Chronic infection with a tissue-invasive helminth attenuates sublethal anaphylaxis and reduces granularity and number of mast cells.

BACKGROUND: Immunoglobulin E (IgE)-mediated anaphylaxis is a potentially fatal condition in which allergy effector cells rapidly discharge pre-formed inflammatory mediators. Treatments that address the immune component of allergic anaphylaxis are inadequate. Helminths have been previously shown to suppress effector cell function; however, their ability to treat pre-existing allergy remains unclear. OBJECTIVE: To evaluate the ability of chronic helminth infection to protect against anaphylaxis in previously sensitized mice. METHODS: A sublethal model of anaphylaxis was used, in which BALB/c mice were sensitized by three intraperitoneal (i.p.) injections of OVA/alum. Temperature drop was then monitored after systemic OVA challenge in uninfected mice and in mice infected chronically with Litomosoides sigmodontis, a tissue-invasive filarial nematode. RESULTS: Litomosoides sigmodontis-infected mice exhibited significantly lower serum levels of mMCP-1 and were less hypothermic at 30-minute post-challenge compared to uninfected OVA-challenged controls. Characterization of anaphylaxis revealed that FcԑR1 and mast cells were required for hypothermia and elevated serum mMCP-1. OVA-IgE and OVA-IgG1 serum levels were not significantly altered by L sigmodontis infection, and experiments with IL-10-/- mice demonstrated that IL-10 was not required for protection against anaphylaxis. However, peritoneal mast cell numbers were significantly lower in infected mice, and those that were present exhibited decreased granularity by flow cytometry and marked depletion of intracytoplasmic granules by light microscopy. Mast cells from infected mice had lower expression of the activation markers CD200R and CD63 and contained significantly lower basal stores of histamine. CONCLUSIONS: Chronic L sigmodontis infection protects against anaphylaxis, likely due to reduction in mast cell numbers and depletion of pre-formed inflammatory mediators in remaining mast cells.

Gain-of-function mutations in CARD11 promote enhanced aggregation and idiosyncratic signalosome assembly.

BENTA (B cell Expansion with NF-κB and T cell Anergy) is a novel lymphoproliferative disorder caused by germline, gain-of-function (GOF) mutations in the lymphocyte-restricted scaffolding protein CARD11. Similar somatic CARD11 mutations are found in lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL). Normally, antigen receptor (AgR) engagement converts CARD11 into an active conformation that nucleates a signalosome required for IκB kinase (IKK) activation and NF-κB nuclear translocation. However, GOF CARD11 mutants drive constitutive NF-κB activity without AgR stimulation. Here we show that unlike wild-type CARD11, GOF CARD11 mutants can form large, peculiar cytosolic protein aggregates we term mCADS (mutant CARD11 dependent shells). MALT1 and phospho-IKK are reliably colocalized with mCADS, indicative of active signaling. Moreover, endogenous mCADS are detectable in ABC-DLBCL lines harboring similar GOF CARD11 mutations. The unique aggregation potential of GOF CARD11 mutants may represent a novel therapeutic target for treating BENTA or DLBCL.

Autophagy and mitochondrial remodelling in mouse mesenchymal stromal cells challenged with Staphylococcus epidermidis.

The bone marrow stroma constitutes the marrow-blood barrier, which sustains immunochemical homoeostasis and protection of the haematopoietic tissue in sequelae of systemic bacterial infections. Under these conditions, the bone marrow stromal cells affected by circulating bacterial pathogens shall elicit the adaptive stress-response mechanisms to maintain integrity of the barrier. The objective of this communication was to demonstrate (i) that in vitro challenge of mesenchymal stromal cells, i.e. colony-forming unit fibroblasts (CFU-F), with Staphylococcus epidermidis can activate the autophagy pathway to execute antibacterial defence response, and (ii) that homoeostatic shift because of the bacteria-induced stress includes the mitochondrial remodelling and sequestration of compromised organelles via mitophagy. Implication of Drp1 and PINK1-PARK2-dependent mechanisms in the mitophagy turnover of the aberrant mitochondria in mesenchymal stromal cells is investigated and discussed.

Sustained generation of neurons destined for neocortex with oxidative metabolic upregulation upon filamin abrogation.

Neurons in the neocortex are generated during embryonic development. While the adult ventricular-subventricular zone (V-SVZ) contains cells with neural stem/progenitors' characteristics, it remains unclear whether it has the capacity of producing neocortical neurons. Here, we show that generating neurons with transcriptomic resemblance to upper layer neocortical neurons continues in the V-SVZ of mouse models of a human condition known as periventricular heterotopia by abrogating Flna and Flnb. We found such surplus neurogenesis was associated with V-SVZ's upregulation of oxidative phosphorylation, mitochondrial biogenesis, and vascular abundance. Additionally, spatial transcriptomics analyses showed V-SVZ's neurogenic activation was coupled with transcriptional enrichment of genes in diverse pathways for energy metabolism, angiogenesis, cell signaling, synaptic transmission, and turnovers of nucleic acids and proteins in upper cortical layers. These findings support the potential of generating neocortical neurons in adulthood through boosting brain-wide vascular circulation, aerobic adenosine triphosphate synthesis, metabolic turnover, and neuronal activity.

Lack of FVIII detection in humans and dogs with an intron-22 inversion challenges hypothesis regarding inhibitor risk.

BACKGROUND: Almost half of severe hemophilia A (HA) cases are caused by an intron-22 inversion mutation (Int22Inv), which truncates the 26-exon F8 mRNA after exon 22. Another F8 transcript, F8B, is initiated from within F8-intron-22. F8B mRNA consists of a short exon spliced to exons 23-26 and is expressed in multiple human cell types. It has been hypothesized that Int22Inv patients have self-tolerance to partial FVIII proteins expressed from these two transcripts. FVIII is expressed in endothelial cells, primarily in liver and lung. Several studies have reported FVIII expression in other cell types, although this has been controversial. OBJECTIVES: To determine if partial FVIII proteins are expressed from intron 22-inverted and/or F8B mRNA and if FVIII is expressed in non-endothelial cells. METHODS: A panel of FVIII-specific antibodies was validated and employed to label FVIII in cells and tissues, and for immunoprecipitation followed by western blots and mass spectrometry-proteomics analysis. RESULTS: Immunofluorescent (IF) staining localized FVIII to endothelial cells in liver sections from non-HA but not HA-Int22Inv dogs. Neither FVIII nor FVIIIB was detected in human PBMCs, B-cell or T-cell lines, or in cell lines expanded from PBMCs, whereas FVIII antigen and activity were readily detected in primary non-hemophilic liver sinusoidal endothelial cells. CONCLUSIONS: If FVIII is expressed in non-endothelial cells, or if partial FVIII proteins are expressed in HA-Int22Inv, the concentrations are below the detection limits of these sensitive assays. Our results argue against promotion of immune tolerance through expression of partial FVIII proteins in Int-22Inv patients.

Live-Cell Imaging of Dynein-Mediated Cargo Transport in Aspergillus nidulans.

Filamentous fungi have been used for studying long-distance transport of cargoes driven by cytoplasmic dynein. Aspergillus nidulans is a well-established genetic model organism used for studying dynein function and regulation in vivo. Here, we describe how we grow A. nidulans strains for live-cell imaging and how we observe the dynein-mediated distribution of early endosomes and secretory vesicles. Using an on-stage incubator and culture chambers for inverted microscopes, we can image fungal hyphae that naturally attach to the bottom of the chambers, using wide-field epifluorescence microscopes or the new Zeiss LSM 980 (with Airyscan 2) microscope. In addition to methods for preparing cells for imaging, a procedure for A. nidulans transformation is also described.

Diverse Enterococcus faecalis strains show heterogeneity in biofilm properties.

Biofilm formation is important for Enterococcus faecalis to cause healthcare-associated infections. It is unclear how E. faecalis biofilms vary in parameters such as development and composition. To test the hypothesis that differences in biofilms exist among E. faecalis strains, we evaluated in vitro biofilm formation and matrix characteristics of five genetically diverse E. faecalis lab-adapted strains and clinical isolates (OG1RF, V583, DS16, MMH594, and VA1128). Biofilm formation of all strains was repressed in TSB+10% FBS. However, DMEM+10% FBS enhanced biofilm formation of clinical isolate VA1128. Crystal violet staining and fluorescence microscopy of biofilms grown on Aclar membranes demonstrated differences between OG1RF and VA1128 in biofilm development over a 48-h time course. None of the biofilms were dispersed by single treatments of sodium (meta)periodate, DNase, or Proteinase K alone, but the biofilm biomass of both OG1RF and DS16 was partially removed by a sequential treatment of sodium (meta)periodate and DNase. Reversing the treatment order was not effective, suggesting that the extracellular DNA targeted by DNase was obscured by carbohydrates that are susceptible to sodium (meta)periodate degradation. Fluorescent staining of biofilm matrix components further demonstrated that more carbohydrates bound by wheat germ agglutinin comprise OG1RF biofilms compared to VA1128 biofilms. This study highlights the existence of heterogeneity in biofilm properties among diverse E. faecalis strains, which may have implications for the design of novel anti-biofilm treatment strategies.

The oligo-acyl lysyl antimicrobial peptide C12K-2ß12 exhibits a dual mechanism of action and demonstrates strong in vivo efficacy against Helicobacter pylori.

Helicobacter pylori has developed antimicrobial resistance to virtually all current antibiotics. Thus, there is a pressing need to develop new anti-H. pylori therapies. We recently described a novel oligo-acyl-lysyl (OAK) antimicrobial peptidomimetic, C(12)K-2ß(12), that shows potent in vitro bactericidal activity against H. pylori. Herein, we define the mechanism of action and evaluate the in vivo efficacy of C(12)K-2ß(12) against H. pylori after experimental infection of Mongolian gerbils. We demonstrate using a 1-N-phenylnaphthylamine (fluorescent probe) uptake assay and electron microscopy that C(12)K-2ß(12) rapidly permeabilizes the bacterial membrane and creates pores that cause bacterial cell lysis. Furthermore, using nucleic acid binding assays, Western blots, and confocal microscopy, we show that C(12)K-2ß(12) can cross the bacterial membranes into the cytoplasm and tightly bind to bacterial DNA, RNA, and proteins, a property that may result in inhibition of enzymatic activities and macromolecule synthesis. To define the in vivo efficacy of C(12)K-2ß(12), H. pylori-infected gerbils were orogastrically treated with increasing doses and concentrations of C(12)K-2ß(12) 1 day or 1 week postinfection. The efficacy of C(12)K-2ß(12) was strongest in animals that received the largest number of doses at the highest concentration, indicating dose-dependent activity of the peptide (P < 0.001 by analysis of variance [ANOVA]) regardless of the timing of the treatment with C(12)K-2ß(12). Overall, our results demonstrate a dual mode of action of C(12)K-2ß(12) against the H. pylori membrane and cytoplasmic components. Moreover, and consistent with the previously reported in vitro efficacy, C(12)K-2ß(12) shows significant in vivo efficacy against H. pylori when used as monotherapy. Therefore, OAK peptides may be a valuable resource for therapeutic treatment of H. pylori infection.

CARD19 Interacts with Mitochondrial Contact Site and Cristae Organizing System Constituent Proteins and Regulates Cristae Morphology.

CARD19 is a mitochondrial protein of unknown function. While CARD19 was originally reported to regulate TCR-dependent NF-κB activation via interaction with BCL10, this function is not recapitulated ex vivo in primary murine CD8+ T cells. Here, we employ a combination of SIM, TEM, and confocal microscopy, along with proteinase K protection assays and proteomics approaches, to identify interacting partners of CARD19 in macrophages. Our data show that CARD19 is specifically localized to the outer mitochondrial membrane. Through deletion of functional domains, we demonstrate that both the distal C-terminus and transmembrane domain are required for mitochondrial targeting, whereas the CARD is not. Importantly, mass spectrometry analysis of 3×Myc-CARD19 immunoprecipitates reveals that CARD19 interacts with the components of the mitochondrial intermembrane bridge (MIB), consisting of mitochondrial contact site and cristae organizing system (MICOS) components MIC19, MIC25, and MIC60, and MICOS-interacting proteins SAMM50 and MTX2. These CARD19 interactions are in part dependent on a properly folded CARD. Consistent with previously reported phenotypes upon siRNA silencing of MICOS subunits, absence of CARD19 correlates with irregular cristae morphology. Based on these data, we propose that CARD19 is a previously unknown interacting partner of the MIB and the MIC19-MIC25-MIC60 MICOS subcomplex that regulates cristae morphology.

Tourniquet-induced lower limb ischemia/reperfusion reduces mitochondrial function by decreasing mitochondrial biogenesis in acute kidney injury in mice.

The mechanisms by which lower limb ischemia/reperfusion induces acute kidney injury (AKI) remain largely uncharacterized. We hypothesized that tourniquet-induced lower limb ischemia/reperfusion (TILLIR) would inhibit mitochondrial function in the renal cortex. We used a murine model to show that TILLIR of the high thigh regions inflicted time-dependent AKI as determined by renal function and histology. This effect was associated with decreased activities of mitochondrial complexes I, II, V and citrate synthase in the kidney cortex. Moreover, TILLIR reduced mRNA levels of a master regulator of mitochondrial biogenesis PGC-1α, and its downstream genes NDUFS1 and ATP5o in the renal cortex. TILLIR also increased serum corticosterone concentrations. TILLIR did not significantly affect protein levels of the critical regulators of mitophagy PINK1 and PARK2, mitochondrial transport proteins Tom20 and Tom70, or heat-shock protein 27. TILLIR had no significant effect on mitochondrial oxidative stress as determined by mitochondrial ability to generate reactive oxygen species, protein carbonylation, or protein levels of MnSOD and peroxiredoxin1. However, TILLIR inhibited classic autophagic flux by increasing p62 protein abundance and preventing the conversion of LC3-I to LC3-II. TILLIR increased phosphorylation of cytosolic and mitochondrial ERK1/2 and mitochondrial AKT1, as well as mitochondrial SGK1 activity. In conclusion, lower limb ischemia/reperfusion induces distal AKI by inhibiting mitochondrial function through reducing mitochondrial biogenesis. This AKI occurs without significantly affecting PINK1-PARK2-mediated mitophagy or mitochondrial oxidative stress in the kidney cortex.

Nde1 is required for heterochromatin compaction and stability in neocortical neurons.

The NDE1 gene encodes a scaffold protein essential for brain development. Although biallelic NDE1 loss of function (LOF) causes microcephaly with profound mental retardation, NDE1 missense mutations and copy number variations are associated with multiple neuropsychiatric disorders. However, the etiology of the diverse phenotypes resulting from NDE1 aberrations remains elusive. Here we demonstrate Nde1 controls neurogenesis through facilitating H4K20 trimethylation-mediated heterochromatin compaction. This mechanism patterns diverse chromatin landscapes and stabilizes constitutive heterochromatin of neocortical neurons. We demonstrate that NDE1 can undergo dynamic liquid-liquid phase separation, partitioning to the nucleus and interacting with pericentromeric and centromeric satellite repeats. Nde1 LOF results in nuclear architecture aberrations and DNA double-strand breaks, as well as instability and derepression of pericentromeric satellite repeats in neocortical neurons. These findings uncover a pivotal role of NDE1/Nde1 in establishing and protecting neuronal heterochromatin. They suggest that heterochromatin instability predisposes a wide range of brain dysfunction.

Genetic inactivation of SARM1 axon degeneration pathway improves outcome trajectory after experimental traumatic brain injury based on pathological, radiological, and functional measures.

Traumatic brain injury (TBI) causes chronic symptoms and increased risk of neurodegeneration. Axons in white matter tracts, such as the corpus callosum (CC), are critical components of neural circuits and particularly vulnerable to TBI. Treatments are needed to protect axons from traumatic injury and mitigate post-traumatic neurodegeneration. SARM1 protein is a central driver of axon degeneration through a conserved molecular pathway. Sarm1-/- mice with knockout (KO) of the Sarm1 gene enable genetic proof-of-concept testing of the SARM1 pathway as a therapeutic target. We evaluated Sarm1 deletion effects after TBI using a concussive model that causes traumatic axonal injury and progresses to CC atrophy at 10 weeks, indicating post-traumatic neurodegeneration. Sarm1 wild-type (WT) mice developed significant CC atrophy that was reduced in Sarm1 KO mice. Ultrastructural classification of pathology of individual axons, using electron microscopy, demonstrated that Sarm1 KO preserved more intact axons and reduced damaged or demyelinated axons. Longitudinal MRI studies in live mice identified significantly reduced CC volume after TBI in Sarm1 WT mice that was attenuated in Sarm1 KO mice. MR diffusion tensor imaging detected reduced fractional anisotropy in both genotypes while axial diffusivity remained higher in Sarm1 KO mice. Immunohistochemistry revealed significant attenuation of CC atrophy, myelin loss, and neuroinflammation in Sarm1 KO mice after TBI. Functionally, Sarm1 KO mice exhibited beneficial effects in motor learning and sleep behavior. Based on these findings, Sarm1 inactivation can protect axons and white matter tracts to improve translational outcomes associated with CC atrophy and post-traumatic neurodegeneration.

Sarm1 deletion reduces axon damage, demyelination, and white matter atrophy after experimental traumatic brain injury.

Traumatic brain injury (TBI) often damages axons in white matter tracts and causes corpus callosum (CC) atrophy in chronic TBI patients. Injured axons encounter irreversible damage if transected, or alternatively may maintain continuity and subsequently either recover or degenerate. Secondary mechanisms can cause further axon damage, myelin pathology, and neuroinflammation. Molecular mechanisms regulating the progression of white matter pathology indicate potential therapeutic targets. SARM1 is essential for execution of the conserved axon death pathway. We examined white matter pathology following mild TBI with CC traumatic axonal injury in mice with Sarm1 gene deletion (Sarm1-/-). High resolution ultrastructural analysis at 3 days post-TBI revealed dramatically reduced axon damage in Sarm1-/- mice, as compared to Sarm1+/+ wild-type controls. Sarm1 deletion produced larger axons with thinner myelin, and attenuated TBI induced demyelination, i.e. myelin loss along apparently intact axons. At 6 weeks post-TBI, Sarm1-/- mice had less demyelination and thinner myelin than Sarm1+/+ mice, but axonal protection was no longer observed. We next used Thy1-YFP crosses to assess Sarm1 involvement in white matter neurodegeneration and neuroinflammation at 8 weeks post-TBI, when significant CC atrophy indicates chronic pathology. Thy1-YFP expression demonstrated continued CC axon damage yet absence of overt cortical pathology. Importantly, significant CC atrophy in Thy1-YFP/Sarm1+/+ mice was associated with reduced neurofilament immunolabeling of axons. Both effects were attenuated in Thy1-YFP/Sarm1-/- mice. Surprisingly, Thy1-YFP/Sarm1-/- mice had increased CC astrogliosis. This study demonstrates that Sarm1 inactivation reduces demyelination, and white matter atrophy after TBI, while the post-injury stage impacts when axon protection is effective.

The stiffness of collagen fibrils influences vascular smooth muscle cell phenotype.

Cells receive signals from the extracellular matrix through receptor-dependent interactions, but they are also influenced by the mechanical properties of the matrix. Although bulk properties of substrates have been shown to affect cell behavior, we show here that nanoscale properties of collagen fibrils also play a significant role in determining cell phenotype. Type I collagen fibrils assembled into thin films provide excellent viewing of cells interacting with individual fibrils. Cells can be observed to extensively manipulate the fibrils, and this behavior seems to result in an incompletely spread stellate morphology and a nonproliferative phenotype that is typical of these cells in collagen gels. We show here that thin films of collagen fibrils can be dehydrated, and when seeded on these dehydrated fibrils, smooth muscle cells spread and proliferate extensively. The dehydrated collagen fibrils appear to be similar to the fully hydrated collagen fibrils in topology and in presentation of beta(1) integrin ligation sites, but they are mechanically stiffer. This decrease in compliance of dehydrated fibrils is seen by a failure of cell movement of dehydrated fibrils compared to their ability to rearrange fully hydrated fibrils and from direct measurements by nanoindentation and quantitative atomic force measurements. We suggest that increase in the nanoscale rigidity of collagen fibrils can cause these cells to assume a proliferative phenotype.

Tools for quantitative and validated measurements of cells.

In this chapter, we describe the preparation of thin films of collagen that can serve as reference materials for assuring reproducible and predictable cell responses. Subtle differences in the molecular-scale characteristics of extracellular matrix proteins, including the supramolecular structure of type 1 collagen, can have tremendous influences on cell state and cell-signaling pathways; therefore the careful control and analysis of the culture surface is critical to assure a relevant and consistent response in cell-based assays. We also describe how cell-phenotypic parameters such as morphology, proliferation, and green fluorescent protein expression can be unambiguously quantified in adherent cells by automated fluorescence microscopy or high content screening. Careful consideration of protocols, and the use of fluorescent reference materials, are essential to assure day-to-day and instrument-to-instrument interoperability. The ability to collect quantitative data on large numbers of cells in homogeneous matrix environments allows assessment of the range of phenotypes that are reproducibly expressed in clonal cell populations. The inherent distribution of responses in a cell population will determine how many cells must be measured to reach an accurate determination of cellular response.

Nelfinavir inhibits proliferation and induces DNA damage in thyroid cancer cells.

The HIV protease inhibitor Nelfinavir (NFV) inhibits PI3K/AKT and MAPK/ERK signaling pathways, emerging targets in thyroid cancers. We examined the effects of NFV on cancer cells that derived from follicular (FTC), papillary (PTC) and anaplastic (ATC) thyroid cancers. NFV (1-20 µM) was tested in FTC133, BCPAP and SW1736 cell lines. The effects of NFV on cell proliferation were determined in vitro using real-time microscopy and by flow cytometry. DNA damage, apoptotic cell death and expression of molecular markers of epithelial-mesenchymal transition (EMT) were determined by Western blot and real-time PCR. Real-time imaging demonstrated that NFV (10 µM) increased the time required for the cell passage through the phases of cell cycle and induced DNA fragmentation. Growth inhibitory effects of NFV were associated with the accumulation of cells in G0/G1 phase, downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). NFV also induced the expression of γH2AX and p53BP1 indicating DNA damage. Treatment with NFV (20 µM) resulted in caspase-3 cleavage in all examined cells. NFV (20 µM) decreased the levels of total and p-AKT in PTEN-deficient FTC133 cells. NFV had no significant effects on total ERK and p-ERK in BRAF-positive BCPAP and SW1736 cells. NFV had no effects on the expression of EMT markers (Twist, Vimentin, E- and N-Cadherin), but inhibited the migration and decreased the abilities of thyroid cancer cells to survive in non-adherent conditions. We conclude that NFV inhibits proliferation and induces DNA damage in thyroid cancer cell lines. Our in vitro data suggest that NFV has a potential to become a new thyroid cancer therapeutic agent.

Thin films of Type 1 collagen for cell by cell analysis of morphology and tenascin-C promoter activity.

BACKGROUND: The use of highly reproducible and spatiallyhomogeneous thin film matrices permits automated microscopy and quantitative determination of the response of hundreds of cells in a population. Using thin films of extracellular matrix proteins, we have quantified, on a cell-by-cell basis, phenotypic parameters of cells on different extracellular matrices. We have quantitatively examined the relationship between fibroblast morphology and activation of the promoter for the extracellular matrix protein tenascin-C using a tenascin-C promoter-based GFP reporter construct. RESULTS: We find that when considering the average response from the population of cells, cell area correlates with tenascin-C promoter activity as has been previously suggested; however cell-by-cell analysis suggests that cell area and promoter activity are not tightly correlated within individual cells. CONCLUSION: This study demonstrates how quantitative cell-by-cell analysis, facilitated by the use of thin films of extracellular matrix proteins, can provide insight into the relationship between phenotypic parameters.

Components of myelin damage and repair in the progression of white matter pathology after mild traumatic brain injury.

White matter tracts are highly vulnerable to damage from impact-acceleration forces of traumatic brain injury (TBI). Mild TBI is characterized by a low density of traumatic axonal injury, whereas associated myelin pathology is relatively unexplored. We examined the progression of white matter pathology in mice after mild TBI with traumatic axonal injury localized in the corpus callosum. Adult mice received a closed-skull impact and were analyzed from 3 days to 6 weeks post-TBI/sham surgery. At all times post-TBI, electron microscopy revealed degenerating axons distributed among intact fibers in the corpus callosum. Intact axons exhibited significant demyelination at 3 days followed by evidence of remyelination at 1 week. Accordingly, bromodeoxyuridine pulse-chase labeling demonstrated the generation of new oligodendrocytes, identified by myelin proteolipid protein messenger RNA expression, at 3 days post-TBI. Overall oligodendrocyte populations, identified by immunohistochemical staining for CC1 and/or glutathione S-transferase pi, were similar between TBI and sham mice by 2 weeks. Excessively long myelin figures, similar to redundant myelin sheaths, were a significant feature at all post-TBI time points. At 6 weeks post-TBI, microglial activation and astrogliosis were localized to areas of axon and myelin pathology. These studies show that demyelination, remyelination, and excessive myelin are components of white matter degeneration and recovery in mild TBI with traumatic axonal injury.

Glucose-deprivation increases thyroid cancer cells sensitivity to metformin.

Metformin inhibits thyroid cancer cell growth. We sought to determine if variable glucose concentrations in medium alter the anti-cancer efficacy of metformin. Thyroid cancer cells (FTC133 and BCPAP) were cultured in high-glucose (20 mM) and low-glucose (5 mM) medium before treatment with metformin. Cell viability and apoptosis assays were performed. Expression of glycolytic genes was examined by real-time PCR, western blot, and immunostaining. Metformin inhibited cellular proliferation in high-glucose medium and induced cell death in low-glucose medium. In low-, but not in high-glucose medium, metformin induced endoplasmic reticulum stress, autophagy, and oncosis. At micromolar concentrations, metformin induced phosphorylation of AMP-activated protein kinase and blocked p-pS6 in low-glucose medium. Metformin increased the rate of glucose consumption from the medium and prompted medium acidification. Medium supplementation with glucose reversed metformin-inducible morphological changes. Treatment with an inhibitor of glycolysis (2-deoxy-d-glucose (2-DG)) increased thyroid cancer cell sensitivity to metformin. The combination of 2-DG with metformin led to cell death. Thyroid cancer cell lines were characterized by over-expression of glycolytic genes, and metformin decreased the protein level of pyruvate kinase muscle 2 (PKM2). PKM2 expression was detected in recurrent thyroid cancer tissue samples. In conclusion, we have demonstrated that the glucose concentration in the cellular milieu is a factor modulating metformin's anti-cancer activity. These data suggest that the combination of metformin with inhibitors of glycolysis could represent a new strategy for the treatment of thyroid cancer.

Adaptive redox response of mesenchymal stromal cells to stimulation with lipopolysaccharide inflammagen: mechanisms of remodeling of tissue barriers in sepsis.

Acute bacterial inflammation is accompanied by excessive release of bacterial toxins and production of reactive oxygen and nitrogen species (ROS and RNS), which ultimately results in redox stress. These factors can induce damage to components of tissue barriers, including damage to ubiquitous mesenchymal stromal cells (MSCs), and thus can exacerbate the septic multiple organ dysfunctions. The mechanisms employed by MSCs in order to survive these stress conditions are still poorly understood and require clarification. In this report, we demonstrated that in vitro treatment of MSCs with lipopolysaccharide (LPS) induced inflammatory responses, which included, but not limited to, upregulation of iNOS and release of RNS and ROS. These events triggered in MSCs a cascade of responses driving adaptive remodeling and resistance to a "self-inflicted" oxidative stress. Thus, while MSCs displayed high levels of constitutively present adaptogens, for example, HSP70 and mitochondrial Sirt3, treatment with LPS induced a number of adaptive responses that included induction and nuclear translocation of redox response elements such as NFkB, TRX1, Ref1, Nrf2, FoxO3a, HO1, and activation of autophagy and mitochondrial remodeling. We propose that the above prosurvival pathways activated in MSCs in vitro could be a part of adaptive responses employed by stromal cells under septic conditions.