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The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed. [ABSTRACT FROM AUTHOR]

The incidence of heatstroke has been increasing. Heatstroke has been shown to affect physiological barrier functions. However, there are few studies of the effect of heat stress on the blood-brain barrier (BBB) function. In this study, we investigated the influence of heat stress on brain microvascular endothelial cells in vivo and in vitro. Heatstroke model mice administered Texas Red-dextran showed leakage outside the brain vessel walls. In addition, trans-endothelial electrical resistance (TEER) value was significantly reduced in induced pluripotent stem (iPS) cell-derived brain microvascular endothelial cells under heat stress by reducing claudin-5 expression. In addition, our results showed that the expression level of P-glycoprotein (P-gp) was increased in iPS cell-derived brain microvascular endothelial cells under heat stress. Furthermore, serum from heatstroke model mice could impair the BBB integrity of iPS cell-derived brain microvascular endothelial cells. These results suggest that BBB integrity was affected by heat stress in vivo and in vitro and provide important insights into the development of new therapeutic strategies for heatstroke patients. [ABSTRACT FROM AUTHOR]

In this study, we report antifungal activity of auroramycin against Candida albicans, Candida tropicalis, and Cryptococcus neoformans. Auroramycin, a potent antimicrobial doubly glycosylated 24-membered polyene macrolactam, was previously isolated and characterized, following CRISPR-Cas9 mediated activation of a silent polyketide synthase biosynthetic gene cluster in Streptomyces rosesporous NRRL 15998. Chemogenomic profiling of auroramycin in yeast has linked its antifungal bioactivity to vacuolar transport and membrane organization. This was verified by disruption of vacuolar structure and membrane integrity of yeast cells with auroramycin treatment. Addition of salt but not sorbitol to the medium rescued the growth of auroramycin-treated yeast cells suggesting that auroramycin causes ionic stress. Furthermore, auroramycin caused hyperpolarization of the yeast plasma membrane and displayed a synergistic interaction with cationic hygromycin. Our data strongly suggest that auroramycin inhibits yeast cells by causing leakage of cations from the cytoplasm. Thus, auroramycin’s mode-of-action is distinct from known antifungal polyenes, reinforcing the importance of natural products in the discovery of new anti-infectives. [ABSTRACT FROM AUTHOR]

Environmental circadian disruption (ECD), characterized by repeated or long-term disruption in environmental timing cues which require the internal circadian clock to change its phase to resynchronize with the environment, is associated with numerous serious health issues in humans. While animal and isolated cell models exist to study the effects of destabilizing the relationship between the circadian system and the environment, neither approach provides an ideal solution. Here, we developed an in vitro model which incorporates both elements of a reductionist cellular model and disruption of the clock/environment relationship using temperature as an environmental cue, as occurs in vivo. Using this approach, we have demonstrated that some effects of in vivo ECD can be reproduced using only isolated peripheral oscillators. Specifically, we report exaggerated inflammatory responses to endotoxin following repeated environmental circadian disruption in explanted spleens. This effect requires a functional circadian clock but not the master brain clock, the suprachiasmatic nucleus (SCN). Further, we report that this is a result of cumulative, rather than acute, circadian disruption as has been previously observed in vivo. Finally, such effects appear to be tissue specific as it does not occur in lung, which is less sensitive to the temperature cycles employed to induce ECD. Taken together, the present study suggests that this model could be a valuable tool for dissecting the causes and effects of circadian disruption both in isolated components of physiological systems as well as the aggregated interactions of these systems that occur in vivo. [ABSTRACT FROM AUTHOR]

Chemotherapy often causes side effects that include disturbances in taste functions. Cyclophosphamide (CYP) is a chemotherapy drug that, after a single dose, elevates murine taste thresholds at times related to drug-induced losses of taste sensory cells and disruptions of proliferating cells that renew taste sensory cells. Pretreatment with amifostine can protect the taste system from many of these effects. This study compared the effects of a single dose (75 mg/kg) of CYP with effects generated by fractionated dosing of CYP (5 doses of 15 mg/kg), a dosing approach often used during chemotherapy, on the taste system of mice using immunohistochemistry. Dose fractionation prolonged the suppressive effects of CYP on cell proliferation responsible for renewal of taste sensory cells. Fractionation also reduced the total number of cells and the proportion of Type II cells within taste buds. The post-injection time of these losses coincided with the life span of Type I and II taste cells combined with lack of replacement cells. Fractionated dosing also decreased Type III cells more than a single dose, but loss of these cells may be due to factors related to the general health and/or cell renewal of taste buds rather than the life span of Type III cells. In general, pretreatment with amifostine appeared to protect taste cell renewal and the population of cells within taste buds from the cytotoxic effects of CYP with few observable adverse effects due to repeated administration. These findings may have important implications for patients undergoing chemotherapy. [ABSTRACT FROM AUTHOR]

The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) functions as a reducing agent involved in many biosynthetic and antioxidant reactions in cells. Therefore, a lots of detection or assaying method of this cofactor are developed and used broadly in various research and application fields. These detection or assay tools, however, have often some problems, such as the low sensitivity, susceptibility to environmental interference and time-consuming pretreatment steps, remaining hurdle to successful quantification of NADPH or its derivatives accurately and immediately. Herein, we present a rapid (assay time < 30 s) and sensitive (detection limit < 2 pmol) detection method of NADPH using metagenome-derived blue fluorescent protein (mBFP), a protein capable of significantly enhancing NADPH fluorescence upon binding to this cofactor. Our method takes advantage of the high specificity of mBFP to NADPH and the immediate fluorescence enhancement upon the addition of mBFP to a solution of interest containing NADPH. We can apply this detection scheme to directly quantitative assessment of NADP(H)-dependent enzyme activities in-vitro, and further accessed to quantitative assay of other nicotine amide cofactors, such as NAD+ and NADH, by coupling assay using NAD(H) kinase. Thus, our method enabled us to quantitatively assess the activity of nicotinamide cofactor-associated enzymes in both bacterial and human cell lysates. [ABSTRACT FROM AUTHOR]

Background: The codon 72 polymorphism in the p53 gene relates to the risk of breast cancer (BC), but this relationship in racially diverse populations is not known. The present study examined the prognostic value of this polymorphism for African American (AA) and Caucasian (CA) BC patients separately and considered the confounding variables of molecular subtypes and somatic mutations in p53. Methods: Tissue sections of BCs from 116 AAs and 160 CAs were evaluated for p53 mutations and genotyped for the codon 72 polymorphism. The relationships of phenotypes to clinicopathologic features were determined by χ2 analyses; patient survival was estimated by Kaplan-Meier univariate and Cox regression multivariate models in a retrospective cohort study design. Results: The proportion of single nucleotide polymorphism (SNP) 72 alleles differed for races. Many cancers of AAs were Pro/Pro, but most for CAs were Arg/Arg. A higher frequency of missense p53 mutations was evident for AAs. There was an interaction between the SNP allele and p53 mutations for AA women only. The proportion of women with both the Pro/Pro allele and a p53 somatic mutation was higher for AA than CA women. The interaction between missense p53 mutations and Pro/Pro had a negative effect on survival, particularly for AAs with luminal cancers. Conclusions: For BCs, the survival effect of SNP72 combined with a p53 missense mutation is dependent on race and molecular subtype. Although such a mutation is a marker of poor prognosis, it is relevant to identify the variant Pro/Pro in the case of AAs, especially those with luminal subtypes of BC. [ABSTRACT FROM AUTHOR]

Cryopreservation is an essential tool to meet the increasing demand for stem cells in medical applications. To ensure maintenance of cell function upon thawing, the preservation of the actin cytoskeleton is crucial, but so far there is little quantitative data on the influence of cryopreservation on cytoskeletal structures. For this reason, our study aims to quantitatively describe cryopreservation induced alterations to F-actin in adherent human mesenchymal stem cells, as a basic model for biomedical applications. Here we have characterised the actin cytoskeleton on single-cell level by calculating the circular standard deviation of filament orientation, F-actin content, and average filament length. Cryo-induced alterations of these parameters in identical cells pre and post cryopreservation provide the basis of our investigation. Differences between the impact of slow-freezing and vitrification are qualitatively analyzed and highlighted. Our analysis is supported by live cryo imaging of the actin cytoskeleton via two photon microscopy. We found similar actin alterations in slow-frozen and vitrified cells including buckling of actin filaments, reduction of F-actin content and filament shortening. These alterations indicate limited functionality of the respective cells. However, there are substantial differences in the frequency and time dependence of F-actin disruptions among the applied cryopreservation strategies; immediately after thawing, cytoskeletal structures show least disruption after slow freezing at a rate of 1°C/min. As post-thaw recovery progresses, the ratio of cells with actin disruptions increases, particularly in slow frozen cells. After 120 min of recovery the proportion of cells with an intact actin cytoskeleton is higher in vitrified than in slow frozen cells. Freezing at 10°C/min is associated with a high ratio of impaired cells throughout the post-thawing culture. [ABSTRACT FROM AUTHOR]

FnCel5A from Fervidobacterium nodosum is one of the most thermostable endoglucanases that have phenomenal characteristics, such as high activity, pH stability, and multi-specificity towards various substrates. However, large-scale thermophilic enzyme production is still a challenge. Herein, we focus on an optimization approach based on response surface methodology to improve the production of this enzyme. First, a Box-Behnken design was used to examine physiochemical parameters such as induction temperatures, isopropylβ-D-1-thiogalactopyranoside concentrations and induction times on the heterogeneous expression of FnCel5A gene in E. coli. The best culture was collected after adding 0.56 mM IPTG and incubating it for 29.5 h at 24°C. The highest enzymatic activity observed was 3.31 IU/mL. Second, an economical "thermolysis" cell lysis method for the liberation of the enzymes was also optimized using Box-Behnken design. The optimal levels of the variables were temperature 77°C, pH 7.71, and incubation time of 20 min, which gave about 74.3% higher activity than the well-established bead-milling cell disruption method. The maximum productivity of FnCel5A achieved (5772 IU/L) illustrated that its production increased significantly after combining both optimal models. This strategy can be scaled-up readily for overproduction of FnCel5A from recombinant E.coli to facilitate its usage in biomass energy production. [ABSTRACT FROM AUTHOR]

Cell populations across nearly all forms of life generally maintain a characteristic cell type-dependent size, but how size control is achieved has been a long-standing question. The G1/S boundary of the cell cycle serves as a major point of size control, and mechanisms operating here restrict passage of cells to Start if they are too small. In contrast, it is less clear how size is regulated post-Start, during S/G2/M. To gain further insight into post-Start size control, we prepared budding yeast that can be reversibly blocked from bud initiation. While blocked, cells continue to grow isotropically, increasing their volume by more than an order of magnitude over unperturbed cells. Upon release from their block, giant mothers reenter the cell cycle and their progeny rapidly return to the original unperturbed size. We found this behavior to be consistent with a size-invariant ‘timer’ specifying the duration of S/G2/M. These results indicate that yeast use at least two distinct mechanisms at different cell cycle phases to ensure size homeostasis. [ABSTRACT FROM AUTHOR]

Particulate matter (PM) air pollution is a global environmental health problem contributing to more severe lung inflammation and injury. However, the molecular and cellular mechanisms of PM-induced exacerbation of lung barrier dysfunction and injury are not well understood. In the current study, we tested a hypothesis that PM exacerbates vascular barrier dysfunction via ROS-induced generation of truncated oxidized phospholipids (Tr-OxPLs). Treatment of human pulmonary endothelial cells with PM caused endothelial cell barrier disruption in a dose-dependent fashion. Biochemical analysis showed destabilization of cell junctions by PM via tyrosine phosphorylation and internalization of VE-cadherin. These events were accompanied by PM-induced generation of Tr-OxPLs, detected by mass spectrometry analysis. Furthermore, purified Tr-OxPLs: POVPC, PGPC and lyso-PC alone, caused a rapid increase in endothelial permeability and augmented pulmonary endothelial barrier dysfunction induced by submaximal doses of PM. In support of a role of TR-OxPLs-dependent mechanism in mediation of PM effects, ectopic expression of intracellular type 2 platelet-activating factor acetylhydrolase (PAFAH2), which specifically hydrolyzes Tr-OxPLs, significantly attenuated PM-induced endothelial hyperpermeability. In summary, this study uncovered a novel mechanism of PM-induced sustained dysfunction of pulmonary endothelial cell barrier which is driven by PM-induced generation of truncated products of phospholipid oxidation causing destabilization of cell junctions. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) is a stimulator of the innate immune system and is routinely used in animal models to study blood-brain barrier (BBB) dysfunction under inflammatory conditions. It is appreciated that both humans and mice have sexually dimorphic immune responses, which could influence the brain’s response to a systemic inflammatory insult. Mouse strain is also an important factor that can contribute to pathophysiological responses to inflammatory stimuli. Therefore, we aimed to test whether BBB disruption and the associated cytokine profiles in response to LPS differed in male and female mice from two mouse strains most commonly used in blood-brain barrier studies: CD-1 and C57BL6/J (C57). Mice were treated with saline, a single injection of 0.3, or 3mg/kg LPS, or three injections of 3mg/kg LPS, and studied 28 hours after the first LPS injection. To assay BBB disruption, we utilized the tracer 99mTc-DTPA. A 23-plex panel of cytokines was assayed in brain and blood of the same cohort of mice, which allowed us to compare differences in the levels of individual cytokines as well as correlations among cytokines and 99mTc-DTPA uptake. We found that only the three-injection dose of LPS induced significant BBB disruption in all sexes and strains. The treatment, strain, and sex, as well as treatment-by- strain and treatment-by-sex interactions significantly contributed to the variance. The mean brain/serum ratios of 99mTc-DTPA in the three-injection LPS group were ranked CD-1 male < CD-1 female < C57 male < C57 female. There were significant sex and strain differences in cytokine profiles in brain and blood, and pro-inflammatory cytokines and chemokines in brain were most strongly correlated with 99mTc-DTPA brain/serum ratios. [ABSTRACT FROM AUTHOR]

CLEC16A is implicated in multiple autoimmune diseases. We generated Clec16a inducible knockout (KO) mice to examine the functional link between CLEC16A auto-inflammation and autoimmunity. Clec16a KO mice exhibited weight loss and thymic and splenic atrophy. Mitochondrial potential was lowered in KO mice splenocytes resulting in aggregation of unhealthy mitochondria in B, T, and NK cells. In Clec16a KO mice we detected disrupted mitophagy in splenic B and T cells. NK cells from Clec16a KO mice exhibited increased cytotoxicity. Incomplete mitophagy was attenuated with PI3K and/or MEK inhibition in Clec16a KO mice. Our results demonstrate a functional link between CLEC16A and disrupted mitophagy in immune cells and show that incomplete mitophagy predisposes the KO mice to inflammation. Taken together, loss of function variants in CLEC16A that are associated with decreased CLEC16A expression levels may contribute to inflammation in autoimmunity through disrupted mitophagy. Drugs modulating mitophagy reverse the process and may be effective in treating and preventing autoimmunity in individuals with risk associated CLEC16A variants. [ABSTRACT FROM AUTHOR]

Increasing evidence indicates that the Golgi apparatus plays active roles in cancer, but a comprehensive understanding of its functions in the oncogenic transformation has not yet emerged. At the same time, the Golgi is becoming well recognized as a hub that integrates its functions of protein and lipid biosynthesis to signal transduction for cell proliferation and migration in cancer cells. Nevertheless, the active function of the Golgi apparatus in cancer cells has not been fully evaluated as a target for combined treatment. Here, we analyzed the effect of perturbing the Golgi apparatus on the sensitivity of the MDA-MB-231 breast cancer cell line to the drugs Actinomycin D and Vinblastine. We disrupted the function of ARF1, a protein necessary for the homeostasis of the Golgi apparatus. We found that the expression of the ARF1-Q71L mutant increased the sensitivity of MDA-MB-231 cells to both Actinomycin D and Vinblastine, resulting in decreased cell proliferation and cell migration, as well as in increased apoptosis. Likewise, the combined treatment of cells with Actinomycin D or Vinblastine and Brefeldin A or Golgicide A, two disrupting agents of the ARF1 function, resulted in similar effects on cell proliferation, cell migration and apoptosis. Interestingly, each combined treatment had distinct effects on ERK1/2 and AKT signaling, as indicated by the decreased levels of either phospho-ERK1/2 or phospho-AKT. Our results suggest that disruption of Golgi function could be used as a strategy for the sensitization of cancer cells to chemotherapy. [ABSTRACT FROM AUTHOR]

Acute respiratory distress syndrome (ARDS) is a life-threatening condition for which there are currently no medical therapies other than supportive care involving the application of mechanical ventilation. However, mechanical ventilation itself can worsen ARDS by damaging the alveolocapillary barrier in the lungs. This allows plasma-derived fluid and proteins to leak into the airspaces of the lung where they interfere with the functioning of pulmonary surfactant, which increases the stresses of mechanical ventilation and worsens lung injury. Once such ventilator-induced lung injury (VILI) is underway, managing ARDS and saving the patient becomes increasingly problematic. Maintaining an intact alveolar barrier thus represents a crucial management goal, but the biophysical processes that perforate this barrier remain incompletely understood. To study the dynamics of barrier perforation, we subjected initially normal mice to an injurious ventilation regimen that imposed both volutrauma (overdistension injury) and atelectrauma (injury from repetitive reopening of closed airspaces) on the lung, and observed the rate at which macromolecules of various sizes leaked into the airspaces as a function of the degree of overall injury. Computational modeling applied to our findings suggests that perforations in the alveolocapillary barrier appear and progress according to a rich-get-richer mechanism in which the likelihood of a perforation getting larger increases with the size of the perforation. We suggest that atelectrauma causes the perforations after which volutrauma expands them. This mechanism explains why atelectrauma appears to be essential to the initiation of VILI in a normal lung, and why atelectrauma and volutrauma then act synergistically once VILI is underway. [ABSTRACT FROM AUTHOR]

Engineered nanomaterials (ENMs) are increasingly incorporated into a variety of commercial applications and consumer products; however, ENMs may possess cytotoxic properties due to their small size. This study assessed the effects of two commonly used ENMs, zinc oxide nanoparticles (ZnONPs) and silver nanoparticles (AgNPs), in the model eukaryote Saccharomyces cerevisiae. A collection of ≈4600 S. cerevisiae deletion mutant strains was used to deduce the genes, whose absence makes S. cerevisiae more prone to the cytotoxic effects of ZnONPs or AgNPs. We demonstrate that S. cerevisiae strains that lack genes involved in transmembrane and membrane transport, cellular ion homeostasis, and cell wall organization or biogenesis exhibited the highest sensitivity to ZnONPs. In contrast, strains that lack genes involved in transcription and RNA processing, cellular respiration, and endocytosis and vesicular transport exhibited the highest sensitivity to AgNPs. Secondary assays confirmed that ZnONPs affected cell wall function and integrity, whereas AgNPs exposure decreased transcription, reduced endocytosis, and led to a dysfunctional electron transport system. This study supports the use of S. cerevisiae Gene Deletion Array as an effective high-throughput technique to determine cellular targets of ENM toxicity. [ABSTRACT FROM AUTHOR]

Following traumatic brain injury (TBI) neurological damage is ongoing through a complex cascade of primary and secondary injury events in the ensuing minutes, days and weeks. The delayed nature of secondary injury provides a valuable window of opportunity to limit the consequences with a timely treatment. Recently, the amyloid precursor protein (APP) and its derivative APP96-110 have shown encouraging neuroprotective activity following TBI following an intracerebroventricular administration. Nevertheless, its broader clinical utility would be enhanced by an intravenous (IV) administration. This study assessed the efficacy of IV APP96-110, where a dose-response for a single dose of 0.005mg/kg– 0.5mg/kg APP96-110 at either 30 minutes or 5 hours following moderate-severe diffuse impact-acceleration injury was performed. Male Sprague-Dawley rats were assessed daily for 3 or 7 days on the rotarod to examine motor outcome, with a separate cohort of animals utilised for immunohistochemistry analysis 3 days post-TBI to assess axonal injury and neuroinflammation. Animals treated with 0.05mg/kg or 0.5mg/kg APP96-110 after 30 minutes demonstrated significant improvements in motor outcome. This was accompanied by a reduction in axonal injury and neuroinflammation in the corpus callosum at 3 days post-TBI, whereas 0.005mg/kg had no effect. In contrast, treatment with 0.005m/kg or 0.5mg/kg APP96-110 at 5 hours post-TBI demonstrated significant improvements in motor outcome over 3 days, which was accompanied by a reduction in axonal injury in the corpus callosum. This demonstrates that APP96-110 remains efficacious for up to 5 hours post-TBI when administered IV, and supports its development as a novel therapeutic compound following TBI. [ABSTRACT FROM AUTHOR]

Cell adhesion and apicobasal polarity together maintain epithelial tissue organization and homeostasis. Loss of adhesion has been described as a prerequisite for the epithelial to mesenchymal transition. However, what role misregulation of apicobasal polarity promotes tumor initiation and/or early progression remains unclear. We find that human low-grade endometrial cancers are associated with disrupted localization of the apical polarity protein Par3 and Ezrin while, the adhesion molecule E-cadherin remains unchanged, accompanied by decreased Notch signaling, and altered Notch receptor localization. Depletion of Par3 or Ezrin, in a cell-based model, results in loss of epithelial architecture, differentiation, increased proliferation, migration and decreased Notch signaling. Re-expression of Par3 in endometrial cancer cell lines with disrupted Par3 protein levels blocks proliferation and reduces migration in a Notch dependent manner. These data uncover a function for apicobasal polarity independent of cell adhesion in regulating Notch-mediated differentiation signals in endometrial epithelial cells. [ABSTRACT FROM AUTHOR]

ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H2O2, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1’s ATPase activity in BER, we disrupted the ALC1 gene and inserted the ATPase-dead (E165Q) mutation into the ALC1 gene in chicken DT40 cells, which do not express PARP2. The resulting ALC1-/- and ALC1-/E165Q cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H2O2, indicating that ATPase plays an essential role in the DNA-damage response. PARP1-/- and ALC1-/-/PARP1-/- cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H2O2, PARP1-/- and ALC1-/-/PARP1-/- cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1’s role in BER in mammalian cells, we disrupted the ALC1 gene in human TK6 cells. Following exposure to MMS and to H2O2, the ALC1-/- TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H2O2, ALC1-/- cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms utilize sulfate for the synthesis of sulfur-compounds including proteins and a sulfolipid, sulfoquinovosyl diacylglycerol. Upon ambient deficiency in sulfate, cells of a green alga, Chlamydomonas reinhardtii, degrade the chloroplast membrane sulfolipid to ensure an intracellular-sulfur source for necessary protein synthesis. Here, the effects of sulfate-starvation on the sulfolipid stability were investigated in another green alga, Chlorella kessleri, and two cyanobacteria, Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942. The results showed that sulfolipid degradation was induced only in C. kessleri, raising the possibility that this degradation ability was obtained not by cyanobacteria, but by eukaryotic algae during the evolution of photosynthetic organisms. Meanwhile, Synechococcus disruptants concerning sqdB and sqdX genes, which are involved in successive reactions in the sulfolipid synthesis pathway, were respectively characterized in cellular response to sulfate-starvation. Phycobilisome degradation intrinsic to Synechococcus, but not to Synechocystis, and cell growth under sulfate-starved conditions were repressed in the sqdB and sqdX disruptants, respectively, relative to in the wild type. Their distinct phenotypes, despite the common loss of the sulfolipid, inferred specific roles of sqdB and sqdX. This study demonstrated that sulfolipid metabolism might have been developed to enable species- or cyanobacterial-strain dependent processes for acclimation to sulfate-starvation. [ABSTRACT FROM AUTHOR]

The transcriptional functions of the class I histone deacetylases (HDACs) HDAC1 and HDAC2 are mainly viewed as both repressive and redundant based on murine knockout studies, but they may have additional independent roles and their physiological functions in human cells are not as clearly defined. To address the individual epigenomic functions of HDAC2, here we utilized CRISPR-Cas9 to disrupt HDAC2 in human cells. We find that while HDAC2 null cells exhibited signs of cross-regulation between HDAC1 and HDAC2, specific epigenomic phenotypes were still apparent using RNA-seq and ChIP assays. We identified specific targets of HDAC2 repression, and defined a novel class of genes that are actively expressed in a partially HDAC2-dependent manner. While HDAC2 was required for the recruitment of HDAC1 to repressed HDAC2-gene targets, HDAC2 was dispensable for HDAC1 binding to HDAC2-activated targets, supporting the notion of distinct classes of targets. Both active and repressed classes of gene targets demonstrated enhanced histone acetylation and methylation in HDAC2-null cells. Binding of the HDAC1/2-associated SIN3A corepressor was altered at most HDAC2-targets, but without a clear pattern. Overall, our study defines two classes of HDAC2 targets in human cells, with a dependence of HDAC1 on HDAC2 at one class of targets, and distinguishes unique functions for HDAC2. [ABSTRACT FROM AUTHOR]

While grid cells in the medial entorhinal cortex (MEC) of rodents have multiple, regularly arranged firing fields, place cells in the cornu ammonis (CA) regions of the hippocampus mostly have single spatial firing fields. Since there are extensive projections from MEC to the CA regions, many models have suggested that a feedforward network can transform grid cell firing into robust place cell firing. However, these models generate place fields that are consistently too small compared to those recorded in experiments. Here, we argue that it is implausible that grid cell activity alone can be transformed into place cells with robust place fields of realistic size in a feedforward network. We propose two solutions to this problem. Firstly, weakly spatially modulated cells, which are abundant throughout EC, provide input to downstream place cells along with grid cells. This simple model reproduces many place cell characteristics as well as results from lesion studies. Secondly, the recurrent connections between place cells in the CA3 network generate robust and realistic place fields. Both mechanisms could work in parallel in the hippocampal formation and this redundancy might account for the robustness of place cell responses to a range of disruptions of the hippocampal circuitry. [ABSTRACT FROM AUTHOR]

Ultrasound (US) stimulated microbubbles (MB) is a new treatment approach that sensitizes cancer cells to radiation (XRT). The molecular pathways in this response remain unelucidated, however, previous data has supported a role for cell membrane-metabolism related pathways including an up regulation of UDP glycosyltransferase 8 (UGT8), which catalyzes the transfer of galactose to ceramide, a lipid that is associated with the induction of apoptotic signalling. In this study, the role of UGT8 in responses of prostate tumours to ultrasound-stimulated microbubble radiation enhancement therapy is investigated. Experiments were carried out with cells in vitro and tumours in vivo in which UGT8 levels had been up regulated or down regulated. Genetically modified PC3 cells were treated with XRT, US+MB, or a combination of XRT+US+MB. An increase in the immunolabelling of ceramide was observed in cells where UGT8 was down-regulated as opposed to cells where UGT8 was either not regulated or was up-regulated. Clonogenic assays have revealed a decreased level of cellular survival with the down-regulation of UGT8. Xenograft tumours generated from stably transfected PC3 cells were also treated with US+MB, XRT or US+MB+XRT. Histology demonstrated more cellular damage in tumours with down-regulated UGT8 in comparison with control tumours. In contrast, tumours with up-regulated UGT8 had less damage than control tumours. Power Doppler imaging indicated a reduction in the vascular index with UGT8 down-regulation and photoacoustic imaging revealed a reduction in oxygen saturation. This was contrary to when UGT8 was up regulated. The down regulation of UGT8 led to the accumulation of ceramide resulting in more cell death signalling and therefore, a greater enhancement of radiation effect when vascular disruption takes place through the use of ultrasound-stimulated microbubbles. [ABSTRACT FROM AUTHOR]

Flap endonuclease 1 (FEN1) is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI) as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T) microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption. [ABSTRACT FROM AUTHOR]

Cytotoxicity is a commonly used in vitro endpoint for evaluating chemical toxicity. In support of the U.S. Tox21 screening program, the cytotoxicity of ~10K chemicals was interrogated at 0, 8, 16, 24, 32, & 40 hours of exposure in a concentration dependent fashion in two cell lines (HEK293, HepG2) using two multiplexed, real-time assay technologies. One technology measures the metabolic activity of cells (i.e., cell viability, glo) while the other evaluates cell membrane integrity (i.e., cell death, flor). Using glo technology, more actives and greater temporal variations were seen in HEK293 cells, while results for the flor technology were more similar across the two cell types. Chemicals were grouped into classes based on their cytotoxicity kinetics profiles and these classes were evaluated for their associations with activity in the Tox21 nuclear receptor and stress response pathway assays. Some pathways, such as the activation of H2AX, were associated with the fast-responding cytotoxicity classes, while others, such as activation of TP53, were associated with the slow-responding cytotoxicity classes. By clustering pathways based on their degree of association to the different cytotoxicity kinetics labels, we identified clusters of pathways where active chemicals presented similar kinetics of cytotoxicity. Such linkages could be due to shared underlying biological processes between pathways, for example, activation of H2AX and heat shock factor. Others involving nuclear receptor activity are likely due to shared chemical structures rather than pathway level interactions. Based on the linkage between androgen receptor antagonism and Nrf2 activity, we surmise that a subclass of androgen receptor antagonists cause cytotoxicity via oxidative stress that is associated with Nrf2 activation. In summary, the real-time cytotoxicity screen provides informative chemical cytotoxicity kinetics data related to their cytotoxicity mechanisms, and with our analysis, it is possible to formulate mechanism-based hypotheses on the cytotoxic properties of the tested chemicals. [ABSTRACT FROM AUTHOR]

Ocular toxoplasmosis is mediated by monocytes infected with Toxoplasma gondii that are disseminated to target organs. Although infected monocytes can easily access to outer blood-retinal barrier due to leaky choroidal vasculatures, not much is known about the effect of T. gondii-infected monocytes on outer blood-retinal barrier. We prepared human monocytes, THP-1, infected with T. gondii and human retinal pigment epithelial cells, ARPE-19, grown on transwells as an in vitro model of outer blood-retinal barrier. Exposure to infected monocytes resulted in disruption of tight junction protein, ZO-1, and decrease in transepithelial electrical resistance of retinal pigment epithelium. Supernatants alone separated from infected monocytes also decreased transepithelial electrical resistance and disrupted tight junction protein. Further investigation revealed that the supernatants could activate focal adhesion kinase (FAK) signaling in retinal pigment epithelium and the disruption was attenuated by FAK inhibitor. The disrupted barrier was partly restored by blocking CXCL8, a FAK activating factor secreted by infected monocytes. In this study, we demonstrated that monocytes infected with T. gondii can disrupt outer blood-retinal barrier, which is mediated by paracrinely activated FAK signaling. FAK signaling can be a target of therapeutic approach to prevent negative influence of infected monocytes on outer blood-retinal barrier. [ABSTRACT FROM AUTHOR]

PSL1a is a lectin from the mushroom Polyporus squamosus that binds to sialylated glycans and glycoconjugates with high specificity and selectivity. In addition to its N-terminal carbohydrate-binding domain, PSL1a possesses a Ca2+-dependent proteolytic activity in the C-terminal domain. In the present study, we demonstrate that PSL1a has cytotoxic effects on mammalian cancer cells, and we show that the cytotoxicity is dependent on the cysteine protease activity. PSL1a treatment leads to cell rounding and detachment from the substratum, concomitant with disruption of vinculin complexes in focal adhesions. We also demonstrate that PSL1a inhibits protein synthesis and induces apoptosis in HeLa cells, in a time- and concentration-dependent manner. [ABSTRACT FROM AUTHOR]

Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future. [ABSTRACT FROM AUTHOR]

The spontaneous and energy-releasing reaction of protein aggregation is typically prevented by cellular quality control machinery (QC). TRIM32 is a member of the TRIM (tripartite motif-containing) ubiquitin E3 ligases, and when overexpressed in cultured cells, readily forms spherical inclusions designated as cytoplasmic bodies (CBs) even without proteasome inhibition. Here, we show that HSP70, a central QC component, is a primary binding factor of overexpressed TRIM32. Contrary to expectation, however, we find that this molecular chaperone facilitates and stabilizes CB assembly depending on intrinsic ATPase activity, rather than preventing CB formation. We also show that the HSP70-TRIM32 complex is biochemically distinct from the previously characterized 14-3-3-TRIM32 phospho-complex. Moreover, the two complexes have opposing roles, with HSP70 stimulating CB formation and 14-3-3 retaining TRIM32 in a diffuse form throughout the cytosol. Our results suggest that CB inclusion formation is actively controlled by cellular QC and requires ATP, similar to protein folding and degradation reactions. [ABSTRACT FROM AUTHOR]

The important process of nutrient uptake in Escherichia coli, in many cases, involves transit of the nutrient through a class of beta-barrel proteins in the outer membrane known as TonB-dependent transporters (TBDTs) and requires interaction with the inner membrane protein TonB. Here we have imaged the mobility of the ferric enterobactin transporter FepA and TonB by tracking them in the membranes of live E. coli with single-molecule resolution at time-scales ranging from milliseconds to seconds. We employed simple simulations to model/analyze the lateral diffusion in the membranes of E.coli, to take into account both the highly curved geometry of the cell and artifactual effects expected due to finite exposure time imaging. We find that both molecules perform confined lateral diffusion in their respective membranes in the absence of ligand with FepA confined to a region μm in radius in the outer membrane and TonB confined to a region μm in radius in the inner membrane. The diffusion coefficient of these molecules on millisecond time-scales was estimated to be μm2/s and μm2/s for FepA and TonB, respectively, implying that each molecule is free to diffuse within its domain. Disruption of the inner membrane potential, deletion of ExbB/D from the inner membrane, presence of ligand or antibody to FepA and disruption of the MreB cytoskeleton was all found to further restrict the mobility of both molecules. Results are analyzed in terms of changes in confinement size and interactions between the two proteins. [ABSTRACT FROM AUTHOR]

S100A7 is expressed in many squamous cell carcinomas (SCCs). Our previous study revealed that S100A7 was dramatically induced in several SCC cells and activation of the Hippo pathway significantly promoted S100A7 in epidermoid carcinoma cells. However, whether the Hippo pathway regulates S100A7 expression in SCCs remains largely unknown. Here, we uncover that S100A7 induction by the Hippo-YAP pathway displays different characteristic in cervical and glossopharyngeal SCC. In well differentiated HCC94 cervical cells and FaDu pharyngeal cells, S100A7 is easily induced by both suspension and dense culture, which is accompanied by an increase in YAP phosphorylation and a decrease in nuclear YAP. Strikingly, these correlations of S100A7 and YAP reverse after recovery of cell attachment or relief from dense culture. Further examination finds that S100A7 induction is significantly repressed by nuclear YAP, which is validated by activation or inhibition of the Hippo pathway via loss- and/or gain-of- LATS1 and MST1 function. Subsequently, we prove that TEAD1 is required for YAP transcriptional repression of S100A7. However, S100A7 is hardly induced in poorly differentiated SiHa cervical cells and NCI-H226 pulmonary cells even in suspension or activation of the Hippo pathway. More importantly, cervical and lingual SCC tissues array analyses show that S100A7 expression displays the positive correlation with pYAP-S127 and the negative correlation with nuclear YAP in the majority of well differentiated but not in poorly differentiated tissues. Collectively, our findings demonstrate that the different induction of S100A7 toward activation of the Hippo pathway mainly depends on the degree of cell differentiation in cervical and glossopharyngeal SCC. [ABSTRACT FROM AUTHOR]

The amyloid hypothesis posits that the production of β-amyloid (Aβ) aggregates leads to neurodegeneration and cognitive decline associated with AD. Aβ is produced by sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretase. While nascent APP is well known to transit to the endosomal/ lysosomal system via the cell surface, we have recently shown that APP can also traffic to lysosomes intracellularly via its interaction with AP-3. Because AP-3 interacts with cargo protein via interaction with tyrosine motifs, we mutated the three tyrosines motif in the cytoplasmic tail of APP. Here, we show that the YTSI motif interacts with AP-3, and phosphorylation of the serine in this motif disrupts the interaction and decreases APP trafficking to lysosomes. Furthermore, we show that phosphorylation at this motif can decrease the production of neurotoxic Aβ 42. This demonstrates that reducing APP trafficking to lysosomes may be a strategy to reduce Aβ 42 in Alzheimer’s disease. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress is caused by accumulation of unfolded and misfolded proteins in the ER, thereby compromising its vital cellular functions in protein production and secretion. Genome wide association studies in humans as well as experimental animal models linked ER stress in intestinal epithelial cells (IECs) with intestinal disorders including inflammatory bowel diseases. However, the mechanisms linking the outcomes of ER stress in IECs to intestinal disease have not been clarified. In this study, we investigated the impact of ER stress on intestinal epithelial barrier function using human colon carcinoma-derived Caco-2 monolayers. Tunicamycin-induced ER stress decreased the trans-epithelial electrical resistance of Caco-2 monolayers, concomitant with loss of cellular plasma membrane integrity. Epithelial barrier disruption in Caco-2 cells after ER stress was not caused by caspase- or RIPK1-dependent cell death but was accompanied by lysosomal rupture and up-regulation of the ER stress markers Grp78, sXBP1 and Chop. Interestingly, several bifidobacteria species inhibited tunicamycin-induced ER stress and thereby diminished barrier disruption in Caco-2 monolayers. Together, these results showed that ER stress compromises the epithelial barrier function of Caco-2 monolayers and demonstrate beneficial impacts of bifidobacteria on ER stress in IECs. Our results identify epithelial barrier loss as a potential link between ER stress and intestinal disease development, and suggest that bifidobacteria could exert beneficial effects on this phenomenon. [ABSTRACT FROM AUTHOR]

Electroporation-induced cell sensitization was described as the occurrence of a delayed hypersensitivity to electric pulses caused by pretreating cells with electric pulses. It was achieved by increasing the duration of the electroporation treatment at the same cumulative energy input. It could be exploited in electroporation-based treatments such as electrochemotherapy and tissue ablation with irreversible electroporation. The mechanisms responsible for cell sensitization, however, have not yet been identified. We investigated cell sensitization dynamics in five different electroporation buffers. We split a pulse train into two trains varying the delay between them and measured the propidium uptake by fluorescence microscopy. By fitting the first-order model to the experimental results, we determined the uptake due to each train (i.e. the first and the second) and the corresponding resealing constant. Cell sensitization was observed in the growth medium but not in other tested buffers. The effect of pulse repetition frequency, cell size change, cytoskeleton disruption and calcium influx do not adequately explain cell sensitization. Based on our results, we can conclude that cell sensitization is a sum of several processes and is buffer dependent. Further research is needed to determine its generality and to identify underlying mechanisms. [ABSTRACT FROM AUTHOR]

The establishment and maintenance of apical-basal polarity is a defining characteristic and essential feature of functioning epithelia. Apical-basal polarity (ABP) proteins are also tumor suppressors that are targeted for disruption by oncogenic viruses and are commonly mutated in human carcinomas. Disruption of these ABP proteins is an early event in cancer development that results in increased proliferation and epithelial disorganization through means not fully characterized. Using the proliferating Drosophila melanogaster wing disc epithelium, we demonstrate that disruption of the junctional vs. basal polarity complexes results in increased epithelial proliferation via distinct downstream signaling pathways. Disruption of the basal polarity complex results in JNK-dependent proliferation, while disruption of the junctional complex primarily results in p38-dependent proliferation. Surprisingly, the Rho-Rok-Myosin contractility apparatus appears to play opposite roles in the regulation of the proliferative phenotype based on which polarity complex is disrupted. In contrast, non-autonomous Tumor Necrosis Factor (TNF) signaling appears to suppress the proliferation that results from apical-basal polarity disruption, regardless of which complex is disrupted. Finally we demonstrate that disruption of the junctional polarity complex activates JNK via the Rho-Rok-Myosin contractility apparatus independent of the cortical actin regulator, Moesin. [ABSTRACT FROM AUTHOR]

Protein Kinase C (PKC) plays a significant role in thrombin-induced loss of endothelial cell (EC) barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue–specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin), dominant negative PKCδ construct and PKCδ silencing (siRNA). In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ) and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis. [ABSTRACT FROM AUTHOR]

Cell-free protein synthesis (CFPS) is a powerful tool for the preparation of toxic proteins, directed protein evolution, and bottom-up synthetic biology. The transcription-translation machinery for CFPS is provided by cell extracts, which usually contain 20–30 mg/mL of proteins. In general, these cell extracts are prepared by physical disruption; however, this requires technical experience and special machinery. Here, we report a method to prepare cell extracts for CFPS using a biochemical method, which disrupts cells through the combination of lysozyme treatment, osmotic shock, and freeze-thaw cycles. The resulting cell extracts showed similar features to those obtained by physical disruption, and was able to synthesize active green fluorescent proteins in the presence of appropriate chemicals to a concentration of 20 μM (0.5 mg/mL). [ABSTRACT FROM AUTHOR]

Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo. [ABSTRACT FROM AUTHOR]

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy. [ABSTRACT FROM AUTHOR]

In S334ter-line-3 rat model of Retinitis Pigmentosa (RP), rod cell death induces the rearrangement of cones into mosaics of rings while the fibrotic processes of Müller cells remodel to fill the center of the rings. In contrast, previous work established that DL-alpha-aminoadipic-acid (AAA), a compound that transiently blocks Müller cell metabolism, abolishes these highly structured cone rings. Simultaneously, adherens-junction associated protein, Zonula occludens-1 (ZO-1) expression forms in a network between the photoreceptor segments and Müller cells processes. Thus, we hypothesized that AAA treatment alters the cone mosaic rings by disrupting the distal sealing formed by these fibrotic processes, either directly or indirectly, by down regulating the expression of ZO-1. Therefore, we examined these processes and ZO-1 expression at the outer retina after intravitreal injection of AAA and observed that AAA treatment transiently disrupts the distal glial sealing in RP retina, plus induces cones in rings to become more homogeneous. Moreover, ZO-1 expression is actively suppressed after 3 days of AAA treatment, which coincided with cone ring disruption. Similar modifications of glial sealing and cone distribution were observed after injection of siRNA to inhibit ZO-1 expression. These findings support our hypothesis and provide additional information about the critical role played by ZO-1 in glial sealing and shaping the ring mosaic in RP retina. These studies represent important advancements in the understanding of retinal degeneration’s etiology and pathophysiology. [ABSTRACT FROM AUTHOR]

Background: Poor central nervous system penetration of cytotoxic drugs due to the blood brain barrier (BBB) is a major limiting factor in the treatment of brain tumors. Most recurrent glioblastomas (GBM) occur within the peritumoral region. In this study, we describe a hyperthemic method to induce temporary disruption of the peritumoral BBB that can potentially be used to enhance drug delivery. Methods: Twenty patients with probable recurrent GBM were enrolled in this study. Fourteen patients were evaluable. MRI-guided laser interstitial thermal therapy was applied to achieve both tumor cytoreduction and disruption of the peritumoral BBB. To determine the degree and timing of peritumoral BBB disruption, dynamic contrast-enhancement brain MRI was used to calculate the vascular transfer constant (Ktrans) in the peritumoral region as direct measures of BBB permeability before and after laser ablation. Serum levels of brain-specific enolase, also known as neuron-specific enolase, were also measured and used as an independent quantification of BBB disruption. Results: In all 14 evaluable patients, Ktrans levels peaked immediately post laser ablation, followed by a gradual decline over the following 4 weeks. Serum BSE concentrations increased shortly after laser ablation and peaked in 1–3 weeks before decreasing to baseline by 6 weeks. Conclusions: The data from our pilot research support that disruption of the peritumoral BBB was induced by hyperthemia with the peak of high permeability occurring within 1–2 weeks after laser ablation and resolving by 4–6 weeks. This provides a therapeutic window of opportunity during which delivery of BBB-impermeant therapeutic agents may be enhanced. Trial Registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Background and Objectives: The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs) are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium. Methodology/Principal Findings: Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2), membrane type 1-MMP (MT1-MMP) and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohistochemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime. Conclusions/Significance: MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and activation, tight junction protein cleavage, and subsequent surface cell desquamation and renewal may be orchestrated by nocturnal circadian signals. [ABSTRACT FROM AUTHOR]