

OBJECTIVE: We aimed at studying the mechanism of MOB1 inhibiting the proliferation and metastasis of colorectal cancer (CRC), to provide a new guidance for the early diagnosis and treatment of CRC. METHODS: MOB1 expression level in 68 pairs of CRC tissues and adjacent ones was detected by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and the associations between the expression level of MOB1 and the clinicopathological indicators as well as the prognosis of CRC patients were analyzed. After constructing CRC cell lines that stably overexpressing or silencing MOB1, the changes of cell proliferation and metastasis ability were examined by Cell Counting Kit (CCK-8) and Transwell assay. In addition, the interaction between MOB1 and PAK2 and how the these two genes affect the biological functions of CRC cell lines were investigated by luciferase assay, qRT-PCR and Western Blot experiments. RESULTS: Our data showed that MOB1 expression level in CRC tissues was remarkably lower than that in adjacent ones. In comparison to patients of the group of high MOB1 expression, these patients of low MOB1 expression group showed higher incidence of distant or lymph node metastasis and lower survival rate. Cell functional experiments revealed that overexpression of MOB1 markedly attenuated the proliferation and migration ability of CRC cell lines compared to the NC group; In contrast, knockdown of MOB1 enhanced the above-mentioned cell abilities compared to anti-NC group. Luciferase assay verified an interaction between MOB1 and PAK2; and Western blot analysis showed a negative correlation between the expression of the MOB1 and PAK2 protein levels in CRC tissues. Subsequently, we demonstrated that MOB1 interacted with PAK2 to regulate its expression and affected the proliferation and migration capacity of CRC cell lines in vitro. CONCLUSION: In summary, the lowly expressed MOB1 in CRC tissues and cell lines may accelerate the proliferation and migration through modulating PAK2 expression.

Retraction Note: Mir-655 up-regulation suppresses cell invasion by targeting pituitary tumor-transforming gene-1 in esophageal squamous cell carcinoma.

The Editor-in-Chief has retracted this article [1] because Figure 3a overlaps with Figure 2 in [2]. An investigation by Zhengzhou University has confirmed this. The data reported in this article are therefore unreliable. There is also considerable text overlap with a previously published article [3]. Guoqiang Zhao does not agree with this retraction. The other authors have not responded to correspondence from the editor about this retraction.

Mcl-1 inhibits Mff-mediated mitochondrial fragmentation and apoptosis.

Myeloid cell leukemia-1 (Mcl-1) is involved in the regulation of mitochondrial fission and fusion. This report aims to explore whether Mcl-1 can interact with mitochondrial fission factor (Mff) and regulate Mff-mediated mitochondrial fragmentation and apoptosis. Fluorescence images of living cells coexpressing YFP-Mff and CFP-Mcl-1 showed that Mcl-1 markedly inhibited Mff-mediated mitochondrial fragmentation and apoptosis, suggesting that Mcl-1 played a key role in inhibiting mitochondrial fission. The cells coexpressing YFP-Mff and CFP-Mcl-1 exhibited consistent fluorescence resonance energy transfer (FRET) efficiency with that of the cells coexpressing CFP-Mcl-1 and YFP, demonstrating that Mcl-1 did not directly bind to Mff on mitochondria. Collectively, Mcl-1 inhibits Mff-mediated mitochondrial fission and apoptosis not via directly binding to Mff on mitochondria.

Stoichiometry and regulation network of Bcl-2 family complexes quantified by live-cell FRET assay.

The stoichiometry and affinity of Bcl-2 family complexes are essential information for understanding how their interactome network is orchestrated to regulate mitochondrial permeabilization and apoptosis. Based on over-expression model system, FRET analysis was used to quantify the protein-protein interactions among Bax, Bcl-xL, Bad and tBid in healthy and apoptotic cells. Our data indicate that the stoichiometry and affinity of Bcl-2 complexes are dependent on their membrane environment. Bcl-xL, Bad and tBid can form hetero-trimers in mitochondria. Bcl-xL binds preferentially to Bad, then to tBid and Bax in mitochondria, whilst Bcl-xL displays higher affinity to Bad or tBid than to itself. Strikingly, Bax can bind to Bcl-xL in cytosol. In cytosol of apoptotic cells, Bcl-xL associates with Bax to form hetero-trimer with 1:2 stoichiometry, while Bcl-xL associates with Bad to form hetero-trimer with 2:1 stoichiometry and Bcl-xL associates with tBid to form hetero-dimer. In mitochondria, Bcl-xL associates with Bax/Bad to form hetero-dimer in healthy cells, while Bcl-xL associates with Bad to form hetero-tetramer with 3:1 stoichiometry in apoptotic cells.

Quantifying the inhibitory effect of Bcl-xl on the action of Mff using live-cell fluorescence imaging.

Mitochondrial fission regulates mitochondrial function and morphology, and has been linked to apoptosis. The mitochondrial fission factor (Mff), a tail-anchored membrane protein, induces excessive mitochondrial fission, contributing to mitochondrial dysfunction and apoptosis. Here, we evaluated the inhibitory effect of Bcl-xl, an antiapoptotic protein, on the action of Mff by using live-cell fluorescence imaging. Microscopic imaging analysis showed that overexpression of Mff induced mitochondrial fragmentation and apoptosis, which were reversed by coexpression of Bcl-xl. Microscopic imaging and live-cell fluorescence resonance energy transfer analysis demonstrated that Bcl-xl reconstructs the Mff network from punctate distribution of higher-order oligomers to filamentous distribution of lower-order oligomers. Live-cell fluorescence resonance energy transfer two-hybrid assay showed that Bcl-xl interacted with Mff to form heterogenous oligomers with 1 : 2 stoichiometry in cytoplasm and 1 : 1 stoichiometry on mitochondria, indicating that two Bcl-xl molecules primarily interact with four Mff molecules in cytoplasm, but with two Mff molecules on mitochondria.

BCL-XL directly retrotranslocates the monomeric BAK.

BCL-XL, an anti-apoptotic BCL-2 family protein, potently inhibits BAK oligomerization and the formation of toxic mitochondrial pores in response to cellular stress. This report aims to explore which form of mitochondrial monomeric and oligomerized BAK can be retrotranslocated by BCL-XL. Fluorescence imaging of living cells co-expressing CFP-BCL-XL and YFP-BAK showed that BCL-XL markedly inhibited mitochondrial BAK oligomerization and resulted in partial cytosolic BAK distribution. Live-cell fluorescence resonance energy transfer (FRET) analyses showed that BAK auto-oligomerized on mitochondria and BCL-XL physically sequestrated monomeric BAK to prevent BAK oligomerization. Fluorescence loss in photobleaching (FLIP) analyses showed that BCL-XL retrotranslocated the monomeric BAK from mitochondria into cytosol, whereas monomeric BAK reduced the retrotranslocation rate of BCL-XL. Live-cell time-lapse imaging and FLIP experiments in living cells with BAK oligomers displayed that BCL-XL did not depolymerize or retrotranslocate the oligomerized BAK. Collectively, BCL-XL retrotranslocates monomeric instead of oligomerized BAK from mitochondria into cytosol.

Gaussian FRET two-hybrid assays for determining the stoichiometry of hetero-oligomeric complexes in single living cells.

Here we integrate multiple Gaussian-functions analysis into fluorescence resonance energy transfer (FRET) two-hybrid assays (Gaussian FRET two-hybrid assay) to determine the stoichiometric ratios of intracellular hetero-oligomers in single living cells. This method adopts in multiple Gaussian-functions to fit the E-count histograms of both donor- and acceptor-centric FRET efficiency (ED and EA) images of a single cell for obtaining the peak values (EDi and EAi), thus yielding the corresponding stoichiometric ratios (EDi/EAi) of intracellular hetero-oligomers. We performed Gaussian FRET two-hybrid assay for living Hela cells coexpressing different FRET tandem plasmids, and obtained consistent results with the expected values. Gaussian FRET two-hybrid assay for cells coexpressing Bad-CFP and Bcl-XL-YFP reveals that Bcl-XL binds with Bad to form a hetero-oligomeric complex with a stoichiometry of 2:1 on mitochondria.

A novel dual-luciferase assay for anti-HIV drug screening based on the CCR5/CXCR4 promoters.

Acquired immunodeficiency syndrome (AIDS) is a serious worldwide disease caused by infection with the human immunodeficiency virus (HIV). C-C chemokine receptor 5 (CCR5) and C-X-C chemokine receptor 4 (CXCR4) are important coreceptors mediating HIV-1 cell entry. Many new anti-HIV drugs are currently in preclinical and clinical trials; however, drug development has proceeded slowly partly because of the lack of a high-throughput system to screen these drugs. Here, we describe the development of a novel dual-luciferase assay using a CCR5/CXCR4 promoter-driven firefly and Renilla luciferase vector (pGL4.10-RLUC-CCR5/CXCR4). Drugs were screened for the ability to regulate CCR5 and CXCR4 promoter activities. The CCR5 and CXCR4 promoters were inserted separately into the recombinant vector and transfected into the acute T lymphocyte leukemia cell line H9. Treatment of stable transfected cells with four traditional Chinese medicine compounds resulted in the dose-dependent inhibition of the CXCR4 and CCR5 promoter activities. The dual-luciferase reporter assay provides a rapid and direct method to screen anti-AIDS/HIV drugs.