Modulation in the expression levels and protein localization of cell volume and actin cytoskeleton associated proteins by the Slit/ Robo signaling pathway in Glioblastoma multiforme.

Honorable Mention Winner Glioblastoma multiforme (GBM) is the most aggressive and malignant primary brain tumor in adults1. Even after surgical resection, chemotherapy, and radiation, the average patient survival rate is 14 months2. Recent studies indicate that GBM tumors that contact the lateral ventricles within the subventricular zone (SVZ) tend to migrate and recur at distal locations after surgical resection3. One important signaling pathway in Neural Progenitor Cells (NPCs) migration from the SVZ to the olfactory bulbs is the Slit/Robo signaling pathway4. Slit2 proteins are produced by the choroid plexus and create a gradient across ependymal cells. Slit2 proteins then interact with the Roundabout (Robo) receptor on NPCs and acts as a chemo-repelling agent5. This Slit2/ Robo1 signaling pathway is a possible mechanism that contributes to GBM migration from the SVZ to distal locations. The intracellular changes when the Slit/Robo pathway is activated includes changes in cell volume regulation and actin cytoskeleton dynamics6. We then examined how the Slit2/ Robo1 signaling pathway causes changes in the expression of cell volume regulatory genes (AQP4, KCC1, NKCC1, NCC), actin cytoskeleton genes regulatory genes (p-ERM, p-Cofilin), and the localization of actin-associated proteins (FAK, Paxillin, N-WASP, Vinculin). It was found that the Slit/Robo signaling pathway increases the expression levels of genes that contribute to cell volume regulation in GBM cells, and the Slit/Robo signaling pathway increases the phosphorylation of p-Cofilin and p-ERM. This indicates that the Slit2/ Robo1 pathway participates in the regulation of cell volume and actin cytoskeleton dynamics in GBM cells

Determining glioblastoma proteome changes in response to lateral ventricle neural stem cells

Glioblastoma (GBM) is the most common and malignant primary tumor in adults. When GBM tumors are located close to the lateral ventricle they display a more aggressive recurrence pattern and negatively impact patient survival. These findings suggest the involvement of the subventricular zone neurogenic niche in GBM malignancy. To define the inter-cellular communication between neural stem cells and GBM cells, we optimized a tool to determine cell-specific proteomic changes of GBM cells in response to neural stem cell proximity. We cloned the mutated methionyl-tRNA synthetase (MetRS) gene into the lentiviral plasmid MetRS puro. MetRS allows for incorporation of azide-tagged methionine analog azidonorleucine (ANL) into newly formed proteins, effectively labeling proteins synthesized by expressing cells. We utilized the pLKO.1 vector backbone allowing puromycin resistance as a selection method. The MetRS L274 modification was confirmed, as only MetRS-transduced cells of both commercial HEK and primary GBM1A cell lines selectively incorporated ANL. Following verification, we successfully packaged the plasmid into a lentivirus. We transduced primary human fetal neural stem cell (hfNSC) and GBM lines and selected the MetRS-expressing cells by puromycin exposure. After 96 hours, wild type (WT) cells died while successfully transduced cells exhibited resistance and the ANL-compatible MetRS enzyme. Co-cultures consisting of MetRS-transduced GBM and WT hfNSCs were used to simulate a similar environment of glioblastoma neighboring lateral ventricles. Proteome Profiler results showed a significant downregulation of an angiogenesis inhibitor and upregulation of malignancy promoting proteins in GBM1A. Going forward, this analysis method will be used for cell-specific proteomics in vivo

The generation of oligodendroglial cells is preserved in the rostral migratory stream during aging.

The subventricular zone (SVZ) is the largest source of newly generated cells in the adult mammalian brain. SVZ-derived neuroblasts migrate via the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate into mature neurons. Additionally, a small proportion of SVZ-derived cells contribute to the generation of myelinating oligodendrocytes. The production of new cells in the SVZ decreases during aging, affecting the incorporation of new neurons into the OB. However, the age-related changes that occur across the RMS are not fully understood. In this study we evaluate how aging affects the cellular organization of migrating neuroblast chains, the proliferation, and the fate of the newly generated cells in the SVZ-OB system. By using electron microscopy and immunostaining, we found that the RMS path becomes discontinuous and its cytoarchitecture is disorganized in aged mice (24-month-old mice). Subsequently, OB neurogenesis was impaired in the aged brain while the production of oligodendrocytes was not compromised. These findings provide new insight into oligodendrocyte preservation throughout life. Further exploration of this matter could help the development of new strategies to prevent neurological disorders associated with senescence

X-ray determination of compressive residual Stresses in spring steel generated by high-speed water Quenching

Automotive components manufacturers use the 5160 steel in leaf and coil springs. The industrial heat treatment process consists in austenitizing followed by the oil quenching and tempering process. Typically, compressive residual stresses are induced by shot peening on the surface of automotive springs to bestow compressive residual stresses that improve the fatigue resistance and increase the service life of the parts after heat treatment. In this work, a high-speed quenching was used to achieve compressive residual stresses on the surface of AISI/SAE 5160 steel samples by producing high thermal gradients and interrupting the cooling in order to generate a case-core microstructure. A special laboratory equipment was designed and built, which uses water as the quenching media in a high-speed water chamber. The severity of the cooling was characterized with embedded thermocouples to obtain the cooling curves at different depths from the surface. Samples were cooled for various times to produce different hardened case depths. The microstructure of specimens was observed with a scanning electron microscope (SEM). X-ray diffraction (XRD) was used to estimate the magnitude of residual stresses on the surface of the specimens. Compressive residual stresses at the surface and sub-surface of about -700 MPa were obtained.Peer ReviewedPostprint (published version

Use of Mesenchymal Stem Cells in Pre-Clinical Models of Spinal Cord Injury

Spinal Cord Injury (SCI) is a devastating disease that causes disruption of sensorimotor function below the site of injury. Current management is based on surgical decompression of the neural tissue and pharmacotherapy; however, there is no gold standard treatment readily available for patients in the clinic. This indicates that novel therapeutic strategies for the treatment are still needed in the clinical setting. There are several alternatives that are currently under investigation for the treatment of this disease, with increasing focus in regenerative medicine treatments. Mesenchymal stem cells (MSCs) are one of the most promising candidates for stem cell therapy in SCI, as they are easily obtained, have high safety profiles, and help with neural regeneration in SCI mainly via release of trophic factors, neovascularization, and immunomodulation. In this work, authors provide an insight of the available MSC for neural regeneration, their therapeutic role, and the potential MSC-based therapies for SCI

Shear Forces during Blast, Not Abrupt Changes in Pressure Alone, Generate Calcium Activity in Human Brain Cells

Blast-Induced Traumatic Brain Injury (bTBI) describes a spectrum of injuries caused by an explosive force that results in changes in brain function. The mechanism responsible for primary bTBI following a blast shockwave remains unknown. We have developed a pneumatic device that delivers shockwaves, similar to those known to induce bTBI, within a chamber optimal for fluorescence microscopy. Abrupt changes in pressure can be created with and without the presence of shear forces at the surface of cells. In primary cultures of human central nervous system cells, the cellular calcium response to shockwaves alone was negligible. Even when the applied pressure reached 15 atm, there was no damage or excitation, unless concomitant shear forces, peaking between 0.3 to 0.7 Pa, were present at the cell surface. The probability of cellular injury in response to a shockwave was low and cell survival was unaffected 20 hours after shockwave exposure

Age-Related Changes in Astrocytic and Ependymal Cells of the Subventricular Zone

Neurogenesis persists in the adult subventricular zone (SVZ) of the mammalian brain. During aging, the SVZ neurogenic capacity undergoes a progressive decline, which is attributed to a decrease in the population of neural stem cells (NSCs). However, the behavior of the NSCs that remain in the aged brain is not fully understood. Here we performed a comparative ultrastructural study of the SVZ niche of 2-month-old and 24-month-old male C57BL/6 mice, focusing on the NSC population. Using thymidine-labeling, we showed that residual NSCs in the aged SVZ divide less frequently than those in young mice. We also provided evidence that ependymal cells are not newly generated during senescence, as others studies suggest. Remarkably, both astrocytes and ependymal cells accumulated a high number of intermediate filaments and dense bodies during aging, resembling reactive cells. A better understanding of the changes occurring in the neurogenic niche during aging will allow us to develop new strategies for fighting neurological disorders linked to senescence

Resistance against two lytic phage variants attenuates virulence and antibiotic resistance in Pseudomonas aeruginosa

BackgroundBacteriophage therapy is becoming part of mainstream Western medicine since antibiotics of clinical use tend to fail. It involves applying lytic bacteriophages that self-replicate and induce cell lysis, thus killing their hosts. Nevertheless, bacterial killing promotes the selection of resistant clones which sometimes may exhibit a decrease in bacterial virulence or antibiotic resistance.MethodsIn this work, we studied the Pseudomonas aeruginosa lytic phage φDCL-PA6 and its variant φDCL-PA6α. Additionally, we characterized and evaluated the production of virulence factors and the virulence in a Galleria mellonella model of resistant mutants against each phage for PA14 and two clinical strains.ResultsPhage φDCL-PA6α differs from the original by only two amino acids: one in the baseplate wedge subunit and another in the tail fiber protein. According to genomic data and cross-resistance experiments, these changes may promote the change of the phage receptor from the O-antigen to the core lipopolysaccharide. Interestingly, the host range of the two phages differs as determined against the Pseudomonas aeruginosa reference strains PA14 and PAO1 and against nine multidrug-resistant isolates from ventilator associated pneumonia.ConclusionsWe show as well that phage resistance impacts virulence factor production. Specifically, phage resistance led to decreased biofilm formation, swarming, and type III secretion; therefore, the virulence towards Galleria mellonella was dramatically attenuated. Furthermore, antibiotic resistance decreased for one clinical strain. Our study highlights important potential advantages of phage therapy’s evolutionary impact that may be exploited to generate robust therapy schemes

Transitions of cardio-metabolic risk factors in the Americas between 1980 and 2014

Describing the prevalence and trends of cardiometabolic risk factors that are associated with non-communicable diseases (NCDs) is crucial for monitoring progress, planning prevention, and providing evidence to support policy efforts. We aimed to analyse the transition in body-mass index (BMI), obesity, blood pressure, raised blood pressure, and diabetes in the Americas, between 1980 and 2014

Worldwide trends in diabetes since 1980: a pooled analysis of 751 population-based studies with 4.4 million participants

BACKGROUND: One of the global targets for non-communicable diseases is to halt, by 2025, the rise in the age-standardised adult prevalence of diabetes at its 2010 levels. We aimed to estimate worldwide trends in diabetes, how likely it is for countries to achieve the global target, and how changes in prevalence, together with population growth and ageing, are affecting the number of adults with diabetes. METHODS: We pooled data from population-based studies that had collected data on diabetes through measurement of its biomarkers. We used a Bayesian hierarchical model to estimate trends in diabetes prevalence—defined as fasting plasma glucose of 7·0 mmol/L or higher, or history of diagnosis with diabetes, or use of insulin or oral hypoglycaemic drugs—in 200 countries and territories in 21 regions, by sex and from 1980 to 2014. We also calculated the posterior probability of meeting the global diabetes target if post-2000 trends continue. FINDINGS: We used data from 751 studies including 4 372 000 adults from 146 of the 200 countries we make estimates for. Global age-standardised diabetes prevalence increased from 4·3% (95% credible interval 2·4–7·0) in 1980 to 9·0% (7·2–11·1) in 2014 in men, and from 5·0% (2·9–7·9) to 7·9% (6·4–9·7) in women. The number of adults with diabetes in the world increased from 108 million in 1980 to 422 million in 2014 (28·5% due to the rise in prevalence, 39·7% due to population growth and ageing, and 31·8% due to interaction of these two factors). Age-standardised adult diabetes prevalence in 2014 was lowest in northwestern Europe, and highest in Polynesia and Micronesia, at nearly 25%, followed by Melanesia and the Middle East and north Africa. Between 1980 and 2014 there was little change in age-standardised diabetes prevalence in adult women in continental western Europe, although crude prevalence rose because of ageing of the population. By contrast, age-standardised adult prevalence rose by 15 percentage points in men and women in Polynesia and Micronesia. In 2014, American Samoa had the highest national prevalence of diabetes (>30% in both sexes), with age-standardised adult prevalence also higher than 25% in some other islands in Polynesia and Micronesia. If post-2000 trends continue, the probability of meeting the global target of halting the rise in the prevalence of diabetes by 2025 at the 2010 level worldwide is lower than 1% for men and is 1% for women. Only nine countries for men and 29 countries for women, mostly in western Europe, have a 50% or higher probability of meeting the global target. INTERPRETATION: Since 1980, age-standardised diabetes prevalence in adults has increased, or at best remained unchanged, in every country. Together with population growth and ageing, this rise has led to a near quadrupling of the number of adults with diabetes worldwide. The burden of diabetes, both in terms of prevalence and number of adults affected, has increased faster in low-income and middle-income countries than in high-income countries. FUNDING: Wellcome Trust