Insoluble tau aggregates induce neuronal death through modification of membrane ion conductance, activation of voltage-gated calcium channels and NADPH oxidase.
Most neurodegenerative disorders are associated with aggregation and accumulation of misfolded proteins. One of these proteins – tau, is involved in a number of pathologies including Alzheimer’s disease and frontotemporal dementia. Aggregation and phosphorylation of tau have been shown to be a trigger for abnormal signal transduction and disruption of cellular homeostasis. Here, we have studied the effect of extracellular tau at different stages of aggregation in cortical co-cultures of neurons and astrocytes, to understand how this process affects tau pathogenicity. We found that the species formed after prolonged in vitro aggregation of tau (longer than one day) are able to stimulate ROS production through the activation of NADPH oxidase without decreasing the level of the endogenous antioxidant glutathione. The same late insoluble aggregates of tau induced calcium signals in neurons and a gradual increase in the ionic current of artificial membranes. Both tau-induced calcium signals and ROS production in NADPH oxidase were reduced in the presence of the inhibitor of voltage-gated calcium channels (VGCC) nifedipine. This suggests that insoluble aggregates of tau incorporate into the membrane and modify ionic currents, changing plasma membrane potential and activating VGCCs, which induces a calcium influx that triggers ROS production in NADPH oxidase. The combination of all these effects likely leads to toxicity, as only the same insoluble tau aggregates which demonstrated membrane-active properties produced neuronal cell death.
Conserved strategies for pathogen evasion of cGAS-STING immunity.
The cyclic GMP-AMP synthase (cGAS)- Stimulator of Interferon Genes (STING) pathway of cytosolic DNA sensing allows mammalian cells to detect and respond to infection with diverse pathogens. Pathogens in turn encode numerous factors that inhibit nearly all steps of cGAS-STING signal transduction. From masking of cytosolic DNA ligands, to post-translational modification of cGAS and STING, and degradation of the nucleotide second messenger 2’3′-cGAMP, pathogens have evolved convergent mechanisms to evade cGAS-STING sensing. Here we examine pathogen inhibitors of innate immunity in the context of newly discovered regulatory features controlling cellular cGAS-STING activation. Comparative analysis of these strategies provides insight into mechanisms of action and suggests aspects of cGAS-STING regulation and immune evasion that remain to be discovered.
EMA Review of Axicabtagene Ciloleucel (Yescarta) for the Treatment of Diffuse Large B-Cell Lymphoma.
On June 28, 2018, the Committee for Advanced Therapies and the Committee for Medicinal Products for Human Use adopted a positive opinion, recommending the granting of a marketing authorization for the medicinal product Yescarta for the treatment of adult patients with relapsed or refractory diffuse large B-cell lymphoma and primary mediastinal large B-cell lymphoma, after two or more lines of systemic therapy. Yescarta, which was designated as an orphan medicinal product and included in the European Medicines Agency’s Priority Medicines scheme, was granted an accelerated review timetable. The active substance of Yescarta is axicabtagene ciloleucel, an engineered autologous T-cell immunotherapy product whereby a patient’s own T cells are harvested and genetically modified ex vivo by retroviral transduction using a retroviral vector to express a chimeric antigen receptor (CAR) comprising an anti-CD19 single chain variable fragment linked to CD28 costimulatory domain and CD3-zeta signaling domain. The transduced anti-CD19 CAR T cells are expanded ex vivo and infused back into the patient, where they can recognize and eliminate CD19-expressing cells. The benefits of Yescarta as studied in ZUMA-1 phase II (NCT02348216) were an overall response rate per central review of 66% (95% confidence interval, 56%-75%) at a median follow-up of 15.1 months in the intention to treat population and a complete response rate of 47% with a significant duration. The most common adverse events were cytokine release syndrome, neurological adverse events, infections, pyrexia, diarrhea, nausea, hypotension, and fatigue. IMPLICATIONS FOR PRACTICE: Yescarta (axicabtagene ciloleucel) was the first chimeric antigen receptor T-cell therapy to be submitted for evaluation to the European Medicines Agency and admitted into the “priority medicine” scheme; it was granted accelerated assessment on the basis of anticipated clinical benefit in relapsed/refractory diffuse large B-cell lymphoma, a condition of unmet medical need. Indeed, Yescarta showed an overall response rate of 66% and a complete response rate of 47% with a significant duration and a manageable toxicity that compared very favorably with historical controls. Here the analysis of benefits and risks is presented, and specific challenges with this important novel product are highlighted, providing further insights and reflections for future medical research.
Transgenic expression of Sag/Rbx2 E3 causes early stage tumor promotion, late stage cytogenesis and acinar loss in the Kras-PDAC model.
SAG (Sensitive to Apoptosis Gene), also known as RBX2 or ROC2, is a RING component of CRL (Cullin-RING ligase), required for its activity. Our previous studies showed that Sag/Rbx2 co-operated with Kras or Pten loss to promote tumorigenesis in the lung and prostate, respectively, but antagonized Kras to inhibit skin tumorigenesis, suggesting a tissue/context dependent function of Sag. The role of SAG in KRAS-induced pancreatic tumorigenesis is unknown. In this study, we mined a cancer database and found that SAG is overexpressed in pancreatic cancer tissues and correlates with decreased patient survival. Whether Sag overexpression plays a causal role in pancreatic tumorigenesis is unknown. Here, we reported the generation of Sag transgenic mouse model alone (CS), or in combination with KrasG12D, driven by p48-Cre (KCS mice) for pancreatic specific Sag expression. Sag transgenic expression alone has no phenotypical abnormality, but in combination with KrasG12D promotes ADM (acinar-to-ductal metaplasia) conversion in vitro and mPanIN1 formation in vivo at the early stage, and impairs pancreatic functions at the late stage, as evidenced by poor glucose tolerance and significantly reduced α-Amylase activity, and induction of cytogenesis and acinar cell loss, eventually leading to atrophic pancreata and shortened mouse life-span. Mechanistically, Sag transgenic expression altered several key signaling pathways, particularly inactivation of mTORC1 signaling due to Deptor accumulation, and activation of the antioxidant Nrf2-Nqo1 axis. Thus, Sag plays a stage dependent promotion (early) and fate-changing (late) role during Kras-pancreatic tumorigenesis, likely via regulating its key substrates, which control growth-related signal transduction pathways.
Alteration of glycinergic receptor expression in lumbar spinal motoneurons is involved in the mechanisms underlying spasticity after spinal cord injury.
Spasticity is a disabling motor disorder affecting 70% of people with brain and spinal cord injury. The rate-dependent depression (RDD) of the H reflex is the only electrophysiological measurement correlated with the degree of spasticity assessed clinically in spastic patients. Several lines of evidence suggest that the mechanism underlying the H reflex RDD depends on the strength of synaptic inhibition through GABAA (GABAAR) and glycine receptors (GlyR). In adult rats with spinal cord transection (SCT), we studied the time course of the expression of GABAAR and GlyR at the membrane of retrogradely identified Gastrocnemius and Tibialis anterior motoneurons (MNs) 3, 8 and 16 weeks after injury, and measured the RDD of the H reflex at similar post lesion times. Three weeks after SCT, a significant decrease in the expression of GABAA and GlyR was observed compared to intact rats, and the H-reﬂex RDD was much less pronounced than in controls. Eight weeks after SCT, GlyR values returned to normal. Simultaneously, we observed a tendency to recover normal RDD of the H reflex at higher frequencies. We tested whether an anti-inflammatory treatment using methylprednisolone performed immediately after SCT could prevent alterations in GABAA/glycine receptors and/or the development of spasticity observed 3 weeks after injury. This treatment restored control levels of GlyR but not the expression of GABAAR, and it completely prevented the attenuation of RDD. These data strongly suggest that alteration of glycinergic inhibition of lumbar MNs is involved in the mechanisms underlying spasticity after SCI.
Cancer stem cells in Epstein-Barr virus-associated gastric carcinoma.
Cancer stem cells (CSCs) play a decisive role in the development and progression of cancer. To investigate CSCs in Epstein-Barr virus (EBV)-associated carcinoma (EBVaGC), we screened previously reported stem cell markers of gastric cancer in EBV-infected gastric cancer cell lines (TMK1, NUGC3) and identified CD44v6v9 double positive cells as candidate CSCs. CD44v6/v9+/+ cells were sorted from EBVaGC cell line (SNU719) cells and EBV-infected TMK1 cells and these cell populations showed high spheroid forming ability and tumor formation in severe combined immunodeficient mice compared with the respective CD44v6/v9-/- cells. Sphere forming ability was dependent on the NF-kB signaling pathway, which was confirmed by decrease of sphere formation ability under BAY 11-7082. Small interfering RNA-knockdown of latent membrane protein 2A (LMP2A), one of the latent gene products of EBV infection, decreased spheroid formation in SNU719 cells. Transfection of the LMP2A gene increased the sphere-forming ability of TMK1 cells, which was mediated through NF-kB signaling. Together, these results indicate that CD44v6v9 double positive cells are CSCs in EBVaGC that are maintained through the LMP2A/NF-kB pathway. Future studies should investigate CD44v6/v9+/+ cells in normal and neoplastic gastric epithelium to prevent and treat this specific subtype of gastric cancer infected with EBV. Five keywords, for the purposes of indexing: Carcinoma, Epstein-Barr Virus Infections, Neoplastic Stem Cells, Signal Transduction, Stomach Neoplasms.
Baicalein attenuates caspase-independent cells death via inhibiting PARP-1 activation and AIF nuclear translocation in cerebral ischemia/reperfusion rats.
It is reported that baicalein can activate PI3K/AKT pathway, inhibit caspase activation and reduce cerebral infarct volume in middle cerebral artery occlusion (MCAO) rats. However, a caspase-independent mechanism initiated by poly (ADP-ribose) polymerase-1 (PARP-1) activation has been reported to make more contribution to cells death after ischemic stroke. In the present study, we established a cerebral ischemia/reperfusion (I/R) rat model through middle cerebral artery occlusion following reperfusion to investigate the mechanisms of ischemic tissue recovery following baicalein treatment. The data showed that baicalein treatment at dose of 100 mg/kg for 7 days significantly inhibited the release of cytokines, activation of PARP-1, nuclear translocation of apoptosis-inducing factor (AIF) and macrophage migration inhibitory factor (MIF) in cerebral I/R rats, therefore decreased cerebral infarct volume and neurological scores. Then, we further investigated the signal transduction mechanisms of ischemic tissue protection by baicalein in vitro. Following oxygen and glucose deprivation (OGD) in SH-SY5Y cells, the mitochondrial AIF was translocated into nucleus after 12 h. The co-immunoprecipitation analysis showed that the interaction between AIF and MIF was activated by OGD and subsequently resulted in MIF nuclear translocation. Also, the baicalein inhibited apoptosis, reduced oxidative stress, protected mitochondrial function and restored mitochondrial membrane potential in OGD cells. The results obtained from both in vivo and in vitro study demonstrated the PARP-1/AIF pathway involved in mechanisms of baicalein to protect the cerebral tissues from ischemic injury.
Assessing site-specific enhancements imparted by hyperpolarized water in folded and unfolded proteins by 2D HMQC NMR.
Dissolution DNP of hyperpolarized water can be a valuable aid in biomolecular NMR. With suitable optimizations one can utilize it to achieve, under near-physiological conditions, amide group 1H polarizations that are orders of magnitude larger than their thermal counterparts. Suitable experimental procedures can exploit this hyperpolarization to deliver 2D 1H-15N NMR correlations, with good site resolution and enhanced sensitivity. The resulting signal enhancements depend on the exchange rates between the amides and the water protons, thereby yielding diagnostic information about the former’s solvent accessibility. This study applied the “HyperW” NMR method to four proteins, which exhibit a gamut of exchange behaviors. These included PhoA(350-471), an unfolded 122-residue fragment of Alkaline Phosphatase from E. coli; barstar, a fully folded ribonuclease inhibitor from Bacillus amyloliquefaciens; R17, a 13.3 kDa system possessing folded and unfolded forms under slow interconversion; and drkN SH3, an N-terminal signal transduction protein domain whose folded and unfolded forms interchange more rapidly and with temperature-dependent population ratios. For the disordered PhoA4(350-471) fragment 2D HyperW sensitivity enhancements were very high, ≥300× over their thermal counterparts; this was expected due to the fast amide proton exchanges that occur throughout this unfolded protein sequence. Though fully folded, barstar also exhibited substantially-enhanced residues; these, however, were not uniform, and reflected what appeared well-folded but solvent-exposed residues, as confirmed by ancillary CLEANEX experiments. R17 showed in turn the expected superposition of ≥100-fold enhancements for its unfolded form, coexisting with more modest enhancement for the folded counterparts. Unexpected, however, was the behavior of the drkN SH3 fragment, for which HyperW substantially enhanced both folded and unfolded states -but foremost of all certain sites of the folded protein. These preferential enhancements of folded over unfolded amide signals were repeatedly and reproducibly observed and a number of explanations – including three-site exchange magnetization transfers between water protons and amide protons from the unfolded and folded states, the possibility that faster rates of solvent exchange characterize the folded sites than their unfolded counterparts, and cross-correlated relaxation processes from hyperpolarized “structural” waters and labile sidechain protons that bias the enhancements towards the folded state- were considered to account for them.
Where is my infusion pump? Harnessing network dynamics for improved hospital equipment fleet management.
Timely availability of intravenous infusion pumps is critical for high-quality care delivery. Pumps are shared among hospital units, often without central management of their distribution. This study seeks to characterize unit-to-unit pump sharing and its impact on shortages, and to evaluate a system-control tool that balances inventory across all care areas, enabling increased availability of pumps.A retrospective study of 3832 pumps moving in a network of 5292 radiofrequency and infrared sensors from January to November 2017 at The Johns Hopkins Hospital in Baltimore, Maryland. We used network analysis to determine whether pump inventory in one unit was associated with inventory fluctuations in others. We used a quasi-experimental design and segmented regressions to evaluate the effect of the system-control tool on enabling safe inventory levels in all care areas.We found 93 care areas connected through 67,111 pump transactions and 4 discernible clusters of pump sharing. Up to 17% (95% confidence interval, 7%-27%) of a unit’s pump inventory was explained by the inventory of other units within its cluster. The network analysis supported design and deployment of a hospital-wide inventory balancing system, which resulted in a 44% (95% confidence interval, 36%-53%) increase in the number of care areas above safe inventory levels.Network phenomena are essential inputs to hospital equipment fleet management. Consequently, benefits of improved inventory management in strategic unit(s) are capable of spreading safer inventory levels throughout the hospital.
VDAC and its interacting partners in plant and animal systems: an overview.
Molecular trafficking between different subcellular compartments is the key for normal cellular functioning. Voltage-dependent anion channels (VDACs) are small-sized proteins present in the outer mitochondrial membrane, which mediate molecular trafficking between mitochondria and cytoplasm. The conductivity of VDAC is dependent on the transmembrane voltage, its oligomeric state and membrane lipids. VDAC acts as a convergence point to a diverse variety of mitochondrial functions as well as cell survival. This functional diversity is attained due to their interaction with a plethora of proteins inside the cell. Although, there are hints toward functional conservation/divergence between animals and plants; knowledge about the functional role of the VDACs in plants is still limited. We present here a comparative overview to provide an integrative picture of the interactions of VDAC with different proteins in both animals and plants. Also discussed are their physiological functions from the perspective of cellular movements, signal transduction, cellular fate, disease and development. This in-depth knowledge of the biological importance of VDAC and its interacting partner(s) will assist us to explore their function in the applied context in both plant and animal.
Bowel and ureteral assessment by indocyanine green real-time visualization during deep infiltrating endometriosis surgery – Video vignette.
To report the utility of intraoperative use of indocyanine green (ICG) during deep infiltrating endometriosis (DIE) laparoscopic surgery for intestinal and ureteral assessment.A video on the use of ICG to assess bowel perfusion after intestinal resection and on the use of intra-ureteral perfusion of ICG. A 6 Fr ureteral catheter was introduced in to each ureters at cystoscopy and 7 ml ICG solution (1,25mg/ml of distilled water) was injected. The catheter was then removed. Using a near- infrared camera (OLYMPUS Medical System Europa and STRYKER Iberia S.L. Madrid,Spain), the fluorescent ureters could be visualized at laparoscopy. After rectal segmental resection, 4ml of ICG solution was injected intravenously to check the vascularity at the site of transection and the anastomosis further checked at ICG-rectoscopy, using a 12 mm- Kii balloon blunt tip (Applied Medical, Europe) RESULTS: The ICG fluorescence may have a role in helping to identify and follow the course of the pelvic ureter especially when the anatomy is distorted. It has the added advantage ureteral fluorescence is detectable for at least 6 hours after injection of ICG. Intravenous ICG also provided a visual assessment of anastomotic perfusion after discoid and segmental bowel resections at laparoscopy and rectoscopy.ICG enhanced-fluorescence is a feasible, safe and reproducible technology in DIE surgery. It provides a real time evaluation of ureters and bowel. Finally, its adds little to the cost of a standard laparoscopic operation.
The effect of kaempferol and apigenin on allogenic synovial membrane-derived stem cells therapy in knee osteoarthritic male rats.
Based on the anti-inflammatory and anti-oxidant properties of kaempferol and apigenin, we hypothesized that co-injection of these phytochemicals would increase the effectiveness of cell therapy in knee osteoarthritic rats.Anterior cruciate ligament transection (ACLT) was used to induce osteoarthritis (OA). Animals were treated by weekly intra-articular injections of kaempferol (10 or 20 μM) and/or isolated MSCs from synovial membrane (SMMSCs) (3 × 106 cells), a mixture of apigenin (0.1 μM) and kaempferol.