M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells

Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.     

Predicting therapy response in live tumor cells isolated with the flexible micro spring array device

Cells disseminated from primary epithelial tumors into peripheral blood, called circulating tumor cells (CTCs), can be monitored to assess metastases and to provide a surrogate marker of treatment response. Here, we demonstrate how the flexible micro spring array (FMSA) device—a novel microfluidic device that enriches CTCs by two physical parameters: size and deformability—could be used in the rational development of treatment intervention and as a method to study the fundamental biology of CTCs. Cancer cells of different origins were spiked into healthy samples of donor blood to mimic blood samples of metastatic cancer patients. This spiked human blood was filtered using the FMSA device, and the recovered cells were successfully expanded in vitro and in a novel in vivo system. A series of experiments were performed to characterize these cells and to investigate the effect of chemotherapy on the resulting cultures. As few as 20 colon cancer cells in 7.5 mL blood could be isolated with the FMSA device, expanded both in vitro and in vivo and used at 25 cells per well to obtain significant and reliable chemosensitivity data. We also show that isolating a low number of viable patient CTCs and maintaining them in culture for a few weeks is possible. The isolation of viable cancer cells from human blood using the FMSA device provides a novel and realistic means for studying the biology of viable CTCs and for testing drug efficacy on these rare cells—a hypothesis that can be tested in future clinical trials.     

Dual Function of CD81 in Influenza Virus Uncoating and Budding

As an obligatory pathogen, influenza virus co-opts host cell machinery to harbor infection and to produce progeny viruses. In order to characterize the virus-host cell interactions, several genome-wide siRNA screens and proteomic analyses have been performed recently to identify host factors involved in influenza virus infection. CD81 has emerged as one of the top candidates in two siRNA screens and one proteomic study. The exact role played by CD81 in influenza infection, however, has not been elucidated thus far. In this work, we examined the effect of CD81 depletion on the major steps of the influenza infection. We found that CD81 primarily affected virus infection at two stages: viral uncoating during entry and virus budding. CD81 marked a specific endosomal population and about half of the fused influenza virus particles underwent fusion within the CD81-positive endosomes. Depletion of CD81 resulted in a substantial defect in viral fusion and infection. During virus assembly, CD81 was recruited to virus budding site on the plasma membrane, and in particular, to specific sub-viral locations. For spherical and slightly elongated influenza virus, CD81 was localized at both the growing tip and the budding neck of the progeny viruses. CD81 knockdown led to a budding defect and resulted in elongated budding virions with a higher propensity to remain attached to the plasma membrane. Progeny virus production was markedly reduced in CD81-knockdown cells even when the uncoating defect was compensated. In filamentous virus, CD81 was distributed at multiple sites along the viral filament. Taken together, these results demonstrate important roles of CD81 in both entry and budding stages of the influenza infection cycle.     

CRIS—A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending

The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca²⁺ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca²⁺-regulated proteins that—in mature sperm—are involved in flagellar bending.     

A 2200-Year Record of Permafrost Dynamics and Carbon Cycling in a Collapse-Scar Bog, Interior Alaska

Recent high-latitude warming is increasing the vulnerability of permafrost to thaw, which is amplified by local disturbances such as fire. However, the long-term ecological effects and carbon dynamics are not well understood. Here we present a 2200-year record of pollen, plant macrofossils, testate amoebae, and apparent carbon (C) accumulation rates from two peat cores in a collapse-scar bog (thermokarst) near Fairbanks, Alaska. A black spruce ecosystem with low apparent C accumulation rates existed on the site during the first ~1500 years of the record. We identify two thaw events, which are linked to local fires. Permafrost aggraded rapidly following the first thaw, which we attribute to local vegetation feedbacks and a cooler climate. The second thaw event at 525 cal y BP is preceded by a stand-replacing fire, as evidenced by a drastic decline in Picea and an initial increase in Epilobium, Salix, and ericaceous shrubs, followed by a sustained increase in Populus. Locally, the forest does not recover for more than 100 years, and the site has remained permafrost-free for the last 500 years. Following thaw, average apparent C accumulation rates (60 to >100 g C m^?2 y^?1) are 5–6 times higher than average boreal C accumulation rates, indicating that peat C accumulation rates can remain substantially elevated for much more than a century following thaw. The low apparent C accumulation for the formerly forested, permafrost peat (<5 g C m^?2 y^?1) may suggest that C accumulation increases substantially following thaw, but it remains unknown whether deep peat C loss occurred immediately following thaw. Well-preserved Sphagnum peat dominates during this period of rapid accumulation, except for an interval from ~400 to 275 cal y BP which alternates between Sphagnum and vascular plant-dominated peat and wetter, minerotrophic conditions. A decline in Picea pollen during this interval and again ~100 cal y BP suggests a decrease in suitable substrate for tree growth likely attributable to thermokarst expansion on the collapse-scar margin. These findings suggest that the combined effects of fire and thermokarst will result in a long-term reduction of spruce ecosystems in interior Alaska.     

Standardized activities of daily living in presence of sub-acute low-back pain: A pilot study

The aim of this pilot study was to investigate how sub-acute low-back pain (LBP) patients differed with respect to control in movements and muscle activation during standardized tasks representing daily living activities, and explore relationships between cognition and measured motor performance. Linear and nonlinear parameters were computed from kinetics, kinematics and muscle activity recorded for 12 sub-acute patients and 12 healthy matched controls during trunk flexion, sit-to-stand from a chair and lifting a box. Cognitive variables were collected to explore relationships with biomechanical parameters. For trunk flexion, left external abdominal oblique muscle activity level was lower for patients compared with controls (p < 0.05), whereas sample entropy (complexity) was higher (p < 0.05). Normalized mutual information was lower for patients compared with controls for left and right erector spinae (p < 0.05). Level of activity of left external abdominal oblique correlated negatively with cognitive ignoring and positively with catastrophizing (p ? 0.05), and catastrophizing also correlated positively with functional connectivity of abdominal muscles (p < 0.05). Signs of reorganization in muscle activation pointed towards different synergistic actions in trunk muscles in sub-acute LBP patients compared with controls. The interplay with maladaptive cognition suggested that in the subacute stage of LBP, both biomechanical and cognitive factors should be taken into account.     

Spatial distribution of surface action potentials generated by individual motor units in the human biceps brachii muscle

This study analyses the spatial distribution of individual motor unit potentials (MUPs) over the skin surface and the influence of motor unit depth and recording configuration on this distribution. Multichannel surface (13 × 5 electrode grid) and intramuscular (wire electrodes inserted with needles of lengths 15 and 25 mm) electromyographic (EMG) signals were concurrently recorded with monopolar derivations from the biceps brachii muscle of 10 healthy subjects during 60-s isometric contractions at 20% of the maximum torque. Multichannel monopolar MUPs of the target motor unit were obtained by spike-triggered averaging of the surface EMG. Amplitude and frequency characteristics of monopolar and bipolar MUPs were calculated for locations along the fibers’ direction (longitudinal), and along the direction perpendicular (transverse) to the fibers. In the longitudinal direction, monopolar and bipolar MUPs exhibited marked amplitude changes that extended for 16–32 mm and 16–24 mm over the innervation and tendon zones, respectively. The variation of monopolar and bipolar MUP characteristics was not symmetrical about the innervation zone. Motor unit depth had a considerable influence on the relative longitudinal variation of amplitude for monopolar MUPs, but not for bipolar MUPs. The transverse extension of bipolar MUPs ranged between 24 and 32 mm, whereas that of monopolar MUPs ranged between 72 and 96 mm. The mean power spectral frequency of surface MUPs was highly dependent on the transverse electrode location but not on depth. This study provides a basis for the interpretation of the contribution of individual motor units to the interference surface EMG signal.     

Modulation of SERCA in the chronic phase of adjuvant arthritis as a possible adaptation mechanism of redox imbalance

Adjuvant arthritis (AA) is a condition that involves systemic oxidative stress. Unexpectedly, it was found that sarcoplasmic reticulum Ca^{2 +}-ATPase (SERCA) activity was elevated in muscles of rats with AA compared to controls, suggesting possible conformational changes in the enzyme. There was no alteration in the nucleotide binding site but rather in the transmembrane domain according to the tryptophan polar/non-polar fluorescence ratio. Higher relative expression of SERCA, higher content of nitrotyrosine but no increase in phospholipid oxidation in AA SR was found. In vitro treatments of SR with HOCl showed that in AA animals SERCA activity was more susceptible to oxidative stress, but SR phospholipids were more resistant and SERCA could also be activated by phosphatidic acid. It was concluded that increased SERCA activity in AA was due to increased levels of SERCA protein and structural changes to the protein, probably induced by direct and specific oxidation involving reactive nitrogen species.