Mitochondria are organelles that host many metabolic processes, including the generation of energy in the form of adenosine triphosphate (ATP) through oxidative phosphorylation. They contain their own genome, however, the vast majority of mitochondrial proteins are encoded in the nucleus. Nuclei sense changes in mitochondrial functioning via retrograde signaling pathways, resulting in modified gene expression to restore homeostasis. These pathways can be triggered by altered ATP synthesis, increased levels of unfolded or damaged proteins, or changes in the production of reactive oxygen species (ROS), a major by-product of the electron transport chain. Indeed, inhibition of electron transport activity leads to enhanced ROS levels and, somewhat paradoxically, promotes increased lifespan in a range of model organisms. The complex relationship between mitochondrial function, ROS and aging has been a topic of intense research for many years, with particular focus on the retrograde signaling pathways that act as the conduit of information between mitochondria and nuclei .mt) [C. elegans clk-1 null mutants and heterozygous mice display defective oxidative phosphorylation, increased ROS levels and, similar to many other respiration mutants, have extended lifespans [clk-1 null C. elegans, indicating that the nuclear form of CLK- 1 independently affects longevity [mt are reported to promote longevity, their suppression by nuclear CLK-1 would be consistent with its role in limiting lifespan.We have uncovered a novel nuclear role for the respiratory enzyme CLK-1 in maintaining mitochondrial function by regulating the expression of genes that promote ROS homeostasis and dampen the activity of a key stress pathway, the mitochondrial unfolded response (UPRmt) . Importaifespans . It had ongevity . As elevmt. Conversely, decreased ROS lead to a reduction in nuclear CLK-1 and relieve its effects on gene expression allowing ROS levels to recover and the UPRmt to return to basal levels, thus maintaining homeostasis (Figure mt, a pathway that protects against the accumulation of unfolded or misfolded proteins within mitochondria. This suggests that distinct mitochondrial signals can be integrated at the level of nuclear gene expression to provide a unified response.We propose that CLK-1 functions as a rheostat to maintain ROS homeostasis and keep stress-responsive pathways in check. Elevated ROS produced by mitochondria direct a pool of CLK-1 to the nucleus where it regulates the expression of genes that decrease ROS levels and limit activation of the UPRC. elegans UPRmt, the pathway suppressed by nuclear CLK-1, may be similarly regulated. ATFS-1 is targeted to mitochondria and rapidly degraded in the absence of UPRmt induction, while activation of the UPRmt leads to impaired import of newly synthesized ATFS-1 and its redirection to the nucleus where it regulates the expression of genes that restore mitochondrial proteostasis [Important questions concerning the dual mitochondrial and nuclear functions of CLK-1 remain unanswered. Firstly, how do changes in ROS levels lead to the nuclear accumulation of CLK-1? The mitochondrial and nuclear forms of CLK-1 are distinguishable; the mitochondrial form is cleaved at the N-terminus by a protease following import while the nuclear form is the full-length protein, indicating that it has not been imported into mitochondria prior to its translocation to the nucleus. Interestingly, mutation of the mitochondrial targeting sequence in CLK-1 results in its nuclear accumulation independently of ROS levels . This woeostasis . Thus, tA second unresolved issue is the mechanism by which CLK-1 regulates gene expression. This is independent of its role in ubiquinone biosynthesis . CLK-1 iThere is precedent for mitochondrial enzymes regulating nuclear gene expression, including the yeast arginine biosynthesis enzyme Arg5,6 . Our stu


Schistosoma mansoni-endemic settings in Africa indicate it has good sensitivity in detecting infections, but in areas of low prevalence, the POC-CCA can be positive for persons who are egg-negative by Kato-Katz stool assays. We examined the POC-CCA assay for: (a) batch-to-batch stability; (b) intra-reader and inter-reader variability; (c) day-to-day variability compared to Kato-Katz stool assays, and (d) to see if praziquantel (PZQ) treatment converted Kato-Katz-negative/POC-CCA positive individuals to POC-CCA negativity. We found essentially no batch-to-batch variation, negligible intra-reader variability (2%), and substantial agreement for inter-reader reliability. Some day-to-day variation was observed over 5 days of urine collection, but less than the variation in Kato-Katz stool assays over 3 days. To evaluate the effect of treatment on Kato-Katz(−)/POC-CCA(+) children, 149 children in an area of 10–15% prevalence who were Kato-Katz(−) based on 3 stool samples but POC-CCA(+) were enrolled. Seven days after treatment (PZQ 40 mg/kg) samples were again collected and tested. Almost half (47%) POC-CCA positive children turned negative. Those still POC-CCA positive received a second treatment, and 34% of them turned POC-CCA negative upon this second treatment. Most who remained POC-CCA positive shifted each time to a “lesser” POC-CCA “level of positivity.” The data suggest that most Kato-Katz-negative/POC-CCA positive individuals harbor low-intensity infections, and each treatment kills all or some of their adult worms. The data also suggest that when evaluated by a more sensitive assay, the effective cure rates for PZQ are significantly less than those inferred from fecal testing. These findings have public health significance for the mapping and monitoring of Schistosoma infections and in planning the transition from schistosomiasis morbidity control to elimination of transmission.Studies of the urine-based point-of-contact cathodic circulating antigen test (POC-CCA) in  Schistosoma infections to inform programmatic decisions on whom, and how often to treat within endemic areas  cassette assay that detects circulating cathodic antigen (CCA). CCA is derived from the gut of adult schistosomes that are established in the human circulation , 17–22. ensities , 21, 22.nfection , 21, 23.The study protocol was approved by the scientific steering committee (SSC) of the Kenya Medical Research Institute , the Ethical Review Committee (ERC) of KEMRI, the Institutional Review Boards (IRB) of the Centers for Disease Control and Prevention (CDC), the Pontificia Universidad Catolica del Ecuador, Quito, Ecuador, the Aklilu Lemma Institute of Pathobiology, Addis Ababa University of Ethiopia and of the University of Georgia. Parents/guardians, and participating children were informed about the purpose and procedures of the study. Written informed consent was obtained from the parents/guardians and the children participating in the study gave assent prior to study enrollment. All samples obtained in the study were coded and treated confidentially.Most of this study was conducted in schools near the shores of Lake Victoria, western Kenya from January 2013 to April 2014. Studies in areas known not to be endemic for schistosomiasis but endemic for soil-transmitted helminths (STHs) were done in Ethiopia , and inS. mansoni and STHs   for the quantitative determination of Urine samples were collected and tested in the laboratory for positivity and band intensity by the commercially available POC-CCA assay, according to manufacturer’s instructions . Briefly, one drop of urine was added to the well of the testing cassette and allowed to absorb. Once it was absorbed, a drop of buffer (included in the kit) was added into the same well and the assay was allowed to develop for 20 min, at which time the results were read. The tests were considered invalid if an internal control band did not appear or if the tests were left to develop for more than 25 min after addition of the buffer before being read. In these few cases, the samples were re-run with a new test cassette and scored appropriately. To score the intensity of the POC-CCA assay results, the intensity of the test band was compared to that of the control band. Positive results were given a score of “trace” if the band was barely visible, 1+ if the test band was less intense than the control band, 2+ if the test band was of equal intensity as the control band, and 3+ if the test band was more intense than the control band.S. mansoni infections or had infections that ranged from low to moderate intensity based on Kato-Katz stool examinations. Each of the 3 readers read the 10 urine samples using 10 POC-CCA cassettes from Box # 1. The urine samples were shuffled and then read again using 10 cassettes from Box # 2; this retesting of the 10 test urines continued using cassettes from the remaining boxes, 10 per box, to Box # 5.Five separate batches of CCA test kits were used for this study. Three readers were provided with 10 urine samples from subjects who either did not have Five readers were provided with 10 urines that ranged from negative to moderately positive, as described above. In Round 1, each reader read urine results using 10 individual POC-CCA cassettes read at 20 min. For Round 2, the cassettes were randomly rearranged, and each reader re-read and re-scored the same (masked) cassettes for a second time within ~5 min, to keep within the time limit for valid cassette readings.Five readers were provided with 30 urines that ranged from negative to moderately positive, as described above. The five readers read the urines on POC-CCA cassettes independently and the results were recorded without discussion.Seventy-three participants were recruited for this study. Five consecutive urine samples for POC-CCA and three consecutive stool samples for Kato-Katz were collected and assessed to determine if there were any fluctuations in the POC-CCA or Kato-Katz readings based on the different days of collection.One hundred forty-nine children from schools that had received two or three rounds of annual MDA with PZQ and who were POC-CCA positive but Kato-Katz negative (based on a single urine sample and three stool samples) were enrolled at baseline, and treated with PZQ based on the POC-CCA positive results. Seven days later, a single urine and stool sample were collected and tested by POC-CCA and Kato-Katz, respectively. Children who were still POC-CCA positive were treated for a second time, and 7 days later urine and stool samples were again collected and assayed. Children who were still POC-CCA positive were again treated for a third time without further testing. A schematic diagram of the post-treatment study is shown in Figure Data collected from evaluations of school children included egg intensity readings and POC-CCA cassette band readings and were entered into an Excel database and analyzed using SAS statistical software . Fleiss kappa statistical analysis  was usedWhen different batches of the cassettes provided by the manufacturer were tested on the same 10 urine samples, there was no real variation between them. Nine of 10 samples showed the same results across all 5 batches for all 3 readers. Only one sample gave varying results when cassettes were taken from different batches, being negative for some lots and trace (positive) on others. This same variation occurred for all three readers.When 5 trained readers read two rounds of the same 10 specimens, there was only 1 reading of the 50 pairs of repeat readings that was not replicated. That one was read as negative the first time and as trace (positive) when it was re-read by one of the readers. This corresponds to a 2% intra-reader variation.P < 001). This indicates substantial agreement between the five different readers.When 5 trained readers were evaluated in regard to the reliability of their readings of the same 30 specimens and the results analyzed by Fleiss kappa statistics the overall Fleiss kappa value for inter-reader agreement of the POC-CCA scores was 0.70 . As per the manufacturer’s instructions, “Trace” readings were considered positive. For each assay, prevalence estimates based on single tests did not vary significantly between days, ranging between 81 and 88% for the five consecutive POC-CCA and between 41 and 44% for three consecutive Kato/Katz tests. Almost all (94%) of children were positive for at least one of the five POC-CCA tests while 70% were positive for the at least one of Kato/Katz tests  to evaluate When the first three CCA consecutive tests were taken together, 82% of participants had consistent daily results compared to only 49% of participants with consistent daily results by Kato-Katz assays. Mixed results  were observed in 18% of the participants for the POC-CCA tests but in 48% of Kato-Katz tests. Thus, while there was some day-to-day variation for both assays, the Kato-Katz varied much more in terms of diagnosing the presence of infection than the POC-CCA . These analyses indicated that the POC-CCA assay provided high levels of both sensitivity and specificity, as shown in Table When the POC-CCA assay was tested in areas in Ethiopia and Ecuador that had never been endemic for schistosomiasis but were endemic for STH, only one presumably false positive test was found among 100 participants in Ethiopia , while nS. mansoni infection in the study population. The estimated specificity for both POC-CCA and Kato-Katz assays approached 100%.A Bayesian latent class model (BLCM) was developed using data from all 73 subjects in this part of the study, assessing the most likely performance characteristics of 1 up to 5 daily POC-CCA results, and 1 up to 3 daily Kato-Katz stool results, guided by the POC-CCA specificity data Ethiopian and Ecuadorian children described above Figure . By thisInfection intensity using POC-CCA can be determined only semi-quantitatively by comparing the density of the test line that develops to the control line provided in the cassette. Yet, we found a good correlation between egg intensity determined by Kato-Katz as compared to POC-CCA scores based on readings the intensities of the positive bands. All the 10 participants who had moderate or high egg counts by Kato-Katz had an unambiguously positive PCO-CCA result. There was a significant correlation between a participant’s mean EPG and their CCA score on all of the tested days Figure .S. mansoni prevalence (by Kato-Katz) following 2 or 3 rounds of MDA with PZQ. One week after testing, 126 of these children were treated with PZQ. Of those not lost to follow-up, 55 of 117 (47%) became POC-CCA negative on repeat testing performed 1 week post-treatment. Those who remained POC-CCA positive (and were available) were treated a second time, and 7 days later, 19 of 56 (34%) children were negative based on follow-up POC-CCA testing at that time. The Kato-Katz assays of these children, who were selected as Kato-Katz negative, remained negative after each treatment round.For this phase of the study, 149 children who had tested negative by Kato-Katz (3 stools/2 slides each) but positive by POC-CCA (tested on a single urine) were selected from an area that had achieved 10–15% overall S. mansoni infection. The sensitivity of the assay used to evaluate prevalence in these different phases becomes critical, as the current Kato-Katz test is known to become significantly less sensitive as egg count intensities decrease . We had previously observed that when non-technician field workers were minimally trained and then asked to read multiple POC-CCA tests, there was considerable variation in the readings. Since there was little or no variability seen when trained readers (laboratory technicians) were used, this highlights the need for adequate training prior to use of the POC-CCA assay.Day-to-day variability in per-subject prevalence, based on readings from 3 successive days of Kato-Katz or 5 days of POC-CCA, was minimal and almost all variation occurred at the very low intensity end of the CCA test spectrum, involving “trace” readings. Also, based on the first 3 days of testing run in parallel, the day-to-day variability of the POC-CCA readings (18%) was much less than those of the Kato-Katz (48%).Schistosoma infection are likely to be significantly overestimated, based on the post-treatment inaccuracy of a lower-sensitivity assay, the Kato-Katz. PZQ is typically said to produce 70–90% cure rates as evaluated by Kato-Katz assays  yielded only one “trace” POC-CCA positive (from Ethiopia) child out of 243 children tested, confirming the high specificity of this assay.S. mansoni. While not a perfect test (as seen in the day-to-day variability) the POC-CCA assay appears to be highly suitable for initial mapping of S. mansoni areas, and for subsequent post-treatment programmatic decision making. The fact that it indicates higher prevalences than the less sensitive Kato-Katz in areas with low-average intensity should now be factored into how treatment decisions will be made. Our data bring up the possibility that many people in endemic areas are harboring low levels of worms that are producing few, if any, eggs on a daily basis. This raises an issue that is seldom voiced, but may be quite important in programmatic thinking. What are the consequences of such a scenario? If few or no eggs are being released every day, does that level of infection still lead to morbidity? If few or no eggs are being released, it would seem that the implications for transmission would be important. We do not yet have answers to these questions, but believe they need to be discussed. They will continue to arise as the POC-CCA and other more sensitive assays are introduced and employed more widely in the field.In conclusion, our data show that most  Kato-Katz-negative/POC-CCA positive individuals in endemic areas are likely to harbor low level infections with The study was designed by DC, NK, CK, CC, and PM. EO and PC coordinated field activities and laboratory work. NK and CK analyzed the data. All authors actively contributed to the interpretation of the findings, data compilation, and manuscript preparation. All authors read and approved the final manuscript.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.


The neuropsychological features and neuropathological progression patterns associated with rapidly evolving cognitive decline or dementia in Parkinson's disease (PD) remain to be elucidated.Fifty-three PD patients without dementia were recruited to participate in a 3-year longitudinal cohort study. The patients were grouped according to the Clinical Dementia Rating (CDR). Group-wise comparisons were made with regard to demographic characteristics, motor symptoms, neuropsychological performances and 18F-fluorodeoxyglucose positron emission tomography.Patients who had memory-plus cognitive impairment  exhibited higher age at onset, visuoperceptual impairment, non-tremor-dominant motor disturbance, rapid symptomatic progression and posterior neocortical hypometabolism. In patients who were cognitively unimpaired and those who had memory-dominant cognitive impairment , the posterior neocortex was relatively unaffected until a later stage of the disease.These results suggest that visuoperceptual impairment and the early involvement of the posterior neocortex may be risk factors for rapid symptomatic progression and dementia in PD. The cognitive features of Parkinson's disease (PD) are heterogeneous and can be categorized into several major subtypes. Previous morphometric MRI and 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) studies have demonstrated greater frontal, temporal and occipital gray matter volume reduction and greater frontal and parietal cortical hypometabolism in PD patients with dementia or mild cognitive impairment (MCI) compared with cognitively unimpaired patients. All procedures in this study followed the clinical study guidelines of Tohoku University Hospital and were approved by its ethics committee. The patients gave written informed consent after receiving a detailed explanation of the study. When the patients had a compromised ability to consent, their family members gave consent on behalf of the patients.t = 1.6, p = 0.1) or sex  between the patient and control groups.We analyzed 55 patients with PD without dementia  who participated in a 3-year longitudinal study at Tohoku University Hospital. Details of the study design have been described elsewhere. Measuring cognitive changes is challenging because there is no very reliable change measures. Practice effects associated with the repeated administration have a great impact on neuropsychological test performance, yielding spurious cognitive improvement over time. The CDR, which was designed to provide a rating scale for subjects from normal cognition through various stages of dementia, is widely considered to be a reliable scale for staging the severity of cognitive dysfunction. The primary aim of the current study is to discover clinical features and distinctive brain metabolic patterns of patients who have rapid cognitive deterioration. To this end, we first focused on 40 patients who were cognitively unimpaired (CDR 0) at baseline. Among these patients, 26 patients were cognitively unchanged over 3 years , 7 worsened only in the memory domain (memory-only converters) and 6 worsened in the memory and non-memory domains (memory-plus converters). The remaining patient, who showed deterioration only in a non-memory domain, was excluded from the analyses. Second, we analyzed patients whose overall CDR scores were 0.5 at baseline to investigate longitudinal brain metabolic changes after PD patients developed mild cognitive deficits. Eight patients who scored ≥0.5 only in the memory domain at baseline (baseline memory-only) and 6 patients who scored ≥0.5 in the memory and other domains (baseline memory-plus) were recruited for the study. We speculated that the baseline memory-only and the baseline memory-plus patients may represent the clinico-pathological stages following the memory-only converters and the memory-plus converters, respectively. We conducted group comparisons separately among the groups of baseline CDR 0, specifically non-converter, memory-only converter and memory-plus converter patients, and between the groups of baseline CDR 0.5, specifically baseline memory-only and baseline memory-plus patients, because our interest was in longitudinal changes in clinical symptoms and brain glucose metabolism. and The Mini-Mental State Examination (MMSE) and the Word Recall subtest of the Alzheimer’s Disease Assessment Scale (ADAS) were used to assess general cognitive function and episodic memory, respectively.  and Group-wise comparisons of demographic data and baseline scores and progression rates of the cognitive and motor measures were analyzed using the statistical methods described in the captions of http://www.fil.ion.ucl.ac.uk/spm/). All images were normalized onto the standard FDG template with nonlinear warping algorithms, reconstructed into 2 mm3 isotropic voxels and smoothed with 10 mm full width at half-maximum. Global normalisation was performed using proportional scaling, and threshold masking was set at 0.8. Cross-sectional comparisons between the patient groups and the controls were performed using t-test. Two-way repeated-measures ANOVA was used for cross-sectional and longitudinal comparisons of the patient groups. Age and sex were included as nuisance variables in all of the comparisons. The UPDRS part III score was included as a nuisance variable in the comparisons among the patient groups. The statistical threshold was set at an uncorrected p<0.001 at the voxel level and at 20 voxels at the cluster level.The mean interval between the clinical assessments and the positron emission tomography (PET) scan was 4.6 days. Each patient had fasted, and dopaminergic medication had been discontinued for at least 5 hours before the scan. Scanning was performed after an injection of 185–218 MBq 18F-fluorodeoxyglucose (FDG). After an FDG-uptake period of 1 hour, a 20-minute scan was acquired while the patient was at rest. Details of the scanning procedures have described elsewhere. . The neuropsychology-based classification according to the MDS PD-MCI criteria exhibited a spurious improvement over 3 years in 5 of the 12 patients who were classified to PD-MCI at baseline, whereas such an effect was observed only in 1 of the 11 patients who scored 0.5 on the baseline CDR . The baseline performance of the backwards digit-span was worse in the memory-only converters than it was in the non-converters . No group differences were observed in baseline MMSE or baseline ADAS word recall. There were no significant differences in the progression rate on any of the cognitive tests.No significant difference was observed in the baseline UPDRS part III among the three groups. The progression rate of the UPDRS part III was greater in the memory-plus converters than it was in the non-converters and the memory-only converters . The UPDRS non-tremor score was higher in the memory-plus converters than it was in the other groups , and no significant main effect of time or interaction between motor subtypes and times was observed.The results are summarized in Compared with the controls, the non-converters and memory-only converters exhibited patchy, discrete areas of hypometabolism in the frontal, temporal and occipital cortices at baseline .There are a number of limitations in the current study. First, although we claim that the memory-plus converters represent the rapidly progressive clinical subtype, no significant metabolic changes over 3 years were observed in this patient group. The following reasons can be suggested for this negative finding: (1) the small sample size may have result in a low statistical power; and (2) diffuse metabolic decline across the entire cerebral cortex may have obscured by the proportional scaling in the PET analysis. Consistent with the latter, a supplementary PET analysis in which a cerebellar reference was used instead of the proportional scaling demonstrated a CMRglc reduction over 3 years in the preforntal cortex in the memory-plus converters  cognitive changes. Our findings and discussion should be examined by studies with longer follow-up periods.Finally, the reduction in glucose metabolism may reflect not only neurodegeneration itself but also the remote effects of lesions in distant neural structures. In addition, because FDG-PET is unable to differentiate between Alzheimer's disease-related and Lewy-related pathologies, further studies utilizing amyloid-PET and other neuroimaging techniques are necessary to examine these issues.Figure S1The results of a cerebellar-referenced PET analysis for the patient groups with baseline CDR 0 . A two-way repeated-measures ANOVA with variables of no interest of age, sex and UPDRS part III score was used. The statistical threshold was set at an uncorrected p<0.001 at the voxel level and at 20 voxels at the cluster level.(TIF)Click here for additional data file.


We developed a microfluidic model of microcirculation containing both blood and lymphatic vessels for examining vascular permeability. The designed microfluidic device harbors upper and lower channels that are partly aligned and are separated by a porous membrane, and on this membrane, blood vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs) were cocultured back-to-back. At cell-cell junctions of both BECs and LECs, claudin-5 and VE-cadherin were detected. The permeability coefficient measured here was lower than the value reported for isolated mammalian venules. Moreover, our results showed that the flow culture established in the device promoted the formation of endothelial cell-cell junctions, and that treatment with histamine, an inflammation-promoting substance, induced changes in the localization of tight and adherens junction-associated proteins and an increase in vascular permeability in the microdevice. These findings indicated that both BECs and LECs appeared to retain their functions in the microfluidic coculture platform. Using this microcirculation device, the vascular damage induced by habu snake venom was successfully assayed, and the assay time was reduced from 24 h to 30 min. This is the first report of a microcirculation model in which BECs and LECs were cocultured. Because the micromodel includes lymphatic vessels in addition to blood vessels, the model can be used to evaluate both vascular permeability and lymphatic return rate. Microcirculation plays a crucial role in the supply of oxygen and nutrients from the blood to extravascular tissues. The microcirculation system consists of blood and lymphatic vascular capillaries and the interstitium, and functional disorders of the microcirculation system are strongly related to, for example, inflammatory responses, swelling, and tumor. When an injury occurs in a body, the inflammatory response, which is induced by released inflammation-promoting agents such as histamine or cytokines, affects the microvascular system . InflammTo date, the physiological mechanism underlying microcirculation has been investigated using numerous experiments involving animals and cultured cells. In experiments conducted using small animals, blood and lymph flows were measured by observing the movements of erythrocytes and leukocytes in the vessels . VasculaConversely, blood flow in the human body has been measured using noninvasive techniques. In vivo vascular imaging has been performed using imaging methods such as computed tomography, magnetic resonance imaging, optical coherence tomography, and confocal laser-scanning microscopy, and using Doppler ultrasound imaging, a non-optical method . HoweverCompared to animal experiments, experiments on cultured normal human microvascular endothelial cells or normal human lymphatic microvascular endothelial cells are conducted more widely because the behavior of these cells can be monitored in detail. The effects of vascular or lymph flow have also been examined using endothelial cell monolayers cultured on glass slides or petri dishes under various flow rates of culture media ,12. The Recent advances in microfluidic technologies have opened the door for creating in vitro cell-culture formats that are more realistic than those used previously. The size scale of the microchannels in microfluidic devices is close to that of the microvascular system and these microchannel-based cell cultures can be used as perfusion cultures, and, consequently, microvascular models have been realized. In a vascular model, endothelial cells have been stimulated under flow conditions mimicking vascular flow ,18, and In this study, our aim was to develop a microcirculation model featuring both blood and lymphatic vessels for the purpose of examining vascular permeability. We focused the interaction between blood vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs), and in the newly developed device, the flow conditions mimicked vascular and lymphatic flow and molecules moved from blood vessels to lymphatic vessels through an ECM hydrogel.et al. 46][[46][2+-d[[46][2+-lar gaps . Thus, wThe binding of fluorescent-histamine to the BEC or LEC surface was examined using cells cultured in the single-channel device for 24 h under a 6-μL/h continuous-flow condition; the results are shown in 2+ levels. Whereas intracellular Ca2+ is maintained at a low level under quiescent conditions, histamine stimulation induces an increase in the level of intracellular Ca2+ by triggering the movement of Ca2+ from the lumen of the endoplasmic reticulum or the extracellular compartment into the cytosol through Ca2+ channels. To examine the behavior of intracellular Ca2+, we measured the fluorescence signal of the Ca2+ indicator Fura2  after adding the recording medium containing histamine-2HCl. The results of Ca2+ imaging are shown in 2+ level was already high before histamine stimulation. Intracellular Ca2+ was maintained at a higher level in the cells in the microdevice than in the cells in the 5-mm-diameter well , but the localization of VE-cadherin and claudin-5 changed when the histamine concentration was increased, and then the cell-cell junctions were gradually disrupted .We also performed immunofluorescence staining for VE-cadherin and claudin-5 after histamine stimulation of cells cultured in the 5-mm-diameter wells. Both BECs and LECs cultured in these wells showed a response to histamine stimulation that was distinct from that observed in the case of cells cultured in the microdevice . The expThe aforementioned results agreed with those shown in −6 cm/s, which is close to the value reported previously for a microfluidic microvessel model (4.8 × 10−6 cm/s for 20-kDa FITC-dextran) [Because histamine stimulation affected cell-cell junctions in both BECs and LECs cultured in the microdevice, permeability tests were performed to examine the functions of the cells. In these tests, 100 mM histamine and the fluorescent tracers TRITC-dextran and LY (10 μM each) were added to the culture medium that was introduced into the upper channel (BEC channel) by using the syringe pump. The lower channel (LEC channel) was filled with culture medium containing 100 mM histamine. dextran) . The resNext, the permeability test was conducted using habu snake venom. In the human body, a bite by this snake causes organ damage, because habu snake venom induces hemorrhage, myonecrosis, and edema. The most-accepted therapy for snake-bite patients is immediate administration of an anti-venom antiserum, which neutralizes the toxic activities of snake venom. The anti-hemorrhagic potency test is a major test used for measuring anti-venom activity, and in this test, rabbits are used :}. The vPermeation tests were conducted using the same format as that used for the histamine assay, but the test sample was now habu snake venom. Compared to histamine stimulation , treatmeIn a previous study, BODIPY-FL-labeled casein was used in a habu snake venom activity test conducted without either animals or cells . In thisA microcirculation model including both blood and lymphatic vessels was developed for examining vascular permeability. Claudin-5 and VE-cadherin were detected at cell-cell junctions in both BECs and LECs. The permeability coefficient measured here was lower than the value reported for isolated mammalian venules, which suggests that the BECs and LECs cultured in the microdevice retained their barrier functions due to the formation of tight endothelial junctions. Moreover, our results showed that flow cultures promoted the formation of endothelial cell-cell junctions. Histamine, an inflammation-promoting substance, induced changes in the localization of proteins associated with tight and adherens junctions and an increase in vascular permeability in the microdevice. These results indicate that both BECs and LECs appear to maintain their functions in the microfluidic coculture platform. The vascular damage induced by habu snake venom was successfully assayed using the microcirculation device, and the assay time was reduced from 24 h to 30 min.This is the first report of a microcirculation model in which BECs and LECs were cocultured, and this model can potentially promote microcirculation research. Because the micromodel to realize application of this bioassay includes lymphatic vessels in addition to blood vessels, both vascular permeability and lymphatic absorption can be evaluated using this model. Therefore, the device could be used for research on immune surveillance, lymphedema, inflammation, and tumor metastasis, and could potentially also be used in the development of anti-inflammatory drugs as well as anticancer agents. Studies on drug-delivery systems (DDSs) have previously investigated the enhanced permeation and retention effect, which is caused by the presence of imperfect tumor blood vessels and poor lymphatic drainage in tumor tissues ,55, and S1 Fig(TIF)Click here for additional data file.S2 FigThe bifurcation of the microchannels is shown. Red: upper channel. Green: lower channel.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 FigThe arrow indicates a gap in the cell layer.(TIF)Click here for additional data file.


Cluster headache is a severe, disabling disorder with pain that ranks among the most severe known to humans. It is associated with accompanying autonomic symptoms ipsilateral to the pain and a sense of restlessness or agitation. Patients with cluster headaches have few therapeutic options, and a further 10-20% develop drug-resistant attacks. The often brief duration of cluster attacks makes abortive therapy a challenge, and preventive medications are almost always provided to patients.Although NMDA-R activation by glutamate has been hypothesized to play a role in the pathophysiology of primary headache disorders, its role is still not fully understood. In fact, the trigeminovascular nociceptive transmission from primary afferents through the trigeminal nucleus caudalis and on to other parts of the CNS involves both the NMDA and non-NMDA glutamate receptors. The kynurenine pathway (KP), accounting for more than 90% of the tryptophan metabolism, generates neuroactive compounds that are able to interact with glutamate receptors both in the central and in the peripheral nervous systems. Among the KP metabolites, Kynurenic Acid (KYNA) and Quinolinic Acid (QUINA) have been shown to interact with ionotropic glutamate receptors. QUINA acts as an orthosteric agonist at the GluN2 subunits of NMDA receptors and it might regulate endogenous glutamate release and uptake inhibition and lipid peroxidation. In contrast, KYNA acts as a competitive antagonist at the glycine site on the GluN1 subunit of NMDA receptors, thereby inhibiting NMDA receptor function. KYNA also inhibits the kainate and the amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptors, and has a non-competitive inhibitory action on the a7-nicotinic acetylcholine receptors[In this preliminary study we enrolled 14 cluster headache patients  and 15 age-matched healthy controls . We developed a HPLC tandem mass spectrometry method to assess the serum concentrations of KYNA, QUINA, Anthranilic acid and Kynurenine. Kynurenine serum levels resulted not significantly different between the groups , while both QUINA, KYNA and Anthranilic Acid were significantly reduced in cluster headache patients with respect to healthy controls . These results highlight that the endogenous regulation of the glutamatergic transmission in cluster headache might play an important role in its pathophysiology.Written informed consent to publication was obtained from the patient(s).


Cognitive training, including fast simple numerical calculation (FSNC), has been shown to improve performance on untrained processing speed and executive function tasks in the elderly. However, the effects of FSNC training on cognitive functions in the young and on neural mechanisms remain unknown. We investigated the effects of 1-week intensive FSNC training on cognitive function, regional gray matter volume (rGMV), and regional cerebral blood flow at rest (resting rCBF) in healthy young adults. FSNC training was associated with improvements in performance on simple processing speed, speeded executive functioning, and simple and complex arithmetic tasks. FSNC training was associated with a reduction in rGMV and an increase in resting rCBF in the frontopolar areas and a weak but widespread increase in resting rCBF in an anatomical cluster in the posterior region. These results provide direct evidence that FSNC training alone can improve performance on processing speed and executive function tasks as well as plasticity of brain structures and perfusion. Our results also indicate that changes in neural systems in the frontopolar areas may underlie these cognitive improvements. This study focused on training to improve performance on a fast simple numerical calculation (FSNC) task, which involves quickly solving mathematical problems, namely, single-digit addition, subtraction, and multiplication. The ability to complete an FSNC task correlates with processing speed, quantitative ability or knowledge, and general intelligence . PreviouHowever, some questions related to these studies remain to be answered. First, these studies used multiple training protocols such as reading and arithmetic involving simple and more complex numerical calculations , 3 or a This study aimed at investigating the effect of FSNC training on cognitive functions and neural systems in healthy young adults. Considering the transfer effects brought about by the FSNC training, it is important to investigate the extent of and neural mechanisms underlying these FSNC training effects. Among FSNC training-affected neural mechanisms, we focused on changes in regional gray matter volume (rGMV) and regional cerebral blood flow during rest (resting rCBF). Using imaging analyses, we determined whether the effects of FSNC training extend beyond task-specific functional activation in the brain and, if so, the areas in which such changes occur.Based on the results of our previous study , we hypoUsing (a) various psychological measures, such as arithmetic measures, processing speed, and executive function, (b) rGMV analysis with voxel-based morphometry (VBM), and (c) resting rCBF analyses, we investigated the effects of 1-week intensive (up to 4 h/day) adaptive FSNC training on these variables in healthy young adults. Training consisted of three paper-and-pencil FSNC tasks  as well as three computerized FSNC tasks . We inclThe study reported here was implemented in conjunction with and shared control group participants with our previous study which investigated the effects of training on processing speed . Sixty-tGroup assignments were performed in the following manner as described below. In 3 months, there were six experimental periods, each lasting 8 days. Among these six periods, the first, third, and fifth experimental periods involved PS training and no intervention. The rest of the experimental periods involved FSNC training and no intervention. The periods for the FSNC (and the nonintervention control group) and the periods for training on processing speed (and the nonintervention control group) were rotated. We could perform one type of intervention in one experimental period because of the limitation of several types of experimental resources. Subjects chose which period they participated at, but they did not know there were two types of training in the experiment. They were randomly assigned to intervention groups or the no-intervention group. Therefore, neither subjects nor experimenters could decide which groups subjects could be assigned to, and distribution amongst the three groups was arbitrary.P > 0.1, two-tailed t-tests) in basic background characteristics such as age, sex, and scores on Raven's Advanced Progressive Matrix  Arithmetic tasks, similar to the ones constructed by Grabner et al. , measure[B] These included the following: Raven's Advanced Progressive Matrices , a nonve[D] These included the following: a (computerized) digit span task, a verbal working memory task ; [E] a [F] The test used was the Tanaka B-type intelligence test . Type 3Bhttp://dx.doi.org/10.1155/2016/5940634). During each task, the participants were instructed to complete as many tasks as possible in 1 min. Four tasks were performed in a fixed order, but the order of the task did not affect the performance of each task [[G] The task used was the Stroop task (Hakoda's version) , which mach task . We used[H] The S-A creativity test  is used Other than these cognitive tests, we collected several questionnaires designed to assess the traits or states of the participants, but these are not reported here. In most cases, these were self-report questionnaires evaluating participant behavior in daily life. They were designed to assess the traits of the participants and not the effect of the five-day intervention. Other than the self-report questionnaires, all neuropsychological assessments were performed by postgraduate and undergraduate students blinded to the group membership of the participants.P < 0.05) [Behavioral data were analyzed using SPSS 16.0 . Effects of FSNC training on each measure were analyzed by comparing the FSNC training group and no-intervention group using one-way analyses of covariance (ANCOVAs). In these ANCOVAs, the differences between pre- and posttest measures were computed by subtracting the preintervention value from the postintervention value and were entered as dependent variables. Also, in ANCOVAs of the psychological measures, the pretest scores were entered as covariates to exclude the possibility that any preexisting differences between groups in the measures would affect the results of each measure . Repeated measure analyses of variance (ANOVAs) have no superiority over this design as far as we know, but an obvious inferiority of ANCOVA is that repeated measures ANOVAs cannot correct the effects of pretest scores and thus cannot control the preexisting difference in the measures between the groups. Because the superiority  of intervention training was our primary interest, in our behavioral analysis, test-retest changes in the group of interest were compared to those in the control group using one-tailed tests ( < 0.05) , 33. How < 0.05) , 34).We reported results that were only significant at the level of uncorrected data for multiple comparisons. This was partly because this study's investigation of cognitive functions had an exploratory nature (administration of a wide range of cognitive tests of major cognitive functions regardless of the existence of strong a priori hypotheses). This was also partly because we followed the customs of the field in not performing a correction for multiple comparisons , 35–41. However, this study had a smaller sample size than some previous studies  involvinP values of the significant results in the confirmatory analysis of this subsection).In this previous study , the preT1-weighted structural images  were acquired. Arterial Spin Labeling (ASL) was performed to measure resting CBF. It was performed with quantitative signal-intensity targeting by alternating the radio-frequency pulse labeling of arterial regions (QUASAR), a pulsed ASL method [All MRI data acquisition was performed using a 3-T Philips Achieva scanner. Using a MPRAGE sequence, high-resolution L method . DetailsL method –46. The L method . During  in vivo study of human brain structures that can detect changes in rGM caused by training [T1-weighted structural images obtained using this scanner in our previous study [T1-weighted structural images from each subject were segmented into GM and white matter partitions using the abovementioned custom GM and white matter prior probability maps. The resulting images included extracted GM and white matter partitions in the native space. The GM partition was then normalized to the abovementioned custom GM probability map. The normalization parameters determined from this initial step were applied to the native T1-weighted structural image. These normalized T1-weighted structural data were then segmented into GM and white matter partitions. In addition, we performed a volume change correction (modulation) by modulating each voxel with the Jacobian determinants derived from spatial normalization, allowing the determination of regional differences in the absolute amount of GM [VBM, a method oftraining , 48, wastraining  and as ptraining  as an extraining . To reduus study , 51. Nexnt of GM . SubsequT1-weighted structural imaging data because T1WIs obtained using our MPRAGE sequence (see above) were incompatible with preprocessing with VBM5/SPM5 and VBM8/SPM8. When VBM5 or SPM5 was used, many apparent segmentation errors occurred, unlike when the optimized protocol of VBM2 was used. Segmentation errors apparent at first glance were not found when VBM2 or VBM8 was used. However, when VBM8 was used, the test-retest reliability of total GMV of 50 participants who participated in a 1-week longitudinal intervention study in which T1WI was taken on the first day of the experiment and 1 week thereafter [T1WIs of certain sequences and VBM5/VBM8. For more extensive discussions related to this issue, please refer to [VBM2 was used instead of VBM5 or VBM8 for the preprocessing of ereafter  was 0.74ereafter , 30, 51.refer to .P < 0.05, corrected for multiple comparison (FWE) at the nonisotropic adjusted cluster level [P < 0.0025. Nonisotropic adjusted cluster size tests can and should be applied when cluster size tests are applied to nonstationary data , such as VBM data [P < 0.01 seems to lead to too many false positives, whereas that of P < 0.001 seems to lead to slight conservativeness [In the group-level analysis, we tested for a change in rGMV between the first and second time points by comparing the training and control groups . The statistical significance level was set at er level  with an VBM data . In thistiveness .Furthermore, we investigated whether preexisting differences in rGMV (in the preintervention scan) existed between the training and control groups at the whole brain level using ANOVA. In all of these group analyses of morphological data, we included only voxels with a GM value >0.10 to avoid the possibility of partial volume effects around the borders between GM and WM as well as those between GM and the cerebrospinal fluid (CSF).We did not control the global signals across all brain imaging analyses, as was the case for almost all intervention imaging studies.R1 = 1/T1) of each subject were obtained using dedicated software running on IDL   [Maps of raw resting rCBF and the longitudinal relaxivity (lorado)  .T1-weighted images acquired using the MPRAGE sequence were incompatible with VBM5 and SPM5 preprocessing and resulted in numerous apparent segmentation errors.Preprocessing and data analysis were performed using SPM5 implemented in Matlab, except in the segmentation procedure (see below), where SPM2 was used. This approach was used because the T1-weighted images into GM, WM, and CSF. Then, using the segmented GM and WM images as masks, we removed the parts that did not belong to the GM and WM images from the T1-weighted image and created images that solely consisted of GM and WM (designated “GM + WM T1-weighted image”).We segmented the R1 maps from the pre- and post-MRI scans of each subject, which lack the skull and skin section of the head and retain their alignment with the rCBF maps of each subject, were coregistered to the GM + WM T1-weighted image from the pre-MRI scan of each subject using the within-subject registration method. Asymmetry-induced bias was avoided during preprocessing of ASL images using the third structural image for the registration of pre- and post-ASL images .T1-weighted structural image from the pre-MRI scan of each subject, which maintained its alignment with the GM + WM T1-weighted image from the pre-MRI scan and rCBF maps from the pre- and post-MRI scans, was then normalized to our original template of the T1-weighted structural image which was established in our previous study on images of young adults taken with our scanner [The raw  scanner .3 voxels. The processed normalized rCBF maps from the pre-and post-MRI scans were then spatially smoothed using a Gaussian kernel of 12 mm FWHM. Finally, the signal change in resting rCBF between the pre- and post-scan images was computed at each voxel by subtracting the former image from the latter for each subject. The maps representing resting rCBF from the pre-MRI scan and those representing changes in resting rCBF from the pre-MRI scan to the post-MRI scan were subjected to the group-level analysis (see below). The total CBF in the whole brain may change through these processes, as was the case with when total signals in BOLD signal images and FA images change through normalization procedures [Using the parameters for this normalizing procedure, rCBF maps from the pre- and post-MRI scans of each subject were spatially normalized to create images with 2 × 2 × 2 mmocedures , 57, it In the group-level imaging analysis, we tested for group-wise differences in changes in resting rCBF. We performed voxel-wise ANCOVAs using the differences in each measure between the pre- and post-MRI scan values at each voxel as dependent variables and the pre-MRI scan values at each voxel as independent variables. These voxel-wise ANCOVAs were performed using Biological Parametrical Mapping (BPM) , which iP < 0.05, after correction for multiple comparisons (FWE) at cluster size with a voxel-level cluster-determining threshold of P < 0.0025, uncorrected, were considered statistically significant in this analysis.Regions of significance for the ASL analysis were inferred using cluster-level statistics of the standard SPM method . Only clWe next investigated whether there was an association between changes in performance on training tasks and neural changes where the effects of FSNC training were observed through simple regression analyses. Individual performance change was calculated as follows. First, each task's best performance  of blocks in which participants answered correctly in more than half of the trials; in the paper-and-pencil tasks: the largest number of items completed in a single trial) of the first training day and that of the last training day were calculated across all six training tasks. Then, the ratio of change in these performances was calculated. Finally, the mean ratio of the three computerized tasks was averaged and that of three paper-and-pencil tasks was averaged and evaluated as the degree of the performance increase during the course of training. Next, we extracted the mean value of the pre- to posttraining changes in rGMV or resting rCBF in each of the significant clusters identified above. Then, simple regression analyses were performed to determine the association between the improvements in performance (either computerized tasks or paper-and-pencil tasks) on FSNC training tasks, and the neural changes of each cluster were calculated as described above. We employed one-tailed analyses to investigate the association between the increases in performance on FSNC training tasks and mean changes in each cluster in directions in which FSNC training effects were seen because that was our sole hypothesis and interest. In other words, when FSNC training resulted in a decrease in neural values, the associations between individual task performance increase and mean decrease in neural values for the cluster were investigated and vice versa.t-test, P < 0.001 for all six training tasks; Practice resulted in a significant increase in performance across all six training tasks  of blocks, in which participants answered correctly in more than half of trials in the day, was decreased; in the paper-and-pencil tasks: the number of items completed in a single trial was increased) from the first to the last day of training , a complex arithmetic (multiplication) task , a processing speed measure , and an executive function task  task , and the results of a complex arithmetic (multiplication) task and an executive function task (reverse Stroop task) still showed a nearly significant tendency .We reported results with significant values that were uncorrected for multiple comparisons for the reasons described in Methods. However, even when correction for multiple comparisons was performed using FDR, the results of a simple arithmetic (multiplication) task and a processing speed measure (Color-Word task) remained significant . Moreover, in this second behavioral analysis, the FSNC training group showed tendency of pre- to posttest increases in performance on the Stroop task . No other changes were observed in the significance of the other tests (RAPM and a creativity measure) used in our previous study [P values for the comparisons between the FSNC training group and the two control groups in the cases where data from the two control groups were available, in addition to the P values for the comparisons between the FSNC training group and one control group in the present experiment in the cases where data from the previous study was not available, three of the significant uncorrected results (uncorrected) in this subsection  remained significant even after the correction of multiple comparisons  and the result of the complex arithmetic task was also close to significance . The results of this comparison further support the significance of the conclusions drawn in this study.In the present analysis, comparison of the combined control group (the data from the no-intervention group in the experiment of the present study and the active control group from the previous study) and the FSNC training group showed that all significant results in the first behavioral analysis remained significant . This analysis revealed that FSNC training resulted in a statistically significant greater decrease in rGMV around the right frontopolar area .Furthermore, whole-brain ANOVA showed no significant regional differences in rGMV between the training and control groups before the intervention  to determine whether any weak but widely significant results could be observed. This analysis revealed a statistically significant FSNC training-related (training group versus control group) increase in resting rCBF in (a) a large cluster that included the precuneus, left postcentral gyrus, right middle temporal gyrus, left inferior parietal lobule, right superior occipital lobe, paracentral lobule, posterior cingulate cortex, right parahippocampal gyrus, and left superior parietal lobule , it is difficult to conclude something regarding the overlap of the cluster of rGMV and cluster of ASL in the frontal area. For statistical values, see We next compared changes in resting rCBF in the training and control groups. However, no region showed statistically significant changes at a threshold of  reasons . Thus, wl lobule  and (b) l lobule . No regi∗2 = 4 analyses). The results only revealed the tendency of the negative correlation between rGMV change in the cluster of the right OFC and computerized FSNC tasks' performance change . The result may suggest an association between an rGMV decrease on the right OFC and an increase in FSNC training tasks performance; however, due to the nonsignificant tendency of the results, we cannot draw conclusions.Simple regression analyses that tested correlations between improvements in the performance of computerized FSNC training tasks and paper-and-pencil FSNC training tasks and the amount of rGMV and resting rCBF changes in the significant clusters identified in this study's analyses (see above) were also performed  . The forDecreases in rGMV observed after just 1 week of intensive (such as four hours per day) cognitive training are consistent with those observed in two of our previous studies of cognitive training , 22. DecFSNC training may increase resting rCBF in the frontopolar and posterior regions through changes in the capillary network and increases in metabolic demand. The posterior parietal, posterior temporal, and occipital regions are recruited during simple numerical calculation . In partConsistent with our hypothesis, FSNC training led to improved performance on executive function (reverse Stroop task), processing speed, and simple and complex arithmetic tasks. Our previous study showed that a brain training game including FSNC improved executive function and processing speed . This stThis study has the same limitations as our previous studies of cognitive training , 19, 22.The executive function and frontopolar areas affected by FSNC training play important roles in higher order cognitive functioning in humans , 75. ThuSupplementary materials contain the table which described the data of the active control group in our previous study , the figure which showed the schema of the stroop tasks that we used , and the figure which showed the areas of regional cerebral blood flow during rest .


Cannabis is the most frequently used illicit substance in the United States resulting in high rates of cannabis use disorders. Current treatments for cannabis use are often met with high rates of lapse/relapse, tied to (1) behavioral health factors that impact cannabis use such as poor sleep, and (2) access, stigma, supply, and cost of receiving a substance use intervention.This pilot study examined the feasibility, usability, and changes in cannabis use and sleep difficulties following mobile phone–delivered Cognitive Behavioral Therapy for Insomnia (CBT-I) in the context of a cannabis cessation attempt.Four male veterans with DSM-5 cannabis use disorder and sleep problems were randomized to receive a 2-week intervention: CBT-I Coach mobile app (n=2) or a placebo control (mood-tracking app) (n=2). Cannabis and sleep measures were assessed pre- and post-treatment. Participants also reported use and helpfulness of each app. Changes in sleep and cannabis use were evaluated for each participant individually.Both participants receiving CBT-I used the app daily over 2 weeks and found the app user-friendly, helpful, and would use it in the future. In addition, they reported decreased cannabis use and improved sleep efficiency; one also reported increased sleep quality. In contrast, one participant in the control group dropped out of the study, and the other used the app minimally and reported increased sleep quality but also increased cannabis use. The mood app was rated as not helpful, and there was low likelihood of future participation.This pilot study examined the feasibility and initial patient acceptance of mobile phone delivery of CBT-I for cannabis dependence. Positive ratings of the app and preliminary reports of reductions in cannabis use and improvements in sleep are both encouraging and support additional evaluation of this intervention. Cannabis is the most frequently used illicit substance in the United States , with raIn light of the relatively poor outcomes associated with standard cannabis use disorder treatments, there has been increasing interest in identifying malleable factors associated with heightened relapse risk among individuals dependent on cannabis, specifically for the purposes of intervention development and refinement -12. IndeFrom these findings, the evaluation of existing sleep treatments as clinical tools to aid in the treatment of cannabis use disorders has been proposed, with the aim of improving sleep and reducing individual “self-medication” with cannabis . InitialCognitive Behavioral Therapy for Insomnia (CBT-I) is the leading behavioral treatment for sleep disturbances ,21. AlthGiven identified barriers to substance use disorder care that include access, stigma, supply, and cost -27, a moThis pilot study aimed to begin addressing these gaps in the literature by determining the feasibility of providing CBT-I to individuals with a cannabis use disorder engaged in a cessation attempt through a mobile phone app. CBT-I was administered via mobile app instead of in-person sessions as this would allow for greater reach of the intervention  and consistent fidelity of the intervention. For the purposes of this feasibility study, we sought to examine mobile app engagement, user-friendliness, and use and future interest in use, in order to provide initial data regarding the feasibility and usability of this technology among veterans with a cannabis use disorder. As a secondary aim, we examined preliminary trends in the impact of app use on cannabis and sleep outcomes. Specifically, we hypothesized that receiving CBT-I through an app, within the context of a self-guided cannabis cessation attempt, would be acceptable to study participants and utilization of the app would correspond with a reduction in cannabis use and an improvement in sleep quality.In terms of recruitment, 40 individuals contacted the lab expressing interest in the study. Individuals leaving a message were contacted up to four times for the purpose of conducting an initial phone screening. Of the original 40 individuals, 28 veterans who contacted the lab were screened over the phone. Of these, 12 individuals were screened out because they did not report current cannabis use, 6 were not veterans, 5 reported no interest in quitting, and 1 individual was eligible but refused to participate. This resulted in a total sample of 4 individuals.Study participants (N=4) were adult veterans (18 and over) who reported (1) current sleep problems, defined as a total score of 5 or greater on the Pittsburgh Sleep Quality Index (PSQI) , (2) a cThe Structured Clinical Interview-Non-Patient Version (SCID I-N/P) for DSM-IV  was admiA Mobile App Use Measure (MAUM) was developed to gather information about participant use of both the experimental  and control (mood-tracking) apps between baseline and 2-week follow-up, as neither app recorded use data. The MAUM prompted participants to report on (1) frequency of app use, (2) length of use per occasion, (3) components of the app that were used most often, (4) user-friendliness of the app, (5) helpfulness of the app, (6) likelihood of future app use, (7) use of the app for sleep improvement, and (8) use of the app for cannabis cessation. The MAUM was administered at 2-week follow-up.Sleep quality at baseline and 2-week follow-up was assessed with the 19-item Pittsburgh Sleep Quality Index (PSQI) . Partici-Back Interview (TLFB) [The Timeline Followw (TLFB)  was admiw (TLFB) ), with tw (TLFB) ,38. Due w (TLFB)  was usedCognitive behavioral therapy for insomnia (CBT-I) was administered via the CBT-I Coach mobile app for iOS . The conA self-monitoring mobile app  was provParticipants were recruited from a VA hospital in the San Francisco Bay Area via flyers placed throughout the hospital campus and by referrals from clinical staff. Recruitment materials were designed to target veterans interested in making a cannabis cessation attempt. Interested individuals contacted the research lab and completed a brief phone screen to assess for initial eligibility. Those meeting initial eligibility criteria were scheduled for a baseline appointment 1 day prior to the date that they would be willing to make an independent, self-guided, cannabis cessation attempt.On arrival for the baseline appointment, individuals provided written informed consent. Individuals consented to participate in a cannabis cessation attempt and to engage with a behavioral program via mobile app. The inclusion of a sleep intervention was not discussed in recruitment or consenting procedures. Following informed consent, participants were interviewed with the SCID I-N/P and then completed a battery of self-report measures. Following the completion of the baseline assessment, participants were randomly assigned, by coin flip, to receive either the CBT-I  or mood-tracking app (control condition). Following randomization, participants were then loaned an iPod touch with charger, trained on how to use their assigned mobile app , and instructed to (1) make a cannabis cessation attempt beginning the following morning, and (2) use their assigned mobile app during the first 2 weeks of their cessation attempt. At the end of the baseline assessment, contact information was obtained for each participant to allow for reminder calls regarding app use and follow-up appointment date. Participants were then scheduled for their 2-week follow-up and compensated US $45 for completion of the baseline appointment.Two weeks following baseline, participants returned to the research laboratory to complete a self-report assessment battery that was similar to the baseline assessment, with the exception of the SCID I-N/P and addition of the MAUM. Following the 2-week follow-up assessment, participants were instructed to relinquish their iPod touch devices and were compensated US $45. All study procedures were approved by the VA Palo Alto and Stanford Institutional Review Boards.Based on the small sample size of this pilot study, study outcomes  were examined qualitatively for each participant separately.Information regarding baseline and follow-up scores on study variables can be found in Participant 1 was a 42-year-old single Caucasian male veteran who met DSM-5 criteria for current cannabis use disorder and alcohol use disorder. Participant 1 was a non-smoker and did not meet criteria for any mood or anxiety disorders. He was a novice with mobile technology and did not own a smartphone. As noted in the Method section, the MSHQ was administered instead of the TLFB for this participant. Based on the number of reported days using cannabis during the prior month, coupled with the quantity reported, we were able to extrapolate this participant’s cannabis use during the 30 days prior to baseline see . From thParticipant 1 was randomly assigned to receive CBT-I coach. He reported using the mobile app daily over the 2-week course of his cessation attempt. On average, Participant 1 used the app for 5 minutes at a time and typically interacted with the app in the morning. He reported the most helpful aspect of the app as allowing him to track and view his sleep over the 2-week period. He reported the app as user-friendly and indicated interest in using it in the future. Participant 1 also reported that the app helped his sleep because it provided daily accountability to the intervention components. He also noted that it helped with the cessation attempt because it made him more aware of when and how much cannabis he was using. At 2-week follow-up, Participant 1 had self-reported no cannabis use during the 2-week study period. His total PSQI score did not change from baseline ; however, his sleep efficiency increased from 58% to 80%, bringing it to just below the normal range (85%). At follow-up he reported using medication (prescription or non-prescription) less than once a week for sleep.Participant 2 was a 54-year-old widowed black/non-Hispanic male veteran who met DSM-5 criteria for current cannabis use disorder and alcohol use disorder. He did not meet criteria for any mood or anxiety disorders. Participant 2 was a tobacco smoker, consuming an average of 4 cigarettes per day. His mean cannabis use over the 90 days prior to the baseline appointment was 0.71  and hisParticipant 2 was randomly assigned to the control condition (mood-tracking app). This participant dropped out of the study , thus follow-up data are not available.Participant 3 was a 65-year-old married black/Hispanic male veteran who met DSM-5 criteria for cannabis use disorder. He did not meet criteria for any mood or anxiety disorders. Participant 3 was also a novice with mobile apps and did not own a smartphone. His mean cannabis use over the 90 days prior to the baseline appointment was 0.18  over the 2-week cessation period. Participant 3 reported the app as extremely user-friendly, though he indicated that he would not use it in the future and felt that it did not help at all with his cannabis cessation attempt or sleep. On follow-up, Participant 3 reported an increase in cannabis use , as comParticipant 4 was a 27-year-old divorced Caucasian male veteran of recent conflicts in Iraq and Afghanistan. SCID I-N/P assessment indicated that he met criteria for current PTSD and cannabis use disorder; he was a tobacco non-smoker. Participant 4 had the most experience (of the study participants) with mobile technology, and owned an Android smartphone. He reported the highest amount of cannabis use in the 90 days prior to baseline relative to other participants, with a mean use of 4.51  and staff costs.Overall, our sample consisted of novices in terms of mobile technology and mobile app use. Though Participant 4 had experience with using mobile apps, all other participants reported not owning a smartphone or having previous experience with mobile apps. However, all were able to use the apps after a brief explanation. The responses and engagement with the mobile apps was found to vary by treatment condition. Participant 3 (control) reported minimal use of the app (scoring a 1 out of a possible 4). Follow-up assessment of app use found that this individual did not find the mood tracking app helpful for sleep (reporting 0 out of 4) or cannabis cessation (reporting 0 out of 4) and therefore reported little interest in engaging with the app and low likelihood of using it in the future (reporting 0 out of 4). However, for those who received the CBT-I app (Participants 1 and 4), daily use was reported over the 2-week study period, with an average of 5-10 minutes spent with the app per use session.Participants assigned to the CBT-I app reported that the most helpful portions of the app were the sleep logs and the exercises for improving sleep. Both reported the app to be very user-friendly (scoring 3 out of 4), found that it was very helpful in improving sleep (scoring 3 out of 4), and reported strong interest in using an intervention app in the future (scoring 3 out of 4). All loaned iPod touches were returned to the study team after completion of the study. Although Participant 2 did not complete the 2-week follow-up, he did return to the laboratory to return his iPod touch.This pilot study served as a feasibility test of mobile-app delivered CBT-I (and mood-tracking control) for both cannabis and sleep outcomes among individuals engaged in a cannabis cessation attempt. This study indicates that individuals who received the CBT-I app reported daily engagement with the app, while individuals in the control condition either dropped out of the study or reported minimal use. Importantly, regardless of condition, this study provided support for loaning out iOS devices for the purposes of treatment, as all devices were returned following study completion.Findings support the initial feasibility of providing CBT-I via mobile app to improve outcomes among individuals engaged in a cannabis cessation attempt. Coupled with theoretical and empirical work that has highlighted the pivotal role of sleep in the maintenance and treatment of cannabis use disorders , includiAlthough our investigation served as a novel extension of prior empirical and theoretical work, it was not without limitations. First, the small number of participants, all of whom were male, enrolled in this preliminary study limits inference regarding generalizability of findings. Future work would benefit from the replication and extension of this pilot study to a larger and more gender diverse sample. Future work would benefit from focusing recruitment efforts to target female participants. Second, while we screened 40 individuals, due to our inclusion criteria, only four were enrolled. This may have biased the randomness of our sample. Third, given the scope of the study, follow-up data were collected only 2-weeks post-baseline. As the impact of cannabis cessation on sleep can last for over 30-days post-cessation , future This study provides initial evidence for the feasibility of CBT-I for cannabis dependence delivered through a mobile phone app. Findings support the use of technology to enable easy, affordable access to treatment for substance use disorders -45, and


The HgCl2-treated 5-week-old group exhibited worse emotional memory performance in PA, worse spatial learning and memory performances in MWM. There were no significant differences between the groups of 5-month-old rats in LA, MWM or PA. However, the DPOAE tests worsened in the mid- and high-frequency hearing thresholds. The HgCl2-treated 5-month-old group exhibited the most hearing loss of all groups. Our results convey that mercury exposure in young rats may worsen learning and memory performances as well as hearing at high-frequency levels. While there was no statistically significant difference in the behavior and learning tests in adult rats, the DPOAE test produced poorer results. Early detection of effects of mercury exposure provides medicals team with an opportunity to determinate treatment regimens and mitigate ototoxicity. DPOAE test can be used in clinical and experimental research investigating heavy metal ototoxicity. We investigated the effects of inorganic mercury exposure during gestational/lactational periods on the behaviour, learning and hearing functions in a total of 32, 5-week-old and 5-month-old WAG/Rij rats . We evaluated the rats in terms of locomotor activity (LA), the Morris-water-maze (MWM) test and the passive avoidance (PA) test to quantify learning and memory performance; we used distortion product otoacoustic emission (DPOAE) tests to evaluate hearing ability. There were no significant differences between the 5-week-old rat groups in LA, and we detected a significant difference (p < 0.05) in the HgCl Until WAG/Rij rats are preferred as an animal model for absence epilepsy in humans and they have many EEG and behavioural characteristics that are common with or similar to human absence epilepsy. The WAG/Rij strain of rats has been used for many studies of genetic epilepsy, colorectal cancer, and learning and memory performance  of the offspring based on behavioural and DPOAE tests for possible cognitive dysfunctions, spatial learning, emotional memory performances and hearing loss when they reached 5 weeks and 5 months of age to observe age-dependency. We designed this experiment so that female rats were continuously given HgClWe obtained male and female WAG/Rij strained rats from Kocaeli University, Experimental Medical Research and Application Center . During the treatment, the rats had unlimited access to standard rat chow and tap water and they were kept at a density of two to three animals per cage at constant humidity, light cycle (12:12 h light-dark), and room temperature. Ethics approval was granted by the Kocaeli University Animal Research Ethics Committee (2014-11) . The female rats were mated individually with the male rats. Mating was verified via observation of spermatozoa in a vaginal smear, and this time was designated as Day 0 of pregnancy. Exposure was begun at pregnancy Day 0. The females were then placed in separate polycarbonate cages. The mothers received injections during their gestational and lactational periods.Totally 32 offspring rats were equally divided into 4 groups as; 5 week-old control group, 5 week-old mercury group, 5 month-old control group, 5 month-old mercury group. Mean weights of the 5 week-old rat and 5 month old rats were 63.80 ± 1.3 g, 69.30 ± 2.6 g; 235.2 ± 6.8 g, 238.4 ± 12.3 g respectively.st dose, 0.07 µg/kg/day as subsequent dose im, to cover daily loss). We made the injections between 9:00 am and 10:00 am, and doses were selected according to previous studies  Chloride was purchased from Sigma-Aldrich M1136 (USA). The rats were divided into two groups: control  and treated with mercury chloride during gestational and lactational periods .The dams were checked every morning for the presence of newborn offspring; when the pups were first observed was considered to be Postnatal Day 0 (PND 0). The pups remained with their mothers until they were weaned and given drinking water (PND 30). A computerized animal activity monitoring system  that is tracked and observed via an open field activity software package and comprises a Plexiglas chamber was used to assess locomotor activity (LA). The Plexiglas chamber (40 x 40 x 35 cm) had pairs of infrared photo beams and detectors assembled vertically every 4.5 cm and horizontally every 2.5 cm. The software program was used to detect and record the interruptions of the photocell beams. The total LA was expressed in terms of the sum of stereotypic, ambulatory, and vertical activities. We monitored the activity for 5 min continuously following acclimatisation to the test room illuminated for a period of one hour with two 36-Watt overhead fluorescent tubes with a light intensity of 18-45 lx on the cage floors and 190-220 lx in the centre of the room .Animals learn to refrain from a formerly accustomed behaviour, namely, passing from the light chamber to the dark chamber, in the passive avoidance (PA) test . The raOn the first day , we placed the rats in the light compartment individually and allowed them to explore this light compartment. After 10 sec, the system automatically opened the door between the two compartments. At this stage, the animal was able to pass into the dark compartment freely.We conducted the acquisition trial 15 min following the pre-acquisition trial. We placed the rats in the light compartment. The system automatically opened the door dividing the two compartments after waiting for a 30-second-long adaptation period. The door automatically closed when a rat completely entered the dark compartment (four paws in) and for a period of 3 sec, an electric shock of 0.5 mA was applied on the rat's feet via the grid floor. We then took the animals from the dark compartment and placed them in their home cages. The system recorded the time that the animals took to enter the dark compartment. We discarded all the animals that failed to cross from the light compartment to the dark one within 300 sec from the experiment. We cleaned both compartments between each training session to eliminate olfactory cues.24 h after training, we returned animals to the light compartment and recorded their latency to enter the dark compartment (four paws in) to evaluate memory. We did not apply any footshocs in this trial. When a rat did not make its way entirely into the dark compartment within 300 sec, we returned it to its cage and recorded a maximum latency of 300 sec for that rat. The latency to enter the dark compartment was used as a criterion for retention performance of step-through avoidance behaviors.We used a circular pool (150 cm in diameter) that was filled with water at 25 °C as the water maze. To make the platform invisible, we placed small white pieces of plastic in the pool . The poThe maze was separated into four quadrants, and three equally spaced points around the edge of the pool were utilized as starting positions. We varied the order of the release positions throughout the experiment. During the acquisition sessions, we placed an escape platform (10 cm in diameter) in one of the quadrants 1 cm below the water surface. For five daily sessions , we trained the rats in the Morris water maze (MWM). We conducted all the five consecutive daily sessions between 9:00 am and 12:00 pm.For each acquisition trial, we placed a rat in the pool from one of the randomly chosen three locations with its head facing toward the wall of the pool. The initiation of a trial was marked with the release of a rat into the pool. When a rat found the platform and climbed onto it, we terminated the trial and recorded the mean escape latency. The trial length was limited to a maximum of 60 sec. When a rat failed to climb onto the platform within 60 sec, we terminated the trial and the rat was guided to the platform by the experimenter using their hand.The inter-trial interval was 30 sec. We left the rat on the escape platform before starting the next trial during the inter-trial interval. We placed the rat in the pool anew from a different location after the inter-trial interval, and upon its release, the next trial began. We returned the rat into its cage at the end of each session. We observed that as the animal learned the location of the hidden platform, escape latency typically declined during acquisition. In these cases, we recorded an escape latency of 60 sec.We employed a 'probe trial' twenty-four hours following the final acquisition session to determine the rats' recollection of the location of the hidden platform. During this trial, we removed the platform from the maze and allowed each rat to search the pool for 60 sec. Within this period, we anticipated that the animals were to spend comparatively more time looking for the quadrant that previously contained the hidden platform compared to they would for the other three quadrants.The DPOAE test is a hearing assessment method that evaluates the outer hair cell function of the cochlea. With this technique, a wide frequency range of the cochlea can be evaluated by providing two different sound stimuli termed F1 and F2 to the external ear channel to obtain a frequency-specific response from the cochlea. High frequencies are more likely to suffer from ototoxicity because most ototoxic changes first affected the basal turn, or high-frequency region, of the cochlea before progressing to lower-frequency regions. In the current study, we studied frequencies between 4 and 10 kHz. 1 - F2) were performed in the General Diagnostic mode, and the F1/F2 frequency ratio was set to be 1.22 to obtain the most powerful responses. We performed the measurements at 4004, 4502, 5000, 5596, 6299, 7099, 7993, 9004 and 10000 Hz F2frequencies at 6 points per octave.The animals were anaesthetised via an intraperitoneal injection of xylazine  5 mg/kg and ketamine  50 mg/kg prior to the DPOAE measurements. We determined the depth of anaesthesia with the pedal reflex and to maintain anaesthesia and we administered a half dose of this initial cocktail as required. After an otoscopic examination to rule out possible middle-ear pathologies, we recorded DPOAE using the smallest probe in a silent room. We used a standard commercial MI 34 OAE apparatus cochlear emission analyser  to elicit the DPOAEs from each ear of the experimental animals. The cubic difference distortion products . Moreover, the HgCl2-exposed 5-month-old rats exhibited the least total activity of all of the groups. We compared to a same age-matched control group as an HgCl2-exposed group and a control group.The LA results of all of the experimental groups are listed in Table 12-exposed 5-week-old rats, the HgCl2-exposed 5-month-old rats, the 5-week-old rats and the 5-month-old WAG/Rij rats . Latency to enter the dark compartment was decreased in both the HgCl2-exposed groups (5-week-old and 5-month-old) compared with their own control groups. Latency to enter the dark compartment was recorded for the HgCl2-exposed rats exhibited a larger decrease than their own control groups  and 5-month-old (p > 0.05) groups. The HgCl Figure 1.2-exposed groups compared with their own control groups . The time spent in the right quadrant in both the 5-week-old (p < 0.05) and 5-month-old (p > 0.05) HgCl2-exposed rats was more decreased than these groups' own control groups  and as 2-exposed group exhibited no different emotional and spatial memory performances on either the PA or MWM tests compared with their age-matched controls at 5 months. Day et al.  propose2 induce behavioural, cognitive impairment and hearing-threshold deficits in 5-week-old rats and progressive hearing thresholds deficits in 5-month-old rats.One of the inorganic forms of mercury is mercury chloride. While mercury is most commonly seen in inorganic form , the math gestational day in rats had negative impact on the PA task when the animals were 8 weeks old, which shows that exposure during a crucial period of development of the CNS to methyl mercury leads to learning and memory deficits. Exposure to mercury in different forms may provoke diverse impacts on the CNS. In this sense, our current findings suggest that chronic HgCl2-treated animals exhibited worse emotional and spatial memory performances on the PA and MWM tests compared with their age-matched controls at 5 weeks. However, we found no significant alterations in the LA test. Non-significant LA results do not imply that mercury does not have detrimental and toxic effects. Brookes and Kristt  showed stt (1989) demonstal. 2011) detecte11 detectal.  or in r2 at 0.025-25 μM) not only caused neuronal degeneration but also perturbed neuronal excitability. Basu et al.  found t012 foundal. (2007).AMPA receptor (AMPAR) membrane trafficking regulation is critical with regards to synaptic plasticity as is the case for learning and memory . The acFurthermore, gradually decreased levels of mercury and the ameliorating neuronal development of young rats after the end of inorganic mercury exposure might contribute to normalising cognitive and spatial functions in 5-month-old rats because the behavioural test results of 5-month-old rats were not statistically significant.On the other hand, our results showed that there are seriously progressive effects of gestational and lactational inorganic mercury exposure. Beyond all of the behavioural tests we conducted, these seriously progressive effects may be disclosed by the DPOAE test.The ototoxic effect of mercury has been studied previously using objective and subjective test methods; the cochleotoxic effect of mercury is a less-studied topic. We determined the cochleotoxicity using DPOAE tests. Otoacoustic emissions can be suppressed in part centrally through the superior olivary complex. Lateral and medial olivocochlear bundles' axons project from the superior olive and leave the brainstem as a ventral component to the inferior vestibular nerve. As the Oort's vestibulocochlear anastomosis, they join the cochlear nerve. Axons of the lateral olivocochlear bundle synapses with afferent neurons from the cochlea. Axons of the medial olivocochlear bundle terminate at the base of cell bodies of the outer hair cells. It is generally believed that the medial efferents counteract the amplifying effects of the outer hair cells. This effect is probably mediated by acetylcholine .+/K+-ATPase activities of the brain regions and cochlear lateral wall .Igarashi et al. (1992) carried2 group exhibited poorer test results for all frequencies compared with the control group. The differences were statistically significant at higher frequencies. These results imply that exposure to inorganic mercury during gestational and lactation periods results in ototoxicity; this toxicity can be monitored using the DPOAE test, which is an easy and objective test method. Additionally, in the 5-month-old rats, both groups exhibited age-related changes. However, our results demonstrated that the HgCl2-exposed group was more affected by hearing thresholds than the control group. Moreover, the largest hearing loss was recorded in the 5-month-old HgCl2 group. This result can be interpreted as age-related hearing loss being more deteriorated by HgCl2 exposure. Our results indicate that the DPOAE test is more sensitive than the other tests used in this study to reveal inorganic mercury toxicity, particularly for late effects in aged rats.In terms of the results of the 5-week-old rat groups, we noted that the HgCl+/K+-ATPase activities and NOx levels of the brain regions and the cochlear lateral wall. This explanation may be the most appropriate to interpret our results. According to Huang et al. (2011), absorpAdditional studies are necessary to better understand the exact pathophysiological mechanism of long-term mercury exposure and its neurotoxicity. For this purpose, cochlear dissection and brain slices of rats must be obtained and analyzed using immunohistochemistry techniques. Moreover, given the urbanization and industrialization of today's rapidly growing world, it is important to raise public awareness about mercury and limit exposure to mercury as a public health priority.We have shown that chronic exposure to low-dose mercury chloride impairs the performance of different memory types and hearing in WAG/Rij rats that were exposed to mercury chloride during their gestational and lactational periods. Although age-dependent effects of mercury chloride appear to be reversible or tolerable by the behavioural tests, DPOAE test results revealed that there were seriously progressive deleterious effects of mercury chloride in rats exposed to mercury. Medical teams may find a chance to identify treatment methods and alleviate hearing changes with the early diagnosis the effects of mercury exposure. In this sense, in experimental and clinical studies that research hearing loss and heavy metal ototoxicity may benefit from DPOAE tests. However, we do not know the exact mechanisms involved in these effects; additional research is accordingly necessary. However, our results are important because information about age-dependent mercury chloride exposure with long-term outcomes on both animal models and humans is very limited in the literature.We thank Si-Ser Hearing Centers for providing us hearing threshold assessment equipment for DPOAE tests.The authors declare that there are no conflicts of interest.