Mitotic catastrophe can be defined as a cell death mode that occurs during or shortly after a prolonged/aberrant mitosis, and can show apoptotic or necrotic features. However, conventional procedures for the detection of apoptosis or necrosis, including biochemical bulk assays and cytofluorometric techniques, cannot discriminate among pre-mitotic, mitotic and post-mitotic death, and hence are inappropriate to monitor mitotic catastrophe. To address this issue, we generated isogenic human colon carcinoma cell lines that differ in ploidy and p53 status, yet express similar amounts of fluorescent biosensors that allow for the visualization of chromatin (histone H2B coupled to green fluorescent protein (GFP)) and centrosomes (centrin coupled to the Discosoma striata red fluorescent protein (DsRed)). By combining high-resolution fluorescence videomicroscopy and automated image analysis, we established protocols and settings for the simultaneous assessment of ploidy, mitosis, centrosome number and cell death (which in our model system occurs mainly by apoptosis). Time-lapse videomicroscopy showed that this approach can be used for the high-throughput detection of mitotic catastrophe induced by three mechanistically distinct anti-mitotic agents (dimethylenastron (DIMEN), nocodazole (NDZ) and paclitaxel (PTX)), and - in this context - revealed an important role of p53 in the control of centrosome number.

Natural killer (NK) cells are important in host to eliminate circulating tumour cells (CTCs) in turn preventing the development of tumour cells into metastasis but the mechanisms are very poorly defined. Here we find that the expression level of miR-296-3p is much lower in the non-metastatic human prostate cancer (PCa) cell line P69 than that in the highly metastatic cell line M12, which is derived from P69. We demonstrate that miR-296-3p directly targets and inhibits the expression of intercellular adhesion molecule 1 (ICAM-1) in the malignant M12. The data from clinical tissue microarrays also show that miR-296-3p is frequently upregulated and ICAM-1 is reversely downregulated in PCa. Interestingly, ectopic expression of miR-296-3p in P69 increases the tolerance to NK cells whereas knockdown of miR-296-3p in M12 reduces the resistance to NK cells, which both phenotypes can be rescued by re-expression or silencing of ICAM-1 in P69 and M12, respectively. These results are also manifested in vivo by the decrease in the incidence of pulmonary tumour metastasis exhibited by knockdown of miR-296-3p in M12 when injected into athymic nude mice via tail vein, and consistently down-expression of ICAM-1 reverses this to increase extravasation of CTCs into lungs. Above results suggest that this newly identified miR-296-3p-ICAM-1 axis has a pivotal role in mediating PCa metastasis by possible enhancing survival of NK cell-resistant CTC. Our findings provide novel potential targets for PCa therapy and prognosis.

Radiotherapy has a critical role in the treatment of small-cell lung cancer (SCLC). The effectiveness of radiation in SCLC remains limited as resistance results from defects in apoptosis. In the current study, we investigated whether using the Bcl-2/Bcl-XL inhibitor S44563 can enhance radiosensitivity of SCLC cells in vitro and in vivo. In vitro studies confirmed that S44563 caused SCLC cells to acquire hallmarks of apoptosis. S44563 markedly enhanced the sensitivity of SCLC cells to radiation, as determined by a clonogenic assay. The combination of S44563 and cisplatin-based chemo-radiation showed a significant tumor growth delay and increased overall survival in mouse xenograft models. This positive interaction was greater when S44563 was given after the completion of the radiation, which might be explained by the radiation-induced overexpression of anti-apoptotic proteins secondary to activation of the NF-κB pathway. These data underline the possibility of combining IR and Bcl-2/Bcl-XL inhibition in the treatment of SCLC as they underscore the importance of administering conventional and targeted therapies in an optimal sequence.

This study was designed to investigate whether delta opioid receptor (DOR) is involved in the neuroprotective effect induced by hypoxic preconditioning (HPC) in the asphyxial cardiac arrest (CA) rat model. Twenty-four hours after the end of 7-day HPC, the rats were subjected to 8-min asphyxiation and resuscitated with a standardized method. In the asphyxial CA rat model, HPC improved the neurological deficit score (NDS), inhibited neuronal apoptosis, and increased the number of viable hippocampal CA1 neurons at 24 h, 72 h, or 7 days after restoration of spontaneous circulation (ROSC); however, the above-mentioned neuroprotection of HPC was attenuated by naltrindole (a selective DOR antagonist). The expression of hypoxia-inducible factor-1α (HIF-1α) and DOR, and the content of leucine enkephalin (L-ENK) in the brain were also investigated after the end of 7-day HPC. HPC upregulated the neuronal expression of HIF-1α and DOR, and synchronously elevated the content of L-ENK in the rat brain. HIF-1α siRNA was used to further elucidate the relationship between HIF-1α and DOR in the HPC-treated brain. Knockdown of HIF-1α by siRNA markedly abrogated the HPC induced upregulation of HIF-1α and DOR. The present study demonstrates that the expression of DOR in the rat brain is upregulated by HIF-1α following exposure to 7-day HPC, at the same time, HPC also increases the production of endogenous DOR ligand L-ENK in the brain. DOR activation after HPC results in prolonged neuroprotection against subsequent global cerebral ischemic injury, suggesting a new mechanism of HPC-induced neuroprotection on global cerebral ischemia following CA and resuscitation.

Disturbance of calcium homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum (ER) are considered contributory components of cell death after ischemia. However, the signal-transducing events that are activated by ER stress after cerebral ischemia are incompletely understood. In this study, we show that caspase-12 and the PERK and IRE pathways are activated following oxygen-glucose deprivation (OGD) of mixed cortical cultures or neonatal hypoxia-ischemia (HI). Activation of PERK led to a transient phosphorylation of eIF2α, an increase in ATF4 levels and the induction of gadd34 (a subunit of an eIF2α-directed phosphatase). Interestingly, the upregulation of ATF4 did not lead to an increase in the levels of CHOP. Additionally, IRE1 activation was mediated by the increase in the processed form of xbp1, which would be responsible for the observed expression of edem2 and the increased levels of the chaperones GRP78 and GRP94. We were also able to detect caspase-12 proteolysis after HI or OGD. Processing of procaspase-12 was mediated by NMDA receptor and calpain activation. Moreover, our data suggest that caspase-12 activation is independent of the unfolded protein response activated by ER stress.

The multinucleated syncytial trophoblast, which forms the outermost layer of the placenta and serves multiple functions, is differentiated from and maintained by cytotrophoblast cell fusion. Deficiencies in syncytial trophoblast differentiation or maintenance likely contribute to intrauterine growth restriction and pre-eclampsia, two common gestational diseases. The cellular and molecular mechanisms governing trophoblast syncytialization are poorly understood. We report here that the proprotein convertase furin is highly expressed in syncytial trophoblast in the first trimester human placentas, and expression of furin in the syncytiotrophoblast is significantly lower in the placentas from pre-eclamptic patients as compared with their gestational age-matched control placentas. Using multiple experimental models including induced fusion of choriocarcinoma BeWo cells and spontaneous fusion of primary cultured cytotrophoblast cells or placental explants, we demonstrate that cytotrophoblast cell fusion and syncytialization are accompanied by furin expression. Furin-specific siRNAs or inhibitors inhibit cell fusion in BeWo cells, as well as trophoblast syncytialization in human placental explants. Furthermore, type 1 IGF receptor (IGF1R) is indicated in this study as a substrate of furin, and processing of IGF1R by furin is an essential mechanism for syncytialization. Finally, using lentivirus-mediated RNAi targeting to mouse trophectoderm, we demonstrate that furin function is required for the development of syncytiotrophoblast structure in the labyrinth layer, as well as for normal embryonic development.

Mitochondrial complex I inhibition has been implicated in the degeneration of midbrain dopaminergic (DA) neurons in Parkinson's disease. However, the mechanisms and pathways that determine the cellular fate of DA neurons downstream of the mitochondrial dysfunction have not been fully identified. We conducted cell-type specific gene array experiments with nigral DA neurons from rats treated with the complex I inhibitor, rotenone, at a dose that does not induce cell death. The genome wide screen identified transcriptional changes in multiple cell death related pathways that are indicative of a simultaneous activation of both degenerative and protective mechanisms. Quantitative PCR analyses of a subset of these genes in different neuronal populations of the basal ganglia revealed that some of the changes are specific for DA neurons, suggesting that these neurons are highly sensitive to rotenone. Our data provide insight into potentially defensive strategies of DA neurons against disease relevant insults.

Human age-related retinal diseases, such as age-related macular degeneration (AMD), are intimately associated with decreased tissue oxygenation and hypoxia. Different antioxidants have been investigated to reverse AMD. In the present study, we describe the antioxidant 17β-estradiol (βE2) and investigate its protective effects on retinal neurons. Fourteen days after ovariectomy, adult Sprague-Dawley rats were exposed to 8000-lux light for 12h to induce retinal degeneration. Reactive oxygen species (ROS) levels were assessed by confocal fluorescence microscopy using 2,7-dichlorofluorescein diacetate. Nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant enzyme mRNA expression were detected by real-time PCR. Western blotting was used to evaluate NRF2 activation. NRF2 translocation was determined by immunohistochemistry, with morphological changes monitored by hematoxylin and eosin staining. Following light exposure, βE2 significantly reduced ROS production. βE2 also up-regulated NRF2 mRNA and protein levels, with maximal expression at 4 and 12h post-exposure, respectively. Interestingly, following βE2 administration, NRF2 was translocated from the cytoplasm to the nucleus, primarily in the outer nuclear layer. βE2 also up-regulated NRF2, which triggered phase-2 antioxidant enzyme expression (superoxide dismutases 1 and 2, catalase, glutaredoxins 1 and 2, and thioredoxins 1 and 2), reduced ROS production, and ameliorated retinal damage. However, the beneficial effects of βE2 were markedly suppressed by pretreatment with LY294002 or ICI182780, specific inhibitors of the phosphatidylinositol 3-kinase-Akt (PI3K/AKT), and estrogen receptor (ER) signaling pathways, respectively. Taken together, these observations suggest that βE2 exerts antioxidative effects following light-induced retinal degeneration potentially via NRF2 activation. This protective mechanism may depend on two pathways: a rapid, non-genomic-type PI3K/AKT response, and a genomic-type ER-dependent response. Our data provide evidence that βE2 is a potentially effective in the treatment of retinal degeneration diseases.

Wearable and skin electronics benefit from mechanically soft and stretchable materials to conform to curved and dynamic surfaces, thereby enabling seamless integration with the human body. However, such materials are challenging to process using traditional microelectronics techniques. Here, stretchable transistor arrays are patterned exclusively from solution by inkjet printing of polymers and carbon nanotubes. The additive, non-contact and maskless nature of inkjet printing provides a simple, inexpensive and scalable route for stacking and patterning these chemically-sensitive materials over large areas. The transistors, which are stable at ambient conditions, display mobilities as high as 30 cm2 V-1 s-1 and currents per channel width of 0.2 mA cm-1 at operation voltages as low as 1 V, owing to the ionic character of their printed gate dielectric. Furthermore, these transistors with double-layer capacitive dielectric can mimic the synaptic behavior of neurons, making them interesting for conformal brain-machine interfaces and other wearable bioelectronics.

Tumor suppressor gene p53 is a critical regulator of the cellular response to DNA damage. To examine the function of p53 in postmitotic CNS neurons, we cultured cerebellar granule cells from 15-day-old wild type and p53-deficient mice, and analyzed changes of protein expression in apoptosis elicited by DNA damage. When cerebellar granule cells from wild type mice were treated with bleomycin, a DNA strand-break inducing agent, neuronal death occurred. In contrast, cells from p53-deficient mice were resistant to bleomycin-induced neuronal death. Furthermore, cells from p53 heterozygous mice showed an intermediate resistance between wild type and p53-deficient mice. These results show that p53 is required for the bleomycin-induced cerebellar granule cell death. To examine which proteins are involved in this apoptosis, we examined changes in protein levels of the Bcl-2 family, including Bcl-2, Bcl-X and Bax. The relative amounts of these proteins did not change after bleomycin treatment, suggesting that the changes in the levels of these Bcl-2 family proteins are not necessary for apoptosis in this system. In contrast, the levels of c-Jun protein significantly increased 6 h after treatment with bleomycin in wild type but not in p53-deficient cerebellar granule cells. These results raise the possibility that c-Jun is required for p53-dependent neuronal apoptosis induced by bleomycin.

We report a novel method for measuring forward and reverse kinetic rate constants, kf0 and kr0, for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion. Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunoglobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are Ackf0 = (2.6 +/- 0.32) x 10(-7) micron 4 s-1 and kr0 = (0.37 +/- 0.055) s-1 for the CD16A-hIgG interaction and Ackf0 = (5.7 +/- 0.31) X 10(-7) micron 4 s-1 and kr0 = (0.20 +/- 0.042) s-1 for the CD16A-rIgG interaction, respectively, where Ac is the contact area, estimated to be a few percent of 3 micron 2.

Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, thereby resulting in the activation of the spindle assembly checkpoint. Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed, that PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors.

Autophagy is a cellular recycling program that retards ageing by efficiently eliminating damaged and potentially harmful organelles and intracellular protein aggregates. Here, we show that the abundance of phosphatidylethanolamine (PE) positively regulates autophagy. Reduction of intracellular PE levels by knocking out either of the two yeast phosphatidylserine decarboxylases (PSD) accelerated chronological ageing-associated production of reactive oxygen species and death. Conversely, the artificial increase of intracellular PE levels, by provision of its precursor ethanolamine or by overexpression of the PE-generating enzyme Psd1, significantly increased autophagic flux, both in yeast and in mammalian cell culture. Importantly administration of ethanolamine was sufficient to extend the lifespan of yeast (Saccharomyces cerevisiae), mammalian cells (U2OS, H4) and flies (Drosophila melanogaster). We thus postulate that the availability of PE may constitute a bottleneck for functional autophagy and that organismal life or healthspan could be positively influenced by the consumption of ethanolamine-rich food.

The BAG family of Hsp70/Hsc70 co-chaperones is characterised by the presence of a conserved BAG domain at the carboxyl-terminus. BAG3 protein is the only member of this family containing also the N-terminally located WW domain. We describe here the identification of adenovirus (Ad) penton base protein as the first BAG3 partner recognising BAG3 WW domain. Ad penton base is the viral capsid constituent responsible for virus internalisation. It contains in the N-terminal part two conserved PPxY motifs, known ligands of WW domains. In cells producing Ad penton base protein, cytoplasmic endogenous BAG3 interacts with it and co-migrates to the nucleus. Preincubation of BAG3 with Ad base protein results in only slight modulation of BAG3 co-chaperone activity, suggesting that this interaction is not related to the classical BAG3 co-chaperone function. However, depletion of BAG3 impairs the cell entry of the virus and viral progeny production in Ad-infected cells, suggesting that the interaction between virus penton base protein and cellular co-chaperone BAG3 positively influences virus life cycle. These results thus demonstrate a novel host-pathogen interaction, which contributes to the successful infectious life cycle of adenoviruses. In addition, these data enrich our knowledge about the multifunctionality of the BAG3 co-chaperone.

The insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) is a member of a highly conserved protein family that is expressed specifically in placenta, testis and various cancers, but is hardly detectable in normal adult tissues. IGF2BP3 has important roles in RNA stabilization and translation, especially during early stages of both human and mouse embryogenesis. Placenta is an indispensable organ in mammalian reproduction that connects developing fetus to the uterine wall, and is responsible for nutrient uptake, waste elimination and gas exchange. Fetus development in the maternal uterine cavity depends on the specialized functional trophoblast. Whether IGF2BP3 plays a role in trophoblast differentiation during placental development has never been examined. The data obtained in this study revealed that IGF2BP3 was highly expressed in human placental villi during early pregnancy, especially in cytotrophoblast cells (CTBs) and trophoblast column, but a much lower level of IGF2BP3 was detected in the third trimester placental villi. Furthermore, the expression level of IGF2BP3 in pre-eclamptic (PE) placentas was significantly lower than the gestational age-matched normal placentas. The role of IGF2BP3 in human trophoblast differentiation was shown by in vitro cell invasion and migration assays and an ex vivo explant culture model. Our data support a role of IGF2BP3 in promoting trophoblast invasion and suggest that abnormal expression of IGF2BP3 might be associated with the etiology of PE.

Repeated exposure to cocaine during adolescence may affect both physical and psychological conditions in the brain, and increase the risk of psychiatric disorders and addiction behaviors in adulthood. Adolescence represents a critical development period for the hippocampus. Moreover, different regions of the hippocampus are involved in different functions. Dorsal hippocampus (dHP) has been implicated in learning and memory, whereas ventral hippocampus (vHP) plays an important role in emotional processing. In this study, the rats that were exposed to cocaine during adolescence (postnatal days, P28-P42) showed higher anxiety-like behavior in the elevated plus maze test in adulthood (P80), but displayed normal spatial learning and memory in the Morris water maze test. Furthermore, repeated exposure to cocaine during adolescence lead to alterations in morphology of pyramidal neurons, activities of astrocytes, and levels of proteins that involved in synaptic transmission, apoptosis, inflammation and addiction in both dHP and vHP of adult rats. These findings suggest that repeated exposure to cocaine during adolescence in rats may elicit morphologic and neurochemical changes in the hippocampus when the animals reach adulthood. These changes may contribute to the increased susceptibility for psychiatric disorders and addiction seen in adults.

Human trophoblast progenitor cells differentiate via two distinct pathways, to become the highly invasive extravillous cytotrophoblast (CTB) cells (EVT) or fuse to form syncytiotrophoblast. Inadequate trophoblast differentiation results in poor placenta perfusion, or even complications such as pre-eclampsia (PE). Cullin1 (CUL1), a scaffold protein in cullin-based ubiquitin ligases, plays an important role in early embryonic development. However, the role of CUL1 in trophoblast differentiation during placenta development has not been examined. Here we show that CUL1 was expressed in CTB cells and EVT in the first trimester human placentas by immunohistochemistry. CUL1 siRNA significantly inhibited outgrowth of extravillous explants in vitro, as well as invasion and migration of HTR8/SVneo cells of EVT origin. This inhibition was accompanied by decreased gelatinolytic activities of matrix metalloproteinase (MMP)-9 and increased expression of tissue inhibitors of MMPs (TIMP-1 and -2). Consistently, exogenous CUL1 promoted invasion and migration of HTR8/SVneo cells. Notably, CUL1 was gradually decreased during trophoblast syncytialization and CUL1 siRNA significantly enhanced forskolin-induced fusion of choriocarcinoma BeWo cells. CUL1 protein levels in human pre-eclamptic placental villi were significantly lower as compared to their matched control placentas. Taken together, our results suggest that CUL1 promotes human trophoblast cell invasion and dysregulation of CUL1 expression may be associated with PE.

Coronavirus disease 2019 (COVID-19) has caused a global pandemic associated with substantial morbidity and mortality. Nasopharyngeal swabs and sputum samples are generally collected for serial viral load screening of respiratory contagions, but temporal profiles of these samples are not completely clear in patients with COVID-19. We performed an observational cohort study at Renmin Hospital of Wuhan University, which involved 31 patients with confirmed COVID-19 with or without underlying diseases. We obtained samples from each patient, and serial viral load was measured by real-time quantitative polymerase chain reaction. We found that the viral load in the sputum was inclined to be higher than samples obtained from the nasopharyngeal swab at disease presentation. Moreover, the viral load in the sputum decreased more slowly over time than in the nasopharyngeal group as the disease progressed. Interestingly, even when samples in the nasopharyngeal swab turned negative, it was commonly observed that patients with underlying diseases, especially hypertension and diabetes, remained positive for COVID-19 and required a longer period for the sputum samples to turn negative. These combined findings emphasize the importance of tracking sputum samples even in patients with negative tests from nasopharyngeal swabs, especially for those with underlying conditions. In conclusion, this work reinforces the importance of sputum samples for SARS-CoV-2 detection to minimize transmission of COVID-19 within the community.

While general anesthetics control pain via consciousness regulation, local anesthetics (LAs) act by decreasing sensation in the localized area of administration by blocking nerve transmission to pain centers. Perioperative intra-articular administration of LAs is a commonly employed practice in orthopedic procedures to minimize patient surgical and post-surgical pain and discomfort. LAs are also co-administered with cellular mesenchymal stromal cell (MSC) therapies for a variety of tissue regenerative and inflammatory applications including osteoarthritis (OA) treatment; however, LAs can affect MSC viability and function. Therefore, finding an improved method to co-administer LAs with cells has become critically important. We have developed a sustained release LA delivery model that could enable the co-administration of LAs and MSCs. Encapsulation of liposomes within an alginate matrix leads to sustained release of bupivacaine as compared to bupivacaine-containing liposomes alone. Furthermore, drug release is maintained for a minimum of 4 days and the alginate-liposome capsules mitigated the adverse effects of bupivacaine on MSC viability.

Chronic pancreatitis (CP) is associated with elevated plasma levels of bacterial lipopolysaccharide (LPS) and we have demonstrated reduced acinar cell autophagy in human CP tissue. Therefore, we investigated the role of autophagy in experimental endotoxin-induced pancreatic injury and aimed to identify LPS in human CP tissue. Pancreatic Atg7-deficient mice were injected with a single sub-lethal dose of LPS. Expression of autophagy, apoptosis, necroptosis, and inflammatory markers was determined 3 and 24 h later utilizing immunoblotting and immunofluorescence. The presence of LPS in pancreatic tissue from mice and from patients and healthy controls was determined using immunohistochemistry, immunoblots, and chromogenic assay. Mice lacking pancreatic autophagy exhibited local signs of inflammation and were particularly sensitive to the toxic effect of LPS injection as compared to control mice. In response to LPS, Atg7Δpan mice exhibited enhanced vacuolization of pancreatic acinar cells, increase in TLR4 expression coupled to enhanced expression of NF-κΒ, JNK, and pro-inflammatory cytokines by acinar cells and enhanced infiltration by myeloid cells (but not Atg7F/F controls). Cell death was enhanced in Atg7Δpan pancreata, but only necroptosis and trypsin activation was further amplified following LPS injection along with elevated pancreatic LPS. The presence of LPS was identified in the pancreata from all 14 CP patients examined but was absent in the pancreata from all 10 normal controls. Altogether, these results support a potential role for metabolic endotoxemia in the pathogenesis of CP. Moreover, the evidence also supports the notion that autophagy plays a major cytoprotective and anti-inflammatory role in the pancreas, and blunting metabolic endotoxemia-induced CP.