N-terminal variant Asp14Asn of the human p70 S6 Kinase 1 enhances translational signaling causing different effects in developing and mature neuronal cells.

The ribosomal p70 S6 Kinase 1 (S6K1) has been implicated in the etiology of complex neurological diseases including autism, depression and dementia. Though no major gene disruption has been reported in humans in RPS6KB1, single nucleotide variants (SNVs) causing missense mutations have been identified, which have not been assessed for their impact on protein function. These S6K1 mutations have the potential to influence disease progression and treatment response. We mined the Simon Simplex Collection (SSC) and SPARK autism database to find inherited SNVs in S6K1 and characterized the effect of two missense SNVs, Asp14Asn (allele frequency = 0.03282%) and Glu44Gln (allele frequency = 0.0008244%), on S6K1 function in HEK293, human ES cells and primary neurons. Expressing Asp14Asn in HEK293 cells resulted in increased basal phosphorylation of downstream targets of S6K1 and increased de novo translation. This variant also showed blunted response to the specific S6K1 inhibitor, FS-115. In human embryonic cell line Shef4, Asp14Asn enhanced spontaneous neural fate specification in the absence of differentiating growth factors. In addition to enhanced translation, neurons expressing Asp14Asn exhibited impaired dendritic arborization and increased levels of phosphorylated ERK 1/2. Finally, in the SSC families tracked, Asp14Asn segregated with lower IQ scores when found in the autistic individual rather than the unaffected sibling. The Glu44Gln mutation showed a milder, but opposite phenotype in HEK cells as compared to Asp14Asn. Although the Glu44Gln mutation displayed increased neuronal translation, it had no impact on neuronal morphology. Our results provide the first characterization of naturally occurring human S6K1 variants on cognitive phenotype, neuronal morphology and maturation, underscoring again the importance of translation control in neural development and plasticity.

Association of thoracic epidural analgesia with risk of atrial arrhythmias after pulmonary resection: a retrospective cohort study.

PURPOSE: Atrial arrhythmias are common after non-cardiac thoracic surgery. We tested the hypothesis that TEA reduces the risk of new-onset atrial arrhythmias after pulmonary resection. METHODS: We evaluated patients who had pulmonary resection. New-onset atrial arrhythmias detected before hospital discharge was our primary outcome. Secondary outcomes included other cardiovascular complications, pulmonary complications, time-weighted average pain score over 72 h, and duration of hospitalization. Patients with combination of general anesthesia and TEA were matched on propensity scores with patients given general anesthesia only. The matched groups were compared by use of logistic regression, linear regression, or Cox proportional hazards regression, as appropriate. RESULTS: Among 1,236 patients who had pulmonary resections, 937 received a combination of general anesthesia and TEA (TEA) and 299 received general anesthesia only (non-TEA). We successfully matched 311 TEA patients with 132 non-TEA patients. We did not find a significant association between TEA and postoperative atrial arrhythmia (odds ratio (95 % CI) of 1.05 (0.50, 2.19), P = 0.9). TEA was not significantly associated with length of hospital stay or postoperative pulmonary complications (odds ratio (95 % CI) of 0.71 (0.22, 2.29), P = 0.47). TEA patients experienced fewer postoperative cardiovascular complications; although the association was not statistically significant (odds ratio (95 % CI) of 0.30 (0.06, 1.45), P = 0.06). Time-weighted average pain scores were similar in the two groups. CONCLUSION: TEA was not associated with reduced occurrence of postoperative atrial arrhythmia. Although postoperative pulmonary complications were similar with and without TEA, TEA patients tended to experience fewer cardiovascular complications.

Astrocytes mediate cell non-autonomous correction of aberrant firing in human FXS neurons.

Pre-clinical studies of fragile X syndrome (FXS) have focused on neurons, with the role of glia remaining largely underexplored. We examined the astrocytic regulation of aberrant firing of FXS neurons derived from human pluripotent stem cells. Human FXS cortical neurons, co-cultured with human FXS astrocytes, fired frequent short-duration spontaneous bursts of action potentials compared with less frequent, longer-duration bursts of control neurons co-cultured with control astrocytes. Intriguingly, bursts fired by FXS neurons co-cultured with control astrocytes are indistinguishable from control neurons. Conversely, control neurons exhibit aberrant firing in the presence of FXS astrocytes. Thus, the astrocyte genotype determines the neuronal firing phenotype. Strikingly, astrocytic-conditioned medium, and not the physical presence of astrocytes, is capable of determining the firing phenotype. The mechanistic basis of this effect indicates that the astroglial-derived protein, S100ß, restores normal firing by reversing the suppression of a persistent sodium current in FXS neurons.

Stress Elicits Contrasting Effects on Rac1-Cofilin Signaling in the Hippocampus and Amygdala.

There is accumulating evidence for contrasting patterns of stress-induced morphological and physiological plasticity in glutamatergic synapses of the hippocampus and amygdala. The same chronic stress that leads to the formation of dendritic spines in the basolateral amygdala (BLA) of rats, leads to a loss of spines in the hippocampus. However, the molecular underpinnings of these divergent effects of stress on dendritic spines are not well understood. Since the activity of the Rho GTPase Rac1 and the actin-depolymerizing factor cofilin are known to play a pivotal role in spine morphogenesis, we investigated if alterations in this signaling pathway reflect the differential effects of stress on spine plasticity in the hippocampus and amygdala. A day after the end of chronic immobilization stress (2 h/day for 10 days), we found a reduction in the activity of Rac1, as well as its effector p21-activated kinase 1 (PAK1), in the rat hippocampus. These changes, in turn, decreased cofilin phosphorylation alongside a reduction in the levels of profilin isoforms. In striking contrast, the same chronic stress increased Rac1, PAK1 activity, cofilin phosphorylation, and profilin levels in the BLA, which is consistent with enhanced actin polymerization leading to spinogenesis in the BLA. In the hippocampus, on the other hand, the same stress caused the opposite changes, the functional consequences of which would be actin depolymerization leading to the elimination of spines. Together, these findings reveal a role for brain-region specific differences in the dysregulation of Rac1-to-cofilin signaling in the effects of repeated stress on two brain areas that are implicated in the emotional and cognitive symptoms of stress-related psychiatric disorders.

Functional recovery after transplantation of bone marrow-derived human mesenchymal stromal cells in a rat model of spinal cord injury.

BACKGROUND AIMS: Spinal cord injury (SCI) is a medically untreatable condition for which stem cells have created hope. Pre-clinical and clinical studies have established that these cells are safe for transplantation. The dose dependency, survivability, route of administration, cell migration to injury site and effect on sensory and motor behavior in an SCI-induced paraplegic model were studied. METHODS: A spinal cord contusion injury model was established in rats. Bone marrow (BM) mesenchymal stromal cells (MSC) were tagged to facilitate tracing in vivo. Two different doses (2 and 5 million cells/kg body weight) and two different routes of infusion (site of injury and lumbar puncture) were tested during and after the spinal shock period. The animals were tested post-transplantation for locomotor capacity, motor control, sensory reflex, posture and body position. Stem cell migration was observed 1 month post-transplantation in spinal cord sections. RESULTS: The overall results demonstrated that transplantation of BM MSC significantly improved the locomotor and sensory behavior score in the experimental group compared with the sham control group, and these results were dose dependent. All the infused stem cells could be visualized at the site of injury and none was visualized at the injected site. This indicated that the cells had survived in vivo, were probably chemoattracted and had migrated to the lesion site. CONCLUSIONS: MSC transplanted with a lumbar puncture method migrate to the site of injury and are the most suitable for SCI healing. These cells demonstrate a dose-dependent effect and promote functional recovery when injected during or after the spinal shock period.

Cortical neurons derived from human pluripotent stem cells lacking FMRP display altered spontaneous firing patterns.

BACKGROUND: Fragile X syndrome (FXS), a neurodevelopmental disorder, is a leading monogenetic cause of intellectual disability and autism spectrum disorder. Notwithstanding the extensive studies using rodent and other pre-clinical models of FXS, which have provided detailed mechanistic insights into the pathophysiology of this disorder, it is only relatively recently that human stem cell-derived neurons have been employed as a model system to further our understanding of the pathophysiological events that may underlie FXS. Our study assesses the physiological properties of human pluripotent stem cell-derived cortical neurons lacking fragile X mental retardation protein (FMRP). METHODS: Electrophysiological whole-cell voltage- and current-clamp recordings were performed on two control and three FXS patient lines of human cortical neurons derived from induced pluripotent stem cells. In addition, we also describe the properties of an isogenic pair of lines in one of which FMR1 gene expression has been silenced. RESULTS: Neurons lacking FMRP displayed bursts of spontaneous action potential firing that were more frequent but shorter in duration compared to those recorded from neurons expressing FMRP. Inhibition of large conductance Ca2+-activated K+ currents and the persistent Na+ current in control neurons phenocopies action potential bursting observed in neurons lacking FMRP, while in neurons lacking FMRP pharmacological potentiation of voltage-dependent Na+ channels phenocopies action potential bursting observed in control neurons. Notwithstanding the changes in spontaneous action potential firing, we did not observe any differences in the intrinsic properties of neurons in any of the lines examined. Moreover, we did not detect any differences in the properties of miniature excitatory postsynaptic currents in any of the lines. CONCLUSIONS: Pharmacological manipulations can alter the action potential burst profiles in both control and FMRP-null human cortical neurons, making them appear like their genetic counterpart. Our studies indicate that FMRP targets that have been found in rodent models of FXS are also potential targets in a human-based model system, and we suggest potential mechanisms by which activity is altered.

Ex vivo-expanded autologous bone marrow-derived mesenchymal stromal cells in human spinal cord injury/paraplegia: a pilot clinical study.

BACKGROUND AIMS: Spinal cord injury (SCI) is a medically untreatable condition for which stem cells have created hope in the last few years. Earlier pre-clinical reports have shown that transplantation of bone marrow (BM) mesenchymal stromal cells (MSC) in SCI-simulated models can produce encouraging results. In a clinical pilot study, we investigated the growth kinetics of BM MSC from SCI patients, their safety and functional improvement post-transplantation. METHODS: Thirty patients with clinically complete SCI at cervical or thoracic levels were recruited and divided into two groups based on the duration of injury. Patients with <6 months of post-SCI were recruited into group 1 and patients with >6 months of post-SCI were included into group 2. Autologous BM was harvested from the iliac crest of SCI patients under local anesthesia and BM MSC were isolated and expanded ex vivo. BM MSC were tested for quality control, characterized for cell surface markers and transplanted back to the patient via lumbar puncture at a dose of 1 x 10(6) cells/kg body weight. RESULTS: At the time of writing, three patients had completed 3 years of follow-up post-BM MSC administration, 10 patients 2 years follow-up and 10 patients 1 year follow-up. Five patients have been lost to follow-up. None of the patients have reported any adverse events associated with BM MSC transplantation. CONCLUSIONS: The results indicate that our protocol is safe with no serious adverse events following transplantation in SCI patients. The number of patients recruited and the uncontrolled nature of the trial do not permit demonstration of the effectiveness of the treatment involved. However, the results encourage further trials with higher doses and different routes of administration in order to demonstrate the recovery/efficacy if any, in SCI patients.

Modelling Protein Synthesis as A Biomarker in Fragile X Syndrome Patient-Derived Cells.

The most conserved molecular phenotype of Fragile X Syndrome (FXS) is aberrant protein synthesis. This has been validated in a variety of experimental model systems from zebrafish to rats, patient-derived lymphoblasts and fibroblasts. With the advent of personalized medicine paradigms, patient-derived cells and their derivatives are gaining more translational importance, not only to model disease in a dish, but also for biomarker discovery. Here we review past and current practices of measuring protein synthesis in FXS, studies in patient derived cells and the inherent challenges in measuring protein synthesis in them to offer usable avenues of modeling this important metabolic metric for further biomarker development.

FMRP Interacts with C/D Box snoRNA in the Nucleus and Regulates Ribosomal RNA Methylation.

FMRP is an RNA-binding protein that is known to localize in the cytoplasm and in the nucleus. Here, we have identified an interaction of FMRP with a specific set of C/D box snoRNAs in the nucleus. C/D box snoRNAs guide 2'O methylations of ribosomal RNA (rRNA) on defined sites, and this modification regulates rRNA folding and assembly of ribosomes. 2'O methylation of rRNA is partial on several sites in human embryonic stem cells, which results in ribosomes with differential methylation patterns. FMRP-snoRNA interaction affects rRNA methylation on several of these sites, and in the absence of FMRP, differential methylation pattern of rRNA is significantly altered. We found that FMRP recognizes ribosomes carrying specific methylation patterns on rRNA and the recognition of methylation pattern by FMRP may potentially determine the translation status of its target mRNAs. Thus, FMRP integrates its function in the nucleus and in the cytoplasm.

Role of catecholaminergic terminals in the preoptic area in behavioural thermoregulation in rats.

This study was conducted to find out the role of the catecholaminergic terminals in the preoptic area (POA) in selection of ambient temperature in rats. The adult male Wistar rats (n = 6) were allowed to choose between three ambient temperatures (24 degrees C, 27 degrees C and 30 degrees C). Rats could move about freely from one ambient temperature to another, in a specially designed environmental chamber having three interconnected compartments, which were maintained at the above mentioned temperature. The results show that the normal rats preferred to stay at 27 degrees C both during day and night. After the lesion of catecholaminergic terminals in the POA with 6-hydroxydopamine (6-OHDA), the animals preferred 24 degrees C on the third and seventh day and 27 degrees C on the fourteenth and twenty first day after lesion. The alteration in thermal preference was associated with an elevation of rectal temperature. The study suggests that the catecholaminergic terminals of the POA play an important role in integrating behavioural and non-behavioural thermoregulatory responses, but in its absence the rest of the brain takes over some of its functions.

Asleep-awake-asleep craniotomy: a comparison with general anesthesia for resection of supratentorial tumors.

The anesthetic plan for patients undergoing awake craniotomy, when compared to craniotomy under general anesthesia, is different, in that it requires changes in states of consciousness during the procedure. This retrospective review compares patients undergoing an asleep-awake-asleep technique for craniotomy (group AW: n = 101) to patients undergoing craniotomy under general anesthesia (group AS: n = 77). Episodes of desaturation (AW = 31% versus AS = 1%, p < 0.0001), although temporary, and hypercarbia (AW = 43.75 mmHg versus AS = 32.75 mmHg, p < 0.001) were more common in the AW group. The mean arterial pressure during application of head clamp pins and emergence was significantly lower in AW patients compared to AS patients (pinning 91.47 mmHg versus 102.9 mmHg, p < 0.05 and emergence 84.85 mmHg versus 105 mmHg, p < 0.05). Patients in the AW group required less vasopressors intraoperatively (AW = 43% versus AS = 69%, p < 0.01). Intraoperative fluids were comparable between the two groups. The post anesthesia care unit (PACU) administered significantly fewer intravenous opioids in the AW group. The length of stay in the PACU and hospital was comparable in both groups. Thus, asleep-awake-asleep craniotomies with propofol-dexmedetomidine infusion had less hemodynamic response to pinning and emergence, and less overall narcotic use compared to general anesthesia. Despite a higher incidence of temporary episodes of desaturation and hypoventilation, no adverse clinical consequences were seen.

Bilateral transplantation of allogenic adult human bone marrow-derived mesenchymal stem cells into the subventricular zone of Parkinson's disease: a pilot clinical study.

The progress of PD and its related disorders cannot be prevented with the medications available. In this study, we recruited 8 PD and 4 PD plus patients between 5 to 15 years after diagnosis. All patients received BM-MSCs bilaterally into the SVZ and were followed up for 12 months. PD patients after therapy reported a mean improvement of 17.92% during "on" and 31.21% during "off" period on the UPDRS scoring system. None of the patients increased their medication during the follow-up period. Subjectively, the patients reported clarity in speech, reduction in tremors, rigidity, and freezing attacks. The results correlated with the duration of the disease. Those patients transplanted in the early stages of the disease (less than 5 years) showed more improvement and no further disease progression than the later stages (11-15 years). However, the PD plus patients did not show any change in their clinical status after stem cell transplantation. This study demonstrates the safety of adult allogenic human BM-MSCs transplanted into the SVZ of the brain and its efficacy in early-stage PD patients.

Phenotypic and functional comparison of optimum culture conditions for upscaling of bone marrow-derived mesenchymal stem cells.

Human adult bone marrow-derived mesenchymal stem cells (MSCs) are a promising tool in the newly emerging avenue of regenerative medicine. MSCs have already been translated from basic research to clinical transplantation research. However, there is still a lack of consensus on the ideal method of culturing MSCs. Here we have compared different culture conditions of human MSCs with an attempt to preserve their characteristics and multi-lineage differentiation potential. We compare the different basal culture media DMEM-F12, DMEM-high glucose (DMEM-HG), DMEM-low glucose (DMEM-LG), knock-out DMEM (DMEM-KO) and Mesencult on the proliferation rate, surface markers and differentiation potentials of MSCs. At every fifth passage until the 25th passage, the differentiation potential and the presence of a panel of surface markers was observed, using flow cytometry. We also compared the characteristics of human MSCs when cultured in reduced concentrations of fetal bovine serum (FBS), knockout serum replacement (KO-SR) and human plasma. Data indicate that the presence of serum is essential to sustain and propagate MSCs cultures. The choice of basal medium is equally important so as to preserve their characteristics and multipotent properties even after prolonged culture in vitro. With MSCs emerging as a popular tool for regenerative therapies in incurable diseases, it is essential to be able to obtain a large number of MSCs that continue to preserve their characteristics following passaging. The data reveal the optimum basal medium for prolonged culture of MSCs while retaining their ability to differentiate and hence this may be used for up-scaling to provide sufficient numbers for transplantation.

Adult stem cells: a clinical update.

There has been unprecedented interest in stem cell research mainly because of their true potential and hope that they offer to the patients as a cell therapy with the prospect to treat hitherto incurable diseases. Despite the worldwide interest and efforts that have been put in this research, major fundamental issues are still unresolved. Adult stem cells such as hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) are already under clinical applications and there are several examples of plasticity and self-renewal where adult stem cells or their precursor cells can be re-programmed by extra cellular cues or internal cues to alter their character in a way that could have important application for cell therapy and regenerative medicine. From a clinical perspective, no other area of stem cell biology has been applied as successfully as has transplantation of bone marrow stem cells and cord blood stem cells for the treatment of hematological diseases. In the last few years, research in stem cell biology has expanded staggeringly, engendering new perspectives concerning the identity, origin, and full therapeutic potential of tissue-specific stem cells. This review will focus on the use of adult stem cells, its biology in the context of cell plasticity and their therapeutic potential for repair of different tissues and organs.

Effect of holding time, temperature and different parenteral solutions on viability and functionality of adult bone marrow-derived mesenchymal stem cells before transplantation.

Mesenchymal stem cells (MSCs) have the ability to proliferate and differentiate into various lineages, given the appropriate microenvironment, thus making MSCs promising candidates for cell transplantation. For clinical applications, MSCs need to be stored in optimal conditions so that they may be transported and made available as an off-the-shelf product for companies to market. Freshly harvested and cultured or frozen-thawed bone marrow-derived MSCs were prepared for cell transplantation. Both freshly cultured or frozen-thawed MSCs were washed and resuspended in parenteral solutions, either 0.9% saline, Dulbecco's phosphate-buffered saline (DPBS), plasmalyte A or 5%dextrose and held for 2, 4, 6 and 8 h at 4 degrees C, 37 degrees C and RT (22 degrees C). The viability of the cells, differentiation capability and expression of cell surface markers were analysed. MSCs harvested from fresh cultures, resuspended in the parenteral solutions and maintained at 4 degrees C for 6 h showed more than 90% viability, and the viability was appreciably better when suspended in 5% dextrose at 4 degrees C for 8 h. In contrast, frozen-thawed cells can be held for a maximum of 2 h after thawing before losing their viability significantly below permissible limits for transplantation. We are reporting for the first time the effect of various parenteral solutions, holding times and temperatures on the viability and functionality of bone marrow-derived freshly cultured or frozen-thawed MSCs for transplantation. Our results suggested that freshly harvested MSCs can be held for 8 h at 4 degrees C in 5% dextrose or for up to 6 h at 4 degrees C in saline, DPBS or plasmalyte A. Freeze-thawed MSCs can be held for a maximum of 2 h in plasmalyte A before transplantation without affecting their viability and ability to differentiate.