The GABAergic neuroactive steroid -3-hydroxy-pregnan-20-one  is decreased in various brain regions of C57BL/6J mice following exposure to an acute stressor or chronic intermittent ethanol (CIE) exposure and withdrawal. It is well established that there are complex interactions between stress and ethanol drinking, with mixed literature regarding the effects of stress on ethanol intake. However, there is little research examining how chronic ethanol exposure alters stress responses. The present work examined the impact of CIE exposure and withdrawal on changes in brain levels of 3α,5α-THP, as well as hormonal and behavioral responses to forced swim stress (FSS). Adult male C57BL/6J mice were exposed to four cycles of CIE to induce ethanol dependence. Following 8 h or 72 h withdrawal, mice were subjected to FSS for 10 min, and 50 min later brains were collected for immunohistochemical analysis of cellular 3α,5α-THP. Behavioral and circulating corticosterone responses to FSS were quantified. Following 8 h withdrawal, ethanol exposure potentiated the corticosterone response to FSS. Following 72 h withdrawal, this difference was no longer observed. Following 8 h withdrawal, stress-exposed mice showed no differences in immobility, swimming or struggling behavior. However, following 72 h withdrawal, ethanol-exposed mice showed less immobility and greater swimming behavior compared to air-exposed mice. Interestingly, cellular 3α,5α-THP levels were increased in the lateral amygdala 8 h and 72 h post-withdrawal in stressed ethanol-exposed mice compared to ethanol-exposed/non-stressed mice. In the paraventricular nucleus of the hypothalamus, stress exposure decreased 3α,5α-THP levels compared to controls following 72 h withdrawal, but no differences were observed 8 h post-withdrawal. There were no differences in cellular 3α,5α-THP levels in the nucleus accumbens shell at either withdrawal time point. These data suggest that there are different mechanisms mediating hormonal, behavioral, and brain responses to stress following CIE exposure. The lateral amygdala appears to be an extremely sensitive brain region exhibiting changes in cellular 3α,5α-THP levels following CIE and exposure to swim stress. It is likely that these changes in cellular 3α,5α-THP levels in the lateral amygdala contribute to the behavioral effects observed following 72 h withdrawal. Chronic intermittent ethanol (CIE) exposure and withdrawal produces ethanol dependence and increases subsequent ethanol consumption in C57BL/6J mice -3-hydroxy-pregnan-20-one  is an endogenous neuromodulator synthesized in the brain, adrenals, and gonads that modulates neuronal activity and behavioral output. It is a potent positive allosteric modulator of GABAChronic ethanol exposure and withdrawal can be considered a potent stressor that may have an impact on subsequent alcohol and stress responding [reviewed in Heilig et al. ]. IncreaNeuroactive steroids alter ethanol intake in a complex manner. Specifically, low doses of 3α,5α-THP increase ethanol self-administration, and higher doses decrease self-administration in C57BL/6J mice  increases circulating, cerebral cortical, and hypothalamic levels of 3α,5α-THP and 3α,5α-THDOC, as well as the precursor pregnenolone in various rat strains  were obtained from Jackson Laboratories  at 7–8 weeks of age. Animals were between 9–11 weeks of age at the beginning of the experiment. All mice were allowed to acclimate to the colony before initial exposure to vapor inhalation chambers. Animals were group-housed 5/cage with free access to food and water. All mice were maintained in a temperature (23 ± 0.06°C) and humidity (40 ± 10%) controlled room with lights on from 0700–1900 h. Animal care followed National Institutes of Health Guidelines under University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee approved protocols.Adult male C57BL/6J mice  were used as the swim test apparatus. The glass cylinders were filled with water (23–25°C) to approximately 20 cm in depth. Cylinders were separated by a visual barrier.Within 1 week of arrival to the animal vivarium, all mice were subjected to mild restraint for blood collection of approximately 20 μl from the submandibular space for assessment of baseline corticosterone levels. Specifically, mice were mildly restrained, poked in the cheek pouch with a lancet , 20 μl blood collected from the submandibular space, and mild pressure applied to the wound area to promote blood clotting. No anesthesia is required for this method of blood collection.Mice were exposed to repeated intermittent ethanol exposure or air for four cycles over 4 weeks as described below. Mice were transported from the colony to the procedure room at approximately 1630 h. Animals were weighed and administered an intraperitoneal injection (0.02 ml/g) of the alcohol dehydrogenase inhibitor pyrazole (1 mmol/kg) combined with saline for control animals or combined with 1.6 g/kg ethanol (8% w/v) and immediately placed in the inhalation chambers . Mice remained in the vapor inhalation chambers for 16 h overnight with room air (control group) or volatized ethanol (ethanol group) delivered to the chambers at a rate of 10 liters/min. These procedures were designed to maintain blood ethanol concentrations (BECs) at 150–250 mg/dl throughout the exposure period. Ethanol (95%) was volatilized by passing air through an air stone (gas diffuser) submerged in ethanol. The following morning, at 0900 h, mice were removed from the vapor inhalation chamber and returned to the home cage for 8 h. One cycle consisted of four 16 h exposures. Following each exposure cycle, mice were returned to the home cage and colony and remained undisturbed for 3 days.2O and 500 mg of NaCl in a sealed 12 × 75 test tube. The sample was volatized in a water bath set at 55°C and 1.5 mL of volatized sample was removed from the tube and injected.On the morning following initial exposure to the vapor inhalation chamber and following the third day of each cycle, mice were removed from the chamber and 20 μL of blood was collected from the submandibular space for BEC assessment Table . BECs weMice were exposed to four cycles of CIE vapor exposure and subsequently underwent withdrawal from ethanol for 8 h (Experiment 1) or 72 h (Experiment 2) withdrawal.On the day of the FSS exposure, all mice were transported to the procedure room to acclimate for a minimum of 60 min. Control animals were maintained in a room separate from the procedure room where mice were exposed to the forced swim test. The FSS occurred between 1500–1700 h for Experiment 1  or between 0900–1100 for Experiment 2 .At the beginning of the swim stress session, mice were removed from the home cage, weighed and lowered into the apparatus for a 10 min swim session. Following the swim session, the mouse was removed from the swim tank, lightly dried, and 20 μl of blood was collected from the submandibular space for assessment of stress-induced corticosterone levels. The animal was then returned to the home cage and placed on a warming pad for 15–20 min. Immobility, swimming, and struggling were assessed by experimenter-manual scoring after the session, where the experimenter was blind to the condition of the mouse. Immobility was defined as the mouse remaining completely still or with minimal involuntary movements required to remain afloat. Swimming was defined as relatively calm paddling, including movement of all four legs, or just the two hind legs. Struggling was defined as vigorous swimming, thrashing, and/or climbing in an attempt to escape, which usually subsided by the first three minutes after presentation to the inescapable swim tank. After each swim session, the cylinders were emptied, cleaned, and refilled with fresh water for the next subject. The mice remained in the home cage for 50 min.Serum blood samples were centrifuged at 1730 g for 15 min at 4°C. Serum was collected and frozen at −80°C until analysis. Serum corticosterone levels were analyzed using a radioimmunoassay kit according to manufacturer’s instructions .IHC was performed on free-floating sections  using an affinity purified 3α,5α-THP sheep antibody, that has been previously characterized by radioimmunoassay , paraventricular hypothalamic nucleus (−0.58 to −1.06 AP), and lateral amygdala (−1.22 to −1.58 AP). All coordinates were expressed relative to bregma in the Mouse Brain Atlas in Stereotaxic Coordinates , images were captured with a digital camera , and analyzed using Bioquant  image analysis to obtain linear integrated optical density for immunoreactivity assessment. The microscope, camera, and software were background corrected to eliminate non-specific labeling and normalized to preset light levels to ensure fidelity of data acquisition. For each brain region, a threshold is selected based on positive cellular staining of cell bodies. This threshold is then applied to each image and pixel density is quantified. Positive pixel count of immunoreactivity was quantified from a circumscribed field, delineated as a brain region, divided by the area of the region in square millimeters, and expressed as pixels/mmAll data were analyzed separately for each experiment. Corticosterone data were transformed as a difference score (Post Stress—Baseline) and analyzed using a two-way between subjects design ANOVA with Exposure  and Stress  as factors. Behavioral data were analyzed as a two-way mixed model design ANOVA with Exposure  as a between subjects factor and Behavior  as a repeated measure. Immunohistochemical data were transformed and expressed as percent control (air/non-stressed group) values and analyzed using a two-way between subjects design ANOVA with Exposure  and Stress  as factors. In the presence of significant main effects or interactions, Fisher’s planned least significant difference (PLSD) test was conducted to isolate effects. The level of significance was set to a α level ≤0.05.t(252) = 4.5, p < 0.0001). However, by the second cycle through the end of the experiment comparable BECs were observed between the groups  = 10.7, p < 0.0001] and a significant main effect of Cycle ).In general, mice were exposed to comparable ethanol concentrations >170 mg/dl) across the four CIE cycles  = 27.11, p < 0.0001]). In general, ethanol-exposed mice showed greater corticosterone levels in response to FSS compared to air-exposed mice  = 6.60, p < 0.05]). Following 72 h withdrawal  = 153.6, p < 0.0001]).FSS following four cycles of CIE altered circulating corticosterone levels in air-exposed and ethanol-exposed mice 8 h and 72 h post-withdrawal Figure . Followil Figure , FSS incF = 6.4, p < 0.005]). Specifically, ethanol exposed mice showed less immobility (t(72) = 3.3, p < 0.005) and more swimming (t(72) = 2.9, p < 0.05) behavior compared to air-exposed mice  = 2.2, p < 0.05] as supported by an Exposure by Stress interaction . In contrast, FSS did not alter 3α,5α-THP levels in the air-exposed condition. Similarly, 72 h post-withdrawal  = 2.4, p < 0.05] and compared to air-exposed/stressed mice  as supported by an Exposure by Stress interaction . No effect of FSS was observed in the air-exposed mice.Ethanol exposure altered 3α,5α-THP levels in response to FSS in a unique manner in the lateral amygdala at both 8 h and 72 h post-withdrawal Figure , ethanoll Figure , ethanolt(40) = 2.3, p < 0.05] as supported by an Exposure by Stress interaction . When ethanol-exposed mice were stressed, this combination prevented the stress-induced reduction in 3α,5α-THP levels in these mice.In the paraventricular nucleus of the hypothalamus, no changes in 3α,5α-THP levels were observed following 8 h withdrawal in either the control or FSS condition Figure . HoweverIn the nucleus accumbens shell, there were no changes in 3α,5α-THP levels following CIE or FSS exposure following 8 h Figure  or 72 h Overall, this work illustrates that CIE alters the brain and behavioral stress response in a time-dependent manner. Specifically, stress increased 3α,5α-THP levels following CIE exposure at both 8 h and 72 h withdrawal in the lateral amygdala, while stress had no effect on 3α,5α-THP levels in the air-exposed control animals. Behaviorally, an escalation of swimming behavior coupled to less immobility was observed in CIE-exposed mice following 72 h withdrawal, with no change observed following 8 h withdrawal. To our knowledge, these are the first data to show that a history of CIE exposure alters the 3α,5α-THP response to FSS challenge in C57BL/6J mice. Further, we show an association between elevated 3α,5α-THP levels in the lateral amygdala and increased swimming behavior, consistent with increased reactivity to stress after CIE exposure. The lateral amygdala appears to be an extremely sensitive brain region, exhibiting changes in cellular 3α,5α-THP levels following CIE and exposure to swim stress.Chronic ethanol exposure and withdrawal can be considered a potent stressor that may have an impact on subsequent alcohol and stress responding [reviewed in Heilig et al. ]. IncreaSommer et al.  have posThe lateral amygdala serves as a sensory information gating region that receives input from many brain regions and projects to other subregions of the amygdala and reciprocally back to input regions  with no change following 72 h withdrawal .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Finally, we used APP23 mutant mice to study the association between M18L/S and the time-dependent accumulation of common Alzheimer’s disease pathology.Presynaptic terminals contribute to cognitive reserve, balancing the effects of age-related pathologies on cognitive function in the elderly. The presynaptic protein Munc18-1, alternatively spliced into long (M18L) or short (M18S) isoforms, is a critical modulator of neurotransmission. While subtle alterations in Munc18-1 have been shown to cause severe neuropsychiatric disorders with cognitive impairment, little information is known regarding the specific roles of Munc18-1 splice variants. We first investigated functional and anatomical features evidencing the divergent roles of M18L and M18S, and then evaluated their contribution to the full range of age-related cognitive impairment in the dorsolateral prefrontal cortex of a large sample of participants from a community-based aging study, including subjects with no- but not M18S (p = 0.88). Mean M18L in dementia cases was 51 % lower than in NCI cases (p < 0.001), and each unit of M18L was associated with a lower likelihood of dementia . In contrast, M18S balanced across clinical and pathologically diagnosed groups. M18L loss may not be caused by age-related amyloid pathology, since APP23 mice (12- and 22-months of age) had unchanged cortical levels of M18L/S compared with wild-type animals.Using isoform-specific antibodies, M18L was localized to the synaptosomal fraction, with a distribution matching lipid raft microdomains. M18S was found widely across cytosolic and synaptosomal compartments. Immunocytochemical studies identified M18L in perisomatic, GABAergic terminals, while M18S was broadly distributed in GABAergic and glutamatergic terminals. Using regression models taking into account multiple age-related pathologies, age, education and sex, global cognitive function was associated with the level of M18L  contains supplementary material, which is available to authorized users. Common age-related neuropathologies, including Alzheimer’s disease pathology (i.e. neuritic plaques and neurofibrillary tangles), cerebral infarcts, and Lewy bodies, accumulate in the human brain across the lifetime. The susceptibility of cognitive function to decline as growing amounts of neuropathology develop in the forebrain varies greatly between individuals, raising the concept of cognitive reserve –3. LargeN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) [In this context, we previously reported that better cognitive performance, a reduced odds of dementia, and slower rate of cognitive decline over multiple years prior to death were associated with greater densities of specific presynaptic proteins, and enhanced presynaptic protein-protein interactions (PPIs) , 14. Int (SNARE) . However (SNARE) . Previou (SNARE) –19. Toge (SNARE) , 21.unc-18-1  is a strong candidate. First identified by random mutagenesis screening for uncoordinated phenotypes in the worm Caenorhabditis elegans, M18 caught the attention of many research teams when described as a ‘syntaxin-binding protein’ (coining the M18 coding gene as STXBP1) twenty years later [cis-to-trans SNARE conformations, and/or docking the presynaptic vesicle [STXBP1 null mice lack neocortical synaptic activity [Although the biochemical and physiological nature of neurosecretion is well established –24, the rs later –27. Whil vesicle , 28, 29.activity  highlighactivity .STXBP1 gene is alternatively spliced to yield either a long (M18L) or a short (M18S) variant [a and b, respectively. Processing of the final exon in the STXBP1 primary transcript may include (M18L) or skip (M18S) a sequence of 110 bp containing a stop codon [STXBP1 gene splicing have not been described. Although intensive research focuses on M18 activities in regulated exocytosis, the potentially divergent roles of the M18 variants are relatively neglected, assuming no substantial differences between them [In mammals, the  variant , 32, alseen them , 34. Theeen them , compatieen them –38. At aeen them . Howeverde novo mutations affecting STXBP1 gene can cause Ohtahara syndrome in humans [STXBP1 mutations associated with Ohtahara syndrome impair M18–syntaxin-1 interaction (and thereby proper SNARE assembly), compromising cellular availability of functional M18 (haploinsufficiency) [STXBP1 de novo mutations were associated with similar mental retardation without epileptic seizures [It has been reported that n humans –45, a deiciency) , 43. Coniciency) . In addiseizures .n = 32 Alzheimer’s disease cases, although after synaptophysin normalization, the authors concluded that productive synapses in Alzheimer’s disease brains might be enriched in M18 [n = 5 Alzheimer’s disease cases. These two studies did not examine the correlation of M18 with cognitive outcomes, or with the likelihood of dementia; nor were potential differences in M18 splice variants assessed. In addition, the small sample sizes limited the possibility of investigating possible effects of stage of illness, or potential confounders.In the context of aging, two previous studies addressed alterations in M18 protein levels in Alzheimer’s disease postmortem brain. In the first, M18 immunodensity was lower in two cortical areas in d in M18 . A laterd in M18  found inAltogether, clinical and preclinical data demonstrated that M18 is essential for neurosecretion, subtle alterations have a dramatic impact on normal synaptic function and cognition in humans and rodents, and could represent an early sign of synaptic pathology and cognitive impairment. For the present study we hypothesized that cortical downregulation of M18 long and/or short (M18L/S) splice variants could contribute to poorer cognitive performance and increased likelihood of dementia in old age. Due to the scarce data on specific functions of M18 isoforms, we first performed functional and anatomical characterization experiments that evidenced the complementary, rather than overlapping roles of M18L and M18S. Using tissue samples from the community-based Memory and Aging Project (MAP) , corticaWe first used immunoprecipitation (IP) followed by either sodium dodecyl sulfate (SDS) or blue native (BN) polyacrylamide gel electrophoresis (PAGE) to investigate functional binding affinities of M18 isoforms for the SNARE complex. As expected from their target immunogenic sequences Fig. , and alsSubstantial differences in the subcellular localization of M18 variants were also observed Fig. –g. WhileImmunohistochemical analyses of rat brain revealed a differential distribution of M18 variants, particularly in the hippocampus Fig. . M18L waTriple co-immunolabeling with antibodies against M18L, vesicle GABA (VGAT) and glutamate (VGLUT1) transporters showed a high degree of M18L localization within the inhibitory (VGAT-positive) synapses, with little (or possibly no) presence at excitatory (VGLUT1-positive) terminals, in both cortex and hippocampus Fig. –g. In coDemographic, cognitive and pathological variables of MAP participants are summarized in Table Similar numbers of clinically diagnosed MAP participants with no (NCI) or mild (MCI) cognitive impairment, and dementia were observed Table . As expen = 308). Initial inspection of quantitative immunoblotting datasets  revealed large variability in M18L immunodensities among MAP participants (interquartile range = 7–185 %), compared to that of M18S (86–122 %) or β-actin (85–117 %). The immunodensities of the M18 variants showed a non-linear association . Due to the large number of values proximate to zero .We investigated possible alterations in the amounts of M18 splice variants, and their potential association with cognitive function, in samples from the dorsolateral prefrontal cortex (DLPFC) of MAP participants  to address basic associations between M18 variants, Alzheimer’s disease pathology and global cognitive function. Lower cortical immunodensity of M18L, but not M18S, was associated with the severity of Alzheimer’s disease pathology, and lower cognitive scores Fig. . ImportaBased on prior work showing that select presynaptic proteins were related to cognition in a relatively independent manner , we nextp < 0.001) than those with no dementia , while APP23 mice tended to display lower M18S cortical immunodensities regardless of age . These marginal effects were considerably smaller than the M18L loss (−64 %) observed in the DLPFC of MAP participants graded according to Braak’s V–VI stages.We used APP23 transgenic mice to examine the association between M18L and a common Alzheimer’s disease pathology (i.e. amyloid plaques). These animals develop an Alzheimer’s disease-like syndrome via expression of a mutant APP that causes abnormal, age-dependent extra-synaptic amyloid-β accumulation . M18L/S In the present study, variations in frontal cortical M18 levels were associated with cognitive function and the likelihood of dementia in the elderly, beyond the effects of common age-related neuropathologies. Specific loss of M18L (but not M18S), which was found preferentially at GABAergic presynaptic terminals, may initiate synaptic dysfunction and consequent synaptic pathology.2+) may induce a SNARE conformation that favors M18S relative to M18L binding in vitro. Since binding to syntaxin-1 is the major mechanism by which M18 exerts its functions [2+-calmodulin kinase II (CaMKII) activity in the C-terminal tail of M18L, but not M18S, may indicate a differential regulatory mechanism for the M18 variants [We first characterized biochemical, cellular and anatomical aspects of M18 splice variants. Compared to M18S, M18L showed minimal binding affinity for syntaxin-1 (and hence for the SNARE complex) in two functional assays, co-IP and BN-PAGE. A previous study showed similar, but less pronounced, variant-specific binding affinities to syntaxin-1 . Furtherunctions , 28, 29,unctions . Despiteunctions . Of noteunctions . Remarkaunctions . Additiovariants . This Cain situ hybridization showed greatest expression of M18L in cells within the granule and pyramidal layers of mouse hippocampus [Immunohistochemical assays were consistent with tissue fractionation experiments. In contrast to the wide distribution of M18S across subcellular compartments, M18L was restricted to nerve endings and enriched in perisomatic areas. The M18S subcellular and histological distribution was similar to that previously reported for M18 , 53, andpocampus . Combinepocampus , 56. Thipocampus , 45, a dSTXBP1 transcript are unclear, other splicing deficits have been associated with aging related neuropathologies and dementia [We found marked reductions of M18L, but not M18S, in DLPFC of MAP participants displaying clinical dementia and/or a large burden of Alzheimer’s disease pathology. Pairwise correlations between M18L levels, the burden of Alzheimer’s disease pathology, and cognitive function scores were all significant. Accordingly, participants having high M18L cortical density displayed greater cognitive resistance against the accumulation of Alzheimer’s disease pathology, which may directly implicate M18L in the molecular mechanisms of cognitive reserve. Statistical modeling indicated that loss of M18L contributed moderately to a higher likelihood of dementia and to cognitive impairment. Additionally, including synaptophysin as a covariate did not alter the results, suggesting that loss of M18L is an early event in synaptic pathology. The importance of maintaining appropriate splicing balance between M18L/S isoforms is probably best represented by the dramatic endophenotypes observed in murine strains either lacking or overexpressing the M18L variant , 35. Whidementia .The predominant expression of M18L in GABAergic terminals suggests that synaptic deficits may be initiated in inhibitory, rather than excitatory synapses. This could be related to the surprising relative upregulation of glutamatergic terminals observed in MCI . Indeed,The present study identified the M18L isoform as a relatively specific component of the GABAergic presynaptic machinery. In contrast to the alternatively spliced M18S, which is highly abundant and ubiquitously expressed in inhibitory and excitatory neurons, M18L appears restricted to lipid raft microdomains within the presynaptic compartment, and may have a function independent from its interaction with syntaxin-1. Importantly, M18L, but not M18S, is associated with cognitive function in the elderly, and may contribute to the mechanisms of cognitive reserve that protect against dementia related to age-associated pathologies.n = 308 participants were included in the present study. A summary of demographic, cognitive and pathological characteristics is listed in Table MAP recruits volunteers without known dementia, living in Chicago  . Since 1Methodological approaches to systematic cognitive, clinical and neuropathological evaluations were extensively reported , 62. AnnNeuropathological examinations documented Alzheimer’s disease-related pathology , vascular diseases , Lewy bodies, and hippocampal sclerosis , 62. A bApproval from the UBC’s Animal Care Committee was obtained prior to experiments involving laboratory animals. Adult Sprague–Dawley rats were supplied by Charles-River . Novartis Pharma  provided APP23 transgenic mice, overexpressing a variant of human APP carrying the ‘Swedish double mutation’ KM670/671NL . Pentobag for 1 min (P1). Supernatants (S1) were centrifuged at 17,000 × g for 15 min, and the resulting pellets (P2) washed in 0.32 M sucrose and re-centrifuged. P2 fractions were resuspended in 0.32 M sucrose and layered onto a sucrose discontinuous gradient, with 1.2 (bottom), 0.8 M (middle), and 0.32 M (topping) layers. After a 12-h centrifugation at 96,000 × g, the interfaces between 0.8 and 1.2 M , and 0.32–0.8 M  were collected. IF1 was again overlaid on 0.32 M sucrose buffer and centrifuged at 30,000 × g for 20 min. The resulting pellet, containing purified synaptosomes, was resuspended in 1 ml of homogenization buffer supplemented with 0.5 % TritonX. In all steps, aliquots of each fraction were separated for further SDS-PAGE and immunoblotting analyses.Cortical synaptosomes were obtained following standard procedures , and the pellet (P1) was resuspended in 1 ml of the same ice-cold Tris-buffered solution supplemented with 0.5 % TritonX. To achieve a homogenous solution, P1 was gently sonicated . The same centrifugation step was performed and the supernatant (S2) was kept. The pellet (P2) was again resuspended and sonicated in Tris-buffer containing 2 % TritonX, and the centrifugation step was repeated. The subsequent supernatant (S3) was separated and the pellet (P3) homogenized as above in ice-cold Tris-buffer containing 0.5 % SDS. P3 was incubated at 15 °C with rotation for 10 min before a final centrifugation at 31,000 × g and at 12 °C was performed for 1 h. The supernatant (S4), enriched in lipid-raft proteins, was collected, and the final pellet (P4) resuspended as above. All fractions were stored at−80 °C until resolved by SDS-PAGE and immunoblotting.Purification of lipid raft-enriched fraction was based on the TritonX-insoluble, SDS-soluble property of these membrane microdomains. The present method, initially designed for separating cytosolic from insoluble, raft-associated α-synuclein , offers A list of primary antibodies used appears in Additional file Target proteins were immunoprecipitated using protein G-coated magnetic Dynabeads , as reported . In eachGrey matter samples from the middle-frontal gyrus (Brodmann’s area 46/9) of the DLPFC were obtained at autopsies of MAP participants, following a standard atlas . The DLPn = 132 MAP participants) was spaced-loaded in triplicate in all gels to control for correct loading and protein transfer to the membranes , followed by immunoblotting, as previously reported . Quantifsee Fig. , and alsr = 0.80 was required for M18L/S and β-actin immunoreactivity values. Gels not meeting this criterion were discarded and repeated. For each sample, immunoreactivity of M18L/S  was first divided by that of β-actin within the same gel, and then calculated as a percentage of in-gel standards. Mean value between the two different, valid gels was used as a final estimate. This procedures were reported to reduce the experimental variability in studies that used immunoblotting as a quantitative technique for large cohorts of samples [In quantitative studies, membrane stripping and reprobing with anti-β-actin antibody was done for housekeeping and data normalization purposes. Densitometric analyses of the immunoblots were done with ImageGauge software, version 4.22 (Fujifilm). Every gel was run with 14 brain samples , and a molecular weight ladder (Bio-Rad). Gels containing the same subset of samples were assessed at least twice in different days. For quality control, a minimal between-gel Pearson’s correlation coefficient of  samples , 38.g, 30 min, 4 °C), supernatants or IP products were combined with equal volumes of a loading buffer . Samples were loaded in 4–16 % gradient NativePAGE precast gels . Electrophoresis was run under constant voltage (150 V), at 4 °C, with pre-chilled anode  and cathode  buffers. Before transferring proteins and complexes to PVDF membranes, BN-gels were incubated an ice-cold 12 mM Tris, 96 mM glycine buffer, pH 8.3, containing 0.1 % SDS. To remove excess of Coomassie dye, membranes were rinsed in 100 % methanol, followed by immediate rehydration in TBS for 15 min. Following immunoblotting procedures were as described for SDS-PAGE. Molecular markers  were loaded in all gels to estimate protein complex sizes.Originally developed to study mitochondrial membrane complexes , separatForty-μm floating coronal sections (−3.30 to−4.20 relative to bregma) were obtained from paraformaldehyde-fixed rat brain hemispheres . Other fIn quantitative immunoblotting assays, M18L/S immunodensities were first normalized to corresponding β-actin values, and calculated as a percentage of in-gel standards , 38. Linpost hoc test for standardized data, or Kruskal-Wallis followed by Dunn’s post hoc tests for non-transformed values. Experiments involving APP23 mice were analyzed with two-way ANOVA, with genotype and age as independent factors, followed by Dunnet’s post hoc test.Multivariate analyses were performed to survey putative associations between M18 isoforms and cognitive, pathological, neurochemical and/or putative confounding variables. An additional exploratory analysis was performed by ranking participants according to their M18L/S cortical immunodensities and comparing pathology-cognition decay curves across subjects with high (above the 90th percentile) and low (below 10th percentile) M18L/S values. Given the potential effect of cortical M18L levels on cognitive performance, we generated multiple linear regression models with cognitive measures as outcomes and pathological and neurochemical variables as predictors. Additionally, using the same predictors, we built logistic regression models with clinical diagnosis of dementia as the outcome. Linear and logistic models were controlled for sex, age and education. Differences in M18 immunodensities between clinically diagnosed or pathologically graded groups were assessed by ANOVA followed by Bonferroni’s versus excitatory compartments were performed with paired t-tests.For colocalization analyses of confocal imaging, we used an ImageJ 2.0  built-in method , 77. Comp-values < 0.05 were considered significant. Datasets were analyzed and plotted with JMP 10.0.2 , and/or GraphPad Prism 6.0 .All tests were two-tailed, and


CORIN gene variations and their association to preeclampsia in Caucasian and African women. Our study population was composed of 571 pregnant women (295 with preeclampsia and 276 normotensive controls) matched for maternal and gestational age, and ethnic origin. The 22 exons of the CORIN gene were sequenced in a discovery sample (n = 260), where 31 single nucleotide polymorphisms were identified. In a replication sample (n = 311), 4 single nucleotide polymorphisms were tested. Two minor alleles (C for rs2271036 and G for rs2271037) were significantly associated to preeclampsia. Adjusted odds ratios [95% confidence interval] were 2.5 [1.2–3.8] (p = 0.007) and 2.3 [1.5–3.5] (p = 1.3×10−4), respectively. These associations were ethnic-specific, as only found in the Caucasian of subjects . The two single nucleotide polymorphisms are in almost perfect linkage disequilibrium (r2 = 0.93). No specific association was found with severe preeclampsia, early-onset preeclampsia nor fetal growth retardation. In conclusion, this is the first report of a highly significant association between these two single nucleotide polymorphisms in CORIN gene and preeclampsia. Our findings further support the probability of a critical role of corin in preeclamspia pathophysiology at the uteroplacental interface.Preeclampsia is a frequent medical complication during pregnancy. Corin, a serine protease which activates pro-atrial natriuretic peptide, has recently been shown to be involved in the pathophysiology of preeclampsia. The aim of this study was to search for  Preeclampsia (PE) is a frequent medical complication during pregnancy, occurring in 2% to 7% of pregnancies worldwide CORIN knockout mice develop significant increase of systolic blood pressure associated with late-gestational proteinuria, mimicking human PE phenotype Corin is a transmembrane type II serine protease expressed primarily in cardiomyocytes, were it cleaves pro-atrial natriuretic peptide (ANP) into active ANP, a cardiac hormone that regulates blood pressure CORIN gene is located on chromosome 4p12-p13, has 22 exons and spans 244 kb Human CORIN gene variations in large series of human patients with preeclampsia. The aim of our study was to search for CORIN gene variations and their frequency in Caucasian and Sub-Saharan African pregnant women with and without preeclampsia.To date, there are no data on In this study, we used the samples of the “Search for an Association between CX3CR1 and V249I Polymorphisms, Preeclampsia and Endothelial Injury” (ECLAXIR) study, a multicenter case-control study. The objectives and design of the ECLAXIR study are detailed in a previous work where 2 polymorphisms of CXR3R1 were analyzed z-score lower than −1.88, calculated using tables from a Paris area-based population where birthweight was adjusted for gestational age and sex 2.PE was defined as blood pressure ≥140/90 mm Hg occurring after 20 weeks of gestation with previously normal blood pressure, associated with proteinuria ≥0.3 g in a 24-hour urine specimen CORIN (see below), and tested their association with PE. In the replication sample (sample 2), composed of the remaining patients of the ECLAXIR study, we genotyped the most promising Single Nulceotide Polymorphiss (SNPs) identified in the discovery sample, to replicate their association with PE, and strengthen the evidence for a true effect.Cases and controls were recruited as pairs, but as a few subjects were excluded , our final sample was composed of pairs and supplemental unmatched cases. These latter were rematched with one pair using the same criteria as the ones described above. Finally, our study population was divided into 2 samples of nearly same size. In the discovery sample (sample 1), composed of randomly chosen subjects, we searched for genetic variations by sequencing the whole 22 exons of 3EDTA solution, at the time of enrolment . DNA extraction was performed with QIAmp DNA Blood Midi Kit , according to the manufacturer’s instructions. The samples were also stored at −80°C until testing.Written informed consent was obtained from each woman before enrolment and blood sampling. Venous blood was collected in 15% KCORIN gene was first performed in a discovery sample (sample 1) composed of randomly chosen patients within the ECLAXIR study. This was done by means of the VariantSEQr System. The VariantSEQr System protocol consists of 5 steps: (1) PCR amplification of human genomic DNA, (2) purification of PCR product, (3) cycle sequencing, (4) electrophoresis on a sequencer, and (5) data analysis. PCR primers used for the amplification of the amplicon contain a specific sequence of the exon and either universal primer M13 forward (M13F) or M13 reverse (M13R) sequences for subsequent sequencing reaction; all sequences are freely available on www.pubmed.org [Probe section - Homo sapiens RSA (ReSequencing Amplicon) probe]. PCR conditions were defined as: 1 µL of DNA (30 ng/µL), 0.45 µL of each primers (10 µM), 0.15 µL of dNTP (25 mM each), 0.6 µL of MgCl2 (50 mM), 1.5 µL of ), 3 µL of 40% Glycerol in water, 0.15 µL of Taq, water 8.5 µL. All PCR were performed as followed: (i) denaturation: (96°C - 5 min) on initial cycle and (94°C - 30 sec) on rest; (ii) annealing: (60°C - 45 sec); (iii) extension: (72°C - 47 sec - 43 cycles) and 13 min on last cycle. PCR were checked on gel and purified by Sepadex G50. Sequencing was performed by a single universal primer (M13R or M13F). Specifically, a total of 8 kb (exons and intronic regions flanking the exons) were amplified from each of the genomic DNA samples, and sequenced using a capillary-based ABI 3730xl DNA Analyzer . Sequence traces were assembled and aligned for variant calling using Lasergene SeqMan v8.1 . The variants were oriented according to Human Genome 19.As a first step, amplification of the 22 exons of the In a second step, the two most promising SNPs identified in the discovery sample were subsequently genotyped as described above in the replication sample (sample 2), composed of the remaining patients of the ECLAXIR study.www.ensembl.org).Phylogenetic analysis, i.e. comparison of Corin protein sequence with other species, was performed using the Ortheus algorithm as implemented in the ensembl platform (2) between SNPs and tested for Hardy Weinberg equilibrium using the algorithm implemented in Haploview 4.2 Descriptive demographic and clinical data were expressed in percentages or means ± standard deviations (SD). Conditional logistic regression analysis performed with SAS was used to compare qualitative and quantitative variables between cases and controls in both sample 1 and sample 2, and in the combined sample. We estimated population allelic frequencies, pairwise LD  .vs. 8.0% [p<0.0001] and 41.3% vs. 31.5% [p = 0.006], respectively). As expected, the proportion of neonates small for gestational age (SGA) was higher in patients with PE as compared to controls. There were no significant differences between cases and controls for history of thrombosis, pregestational diabetes, primipaternity, connective tissue disease or antiphospholipid antibody.CORIN gene was first sequenced in the discovery sample 1 composed of 260 individuals (116 cases and 144 controls), where 31 polymorphisms wee identified. Twelve were disregarded because their minor allele frequency (MAF) was <1% in patients regardless of their ethnic origin . −5 and p = 0.004, respectively) in the global sample. Among the remaining 12, all displayed a p-value above 0.2 but rs61759670 (p = 0.1).The In a second batch of analysis,  subjects were genotyped for the two SNPs most significantly associated with PE in sample 1 . Note that because of the genotyping technique, i.e. based on sequencing, genotypes were obtained for two additional SNPs in the vicinity of the same exon . Among them, only rs2271036 and rs2271037 were significantly associated to PE  and rs2271037 (GT+GG versus TT genotypes) were 2.5 [1.2–3.8] (p = 0.007) and 2.3 [1.5–3.5] (p = 1.3×10−4) in the combined total sample, respectively.p = 1.1×10−4 for rs2271036 and 3.1 [1.7–5.8], p = 2.1×10−4 for rs2271037  with increased ORs , time of onset , or with the proportion of neonates SGA . Note that because rs2271036 and rs2271037 SNPs are almost in perfect linkage disequilibrium in the Caucasian sample (r2 = 0.93) no multivariate analyses, i.e. including the 2 SNPs in the same model, were conducted.Finally, Among PE cases, rs2271036 and rs2271037 showed no significant association with PE severity (CORIN gene (rs2271036 and rs2271037) and PE in a large sample of 571 matched patients. This association is specifically observed in Caucasian patients originated from Europe.Here, we report for the first time a highly significant association between two SNPs located in the F5 rs6020 and rs6025, F2 rs1799963, ACE rs4646994, AGT rs699, AGTR1 rs5186, CTLA4 rs231775, LPL rs268 and SERPINE1 rs1799889. The ORs [95% IC] range from 1.17 [0.99–1.40] (ACE rs4646994) or 1.17 [1.03–1.33] (SERPINE1 rs1799889) to 2.42 [1.25–4.68] (LPL rs268) To date, more than 150 genes have been described in relation to PE or haemolysis, elevated liver enzymes and low platelets (HELLP) syndrome, a thrombotic microangiopathy that can complicate PE CORIN gene was first described by Pan et al concomitantly in human and mice The CORIN gene and PE in patients from Sub-Saharan Africa, a high-risk population for PE, especially very early-onset PE CORIN introns. Interestingly, rs2271037 affects a very highly conservative site when comparing CORIN sequence between species; the nucleotide differs only in humans and the SNP is associated to a human-specific disease, suggesting subsequent functional impact of the SNP on corin expression or activity. This could be due to impaired splicing as suggested by the close position of the SNP near a splicing site.Corin deficiency was first described in hypertensive heart disease et al also reported corin mRNA and protein expression in uterus samples from pregnant women, which were lower in patients with PE than normal pregnancies et al also showed that corin is expressed in first trimester human implantation sites and is up-regulated with endometrial decidualization et al described for the first time two CORIN mutations in 3 Han Chinese patients, after sequencing CORIN exons in 56 PE patients and 108 normal pregnant women The strong association of rs2271036 and rs2271037 SNPs with PE is suggestive of a contributive role of corin in PE. This statement is in agreement with 2 series of observations. First, corin deficient pregnant mice demonstrate late-gestation proteinuria and hypertension, mimicking human PE phenotype CORIN gene and PE in Caucasian women from Europe, and also the strongest association between 2 SNPs and PE described so far. These findings reinforce the probability of a critical role of corin in PE pathophysiology at the uteroplacental interface. Likewise, understanding how these SNPs can affect corin uterine expression and/or function are questions to be investigated. Further propective studies are also needed to confirm the strength of these associations and their potential to screen for high risk of preeclampsia.This is the first report of a highly significant association between two SNPs in the


APP, PSEN1, PSEN2, GRN, MAPT, and PRNP), in 141 LOAD patients and 179 elderly controls, neuropathologically proven, from the UK. In our cohort, 14 LOAD cases (10%) and 11 controls (6%) carry at least 1 rare variant in the genes studied. We report a novel variant in PSEN1 (p.I168T) and a rare variant in PSEN2 (p.A237V), absent in controls and both likely pathogenic. Our findings support previous studies, suggesting that (1) rare coding variability in PSEN1 and PSEN2 may influence the susceptibility for LOAD and (2) GRN, MAPT, and PRNP are not major contributors to LOAD. Thus, genetic screening is pivotal for the clinical differential diagnosis of these neurodegenerative dementias.The overlapping clinical and neuropathologic features between late-onset apparently sporadic Alzheimer's disease (LOAD), familial Alzheimer's disease (FAD), and other neurodegenerative dementias  raise the question of whether shared genetic risk factors may explain the similar phenotype among these disparate disorders. To investigate this intriguing hypothesis, we analyzed rare coding variability in 6 Mendelian dementia genes  in a cohort composed of 141 late-onset sporadic Alzheimer's disease cases and 179 elderly controls, autopsy proven from the UK.We have used exome sequencing to investigate rare coding variability in Mendelian dementia genes (•PSEN1 (p.I168T) and a rare variant in PSEN2 (p.A237V), both likely pathogenic.We report a novel mutation in •PSEN1 and PSEN2 harbor susceptibility factors for sporadic Alzheimer's disease. By contrast, GRN, MAPT, and PRNP do not play a major role for the development of late-onset sporadic Alzheimer's disease.We conclude that •Genetic screening is therefore pivotal for a clinical differential diagnosis of sporadic late-onset Alzheimer's disease and other neurodegenerative dementias . The APOE ε4 allele is the main risk factor for apparently sporadic AD. In the last 5 years, genome-wide association studies (GWASs) identified several loci, harboring common variants with low risk effect size (OR: 1.2–1.5) (Alzheimer's disease (AD) (OMIM #104310) is the most common cause of progressive dementia in the elderly individuals. Aging and genetic factors play a pivotal role for the disease development. AD incidence increases exponentially from the age of 65 years (1.5% affected) to 80 years and older (30% affected). Twin studies have shown that AD heritability ranges between 60% and 80% . Fully p1.2–1.5) .TREM2 (p.R47H) and PLD3 (p.V232M), and a very rare protective variant in APP (p.A637T)  . In addinical AD .APP, PSEN1, PSEN2, MAPT, GRN, and PRNP) does not influence susceptibility to AD. By contrast, a growing body of evidence highlighted the significant role of rare coding variants in PSEN1 in LOAD  may underlie LOAD pathogenesis, we have analyzed exome sequencing data, in a British cohort composed of 141 LOAD cases without any apparent family history and 179 elderly controls autopsy proven.The overlapping clinical and neuropathologic features between AD and other neurodegenerative dementias  lead to a misdiagnosis in 17%–30% of AD cases . This ra in LOAD . Thus, t22.1Our cohort was composed of 141 independent LOAD (age at onset ≥65 years) cases and 179 elderly (>60 years) unrelated controls, neuropathologically confirmed. These patients were referred as apparently sporadic LOAD cases.All the patients and controls were Caucasian, mostly from the UK  and to a lesser extent from North America. The average age at diagnosis was 76.7 years (range 65–97 years) for the LOAD patients and the mean age of ascertainment was 78 years (range 60–102 years) for the controls .Table 1CWritten informed consent was obtained for each individual and the study was approved by the appropriate institutional review boards.2.2APP, PSEN1, PSEN2, GRN, MAPT, and PRNP loci. Exome-enriched libraries were sequenced on the Illumina HiSeq 2000 using 2 × 100 bp paired end read cycles.Library preparation for next-generation sequencing was performed according to the NimbleGen (Roche NimbleGen v2) and TruSeq (Illumina) sample-preparation protocols. DNA libraries were then hybridized to exome-capture probes with NimbleGen SeqCap EZ Human Exome Library, version 2.0 (Roche NimbleGen) or TruSeq (Illumina). Each capture method covers the 2.3www.picard.sourceforge.net/index.shtml). The Genome Analysis Toolkit was used to recalibrate base quality scores, perform local realignments around indels and to call and filter the variants (gvs.gs.washington.edu/SeattleSeqAnnotation). All variants within the coding regions of APP, PSEN1, PSEN2, MAPT, GRN, and PRNP were annotated for both cases and controls.Sequence alignment and variant calling were performed against the reference human genome (UCSC hg19). Paired end sequence reads (2 × 100 bp paired end read cycles) were aligned using the Burrows-Wheeler aligner . Format variants . VCFtoolvariants . Variant2.4All rare variants identified by whole exome sequencing in the candidate genes were validated by Sanger sequencing.http://bioinfo.ut.ee/primer3-0.4.0/) using UCSC (http://genome.ucsc.edu/) reference sequences NM_000484.3 (APP), NM_000021.3 (PSEN1), NM_000447.2 (PSEN2), NM_001123066.3 (MAPT), NM_002087.2 (GRN), and NM_000311.3 (PRNP).Primers for exons harboring rare variants were designed in Primer3  and electropherograms were visualized in Sequencher software .2.5APOE genotypes comprising the APOE ɛ2, ɛ3, and ɛ4 alleles were assayed using the TaqMan method . SNP-specific primers and probes were designed by ABI (TaqMan genotyping assays).3MAPT c.115–2A>T), 1 low frequency and 1 common coding polymorphisms in PRNP: a 24 bp deletion (rs138688873) and the p.M129V (rs1799990), respectively. In our cohort, 14 LOAD cases (10%) and 11 controls (6%) carry at least one of these rare variants  and 2 in controls (MAPT p.G200E and PRNP p.M134V).We identified 226 variants  and 18 indels (coding and intronic) in the genes studied. Of these, we analyzed the 18 rare coding variants , 1 splice-site mutation , compared to cases , thus, suggesting that rare coding variability in these genes may be well tolerated  and the other in PSEN2 (p.A237V). In contrast, the variants detected in APP, GRN, MAPT, and PRNP are likely tolerated polymorphisms.In our LOAD cohort, we identified a total of 9 rare coding variants in PSEN1 is a deleterious change. First, it clusters in the third transmembrane domain (TM3), on the alpha helix surface, where all the known pathogenic variants have been reported  (PSEN1 p.I168T) was diagnosed at 86 years of age, heterozygous for APOE ε4 allele (ε2ε4), presented an advanced Alzheimer's disease (Braak V), and did not report any positive family history.Several lines of evidence suggest that p.I168T in ix rule) . Second,ix rule) . The patPSEN2 p.A237V has been only recently reported by the ClinSeq pilot study (PSEN1 (p.A231), where 2 causative mutations (p.A231V and p.A231T) have been described in a Dutch, French and Canadian family  do not cluster in any TM domain. In addition, the p.S130L and p.D439A have been found also controls.The other variants detected in our cohort in GRN harbors 4 missense mutations , 2 of which have only been detected in cases (p.R432C and p.R433W). Although the p.R432C variant has been already associated with familial FTD and clinical AD (www.molgendatabase).nical AD , its patMAPT  cluster outside the microtubule binding domain, where most of the pathogenic mutations have been reported up to date (www.molgendatabase). The c.115-2A>T is predicted to alter Tau exon 7 splicing, introducing a nonsense codon within exon 11. We have not detected any difference in exon 7 and in MAPT expression between the splice-site mutation carrier and the other cases and controls. Thus, these findings suggest that c.115-2A>T is not disease related  for the serum and glucocorticoid inducible kinase 1 (SGK1). SGK1 controls neurite outgrowth by depolarizing the microtubules through the serine phosphorylation at codon 214 (The odon 214 .PRNP open reading frame (rs138688873), between the repeat 3 (R3) and repeat 4 (R4). This octapeptide deletion (rs138688873) has been described as a risk factor for neurodegenerative diseases with some controversy  . Thus, suggesting this common variant may be a benign polymorphism.Two cases and no controls carry a 24 bp deletion in the troversy . Furthere (sCJD) , p.M129MAPOE ε4 allele and LOAD in our cohort . Nevertheless, we have not detected any enrichment for the APOE ε4 allele among the patients carrying rare variants in APP, PSEN1, PSEN2, GRN, MAPT, and PRNP . By contrast, APOE ε2 allele did not present a significantly higher frequency in controls compared to cases .Finally, we report a significant association between 4APP, PSEN1, PSEN2, MAPT, GRN, and PRNP) aiming to establish whether rare coding variability in these genes is responsible for an appreciable portion of the LOAD risk.In this study, we tested the hypothesis that the significant phenotypic overlap between sporadic LOAD and other neurodegenerative dementias  may be explained by a common genetic background. Thus, we screened 6 Mendelian dementia genes (PSEN1 (p.I168T) and a rare variant in PSEN2 (p.A237V). These variants are likely pathogenic: (1) both cluster in TM domains, on the alpha helix surface; (2) the literature already reported in the same (PSEN1 p.I168) or homologous residue (PSEN2 p.A237 and PSEN1 p.A231) causative mutations for FAD; and (3) the PSEN1 p.I168T and PSEN2 p.A237V are classified as possible pathogenic, following the algorithm proposed by PSEN2 p.R62H; PRNP rs138688873 and GRN p.R433W) (www.molgendatabase). Second, they cluster outside the reported pathogenic domains . Third, they do not alter the gene expression (MAPT c.115-2A>T).In our LOAD cohort, we found a novel rare variant in GRN p.R432C, the effect of a decreased GRN secretion in AD pathogenesis remains controversial. GRN pathogenic mutations act through a messenger RNA nonsense-mediate decay, interfering with GRN expression and generally are loss of function mutations (LoF) . The only exception to this rule is represented by the pathogenic missense mutations which cluster in the GRN signal peptide domain (GRN p.A9D) (www.molgendatabase). Thus, we suggest, in concert with previous studies (GRN missense mutations mapping outside the signal peptide domain are likely to be well tolerated.Finally, despite the functional consequence of  studies , that GRPSEN1 and PSEN2 contributes to susceptibility for apparently sporadic LOAD. Therefore, sporadic LOAD and FAD may be influenced by the same genes and thus pathogenic mechanisms. On the contrary, rare coding variants in MAPT, GRN, and PRNP are not major players in the development of LOAD. Thus, genetic screening is fundamental for the differential diagnosis of these disparate neurodegenerative dementias.In conclusion, our findings support recent studies, suggesting that rare coding variability in


Busulfan is currently used by many hematologists and oncologists as second-line treatment in patients with BCR-/ABL1-negative MPNs that are intolerant to or developed side effects from hydroxyurea. Furthermore, in a recently published study, use of busulfan in patients with polycythemia vera (PV) was accompanied by complete hematologic and molecular remission.7 In the current retrospective study, we wanted to share our experience with the use of busulfan in patients with BCR-/ABL1-negative MPN.Busulfan  is an alkylating agent that has been used for the treatment of myeloproliferative neoplasms (MPNs). It has been used since 1959, and it is a cell cycle non-specific alkylating agent. Busulfan is approved by the Food and Drug Administration and was commonly used for the treatment of chronic phase chronic myelogenous leukemia (a BCR-/ABL1-positive MPN) before it was displaced by the tyrosine kinase inhibitors (imatinib and other tyrosine kinase inhibitors). Busulfan has also shown significant activity in BCR-/ABL1-negative MPNs, as shown in a number of studies.The current study was approved by the Mayo Clinic Institutional Review Board. Institutional databases from 1970 to 2014 were interrogated to identify informative patients by using the terms MPN, PV, essential thrombocythemia (ET), myelofibrosis (MF) and busulfan (or myleran). Patients' follow-up information was collected till July 2015. Seventy-five patients with full demographic, diagnostic and therapeutic information were identified: 37 patients with ET, 22 with PV, 12 with MF and 4 with MPN unclassifiable. Median age of the study population was 64 years (range 31–91). After a median follow-up of 17 years, 40 patients (53%) have died and leukemic transformation was documented in 4 (5%) patients with median time to leukemic transformation of 86 months (range 12–229).9/l) and platelet count (× 109/l) were 13.6 (9.8–16.9), 10.2 (5–231) and 1113 (593–2062), respectively. After a median follow-up time of 230 months, 15 patients (41%) have died and leukemic transformation was documented in only 1 patient who was also treated with radioactive phosphorous (P32). Leukemic transformation in the particular patient was documented 230 months from the date of diagnosis. Post-treatment, complete blood count was available in 20 patients and revealed median (range) Hgb, WBC and platelet count of 12 g/dl (9.9–16), 7.4 × 109/l (3.1–25) and 267 × 109/l (126–573), respectively.Among 37 patients with ET, 29 (79%) were females and the median (range) age was 67 (33–90) years. At diagnosis, the median (range) hemoglobin (Hgb) (gm/dl), white blood cell count (WBC) (× 109/l (1.2–26.6) and 669 × 109/l (185–2370), respectively. After a median follow-up time of 188 months, 13 (57%) patients have died, and leukemic transformation was documented in 2 patients, one of whom was also treated with P32. Post-treatment, complete blood count was available in 21 patients and revealed median (range) Hgb, WBC and platelet count of 12.9 gm/dl (10–15.2), 7.2 × 109/l (2.8–20) and 303 × 109/l (124–833), respectively.Twenty-two patients with PV were identified, and 14 (61%) were females, and the median (range) age was 64 (46–91) years. At diagnosis, the median (range) Hgb, WBC and platelet count were 17.5 gm/dl (15.1–20.8), 11.5 × 109/l, and 472 × 109/l. Six (50%) patients displayed splenomegaly and 5 of them have had splenic size reduction from 2–20 cm below the costal margin to 0–5 cm after busulfan treatment, 3 were splenectomized and 3 patients did not have palpable spleen. At a median follow-up of 208 months, and 39 months (range 78–401) from the start of busulfan treatment, 10 (84%) patients have died and no leukemic transformation was documented.Twelve patients with MF were identified. The median age was 52 years (31–75) and 5 were females. The median Hgb, WBC and platelet count were 13.6 gm/dl, 14.5 × 109/l, and the drug be held when the platelet counts drop below 150–200 × 109/l.The preferred first-line cytoreductive therapy in patients with MPNs is hydroxyurea. Ruxolitinib (a JAK1/2 inhibitor) was recently approved for use in intermediate or high-risk treatment requiring patients with MF and hydroxyurea refractory/intolerant PV. The current study, as well as many other previously published ones, supports the use of busulfan as an alternative second-line therapy in MPNs, along with interferon-alpha, especially in ET and PV. However, some cautions need to be exercised while using busulfan in MPN. The drug can cause profound and prolonged cytopenias, especially thrombocytopenia. The dose of busulfan may need to be decreased when the platelet level drops below 300 × 10


TP53. However, many of these representative cell lines have not been widely used so it is unclear if these cell lines capture the variability that is characteristic of the disease.The factors driving the onset and progression of ovarian cancer are not well understood. Recent reports have identified cell lines that are representative of the genomic pattern of high-grade serous ovarian cancer (HGSOC), in which greater than 90 % of tumors have a mutation in TP53-mutant HGSOC cell lines  for migration, MMP2 expression, proliferation, and VEGF secretion, behaviors that play critical roles in tumor progression. In addition to comparing baseline variation between the cell lines, we determined how these behaviors changed in response to four growth factors implicated in ovarian cancer progression: HB-EGF, NRG1β, IGF1, and HGF.We investigated six MMP2 expression in at least two cell lines. Growth factor-induced responses demonstrated substantial heterogeneity, with cell lines sensitive to all four growth factors, a subset of the growth factors, or none of the growth factors, depending on the response of interest. Principal component analysis demonstrated that the data clustered together based on cell line rather than growth factor identity, suggesting that response is dependent on intrinsic qualities of the tumor cell rather than the growth factor.Baseline levels of each behavior varied across the cell lines and this variation was comparable to that seen in tumors. All four growth factors impacted cell proliferation or VEGF secretion, and HB-EGF, NRG1β, and HGF impacted wound closure or Significant variation was seen among the cell lines, consistent with the heterogeneity of HGSOC.The online version of this article (doi:10.1186/s12935-015-0263-4) contains supplementary material, which is available to authorized users. TP53, a low rate of other mutations, and extensive DNA copy number changes, is the most aggressive and most common subtype of ovarian cancer, accounting for two-thirds of deaths [Ovarian cancer is the deadliest gynecological cancer in the developed world, with nearly two-thirds of patients diagnosed with advanced, metastatic disease . Most off deaths , 3. Due f deaths , 5, HGSOf deaths . ExtensiMMP2 in peritoneal implants correlates with elevated mortality risk [Unlike other solid tumors, ovarian cancer is not restricted to metastasis through the blood and lymph systems; instead, tumor cells can exfoliate from the ovary and attach to organs in the peritoneum, especially the omentum . This peity risk . Followiity risk . As in oity risk –16.HB-EGF expression has been associated with shorter progression-free survival [IGF1 was associated with shorter progression-free survival [The different stages of HGSOC metastasis are influenced by the presence of growth factors and cytokines in the tumor microenvironment, which in HGSOC includes ascites fluid. For example, heparin-binding EGF-like growth factor (HB-EGF), neuregulin-1 beta (NRG1β), insulin-like growth factor 1 (IGF1), and hepatocyte growth factor (HGF) are all expressed in tumors and found at higher levels in ascites fluid of ovarian cancer patients compared to healthy controls –21. Elevsurvival ; HB-EGF survival . Autocrisurvival  and has survival . Elevatesurvival . In vitrsurvival , 28.MMP2 expression, proliferation, and VEGF secretion in response to HB-EGF, NRG1β, IGF1, and HGF.Therefore, to determine if HGSOC cell lines that have genomic profiles similar to TCGA tumors  demonstrate heterogeneity in the various metastatic processes, we examined migration, Progression in HGSOC is marked by the dissemination of tumor cells throughout the peritoneum , with tuNot surprisingly given the different biological mechanisms involved , differeMMP2 and found that all of the cell lines had detectable levels  two growth factors , (2) one growth factor , or (3) none of the tested growth factors (OV90). In general, OVCAR3 and OVCAR4 showed the most similar patterns of sensitivity to the different growth factors. Most of these effects were modest, with only OVCA432 demonstrating increases in MMP2 of greater than twofold after growth factor stimulation. With respect to the individual growth factors, our results demonstrated that HB-EGF increased MMP2 levels in OVCA432, OVCAR3 and OVCAR4, and NRG1β increased MMP2 expression in Caov4 and OVCA432. Similar to the results for migration  changes in proliferation regardless of which growth factor was used while OVCA432 was strongly sensitive to several growth factors. Of the growth factors, the effects of NRG1β were the most widespread, with large increases in proliferation for Caov3, Caov4, OVCA432, and OVCAR3, a slight increase for OVCAR4, and a slight decrease for OV90. HB-EGF significantly induced proliferation for Caov3, Caov4, and OVCA432, and a slight decrease in proliferation in OVCAR4 was seen. IGF1 induced proliferation in Caov3, OVCA432, and OVCAR3, with a slight increase for OV90. Finally, HGF increased proliferation for Caov3 and OVCAR3 with slight effects on Caov4 and OVCAR4.Elevated cell proliferation is associated with the advanced stages of HGSOC . TherefoProduction of VEGF by tumor cells and other cells in the microenvironment is essential for the development of new blood vessels to support tumor growth ; vesselsMMP2 expression, proliferation, and VEGF secretion. As seen in the scores plot  was performed on the growth factor-induced effects on migration, lot Fig. , cell liWe examined two potential reasons for this result. First, it is possible that the baseline behavior of the cells would influence the growth factor-induced response. For example, it is possible that cells with high basal production would not be able to produce additional VEGF. However, OVCA432 had the second highest basal level of VEGF 587 pg/mL), but was exquisitely sensitive to NRG1β, and nearly quadrupled the amount of VEGF produced . While the results were generally consistent with previous reports for the members of the cell panel , 36, 37 MMP2 levels did not mirror the effects seen for migration, suggesting that unique therapeutic strategies may be required to control these different mechanisms of HGSOC metastasis.Of the individual cellular behaviors examined, the selected growth factors had the strongest effect on proliferation, with three or more cell lines sensitive to each growth factor, and VEGF secretion, where each growth factor impacted at least two cell lines. This may of course result from the high sensitivity and precision of these assays and highlights the importance of considering assay choice when doing in vitro studies. The impact of assay choice was further exemplified by the striking differences observed between the wound and transwell assays for migration. While both assays are commonly used to measure cell migration, they examine different biological mechanisms as wound assays monitor collective cell migration and transwell assays assess single cell motility in response to chemotactic stimuli. Our results indicated that growth factors have a substantially stronger effect on collective migration versus single cell migration in the HGSOC cell lines. In addition, the effects of growth factor treatment on It is of course important to compare our in vitro findings to data from clinical samples in order to determine if these cell lines that are representative of the HGSOC genotype  are alsoFinally, even though a growth factor was capable of impacting a cell behavior in one cell line, there was no guarantee that it would impact another cell line, even if that other cell line was sensitive with respect to a different endpoint. For example, HB-EGF induced proliferation and migration in Caov3 but only proliferation for OVCA432. This is reflected in the clustering by cell type rather than growth factor in PCA. However, neither baseline cellular behaviors nor growth factor receptor levels were predictive of sensitivity. It is possible that variations in the levels of other components of the cellular signaling network besides growth factor receptors could be responsible for the observed heterogeneity. For example, it has been shown that when predicting cell response, dimerization patterns of ErbB1 with other ErbB receptors is important to consider , as is t® 16 HS reagent system, multiplex PCR amplification, capillary electrophoresis (ABI 3500) and GeneMapper (v.4.1) software. All HGSOC cell lines are reported to possess a mutation in TP53  ratio of MCDB 105  and Medium 199  supplemented with 1 % penicillin/streptomycin  and 10 % heat-inactivated fetal bovine serum (Life Technologies). OVCAR3 cells were cultured in RPMI 1640 (Corning) supplemented with 20 % heat-inactivated fetal bovine serum, 1 % penicillin/streptomycin, 1 % sodium pyruvate (Sigma), 0.3 % glucose (Corning), and 10 ng/mL insulin (Sigma).Caov3 (HTB-75), Caov4 (HTB-76), OV90 CRL-11732), OVCAR3 (HTB-161), and MDA-MB-231 were purchased from American Type Culture Collection . OVCA432  were obt1732, OVC2, OVCA432 and OVCAR3 at 105,000 cells/cm2, and OVCAR4 at 121,000 cells/cm2. Cells were then rinsed with PBS and serum-free media was added. After serum starving for 24 h, a scratch was made in each well with a 200 μL pipette tip, cells were washed twice with serum-free media, and the wound was imaged with an Olympus IX51 Fluorescent Microscope . Cells were then treated with vehicle (0.1 % BSA in PBS) or 10 ng/mL recombinant human HB-EGF, NRG1β, IGF1, or HGF  in serum-free media. Forty-eight hours post-treatment, the wound was imaged again, and the percent wound closure was measured using ImageJ .For wound assays, HGSOC cells were stained with 5 μM CellTracker™ Green CMFDA Dye (Life Technologies), plated in 48-well plates at cell densities empirically determined to result in 100 % confluency, and allowed to attach overnight. Specifically, Caov3, Caov4, and OV90 were plated at 132,000 cells/cm® inserts  with 8 μm pores. HGSOC cells were serum starved for 24 h, dissociated using TrypLE Select Enzyme (Life Technologies), and 150,000 cells/well were seeded in serum free media in the top compartments of the transwells inserted in 24-well plates. The bottom compartments contained 500 μL serum free media plus vehicle (0.1 % BSA in PBS) or 10 ng/mL HB-EGF, NRG1β, IGF1, or HGF. All experiments included a positive control of the highly invasive MDA-MB-231 breast cancer cell line [Transwell assays were performed using polycarbonate Transwellell line  in the t2, OVCA432 and OVCAR3 at 24,000 cells/cm2, and OVCAR4 at 26,000 cells/cm2. Cells were then rinsed with PBS and serum-free media was added. After 48 h of serum starvation, conditioned media was collected and assayed at a 1:1 (v/v) ratio with 20 μM Mca-PLGL-Dpa-AR-NH2 fluorogenic MMP substrate  in 96-well, black-walled plates. After a 24 h incubation at 37 °C, MMP activity was measured by quantifying fluorescent intensity at an excitation/emission of 320/405 nm. To determine the impact of growth factors on MMP2 expression, cells were plated in 6-well plates at the same cell densities as the total MMP assay. After overnight attachment, cells were washed with PBS and serum-free media was added. Following 24 h of serum starvation, cells were washed with PBS and vehicle (0.1 % BSA in PBS) or 10 ng/mL HB-EGF, NRG1β, IGF1, or HGF was added in serum-free media. After 24 h of growth factor treatment, mRNA was isolated using the RNeasy Mini Kit  according to manufacturer’s instructions and quantified on a NanoDrop 2000 Spectrophotometer (Thermo Scientific Pierce). cDNA was synthesized from 1 μg RNA using the Superscript III First-Strand Synthesis System kit (Life Technologies) according to manufacturer’s instructions. qRT-PCR was performed on 100 ng cDNA using SYBR Green PCR Master Mix (Life Technologies) and QuantiTect primers for MMP2  or GAPDH . The fold change in gene expression of MMP2 was determined by the ΔΔCt method relative to GAPDH expression.To characterize total levels of active MMPs, HGSOC cells were plated in 12-well plates at cell densities empirically determined to result in approximately 70 % confluency and allowed to attach overnight. Specifically, Caov3, Caov4, and OV90 were plated at 29,000 cells/cmMMP2 expression assay and allowed to attach overnight. Cells were then rinsed with PBS and serum-free media was added. After serum starving for 24 h, cells were washed with PBS and stimulated with vehicle (0.1 % BSA in PBS) or 10 ng/mL HB-EGF, NRG1β, IGF1, or HGF in serum-free media. After 24 h of treatment, cell proliferation was quantified using the Click-iT® EdU Alexa Fluor® 488 flow cytometry assay (Life Technologies) according to manufacturer’s instructions. Cells were incubated with EdU for 6 h prior to sample collection and analyzed on a BD Accuri™ C6 flow cytometer . Samples were gated for the EdU-positive population to determine the percentage of cells that entered S-phase during the EdU incubation.HGSOC cells were plated in 12-well plates at the same cell densities as the MMP2 expression assay and allowed to attach overnight. Cells were then rinsed with PBS and serum-free media was added. After serum starving for 24 h, cells were washed with PBS and vehicle (0.1 % BSA in PBS) or 10 ng/mL HB-EGF, NRG1β, IGF1, or HGF was added in serum-free media. After 24 h of growth factor treatment, conditioned media was collected, and VEGF levels were determined by ELISA  according to the manufacturer’s instructions.HGSOC cells were plated in 12-well plates at the same cell densities as the MMP2, fold change in VEGF). The first principal component captured 47.0 % of the variation; inclusion of a second principal component increased this to 74.7 %. PCA was performed using SIMCA.P + v.12.0.1  with mean-centered and variance-scaled data.Principal component analysis  was perAll data are presented as the mean ± standard deviation (n = 3). Additionally, all experiments were performed at least twice to ensure reproducibility. Statistical significance was determined using Dunnett’s with vehicle samples set as the control. All statistical calculations were performed with JMP 4.1 software , with statistical significance set as p < 0.05.


Concordance rates were compared by age or BMI using correlation analysis, Bland-Altman plots, and intraclass correlation coefficient (ICC). Using BIA data, we established a regression formula to reflect CT-VFA. The mean VFAs by CT and BIA were 131.9 ± 57.3 cm2 and 110.5 ± 33.9 cm2, respectively . The mean difference was 21.4 ± 45.6 cm2, tending to increase with BMI. In women with BMI <25 kg/m2 or age <50 years, the VFAs by BIA were similar to those by CT (ICC = 0.496 in BMI <25 kg/m2 and ICC = 0.638 in age <50 years). However, the difference was greater in men with BMI ≥25 kg/m2 or age ≥50 years. Applying our formula, the difference between estimations decreased to 0.2 ± 38.2cm2. VFA estimated by BIA correlated well with that by CT, but a more accurate formula is needed to match CT data, particularly in older men or subjects with a high BMI.We evaluated the concordance between visceral fat area (VFA) estimated by bioelectrical impedance analysis (BIA) or computed tomography (CT) in Korean subjects with a wide range in age and body mass index (BMI). In 1006 individuals (mean age 55.2 ± 11.8 (19–87) years, mean BMI 26.0 ± 3.5 (17–46) kg/m The prevalence of obesity has increased dramatically over the last several decades worldwide and is now a major public health problem . ObesityIn this context, accurate measurement of the visceral fat area (VFA) is critical in the assessment of cardiometabolic risk for each individual. Currently, abdominal computed tomography (CT) is considered one of the most accurate methods for the assessment of VFA . HoweverThe bioelectrical impedance analysis (BIA) method has been widely used to estimate body composition by analyzing impedance obtained when a current flows through the body ,15. ThisThere have been several experimental trials to estimate VFA in the abdomen using BIA and some of them showed good correlations between VFA estimated by BIA and that measured by CT ,10,16. M®  is one of the most frequently used machines worldwide [® showed a good correlation with VFA measured by CT in small studies [Among many machines using BIA, Inbodyorldwide ,19. The  studies ,16. HoweWe recruited 1006 consecutive Korean subjects, 19–87 years of age  who visited Seoul National University Bundang Hospital (SNUBH) for their medical checkup in the period March 2007 to June 2015. Those who had a malignancy, a history of major surgery on their extremities, chronic kidney disease stage IV or renal replacement therapy, liver cirrhosis with ascites, heart failure with peripheral edema, or severe hypothyroidism were excluded. Patients with fever resulting from an active infection or inflammation, those receiving systemic steroid treatment, and those suffering severe dehydration such as from uncontrolled diabetes mellitus were also excluded. The study protocol was approved by the ethics committee of SNUBH (SNUBH IRB#B-1503/292-106) and all participants provided written informed consent.® testing. The BMI was calculated by dividing body weight (in kg) by the square of the height (in m). Waist circumference was measured in the standing position at the midpoint between the lateral iliac crest and the lowest rib at the end of expiration while the subject was breathing gently. Blood pressure was measured using an automatic blood pressure measurement device. Each subject rested for at least 5 min prior to blood pressure measurements while sitting in a chair with both feet flat on the floor and both arms supported at the level of the heart.Height and body weight were measured by standard protocols at the time of InbodyFasting blood samples were obtained in the morning after a 12-h fast including any medication. The glycated hemoglobin (A1c) level was measured by affinity chromatography . A complete blood cell count analysis was performed using Sysmex XE-2100 . Fasting plasma concentrations of total cholesterol, triglycerides, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, and serum creatinine were measured on a Hitachi 747 chemistry analyzer . Estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease equation . Serum a®; Inbody Co.) for each individual, in a fasting state on the same day as their blood test. The study participants were requested to refrain from smoking, drinking alcohol, and strenuous exercise for 48 h prior to measurement. After the subjects had been guided to stand on the platform of the device, age and gender information were entered into the machine. After confirming that the subject was standing correctly with both arms apart from the body and both feet on the right spots on the platform, a supervisor pushed the start button to perform assessment. Both hands were held at a 45° angle away from the body. The device scans at X-scan uses 1, 5, 50, 250, 500 kHz, and 1 MHz frequencies to analyze intracellular and extracellular fluid values and water content.Abdominal VFA was estimated using three multifrequency BIA machines  and −50 HU pixels was calculated automatically using dedicated software . The CT scan was performed within 15 days of the BIA measurement.p < 0.05 was considered significant. The Kolmogorov-Smirnov Goodness of Fit test was used to confirm the normal distribution of the key variables. Paired t test and intraclass correlation coefficient (ICC) for absolute agreement were used to compare the difference between two methods. Pearson’s correlation coefficient was used to investigate any correlations between VFA measured by CT and estimated by BIA. Analysis of the comparability and agreement levels between the two methods was conducted using the Bland-Altman method. Fisher’s r to z transformation was used to compare the correlation coefficients among subgroups according to the clinical characteristics.Data are presented as the mean ± standard deviation (SD), and X-axis and the observations on the Y-axis. All statistical analyses were performed using SPSS for Windows version 22.0  and R program version 3.2.0 .To estimate VFA from BIA more accurately for referencing against VFA values by CT, multiple linear regression models were developed for each gender using age, BMI, and VFA value obtained from the BIA machine. These independent factors were chosen based on clinical judgments. For continuous factors such as VFA estimated by BIA and BMI, a linear relationship was set after investigating nonlinearity through restricted cubic splines. Potential interaction effects were examined using the backward stepwise selection method and the final model was derived based on the Akaike Information Criterion (AIC) and Bayesian Information Criterion . Calibran = 1006). The mean ± SD (range) age and BMI of the population were 55.2 ± 11.8 (19–87) years and 26.0 ± 3.5 (17–46) kg/m2, respectively. Men had a greater mean waist circumference than women by about 5 cm. Men comprised significantly higher percentages of current or ex-smokers (p < 0.001) and alcohol drinkers (p < 0.001).2 and 110.5 ± 33.9 (24.2–246.6) cm2, respectively. The mean VFA measured by CT (CT-VFA) was significantly larger in men than in women . In contrast, the mean VFA by BIA (BIA-VFA) in women was significantly larger than in men .In this population, VFAs (mean ± SD (ranges)) at the umbilicus level measured by CT and by BIA were 131.9 ± 57.3 (11.0–358.9) cmr = 0.605, p < 0.001) . In contrast, it tended to overestimate VFA in those having a lower CT-VFA (<100 cm2).Using Pearson’s correlation test for comparing the two techniques, the BIA-VFA showed a significant positive correlation with CT-VFA (< 0.001) A. The Bl.001) , and diuretics and thiazolidinedione medications, there were no significant differences in correlation coefficients between groups from Fisher z-tests , and age .To determine whether the concordance rates between CT-VFAs and BIA-VFAs differed by gender, BMI, or age, we divided the study populations into subgroups according to gender, BMI . Among the BMI categories, BIA-VFAs were larger than CT-VFAs in the BMI <20 kg/m2 group. In the groups with BMI ≥20 kg/m2, BIA-VFAs were smaller than CT-VFAs with an increasing trend in the mean difference as BMI increased up to 30 kg/m2. The mean differences in VFA estimates tended to increase along with BMI except in subjects with BMI ≥30 kg/m2  vs. overweight or obese (BMI ≥ 25 kg/m2); and younger (age < 50 years) vs. older (age ≥ 50 years). In men, BIA-VFAs were significantly smaller than CT-VFAs in both age groups (both p < 0.001). In women with BMI ≥ 25 kg/m2 or age ≥ 50 years, BIA-VFAs were also significantly smaller than CT-VFAs (p < 0.001 for both groups). In women with BMI <25 kg/m2 or age <50 years, BIA-VFAs were similar to CT-VFAs . The ICC values showed a similar tendency to the mean differences in VFAs. The highest ICC value was 0.638 in women with age <50 years  + 1.11 × BIA-VFA + (−1.49) × Age + 2.10 × BMI + 2.03 × WC + (−0.02) × BIA-VFA × BMI + 0.08 × age × BMI for men; and y = (−142.77) + 1.40 × BIA-VFA + (−1.29) × Age + (–0.98) × BMI + 2.14 × WC + (−0.03) × BIA-VFA × BMI + 0.07 × age × BMI for women .r = 0.605, p < 0.001). However, the concordance rate between BIA and CT in abdominal VFAs differed according to BMI and age groups by gender. The concordance rate was quite high in women (mean difference = 5.4 ± 39.2 cm2 and ICC = 0.577) whereas there was a larger difference in men (mean difference = 38.2 ± 45.9 cm2 and ICC = 0.438). Differences in the VFAs between the two methods changed with BMI. The BIA method used in this study overestimated VFAs in those with BMI < 20 kg/m2 and underestimated VFAs in those with BMI ≥ 20 kg/m2 with a dose-dependent relationship. Among the age groups, the BIA method estimated abdominal VFAs well in the age range of 19–39 years whereas it underestimated the CT-VFAs in those aged over 40 years.In this study, abdominal VFAs estimated by BIA were significantly correlated with those measured by CT , similar results were obtained (There are also a number of limitations. First, not all of the BIA and CT measurements were performed on the same day. However, when we performed a subgroup analysis using those subjects who underwent BIA and CT measurements within 7 days (obtained . Second,2. The application of a slightly modified formula using simply age, BMI, original VFA, and its interaction terms made the VFA values more accurate for the reference values obtained from CT scans. Further investigation of BIA technology is warranted for better estimating visceral fat depots in the abdomen.Our study has shown that VFA estimated by multifrequency BIA was significantly correlated with VFA measured directly by CT. The BIA method tends to underestimate VFA with increase in BMI or age except for women aged < 50 years or in those with a BMI < 25 kg/m


Moreover, after treatment with Aβ1−42 peptide, 12 DIV primary cultured neurons showed similar changes, to a greater degree than in controls. Voltage-clamp recordings revealed that the voltage-dependent sodium current density of neurons incubated with Aβ1−42 was significantly increased, without change in the voltage-dependent sodium channel kinetic characteristics. Immunohistochemistry and western blot results showed that, after treatment with Aβ1−42, expressions of Nav and Nav1.6 subtype increased in cultured neurons or APP/PS1 brains compared to control groups. The intrinsic neuronal hyperexcitability of APP/PS1 mice might thus be due to an increased expression of voltage-dependent sodium channels induced by Aβ1−42. These results may illuminate the mechanism of aberrant neuronal networks in AD.Aberrant increases in neuronal network excitability may contribute to the cognitive deficits in Alzheimer's disease (AD). However, the mechanisms underlying hyperexcitability are not fully understood. Such overexcitation of neuronal networks has been detected in the brains of APP/PS1 mice. In the present study, using current-clamp recording techniques, we observed that 12 days  Various evidence suggests, that β-amyloid (Aβ) peptides play a causal role in AD's pathogenesis, but the underlying mechanisms remain unclear  play an essential role in excitable cells. They are necessary components required to generate and propagate action potentials , which undergoes abnormal proteolytic processing to generate Aβ  and were maintained on a C57BL/6J background by crossing heterozygous transgenic mice with C57BL/6J breeders. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and their suffering.1−42 peptide  and reverse peptide Aβ42−1  were diluted to 1 mg/ml using sterile PBS and incubated at 37°C, 220 rpm for 48 h allowed to aggregate as described  or reverse peptide Aβ42−1 (5.0 μM) for 24 h at 37°C.Lyophilized Aβ2 inhalation and the cortices were rapidly dissected under sterile conditions in cold PBS. The tissue was digested with 0.125% trypsin-EDTA at 37°C for 30 min. The trypsin solution was replaced with 2 ml 10% DMEM . The digested tissue was gently triturated by suction using a glass pipette flamed on the tip to avoid cellular damage. The cell suspension was filtered through a 74 μm screen mesh, plated on poly-L-lysine-coated coverslips or 6/12-well-plates and incubated in a 37°C, 5% CO2 incubator. After 2 h, the medium was changed to Neurobasal  supplemented with 2% B27, 1% L-glutamine, and 1% penicillin/streptomycin. In accordance with the routine culture, the medium was changed every 2 days until use.P0  C57BL/6J mice or APP/PS1 mice and their littermates were sacrificed by CO42−1 and Aβ1−42 peptides were added to wells for 24 h. After cell treatments the medium was removed and the cells were incubated with red free medium and MTT solution (0.5 mg/ml) for 4 h at 37°C. Finally, the medium was removed and formazan particles were dissolved in DMSO. Cell viability, defined as the relative amount of MTT reduction was determined by spectrophotometry at 570 nm.Cell viability of neurons was determined by the MTT assay. Neurons were plated in 96 well-plates. Aβ2 1.1, CaCl2 2.6, HEPES 10, glucose 10, pH adjusted to 7.4 with NaOH. Patch pipettes were made from borosilicate glass capillaries  using a micropipette puller . Pipette resistance ranged from 3 to 5 MΩ. Stimulation and data acquisition were performed using the EPC-10 patch-clamp amplifier and Pulse program . Membrane currents were filtered at 2 kHz and digitized at 10 kHz.Electrophysiological measurements were performed on pyramidal cells. Action potentials or sodium currents were recorded at room temperature using whole-cell patch-clampings. The extracellular solution contained the following (in mM): NaCl 150, KCl 5, MgCl2ATP 2, pH adjusted to 7.2 with KOH. Cells were held at −70 mV, then peak amplitude , threshold , and action potential firing  were recorded and measured.Action potentials recordings were made using the current-clamp mode. The intracellular solution contained the following (in mM): KCl 65, KOH 5.0, KF 80, HEPES 10, EGTA 10, Na2 2, Na2ATP 2, EGTA 10, HEPES 20, pH adjusted to 7.2 with Tris-HCl. Cells were held at −70 mV and stepped to a range of potentials (−60 to +60 mV in 10 mV increments) for 12 ms each. Peak inward currents (I) were plotted as a function of depolarizing potential to generate I–V curves. Activation curves were obtained by converting current (I) to conductance (G) at each voltage (V) using the equation G = I/(V − Vrev), where Vrev is the reversal potential, which was determined for each cell individually. Activation curves were then fit with the Boltzmann function in the form of G/Gmax = 1/{1 + exp [(V1∕2 − V)/κ]}, where Gmax is the maximal sodium conductance, V1∕2 is the half-maximal activation potential, V is the test potential, and κ is the slope factor. Steady-state fast inactivation was achieved with a series of 500 ms prepulses (−120 to −10 mV in 10 mV increments), and the remaining available channels were activated by a 12 ms test pulse to 0 mV. Peak inward currents obtained from steady-state fast-inactivation protocols were normalized to the maximal peak current (Imax) and fit with Boltzmann functions: I/Imax = 1/{1 + exp [(V − V1∕2)/κ]}, where V represents the inactivating prepulse potential, and V1∕2 represents the mid-point of inactivation curve.Sodium currents were recorded using the voltage-clamp mode. The intracellular solution contained the following (in mM): CsCl 140, MgClt-test.Data were analyzed using Pulsefit 8.6 and Origin 7.5, and presented as means ± SEM. The Kruskal–Wallis non-parametric test was used to analyze current density data. One-way ANOVA was used to assess the statistical significance of changes in characteristics of channel activation and inactivation. Statistical comparisons were performed by Student's All cells were lysed in RIPA buffer (with 1% PMSF), and total protein concentrations were determined with a BCA Protein Assay Kit (TransGen). Total protein (10–20 mg) for each sample was loaded into precast 8% SDS-PAGE gels and run with running buffer. Gels were transferred onto PVDF membranes (Millipore). Antigen-specific primary antibodies  were incubated overnight at 4°C and detected with species-specific horseradish-peroxidase-labeled secondary antibodies. An ECL Western Blotting Detection kit (TIANGEN) was used to obtain a chemiluminescence signal, which was detected using Gel Imaging System (Bio-Rad). Band quantification was performed using a Gel-Pro software. Bands of interest were normalized to actin- or tubulin- for a loading control.Total RNA of cultured neurons was prepared by using the Trizol Reagent . The Superscript TM-III kit  was used for reverse-transcribed with oligo dT and 2.5 mg total RNA. Primer sequences were as follows: NM_019266 (SCN8A), and NM_031144 (β-actin). SCN8A forward 5′-CTG GAG AAT GGA GGC ACA CAC-3′, reverse 5′-ACG CTG CTG CTT CTC CTT GTC-3′; and β-actin forward 5′-CGT TGA CAT CCG TAA AGA CCT-3′, reverse 5′-TCA GGA GGA GCA ATG ATC TTG-3′. The resulting cDNA PCR amplification was performed by using the following protocol: 95°C for 10 s followed by 50 cycles of 95°C for 5 s and 60°C for 31 s, and verified by 2.0% agarose gel electrophoresis. Images were captured by using Gel Imaging System (Bio-Rad). The amplicon size of each gene was108 and 144 bp, respectively.Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton-x-100 for 5 min, and incubated with 5% BSA for 60 min at room temperature to block non-specific binding. Without washing, the diluted primary antibodies  were added and incubated at 4°C overnight. After three washes with PBS, cells were incubated with the corresponding secondary antibodies at room temperature for 1 h. For immunohistochemistry, cells were stained with DAB kit (Vector Laboratories) according to the instructions of the manufacturer for peroxidase labeling. Images were acquired from a fluorescence microscopy .Quantitative analysis of mean fluorescence intensities (MFIs) of immunoreactive neurons was performed using Image J software . MFI per square micrometer was calculated by dividing the MFI units by the area of outlined regions.in vitro) primary cultured neurons obtained from P0 APP/PS1 mice and littermate wide type mice in current-clamp mode. Frequency, threshold, and peak amplitude of action potentials (AP) were examined by different protocols. AP firing frequencies (f) elicited by increasing depolarizing currents  were significantly increased in neurons from APP/PS1 mice compared to WT mice . We used a depolarizing current  to induce a single firing of an AP. Threshold potential (Vthreshold) and amplitude of action potential (Vpeak) were recorded and compared with WT. We found that thresholds of neurons from APP/PS1 mice were significantly decreased compared to WT mice , but the peak amplitude was not . These results suggested that the excitability of mature neurons obtained from APP/PS1 mice was increased.To investigate change in intrinsic excitability in APP/PS1 mice, whole-cell patch-clamp recordings were performed on 12 DIV . As described previously, whole-cell patch-clamp recordings were performed in current-clamp mode on normal morphological primary neurons which were incubated with Aβ1−42 or Aβ42−1 for 24 h. Frequency, threshold, and peak amplitude of AP were examined. We found AP firing frequencies were significantly increased in neurons after treatment with Aβ1−42 peptide compared to controls . Aβ1−42 also significantly decreased the threshold . But it had no effect on the peak amplitude . These results suggested that incubation in Aβ1−42 increases neuronal excitability in vitro.It is likely that diverse factors contribute to the pathogenesis of AD patients or mice .In mammalian neurons, dense clusters of voltage-gated sodium channels (Nav) at the axonal initial segment and nodes of Ranvier underlie action potential generation and fast propagation ; a V1∕2, Aβ42−1 = −41.71 ± 1.768 mV and a κ of 4.795 ± 1.825 mV (n = 12); and a V1∕2, Aβ1−42 = −41.19 ± 1.052 mV and a κ of 4.155 ± 1.184 mV (n = 12). For steady-state inactivation curves .We next investigated the effect of Aβ Figures . We foune Figure . Single s Figure , the V1∕1−42 leads to an increased neuronal excitability, likely through a Nav current-mediated mechanism.Overall, these results demonstrated that exposure of neurons to Aβ1−42 increased neuronal excitability, perhaps dues to increasing Nav currents, it remained unclear how the Nav currents increased. There was no observable difference in sodium channel kinetic characteristics after Aβ1−42 incubation . We found, that APP/PS1 mice exhibited more Nav1.6 immunoreactivity than WT mice in cortex regions .Morphological methods were also used to test the expression of Nav1.6. The localization of Nav1.6 in neurons was revealed by immunohistochemistry Figure . Consist6 Figure ; n = 3. 1−42 increased the excitability of cultured cortical neurons, and this effect was mediated by overexpression of Nav, with the Nav1.6 subtype perhaps accounting for much of this increase.Taken together, our findings and previous studies suggest that AβIn addition to cognitive deficits, AD patients have an increased incidence of epileptic seizures. This incidence is even higher in patients with early-onset AD who overexpress human APP, the proteolysis of which generates Aβ (Palop and Mucke, threshold decreased in neurons from APP/PS1 mice compared to WT mice. These results confirm previous studies that showed neuronal hyperexcitability in APP/PS1 mice by patch clamp methods. In recent years, many studies demonstrated that Aβ is associated with increased excitability of neurons in vitro and in animal models, leading to hypersynchronous network activity and higher risk for seizures (Minkeviciene et al., In the present study, we investigated the excitability in cultured pyramidal neurons of APP/PS1 mice using patch clamp techniques. We found that the excitability of 12 DIV cultured pyramidal neurons from APP/PS1 mice was significantly greater than in WT littermates. Additionally, AP firing frequencies increased and V+ currents, thereby increasing excitability of hippocampal pyramidal neurons (Good and Murphy, Aβ-induced aberrant excitatory activity might occur through many different mechanisms. Previous studies have shown that Aβ can downregulate A-type K1−42 could be due, at least in part, to an upregulation of the Nav current. As we expected, the peaks of the voltage-dependent sodium current were significantly increased after Aβ1−42 treatment. The increased voltage-dependent sodium current could decrease action potential threshold and increase the probability of action potential generation in response to synaptic excitation, thus increasing the excitability of the neuron. However, Aβ upregulated sodium currents without significantly altering the voltage-dependence of activation and inactivation. We found that Aβ increased the expression of Nav and Nav1.6 in cultured neurons, indicating that the number of Nav channels could be altered by Aβ1−42. These results provide a possible mechanism for the increased excitability of pyramidal neurons previously observed after Aβ treatment. Confirming the causal relationship between the aberrant excitatory activity induced by Aβ and cognitive decline in AD patients would be an important insight into the pathogenesis of AD and provide new therapeutic avenues.Voltage-dependent sodium currents play a critical role in action potential depolarization and firing frequency in many types of neurons (Kim et al., Conceived and designed the experiments: XW, SL. Performed the experiments: XW, XZ, and CJ. Analyzed the data: XW, XZ, ZX, and QM. Contributed reagents/materials/analysis tools: TZ, NL. Wrote the paper: XW, XZ.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.