Maternal behavior and anxiety are potently modulated by the brain corticotropin-releasing factor (CRF) system postpartum. Downregulation of CRF in limbic brain regions is essential for appropriate maternal behavior and an adaptive anxiety response. Here, we focus our attention on arguably the most important brain region for maternal behavior, the hypothalamic medial preoptic area (MPOA).Crhr1) was more abundantly expressed than for subtype 2 , however expression of Crhr1, Crhr2 and CRF-binding protein  mRNA was similar between virgin and lactating rats. Subtype-specific activation of CRFR, predominantly CRFR1, in the MPOA decreased arched back nursing and total nursing under non-stress conditions. Following acute stressor exposure, only CRFR1 inhibition rescued the stress-induced reduction in arched back nursing while CRFR1 activation prolonged the decline in nursing. Furthermore, inhibition of CRFR1 strongly increased maternal aggression in the maternal defense test. CRFR1 activation had anxiogenic actions and reduced locomotion on the elevated plus-maze, however neither CRFR1 nor R2 manipulation affected maternal motivation. In addition, activation of CRFR1, either centrally or locally in the MPOA, increased local oxytocin release. Finally, inhibition of CRFBP (a potent regulator of CRFR activity) in the MPOA did not affect any of the maternal parameters investigated.Within the MPOA, mRNA for CRF receptor subtype 1 (protein: CRFR1, gene: In conclusion, activity of CRFR in the MPOA, particularly of subtype 1, needs to be dampened during lactation to ensure appropriate maternal behavior. Furthermore, oxytocin release in the MPOA may provide a regulatory mechanism to counteract the negative impact of CRFR activation on maternal behavior. •Activation of MPOA CRFR, predominantly CRFR1, impaired maternal care.•Inhibition of MPOA CRFR1 strongly increased maternal aggression.•Activation of MPOA CRFR1 triggered local oxytocin release.•CRF-binding protein in the MPOA did not regulate maternal behavior.•Dampened CRFR1 activity in MPOA is vital to ensure appropriate maternal behavior. While oxytocin and vasopressin typically promote maternal behavior, and are hence up-regulated postpartum e.g. , the corCrh) and the urocortins 1–3, which bind with different affinities to the two CRF receptors : CRFR1 and CRFR2 (Crhbp)  . CRF is  (Crhbp) . It is r (Crhbp) , b, whicp . CRF iDuring the postpartum period, activation of central CRFR strongly impairs maternal care and maternal aggression thereby inducing maternal neglect in lactating rats , 2016a, The BNST and the adjacent medial preoptic area (MPOA), together form the maternal ‘super-region’ for regulating maternal behavior . Given tIn the present study, we hypothesized that like the BNST, the MPOA CRF system is also involved in the regulation of maternal behavior. Hence, we assessed the expression profiles of both CRFR and CRFBP in the MPOA and examined the effects of modulating CRFR or CRFBP in the MPOA under stress and non-stress conditions. Furthermore, as one of the main mediators of maternal behavior in the MPOA is the oxytocin system , we hypo22.1ad libitum. All rats were initially housed in groups of 3–4 (for further details see below). Female rats were mated with sexually experienced male rats and pregnancy was confirmed by the presence of sperm in vaginal smears (pregnancy day (PD) 1). A separate, naïve cohort of rats was used for each experiment, and in each case rats were randomly assigned to the different treatment groups.Virgin female Sprague-Dawley rats  were housed under standard laboratory conditions  with access to water and standard rat chow For experiment 1, virgin and lactating females were treated similarly; virgins were single-housed 7 days prior to brain collection, consistent with the single-housing period of the lactating rats, which were single-housed on PD 18 and killed 7 days later (lactation day (LD) 4). In experiments 2 and 5, females underwent surgery on PD 18 and were subsequently single-housed to guarantee recovery and undisturbed parturition . In expeFor the maternal defense test, naïve virgin female Wistar rats  were used as intruders at random stages of their estrous cycle. Intruder rats were kept group-housed in a separate room to avoid olfactory recognition .The experiments were approved by the Committee on Animal Health and Care of the local government and conformed with the European Directive (2010/63/EU) on the ethical use of animals. All reasonable efforts were made to minimize the number of rats used and their suffering.2.2All tests were performed between 8 a.m. and 3 p.m. during the light phase of the cycle. In experiments with repeated drug infusion (experiments 2 and 5), rats received the same drug throughout the experiment. After infusion, dams were immediately returned to their home cage.2.2.1Maternal care was monitored before and after drug infusion under non-stress and stress conditions  , b. Obse2.2.2The dams' maternal motivation was tested in the modified pup retrieval test (PRT) as described previously . Briefly2.2.3http://www.jwatcher.ucla.edu/). The following behavioral parameters were scored: total number of attacks, latency to first attack, lateral threat, keep down, and offensive upright as well as non-aggressive behaviors  was confronted with an unknown virgin female intruder in her home cage for 10 min as described previously . The damtion see ).2.2.4Anxiety-related behavior was tested on the elevated plus-maze (EPM) as previously described . Briefly2.32.3.1Crhr1, Crhr2, and Crhbp mRNA expression in the MPOA, 29 virgin and lactating rats were killed in the morning of LD 4 (Crhr mRNA), LD 7 (Crhbp mRNA) or equivalent in virgin rats, i.e. after 7 days of single-housing under basal conditions by conscious decapitation as anesthesia can affect central peptide content as well as activate the HPA axis , mounted on polysine slides, and stored at −20 °C until further processing.To compare the effect of reproductive status on HPA axis . The braCrhr1, Crhr2, and Crhbp mRNA in situ hybridization was performed using an established protocol with previously described cRNA probes for Crhr1, Crhr2 (Crhbp (35S-UTP-labeled cRNA sense probes (negative controls) showed no signal above background.1, Crhr2 , 2011, a2 (Crhbp . Autorad2 (Crhbp ) were an2 (Crhbp . Measure2.3.2(6-33)  20 min before testing. Doses and the lag time between the infusion and behavioral testing were chosen based on previous studies  were implanted bilaterally with 23 G guide cannula targeting the MPOA  under i studies .Maternal care was observed in the same rats under non-stress conditions (LD 1) and stress conditions (LD 7) in their home cage in the colony room. Under non-stress conditions, dams were observed from 8 a.m.–9 a.m., followed by treatment infusion and subsequent observation for a further 120 min. In addition, 5 h after the infusion (2 p.m.–3 p.m.), maternal care was monitored again to assess any potential long-lasting effects of the drug treatment. On LD 7, dams were observed from 8 a.m.–9 a.m. in their colony room before being moved to the test room. At 10 a.m., dams were VEH/drug-infused, exposed to stress using the maternal defense test and immediately afterwards returned to the colony room, where maternal care was observed for a further 60 min to assess the effects of the preceding stressor on maternal care. Additionally, maternal motivation (LD 3), anxiety-related behavior (LD 5), and maternal aggression (LD 7) were assessed as described in 2.2.2.3.3On LD 1, 22 females were implanted with a 21 G guide cannula targeting the left lateral ventricle  and a mOn LD 3, the inflow adapter of the microdialysis probe was connected via PE-20 tubing to a syringe mounted onto a microinfusion pump. The outflow adapter was attached to a 1.5 ml collection tube containing 10 μl 0.1 N HCl. The probe was flushed at a rate of 3.3 μl/min with sterile Ringer's solution (pH 7.4) for 90 min before 30 min sample collections commenced. Starting at 10 a.m., samples 1 and 2 were collected under basal conditions. Afterwards, dams were infused ICV with either (i) VEH , (ii) CRFR1 agonist h/rCRF (1 μg/5 μl), or (iii) CRFR2 specific agonist hUcn3 (3 μg/5 μl). Samples 3–5 were collected during the 90-min period following drug injection. Additionally, maternal care was observed throughout the entire sampling period. All microdialysates were immediately frozen on dry ice and stored at −80 °C until quantification of oxytocin (see 2.4).2.3.4Separate groups of lactating rats (n = 14) were implanted unilaterally with a microdialysis probe targeting the right MPOA on LD 1. Microdialysis was performed on LD 3 as described in 2.3.3 for experiment 3, except that the perfusion rate was 1 μl/min .Starting at 10 a.m., samples 1 and 2 were collected under basal conditions 30 min apart. Next, the syringes containing Ringer's solution were swapped for those filled with either (i) VEH  or (ii) CRFR1 agonist h/rCRF (0.05 μg/μl/min) for 30 min (sample 3). Syringes were then switched back to those containing sterile Ringer's solution and samples 4 and 5 were collected 30 min apart. Microdialysate samples were immediately frozen on dry ice and stored at −80 °C until quantification of oxytocin (see 2.4).2.4Oxytocin peptide content was measured in lyophilized dialysates by a highly sensitive and selective radioimmunoassay .2.52 overdose; experiment 3: LD 4, CO2 overdose; experiment 4: LD 3, CO2 overdose), and brains were collected and flash frozen in n-methylbutane for histological verification of correct cannula placement. To identify implantation sites, 0.5 μl ink was infused post-mortem into the brain via the cannula . Brains were then removed, flash frozen, cut in 40 μm coronal sections, and mounted on slides. Brains were checked for potential diffusion outside the MPOA according to an established protocol , or two-way repeated measures (RM) ANOVA (factors: time x treatment) followed by Fisher's LSD post hoc test. For all statistical analyses, the software package SPSS 24.0  was used. Data are presented as group means ± SEM, and p ≤ 0.05 was considered statistically significant.Data were analyzed using either an independent 33.1Crhr1 and Crhr2 mRNA expression in the MPOA did not differ between virgin and lactating rats .Non-maternal behavior: Significant interactions were found for overall off-nest behaviors , locomotion , and self-grooming  showed that ABN decreased after the infusion/maternal defense test in all groups , except the CRFR1 antagonist- (p = 0.7) and CRFR2 agonist-treated dams (p = 0.1) at t 0 min versus basal.Nursing: Nursing significantly changed over time between the groups  and self-grooming .3.2.44,29 = 5.36, p < 0.01; 4,29 = 9.89, p < 0.01; The number of attacks  and treatment , but not treatment (data not shown). CRFR2 agonist-treated dams showed less nursing at t 0 min compared to the previous time point (p = 0.05).Other maternal behavior: LG differed over time between the treatment groups .3.3.2No significant differences were found for ABN, nursing, pup retrieval/mouthing, and LG (data not shown).3.3.3No differences were found in aggressive behaviors between the groups (data not shown).3.3.44,72 = 4.19, p < 0.01) and treatment , maternal motivation, maternal aggression, or anxiety-related behavior (data not shown).4The brain CRF system is a potent regulator of maternal behavior. In brain areas such as the lateral septum  and the Crhr1 mRNA is predominantly expressed in the MPOA compared to Crhr2. However, we did not find a significant difference in expression between virgin and lactating rats suggesting that CRFR1 are not down-regulated postpartum as a potential adaptive mechanism to ensure reduced CRFR1 signal transduction.Here, we advanced our current knowledge of the effects of an active CRF system in limbic brain areas to the hypothalamic MPOA, one of the most important brain regions for maternal behavior . We provUnder non-stress conditions, ABN and total nursing were reduced following infusion of VEH or either CRFR agonist into the MPOA, which is consistent with our previous findings in the BNST suggesting that the infusion might be perceived as a mild stressor , 2016a. Under stress conditions, nursing was decreased while LG was increased in the CRFR1 agonist-treated dams. LG is generally considered a positive and beneficial behavior by the dam directed towards the pups , which iMaternal motivation, as tested using the pup retrieval test, was not affected by CRFR manipulation, similar to findings following central and intra-BNST manipulations of CRFR in lactating rats , 2016a aDuring the maternal defense test, there was a robust increase in maternal aggression only in the dams administered the CRFR1 antagonist into the MPOA, indicating active CRFR1 during the maternal defense test and thus, a prominent role for MPOA CRFR1 in the regulation of maternal aggression. To our knowledge, this is the first time a direct involvement of the MPOA in modulating maternal aggression via a neurotransmitter system has been demonstrated. To date, the MPOA has only been reported to show increased neuronal activity upon an aggressive encounter in lactating mice  and ratsIn addition to maternal behavior, stimulation of CRFR1 activity within the MPOA had an anxiogenic effect. Indeed, the CRFR1 agonist, CRF, is well known to have anxiogenic actions in lactating females , 2014 anCrh mRNA (Crh transcription (Since previous studies have demonstrated neuronal interactions between the CRF and oxytocin systems , we inveCrh mRNA  and streCrh mRNA , to delacription , and to cription . Moreovecription  and suppcription , neithercription . Thus, icription ; activatCrhr1 and Crhr2 mRNA levels are unchanged and Crh mRNA expression is in fact elevated in lactation (Crhbp mRNA expression during lactation that would support such a role. It is unlikely that the dose used was too low to fully inhibit CRFBP in the MPOA, as the same dose administered into the BNST elicits behavioral changes (Given that actation , we postactation , might b changes , and theIn conclusion, we report that activation of CRFR, particularly CRFR1, in the MPOA impedes maternal care and maternal aggression and increases maternal anxiety. Furthermore, CRFBP does not appear to be involved in the crucial down-regulation of the CRF system postpartum. Yet, we identified another potential regulatory mechanism involving oxytocin release in the MPOA, which may serve to counteract the effects of CRFR activation, thereby ensuring stable levels of maternal behavior. Interestingly, this mechanism might be disturbed in mothers showing stress-induced maternal neglect and represents a potential target for treating such malfunctions in the postpartum period.None.


Acute ingestion of bitter melon (BM) has been shown to suppress the postprandial glycemic response in diabetics, but its impact on glucose regulation among individuals with impaired glucose tolerance is unclear. Moreover, one's glucose tolerance level may influence the effectiveness of BM. This study aimed to examine the acute effects of a beverage containing BM extract on blood glucose regulation during an oral glucose tolerance test (OGTT) among prediabetics.glu) during D3 was lower than D2. To compare the acute effects of the beverage among individuals with varying glucose tolerance levels, subjects were grouped by their glucose response pattern—Fastpeak (peak glucose (Glupeak) at 30 min postglucose (30P)) and Slowpeak (Glupeak after 30P).Ten prediabetic adults completed two OGTTs—glucose only (D2) and glucose+BM (D3). Responders were identified as subjects whose area under the glucose curve (AUCn=5) experienced a 13.2% reduction in AUCglu : −18.1% to −8.3%), 12.2% reduction in mean glucose (95% CI: −17.3% to −7.0%) and 10.6% reduction in Glupeak (95% CI: −17.5% to −3.7%); plasma glucose was reduced by 9.1% at 30P (95% CI: −15.6% to −2.6%), −24.0% at 60P (95% CI: −36.8% to −11.2%) and −20.0% at 90P (95% CI: −35.8% to −4.2%) during D3. No between-trial differences were noted for Fastpeak or Slowpeak.During D3, responders (Acute ingestion of BM prior to the second OGTT (D3) led to a reduced postprandial glucose response in 50% of the subjects but did not affect the insulin response. Furthermore, the effectiveness of the beverage was seemingly uninfluenced by the subjects' glucose tolerance level. Although BM has shown to aid blood glucose management in diabetics, it remains uncertain why only a portion of subjects responded positively to the BM extract in the current study. Momordica charantia, also known as bitter melon (BM), has been purported to have an antihyperglycemic effect, which may serve as a treatment option for managing diabetes mellitus.4 Further, the ingestion of BM has been shown to elicit an acute hypoglycemic effect8 and improve the postprandial glycemic response during oral glucose tolerance testing among diabetics.10 Conversely, others have reported no significant reduction in postprandial blood glucose concentrations during glucose tolerance testing among non-diabetics,12 highlighting the notion that one's ability to maintain blood glucose homeostasis may determine the efficacy of BM. In support, previous studies have shown the presence of responders and non-responders to BM ingestion.1314 Individuals with prediabetes suffer from moderate glucose dysregulation via impaired glucose tolerance and/or impaired fasting glucose15 and are at risk for developing type 2 diabetes mellitus if left untreated.16 Moreover, persistently elevated blood glucose levels are associated with many other comorbidities, such as kidney disease, neuropathies and macrovascular complications.16 Nevertheless, it is possible for one to convert to a healthy, normoglycemic state through certain lifestyle, pharmacological and/or nutritional interventions16; thus the ingestion of an antihyperglycemic agent such as BM may help to minimize the risk of disease progression. Therefore, the purpose of this investigation was to examine the efficacy of BM extract to aid blood glucose regulation in a group of prediabetic adults.Although there have been a number of studies examining the antidiabetic properties of BM in both animals and humans, minimal research has examined these effects with regard to prediabetics. In fact, evidence of the effects of BM on glucose regulation in prediabetics has merely been inferred from studies among diabetics.−1 and/or hemoglobin A1c level between 5.7% and 6.4% volunteered to participate in this single-blinded, cross-over design investigation. Prior to data collection, all subjects completed confidential health questionnaires to assess health status and possible risk factors. Additionally, all subjects provided written consent prior to any data collection. Subjects were asked to avoid participation in any other clinical/investigational trials throughout the duration of this experiment. The New England Independent Review Board's approval was obtained prior to data collection.Ten prediabetic adults with fasting plasma glucose concentrations between 99 and 126 mg dlAll subjects were asked to visit the University's Human Performance Laboratory on three separate occasions. During the first visit (D1), laboratory staff provided a thorough explanation of all procedures, expectations and potential risks of study participation; determined subject eligibility; and assessed body composition via air displacement plethysmography . On the second visit (D2), subjects reported to the laboratory following an 8–10-h fast and underwent an oral glucose tolerance test (OGTT). Following a minimum of 96 h, subjects reported to the laboratory (D3) following an 8–10-h fast and underwent an additional OGTT. During D3, subjects consumed a beverage containing 1.25–3 g of BM extract 30 min prior to the OGTT.During each experimental trial, subjects were asked to ingest a 75-g glucose beverage  within a 5-min period. For the duration of the standard 2-h OGTT, blood samples were collected every 30 min.During each experimental trial, blood samples were collected at six time points. During D2, blood samples were collected upon arrival (BL); after 30 min, another sample was taken immediately prior to glucose intake (PRE), then again 30, 60, 90 and 120 min after glucose intake . During D3, the BM beverage was consumed promptly after the BL blood draw; and all subsequent blood draws followed the same protocol as D2. In between blood draws, participants remained inactive  in the Human Performance Laboratory. All blood draws were obtained following a 15-min equilibration period wherein subjects were instructed to lie in a supine position. All blood samples were obtained using a Teflon cannula placed in a superficial forearm vein using a three-way stopcock with a male luer-lock adapter and plastic syringe. The cannula was maintained patent using an isotonic saline solution .2EDTA . Samples were subsequently centrifuged at 3000 r.p.m. for 15 min at 4 °C. The resulting supernatants were aliquoted into separate micro-centrifuge tubes and frozen at −80 °C for later analyses.At each time point, blood samples were collected into two 6 ml Vacutainer tubes—one untreated and the other coated with Kglu and AUCins, respectively) were calculated using a standard trapezoidal method. To eliminate interassay variance, all samples were analyzed in duplicate by a single technician. The coefficient of variation for each assay was 0.87% for glucose and 8.24% for insulin.Plasma glucose was assessed using the glucose oxidase method via an automated analyzer . Serum insulin concentrations were determined via commercially available enzyme-linked immunosorbent assay kits  and a spectrophotometer . Following analyses, total area under the glucose and insulin curves  and Slowpeak (peak glucose concentration observed after 30P).Subjects were grouped according to their distinct response to the initial OGTT based on previously published methods;Acute effects of the study beverage on biochemical measures among all prediabetic subjects were analyzed using separate two-way, repeated-measures  × time (BL vs PRE vs 30P vs 60P vs 90P vs 120P)) analyses of variance (ANOVAs).13 Therefore, further analyses examined the effects of the study beverage among subjects who were determined to be responders—defined as individuals whose total AUCglu during D3 was lower than D2. Separate three-way, repeated-measures (group (responders vs non-responders) × trial (D2 vs D3) × time (BL vs PRE vs 30P vs 60P vs 90P vs 120P)) ANOVAs were used to assess the acute effects of the study beverage on the postprandial glucose and insulin responses among responders.Previous research suggests that BM may lower postprandial glucose in a portion of its consumers.peak vs Slowpeak) ×  × time (BL vs PRE vs 30P vs 60P vs 90P vs 120P)) ANOVAs.Acute effects of the study beverage on biochemical measures among subjects with distinct glucose responses were analyzed using separate three-way, repeated-measures , paired-samples t-tests were used. Results were considered significant at an alpha level of P⩽0.05, while trends toward significance were acknowledged at P⩽0.10. Data were analyzed via IBM SPSS Statistics for Windows . All data are reported as mean±s.d.In the event of significant interactions, least significant difference peak and Slowpeak , but Fastpeak tended to be younger than Slowpeak . When comparing responders and non-responders, no significant differences were noted for percentage of body fat (P=0.491), body mass index (P=0.948) or age (P=0.143).Subject characteristics are displayed in P=0.732), but significant time effects were noted (P<0.001). Plasma glucose was significantly elevated from BL (95% CI: 100.0–115.1) at 30P , 60P  and 90P . No significant between-trial differences were observed for AUCglu (P=0.723), mean plasma glucose  or peak plasma glucose .Glycemic responses during each experimental trial are displayed in n=5) and non-responders (n=5), a significant group × trial × time interaction was observed for plasma glucose concentrations  but not for non-responders (P=0.144). Additionally, no significant group × time interactions were noted during either trial ; but significant time effects were observed (P=0.001). Plasma glucose concentrations were elevated from BL (95% CI: 100.2–114.9) at 30P , 60P  and 90P . Furthermore, significant group × trial interactions were noted at 30P (P=0.002), 60P (P=0.003) and 90P (P=0.002). Compared with D2, responders displayed significantly lower plasma glucose concentrations at 30P , 60P  and 90P  during D3.When assessing the effects of the BM beverage among responders , Glumean  and Glupeak . When assessing non-responders, AUCins and mean serum insulin (Insmean) during D3 tended to be higher than D2  while peak serum insulin (Inspeak) was significantly greater .In comparison to D2, responders displayed significantly lower AUCpeak and Slowpeak), no significant group × trial × time interaction was observed for plasma glucose concentrations (P=0.168). Similarly, no significant trial × time interaction was observed for either group . A significant group × time interaction was noted during D2 , PRE , 30P , 60P , 90P  and 120P  compared with Slowpeak; however, no significant group × time interaction was noted during D3 (P=0.208). Furthermore, no significant group × trial interactions were noted at any time point.When examining the effects of BM among subjects of differing levels of glucose tolerance , Glumean  or Glupeak .No significant between-trial differences were observed among AUCP=0.307), but significant time effects were noted (P=0.033). Serum insulin was significantly elevated from BL (95% CI: 0.5–3.1) at 30P , 60P , 90P  and 120P . No significant between-trial differences were observed for total AUCins (P=0.836), Insmean (P=0.839) or Inspeak (P=0.126).When assessing all subjects, no significant trial × time interaction was observed for serum insulin concentrations (n=5) and non-responders (n=5), no significant group × trial × time interaction was observed for serum insulin concentrations (P=0.276). Similarly, no significant trial × time interaction was observed for responders (P=0.260) or non-responders (P=0.695). Additionally, no significant group × time interactions were noted during either trial ; but significant time effects were observed (P=0.030). Serum insulin concentrations were elevated from BL (95% CI: 0.4–3.2) at 30P , 60P , 90P  and 120P . Furthermore, no significant group × trial interactions were noted at any time point.When assessing the effects of the BM beverage among responders , Insmean  or Inspeak .No significant between-trial differences were observed among AUCP=0.637). Similarly, no significant trial × time interaction was observed for either group , and no group × time interaction was observed for either trial . However, significant time effects were noted (P=0.037); serum insulin concentrations were elevated from BL (95% CI: 0.4 to 3.3) at 30P , 60P , 90P  and 120P . No significant group × trial interactions were noted at any time point.When examining the effects of BM among subjects of differing levels of glucose tolerance, no significant group × trial × time interaction was observed for serum insulin concentrations , Insmean  or Inspeak .No significant between-trial differences were observed among AUCglu (−7.6% to −17.1%), Glumean (−6.5% to −17.0%) and Glupeak (−2.1% to −15.6%) during D3. However, when the acute glycemic effects of the beverage were compared across subjects with distinct levels of glucose tolerance , the beverage did not promote an improved glucose response for either group. Interestingly, any observed improvements in the glycemic response occurred independently of an augmented insulin response.The results of this study indicated that consuming a beverage containing BM extract prior to an OGTT attenuated the postprandial glucose response in 50% of prediabetic participants. The subjects that experienced a reduction in the postprandial glucose response during an OGTT following ingestion of the BM beverage  displayed lower AUC10 However, a number of the investigations demonstrating the efficacy of BM as a hypoglycemic agent have acknowledged the presence of responders and non-responders.13 In this sample of prediabetics, 50% of all subjects experienced a significant improvement in their glycemic response to the OGTT following an acute ingestion of the study beverage. This is evidenced by the reduced total AUCglu (−13.2%±3.9%) and mean glucose concentration (−12.2%±4.1%) compared with the control trial (D2). Acute ingestion of the study beverage 30 min prior to the OGTT also attenuated the postprandial increase in blood glucose during D3 compared with D2, indicated by a reduced Glupeak concentration (−10.6%±5.6%) and lower concentrations at 30P (−9.1%±5.2%), 60P (−24.0%±10.3%) and 90P (−20.0%±12.7%). Similarly, Leatherdale et al.6 observed significant reductions in plasma glucose concentrations and incremental AUCglu among non-insulin-dependent, type 2 diabetics during the initial 90 min of a 120-min OGTT when 50 ml of BM was consumed. Furthermore, others have reported an improved postprandial glycemic response—that is, lowered blood glucose levels—among type 2 diabetics when 100 ml of BM was consumed at least 30 min prior to glucose administration.19BM has been previously shown to enhance glucose tolerance in diabetics during an OGTT following a single-dose treatment.et al.11 observed no significant improvement in the glycemic response during an OGTT when non-diabetic, overweight men consumed 100 mg kg−1 of BM juice. In addition, no improvement in plasma glucose levels were reported in healthy rats consuming 0.6 g kg−1 of BM extract during an OGTT.12 Furthermore, ingestion of 100 ml of BM juice did not improve the glycemic response to a glucose load among insulin-dependent diabetic rats.19 Although these opposing conclusions may be due to different forms, dosage and/or preparation of the BM , the inconsistent results may also highlight the individual nature of glucose metabolism. In the present study, subjects displayed two distinct response patterns to the OGTT—those whose highest glucose concentration was observed 30 min after glucose ingestion and those whose highest glucose concentration was observed sometime after the 30-min mark. As the shape of the glucose curve during glucose testing has been suggested to provide metabolic information regarding one's level of glucose tolerance20—where greater complexity of the response curve is associated with improved glucose tolerance21—this sample appears to represent individuals with slightly impaired (Fastpeak) and more extensively impaired (Slowpeak) metabolic condition. Although we were able to classify metabolic responses as Fastpeak and Slowpeak, neither group experienced a significant change in the postprandial glycemic response during an OGTT when the study beverage was consumed 30 min prior to glucose ingestion in the present study.In contrast, some studies have reported no significant impact of BM on blood glucose concentrations during OGTT. Specifically, Kasbia in vivo22 thereby enhancing glucose disposal, others posit that its hypoglycemic effects are due to enhancement of glucose metabolism, augmenting tissue sensitivity to glucose and/or inducing translocation of glucose transporter isoform 4 (GLUT4).24 In addition, BM may also inhibit α-glucosidase activity within the small intestine, thus reducing glucose digestion and absorption along the alimentary canal.23 Moreover, a primary chemical constituent of BM, polypeptide-p, has demonstrated insulin-mimetic hypoglycemic properties.22 Previous research has shown an acute hypoglycemic effect of BM ingestion with22 and without26 an amplified insulin response, suggesting that the efficacy of BM may be due to the combined effect of multiple mechanisms. In the present study, BM failed to produce a significant insulinogenic effect suggesting that the acute improvement in glucose tolerance may be attributable to extra-pancreatic mechanisms such as increased GLUT4 activity or enhanced glucose uptake in the skeletal muscle and liver. As the aforementioned explanations were beyond the scope of this investigation, future research may consider examining the acute effects of BM ingestion on a molecular level to assess its efficacy with regard to subclinical, prediabetic individuals.While some hypothesize that BM acts as an insulin secretagogue The present results suggest that the acute ingestion of a beverage containing BM extract may promote an improvement in glucose tolerance during an OGTT among some individuals with impaired glucose regulation. Similar to previous findings, the observed reduction in postprandial glycemia occurred independently of an altered insulin response. Further research is needed to fully elucidate the effects of BM on glucose regulation in prediabetic adults.


Pentraxins are a family of highly conserved secreted proteins that regulate the innate immune system, including monocytes and macrophages. C-reactive protein (CRP) is a plasma protein whose levels can rise to 1000 μg/ml from the normal <3 μg/ ml during inflammation.We find that CRP inhibits proliferation of the human myeloid leukemia cell line Mono Mac 6 with an IC50 of 75 μg/ ml by inducing apoptosis of these cells. The related proteins serum amyloid P (SAP) and pentraxin 3 (PTX3) do not inhibit Mono Mac 6 proliferation. CRP has no significant effect on the proliferation of other leukemia cell lines such as HL-60, Mono Mac 1, K562, U937, or THP-1, or the survival of normal peripheral blood cells. The effect of CRP appears to be dependent on the CRP receptor FcγRI, and is negatively regulated by a phosphatidylinositol −3-kinase pathway.These data reveal differential signaling by pentraxins on immune cells, and suggest that CRP can regulate the proliferation of some myeloid leukemia cells. Pentraxins are a family of highly conserved secreted proteins that regulate the innate immune system, including cells of myeloid lineage such as neutrophils, monocytes, and macrophages –3. In heSAP and PTX3 can affect tumors, either by regulating cancer-related inflammation, angiogenesis, or directly inhibiting cancer cell growth and differentiation , 23–26. In this report, we find that CRP inhibits the proliferation, and induces apoptosis of, Mono Mac 6 cells, but has no effect on the survival of normal peripheral blood cells. The effect of CRP on Mono Mac 6 cells appears to be dependent on CD64 (FcγRI) and the IgA receptor , and is negatively regulated by a phosphatidylinositol-3-kinase (PI3K) dependent pathway. These data reveal differential signaling by pentraxins on immune cells, and suggest that CRP may be a novel regulator of some subtypes of leukemia.Human peripheral blood was collected into heparin tubes  from healthy adult volunteers who gave written consent and with specific approval from the Texas A&M University human subjects Institutional Review Board. Peripheral blood mononuclear cells (PBMC) were isolated from the blood using Ficoll-Paque Plus , as described previously , 29. HL-4/well) and PBMCs (2 × 105/well), in the presence or absence of pentraxins, were plated in 96-well plates  in 200 μl RPMI 1640 containing 5% BCS. The inhibitors PP2, PP3, U0126, Wortmannin, H-89, and okadaic acid were from EMD. LY294002 and the Syk inhibitor were from BioVision . NSC23766, ML141, and Ras inhibitory peptide were from Cayman Chemicals . SP600125 and an aqueous solution of 100 mM sodium vanadate were from NEB . Inhibitors, apart from vanadate, were dissolved in DMSO at 10–100 mM, and diluted to final concentrations in RPMI culture medium. At 48 h, the cells were resuspended, stained with 5 μg/ml propidium iodide  following [Purified human SAP , purified human CRP  and mammalian NSO cell-derived recombinant human PTX3  were diluted with RPMI culture medium. As commercial SAP preparations contain 0.1% azide, we buffer-exchange the SAP into 20 mM sodium phosphate, pH 7.4, as described previously . Leukemiollowing  and thenMono Mac 6 cells were cultured with CRP for 48 h and then fixed and stained as described previously . Mono Mav/v) Tween 20 (Thermo). Antibodies were detected with 1 μg/ml peroxidase conjugated goat Fab2 anti-rabbit or anti-mouse antibodies (Jackson ImmunoResearch), as described previously [For cell extracts, cells were lysed in RIPA buffer (Thermo) containing protein and phosphatase inhibitor cocktail  for 30 min on ice. Samples were clarified by centrifugation at 14,000 x g for 20 min at 4 °C. Supernatants were collected, mixed with Laemmli sample buffer, separated on 4–20% TRIS-glycine gels (Lonza), and transferred to PVDF membrane (EMD) and blocked, as described previously . Membranpropidium iodide and analyzed by flow cytometry. CRP and mouse monoclonal antibodies to CD32 (clone Fun2), CD64 (clone 10.1), or CD89 (clone A59) were also added to Mono Mac 6 cells in the presence or absence of 10 μg/ml donkey anti-mouse antibodies (Jackson ImmunoResearch) to cross-link the antibodies. At 48 h, the cells were resuspended and stained with 5 μg/ml propidium iodide and analyzed by flow cytometry.Mouse monoclonal antibodies to CD16 , CD32 , CD64 (clone 10.1), and CD89 (clone A59), mouse IgG1, IgG2a, and IgG2b , and human Fc blocker (BD Biosciences) or human Fc receptor binding inhibitor (eBioscience) diluted to a final concentration of 10 μg/ml were added to Mono Mac 6 cells in the presence or absence of CRP. At 48 h, the cells were resuspended and stained with 5 μg/ml Mono Mac 6 cells were cultured with or without 75 μg/ml CRP for 48 h. Cells were then collected by centrifugation and resuspended in, and permeabilized with, ice cold 70% ethanol, and then stored at −20 °C for 24 h. After permeabilization, cells were collected by centrifugation, resuspended in PBS, and stained with 5 μg/ml 7-AAD  and analyzed by flow cytometry .p < 0.05.Statistical analysis was performed using Prism . Differences between two groups were assessed by t test, or between multiple groups using analysis of variance (ANOVA) with Dunnett’s post-test. Significance was defined as Since pentraxin receptors are expressed on monocytes, an intriguing possibility is that pentraxins might affect the proliferation or viability of some monocyte-derived leukemia cell lines. To test this, we added pentraxins to the monocyte-derived leukemia cell lines HL-60, Mono Mac 1, Mono Mac 6, K562, U937, and THP-1. In the absence of added pentraxins, all five cell lines proliferated as previously reported –34 Fig.. 6.25 toTo determine if CRP affects normal cells in addition to Mono Mac 6 cells, we added CRP to freshly isolated PBMCs from healthy donors and counted total viable cells. After 48 h, there was no significant effect on cell numbers Fig. . Mono MaCRP interacts with the innate immune system through its binding to receptors such as CD16, CD32a, CD64, and CD89 , 18, 21.In some cases, isotype IgG or anti-receptor antibodies can block the binding of a ligand to the receptor and prevent receptor activation by the ligand , 46. To To determine if CRP affects FcR expression on Mono Mac 6 cells, 75 μg/ml CRP was added to Mono Mac 6 cells and CD32, CD64, and CD89 expression was examined after 48 h. CRP had no significant effect on CD32 expression, increased CD89 expression, and decreased CD64 expression  anti-mouse antibodies to cross-link the mouse antibodies and counted viable cell numbers after 48 h. CRP inhibited the proliferation of Mono Mac 6 cells  and ERK . To deteTo elucidate the intracellular signaling pathways involved in the inhibition of proliferation by CRP, Mono Mac 6 cells were cultured with or without 75 μg/ ml CRP in the presence or absence of intracellular signaling inhibitors. At concentrations that did not inhibit cell proliferation in the absence of CRP, inhibitors of Src kinases (PP2), MEK1/2 (U0126), ras (peptide inhibitor), rac (NSC23766), Cdc42 (ML141), Syk (syk inhibitor), JNK (SP600125), or the serine/threonine protein phosphatases inhibitor okadaic acid did not significantly affect the ability of CRP to inhibit Mono Mac 6 proliferation Fig. . CRP regKi-67 is a cell proliferation marker, and caspase-3 is a key mediator of apoptosis , 53. To To elucidate how CRP slows Mono Mac 6 proliferation, we examined cells after 48 h of incubation with 75 μg/ml CRP. CRP caused many cells to be smaller than control cells Fig.  a-b. CRPPentraxins are involved in regulating many aspects of the innate immune system, such as adhesion, differentiation, and polarization of monocyte/macrophages. In this report, we observed that CRP, but not SAP or PTX3, inhibited the proliferation of, and induced apoptosis of, the acute myeloid leukemia (AML) cell line Mono Mac 6. These data suggest that CRP might regulate the proliferation of some AML cells.We found that the IC50 of CRP on Mono Mac 6 is 75 μg /ml. In healthy humans, the plasma levels of CRP are low < 2 μg/ml), whereas during inflammation, CRP levels may rise to 1000 μg/ml  μg/ml, w. ElevatePentraxins bind to multiple receptors including IgG Fcγ receptors, the IgA receptor FcαR, selectins, and DC-SIGN , 17–21. Although we observed that CRP inhibits the proliferation and induces apoptosis of an AML cell line, others have noted that CRP can promote cell survival in a variety of cell types. CRP promotes myeloma cell survival via a CD32-dependent pathway involving PI3 kinase, ERK, and NF-κB , and CRPTogether, these results suggest that concentrations of CRP that can occur during inflammation inhibit the proliferation and induce apoptosis of at least one AML cell line, and that elevating levels of CRP or treatment with a CRP mimetic may be possible therapies for some forms of AML.


Aipl1−/− mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor β2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of   •Cone photoreceptor precursors form efficiently in mESC retinal organoids•Notch signaling limits temporal competence for cone cell differentiation•Reduced retinoic acid concentrations are required for cone maturation•Cones transplanted into a model of advanced retinal degeneration survive and mature Ali and colleagues characterize the differentiation of cone photoreceptors in mESC retinal organoids, showing substantial numbers of committed cone precursors. Their differentiation is negatively regulated by the Notch pathway, expression of Nrl and Nr2e3, and high concentrations of retinoic acid. Isolated and transplanted into a severely degenerated retina, these cone precursors survive and express markers of advanced maturation. Cone photoreceptors mediate high acuity and color vision in daylight. Cones are a rare subtype in most mammals, constituting approximately 3% of photoreceptors in mice and 5% in humans . Due to +) photoreceptors, enriched for cones compared with postnatal stages  , includiIn early stages of commitment to the cone lineage, co-expression of transcription factors ONECUT1 and OTX2 in retinal progenitors positive for OLIG2 leads to transcriptional activation of thyroid hormone receptor β2 (TRβ2) , an earlAipl1−/− mouse model of end-stage degeneration, in which nearly all host photoreceptors are lost by postnatal day 30 (P30)  . In this+ cone and horizontal cell progenitors decreased markedly between equivalent of embryonic  and neonatal  stages . Conversely, the percentage of OTX2+ cells, which marks all photoreceptor precursors together with bipolar cells , Thrb2 and Rxrg  and an upregulation of cone and rod photoreceptor-specific genes, including Crx, Opn1sw, Nrl, and Rho (p < 0.05), compared with control cultures  remained unchanged (+) were significantly depleted  when Notch inhibition was performed at d16 of differentiation, compared with treatment at d21 (∼E16 versus ∼P2 in vivo). A similar pattern was detected for Rxrg and Opn1sw  at either d16 or d21  and analyzed gene expression in Crx-GFPS at d26 E. Transcd Opn1sw E, indicad Opn1sw F, RECOVEd Opn1sw E, indicaNrl and Nr2e3 with an RFP reporter. Since these genes are only expressed in rod photoreceptor precursors, the effect of the knockdown is limited to postmitotic rods. These constructs mediated very efficient Nrl and Nr2e3 transcriptional silencing, as assessed by qPCR, when packaged into AAV2/9 serotype viral vectors and delivered subretinally into adult mouse retina  photoreceptor precursors derived from embryonic mouse retinas, including the expression of cone markers  and injected them into the subretinal space of adult wild-type mice. When examined 3 weeks post transplantation, we observed that 35% (±18%) of surviving Crx-GFP+ cells co-expressed the all-cone marker RXRγ  was 15% ± 7%  encoding the 2.1-kb fragment of the human red-green (M/L) cone OPSIN promoter , which dgenitors A and S6Bgenitors C and purgenitors D were hind Thrb2 E, suppornd Thrb2 A, but innd Thrb2 C. Strikime point D with tyme point D). This e retina . Initiale retina E–S6H, wie retina H, but sie retina . In the e retina E and S6Fe retina G and S6HAipl1−/− mice, which exhibit rapid photoreceptor loss . Close to the injection site the cells were clustered together and presented morphologies polarized toward the host retina, while further displaced cells were found with neurites extending more horizontally  between days 14 and 16 only. Normoxic conditions were used throughout the culture period. γ-Secretase inhibitors DAPT (Merck Millipore) and RO4929097 (BioVision) or DMSO as a vehicle control were added at a final concentration of 10 μM.E16 CEE line was maintained on gelatin-coated dishes in the presence of leukemia inhibitory factor . Crx-GFP line was cultured in “2i medium” supplemented with 3 μM GSK-3 inhibitor CHIR99021 (Tocris Bioscience) and 10 μM MEK inhibitor PD0325901 (Tebu-bio). The differentiation was performed as previously described . In brieOrganoids were fixed for 30 min in 4% formaldehyde, washed with PBS, and incubated overnight in 20% (w/v) sucrose, prior to embedding in OCT matrix. Tissue was cut into 18-μm cryosections mounted on glass slides, air-dried for 20 min, and kept frozen at −20°C for use in immunostaining. Staining protocol and the antibodies used are listed in + cells were on average >95% pure and >80% viable. Cells were resuspended at a final concentration of 1 × 105 live cells/μL in sterile Hank’s balanced salt solution  and DNase I (50 U/mL) before injection.Organoids were dissociated using a papain-based Neural Tissue Dissociation Kit (Miltenyi Biotec) prior to sorting on a BD Influx Cell Sorter (Becton Dickinson). Flow-sorted GFPFull details of the surgical procedure can be found in K.K. contributed to the conception, design, execution, and analysis of all experiments and writing of the manuscript; A.G.C. contributed to the design and analysis of a number of the experiments; D.G. contributed to the analysis of a number of the experiments; A.N., M.J., S.J.I.B., and M.K., contributed to the maintenance and differentiation of stem cell cultures; Y.D. contributed to histological processing and surgery; A.G. contributed to the design of experiments; R.D.S. contributed to FACS and flow cytometer analysis; R.N.M performed viral purification; A.J.S. contributed interpretation of experiments and manuscript writing; S.D. and Y.I. provided reagents and contributed to revision of the manuscript; J.C.S. contributed to revision of the manuscript and funding; R.A.P. contributed to the conception, design and interpretation of experiments, subretinal surgery, manuscript writing, and funding; E.L.W. contributed to the conception, design and interpretation of experiments, and manuscript writing; R.R.A. contributed to the conception, design and interpretation of experiments, manuscript writing, and funding.


The terminal magnesium fluoride compound, MgF, serves as a hydrogen bond and halogen bond acceptor for indole and C6F5I, and also reacts with Ph3CCl to form a C–F bond. tert-butyl-5-pyrazolyl)hydroborato ligand, , has been employed to obtain the first structurally characterized example of a molecular magnesium compound that features a terminal fluoride ligand, namely MgF, via the reaction of MgMe with Me3SnF. The chloride, bromide and iodide complexes, MgX , can also be obtained by an analogous method using Me3SnX. The molecular structures of the complete series of halide derivatives, MgX  have been determined by X-ray diffraction. In each case, the Mg–X bond lengths are shorter than the sum of the covalent radii, thereby indicating that there is a significant ionic component to the bonding, in agreement with density functional theory calculations. The fluoride ligand of MgF undergoes halide exchange with Me3SiX  to afford MgX and Me3SiF. The other halide derivatives MgX undergo similar exchange reactions, but the thermodynamic driving forces are much smaller than those involving fluoride transfer, a manifestation of the often discussed silaphilicity of fluorine. In accord with the highly polarized Mg–F bond, the fluoride ligand of MgF is capable of serving as a hydrogen bond and halogen bond acceptor, such that it forms adducts with indole and C6F5I. MgF also reacts with Ph3CCl to afford Ph3CF, thereby demonstrating that MgF may be used to form C–F bonds.The bulky tris(3- For example,  ligands provided the first structurally characterized examples of (i) a monomeric zinc hydride compound, [TptBu]ZnH,t,MeBu]ZnOH,]Ga.We have previously employed tris(pyrazolyl)hydroborato ligands, MgF can indeed be obtained readily upon treatment of MgMe3SnF,t,MeBu]MgX can also be obtained by the analogous method using Me3SnX  Å] is considerably shorter than those with bridging fluoride ligands.Ar]Mg(μ-F)(THF)}2 with μ2-bridges are 1.951(2) Å,3(μ3-F)(μ2-TFA)6(OMe)2(py)]3– with a μ3-bridge range from 2.012(5) Å to 2.047(4) Å.4-bridges exhibit even longer Mg–F bonds that range from 2.12 Å to 2.21 Å.t,MeBu]MgF being shorter than other Mg–F bonds, it is also amongst the shortest Mg–X (X ≠ H) bonds listed in the CSD, as illustrated by the magnesium oxide and alkoxide complexes, [{(THF)[BDIAr]Mg}2(μ-O)] [1.8080(5) Å],4N)Mes}2]Mg(OBut)(THF) [1.804(2) Å],2]2Mg (Ar = C6H3Pri2) [1.785(2) Å and 1.790(2) Å].36As would be expected, the Mg–F bond of MgF is characterized by a 19F NMR signal at –169.3 ppm, which is within the range exhibited by the related beryllium and zinc complexes, namely [Tp]BeF (–149 ppm),t,MeBu]ZnF (–207 ppm),p-Tol,Me]ZnF (–219 ppm),Ar]Mg(μ-F) (THF)}2 (–25 ppm) and {[BDIAr]Mg(μ-F)}2 (–26 ppm).19F NMR spectroscopy could be used as a probe of fluoride coordination mode,t,MeBu]MgF (–169.3 ppm) is also comparable to the solid state value for Mg6F2(OMe)10(MeOH)14 (–174.5 ppm), which contains μ4-F atoms.19F NMR chemical shift data do not provide a definitive probe for the fluoride coordination mode in these systems. Nevertheless, 19F NMR data in a comparable region to that of MgF have been reported in protein systems;3-G6P-TSA in 100% H2O buffer exhibits 19F NMR chemical shifts of –147.0, –151.8, and –159.0 ppm.11Spectroscopically, ZnF –9 ppm,21at,MeBu]MgX  have also been determined by X-ray diffraction.C3v symmetry, with a magnesium coordination geometry that is distorted considerably from tetrahedral. Specifically, the τ4 four-coordinate geometry indicesThe molecular structures of MgMe [2.119(3) Å]44In this regard, it is pertinent to note that two sets of covalent radii have been recently proposed by Alvarezt,MeBu]MgX , computationally. Firstly, density functional theory (DFT) geometry optimization calculations reproduce the experimental structures very well, as indicated by the close correspondence between the experimental and calculated Mg–X bond lengths  an ionic contribution to the bonding or (ii) π-bonding. lengths . Secondl lengths .t,MeBu]MgF reacts with Me2Mg to regenerate the methyl derivative, MgMe ,In terms of reactivity, the fluoride compound MgMe . Further3SiX, MgF also reacts with Ph3CCl to afford MgCl and Ph3CF , the chloride and bromide complexes, MgCl and MgBr, also undergo halogen exchange with the heavier Me3SiX derivatives  and Me3SiX , becomes less exoergic upon descending the periodic table. The derived equilibrium constants for the reactions of MgY  with Me3SiI are also listed in In addition to MgF pertains to its ability to participate in intermolecular interactions. In this regard, while fluorine is well recognized as an important structure-directing element by virtue of its ability to bridge two or more metal centers (vide supra),e.g. R–X or X–X, and a Lewis base, and results from the electron density distribution about X being anisotropic, such that it creates a belt of high electron density perpendicular to the covalent bond, but a region of low electron density  in the direction of the bond.i.e. linear A···X–D motifs, where A is the acceptor for the halogen bond and D is the donor. However, despite many structural investigations pertaining to intermolecular interactions involving metal fluoride ligands,t,MeBu]MgF to serve as a hydrogen bond and halogen bond acceptor.Another interesting aspect of the reactivity of [Tpin situ generated [MgF3]– to provide transition state analogues of phosphoryl transfer.1H and 19F NMR spectroscopic data demonstrate that the interaction between MgF and indole involves formation of a 1 : 1 adduct in benzene ,3P)2Ni(C5NF4)F (57.9 M–1),2MF2 ,t,MeBu]MgF must be considered a significant hydrogen bond acceptor.Hydrogen bonding interactions involving magnesium fluoride species are of relevance to the use of  benzene .66 Analyt,MeBu]MgF to participate in halogen bonding interactions has been investigated by a related study using C6F5I. Thus, 19F NMR spectroscopy demonstrates that the fluoride ligand of MgF serves as a halogen bond acceptor  is approximately an order of magnitude smaller than the hydrogen bonding interaction involving indole, but is comparable to the few reports of halogen bonding interactions involving fluoride ligands, namely [κ4-Tptm]ZnF (9.0 M–1) and trans-(R3P)2M(Ar)F .The ability of MgF, has been obtained by the reaction of MgMe with Me3SnF. The chloride, bromide and iodide complexes, MgX, can also be obtained by analogous methods using Me3SnX . Structural characterization by X-ray diffraction demonstrates that, in each case, the Mg–X bond lengths are shorter than the sum of the covalent radii, thereby indicating that there is a significant ionic component to the bonding, which is in accord with density functional theory calculations.In summary, the first structurally characterized example of a molecular magnesium compound that features a terminal fluoride ligand, namely MgF undergoes halide exchange with Me3SiX  to afford MgX. The other halide derivatives MgX undergo similar exchange reactions, but the thermodynamic driving forces are much smaller than those involving fluoride transfer, a manifestation of the often discussed silaphilicity of fluorine. MgF also undergoes metathesis with Ph3CCl to afford Ph3CF, thereby demonstrating that MgF has applications in the formation of C–F bonds.The fluoride ligand of MgF is capable of serving as a hydrogen bond and halogen bond acceptor to indole and C6F5I, respectively. The ability of MgF to participate in hydrogen bonding interactions mimics the involvement of magnesium fluoride species in biological systems.In accord with the highly polarized nature of the Mg–F bond, the fluoride ligand of [TpSupplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


The as-prepared Ag–SnO2/rGO nanocomposite was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and Raman spectrum. The acetylene sensing properties were investigated using different working temperatures and gas concentrations. An optimal temperature of 90 °C was determined, and the Ag–SnO2/rGO nanocomposite sensor exhibited excellent sensing behaviors towards acetylene, in terms of response, repeatability, stability and response/recovery characteristics, which were superior to the pure SnO2 and SnO2/rGO film sensors. The sensing mechanism of the Ag–SnO2/rGO sensor was attributed to the synergistic effect of the ternary nanomaterials, and the heterojunctions created at the interfaces between SnO2 and rGO. This work indicates that the Ag–SnO2/rGO nanocomposite is a good candidate for constructing a low-temperature acetylene sensor.This paper demonstrates an acetylene gas sensor based on an Ag-decorated tin dioxide/reduced graphene oxide (Ag–SnO All reagents were used without further purification.The high-purity graphene oxide (GO) nanosheets (>99%) were supplied by Chengdu Organic Chemicals Co. Ltd. . The GO used was graphene nanosheet, negatively decorated with oxygen functional groups and carboxylic groups, which were located at the sheet surface. The GO suspension was 0.25 wt % concentrated at pH 4.5. Tin chloride pentahydrate  self-assembly technique, which is shown in 2, GO, SnO2 and Ag suspensions. The immersing time here used was 10 min for the polyelectrolytes and 15 min for the SnO2, GO and Ag suspensions. Intermediate rinsing with deionized water and drying with nitrogen gas were required after each monolayer assembly, to reinforce the interconnection between the layers. The film was formed due to the interaction of electrostatic forces between the positively and negatively charged nanoparticles. The first SnO2 layer (positively charged) was designed for the intermediate bonding between PSS (negatively charged) and GO (negatively charged). The Ag–SnO2/rGO nanocomposite sensor was obtained via the thermal reduction of GO into rGO at 220 °C for 5 h in an oven. Furthermore, the pure SnO2 and SnO2/rGO film sensor were fabricated to allow a comparison between the drop-casting and LbL self-assembly methods, respectively.2/rGO sample was carried out with a scanning electron microscope . The X-ray diffraction (XRD) spectrum of the samples was examined with an X-ray diffractometer  using Cu Kα radiation with a wavelength of 1.5418 Å. The lattice fringes of Ag–SnO2/rGO were inspected with a transmission electron microscope . The nanostructural and compositional features of the Ag–SnO2/rGO and SnO2/rGO samples were characterized by Raman spectra .Surface microscopy of the Ag–SnORH), heating voltage (VH), sensor resistance (RS) and protection resistor (RL) made up the simplified circuit. The sensor resistance was recorded using a data logger , connected to a computer via a recommended standard (RS)-232 interface. The sensitivity of the sensor was defined as S = (R0 − Rg)/R0 × 100%, where R0 and Rg were the sensor resistances in dry air and acetylene gas, respectively. The time taken by a sensor to achieve 90% of the total resistance change was defined as the response or recovery time. A schematic of the experimental setup for acetylene sensing is shown in 2/rGO nanocomposite film. SnO2 microspheres and Ag nanoparticles (NPs) attached to the surface of rGO sheets are clearly observed. 2/rGO nanocomposite, and lattice fringe spacings of 0.35, 0.33 and 0.23 nm for rGO, SnO2 and Ag, respectively, are measured. 2 and Ag–SnO2/rGO nanocomposite films. Obvious peaks at 2θ angles of 10.78° and 24.7° are observed for the GO and thermally treated rGO, respectively, which is in agreement with previously published results, and further confirms the successful reduction of GO via thermal treatment [2 indicates several peaks at 26.41°, 33.82°, 37.60°, 51.73° and 65.68°, indexed to the (110), (101), (200), (211) and (301) planes of rutile SnO2, which is in accordance with the data from JCPDS Card no. 41-1445 [2 nanocrystals. Apart from the characteristic peaks attributed to SnO2, the XRD spectrum of the Ag–SnO2/rGO nanocomposite exhibited distinct peaks at 38.10°, 44.37° and 64.17°, which indexed to the (111), (200) and (220) planes of Ag crystallines, respectively [2/rGO nanocomposite, probably because the weak peak of rGO is swamped by the high intensity peak of the SnO2 at the 2θ angle of 26.41° [2/rGO and Ag–SnO2/rGO nanocomposites. The peaks located at 633 cm−1 in the SnO2/rGO and Ag–SnO2/rGO nanocomposites are typical Raman peaks of SnO2. The weak peaks located at 796 and 1588 cm−1 for the two samples are attributed to the Ag NPs. The peaks located at 1363 and 1640 cm−1 are attributed to defects and disorder in the graphite layer.reatment ,30,31. T 41-1445 , and conf 26.41° ,35. Figu2/rGO nanocomposite sensor to 100 ppm acetylene under operating temperatures of 25 to 170 °C. The sensor sensitivity increases and reaches its highest value at 90 °C, and then decreases with any further increase in temperature. This can be explained by the fact that the appropriate operating temperature improves sensor sensitivity, but higher temperatures lower the binding energies of gas molecules and sensing film. An optimal temperature of 90 °C was determined. Therefore, the operating temperature of 90 °C was selected for the sensor in the following experiments.2, SnO2/rGO and Ag–SnO2/rGO film sensors to 50 ppm acetylene at 90 °C. We found that the Ag–SnO2/rGO film sensor yielded the highest sensitivity among the three sensors. Moreover, the Ag–SnO2/rGO film sensor showed a shorter response/recovery time than the other two sensors. A response time and recovery time of 235 and 160 s, respectively, were observed for the Ag–SnO2/rGO film sensor upon exposure to 50 ppm acetylene.2/rGO nanocomposite film sensor upon exposure to acetylene gas, of cumulative concentrations, at 90 °C. The test was performed by exposing the sensor to 5, 10, 50, 100, 150 and 500 ppm of acetylene. The resistance of the Ag–SnO2/rGO nanocomposite sensor decreased with increasing concentrations of acetylene, indicating the n-type semiconductor-like behavior of Ag–SnO2/rGO to acetylene (reducing gas). The inset of Y) and acetylene concentration (X) as Y = 32.09 − 25.42eX/236.4−.2/rGO film sensor with concentrations of 5, 150 and 500 ppm acetylene at 90 °C. There were no significant changes in sensor sensitivity during the repeated exposure/recovery cycles, indicating an acceptable repeatability for acetylene sensing. 2/rGO film sensor towards an acetylene pulse, at concentrations between 0 and 500 ppm, exhibiting good response/recovery behavior. 2/rGO nanocomposite film sensor to concentrations of 5, 150, and 500 ppm acetylene gas, measured every five days for over 30 days. It was clearly shown that the sensor response does not vary significantly with time, confirming that the Ag–SnO2/rGO nanocomposite film sensor has good long-term stability. 2, SnO2/rGO and Ag–SnO2/rGO film devices, measured by applying voltages from −4 to 4 V. It is clearly shown that the measurement results indicate good Ohmic contact to n-type semiconductors for the three devices. A larger current passes through the Ag–SnO2/rGO sensor than that of the SnO2/rGO and SnO2 sensors. This is because the doping of Ag and rGO results in a decrease in the resistance of the Ag–SnO2/rGO film. 2/rGO film sensor exhibited a much higher response and a lower working temperature than that of its MOS-based counterparts. 2/rGO ternary nanocomposite film demonstrated excellent sensing properties towards acetylene gas at low temperatures. Its sensitive mechanism can be attributed to the synergistic effect of the ternary hybrids and the created potential barrier. Pristine SnO2 is an n-type semiconductor and electrons are majority carriers. Graphene, as one of the emerging 2D nanomaterials, has a unique layered structure, a large surface area-to-volume ratio and excellent electrical properties, which greatly facilitate the absorption and diffusion of acetylene gas molecules. 2/rGO nanocomposite film in air and acetylene gas. The oxygen molecules adsorbed on the conduction band of n-type SnO2 are ionized to oxygen negative ions through the trapping of free electrons from the surface of the SnO2 [2H2 (ads) + O− (ads) → CO2 + H2O + e−, which leads to a decrease in the sensor’s resistance [The Ag–SnOthe SnO2 . When thsistance ,28. 2 is beneficial, because it enhances the acetylene-sensing properties. 2/rGO heterojunction. The band-gaps for n-type SnO2 and p-type rGO are 3.6 and 0.4 eV, respectively [2 and rGO, respectively [2 and rGO come into contact with each other, electrons transfer from SnO2 to rGO, and holes flow in the opposite direction until a dynamic equilibrium state is reached, and thus a depletion layer is formed at the interface [2/rGO heterojunction. When the sensor was exposed to acetylene gas, the interaction between adsorbed O− and acetylene molecules released free electrons, with the released free electrons increasing the n-type doping of SnO2. Higher SnO2 doping results in a reduced depletion layer in SnO2, thereby decreasing the sensor resistance during acetylene gas exposure. The formation of a heterojunction at the interface between rGO and SnOectively  and theiectively ,41. Becanterface . Figure 2 and Ag, which is beneficial to the enhancement of gas-sensing. Electrons transfer from SnO2 to Ag NPs and form a highly-resistive “barrier layer” in air, and Ag NPs become centers for electron accumulation. When the sensor is exposed to acetylene gas, electrons transfer from acetylene gas to the Ag NPs and then to SnO2; the highly-resistive “barrier layer” is transformed into a highly-conductive “anti-barrier layer”, which improves the electron mobility and sensing performance of the Ag–SnO2/rGO nanocomposite sensor.The Ag NPs in the nanocomposite have a positive effect on electronic sensitization during gas adsorption ,44. A po2/rGO nanocomposite film was fabricated by layer-by-layer self-assembly technology. Successful preparation of the Ag–SnO2/rGO nanocomposite was characterized and confirmed by means of SEM, TEM, XRD and Raman spectrum. The Ag–SnO2/rGO nanocomposite sensor exhibited excellent sensing behaviors towards acetylene in terms of response, repeatability, stability and response/recovery characteristics at an optimal temperature of 90 °C, which are superior to pure SnO2 and SnO2/rGO film sensors. The underlying sensing mechanism of the Ag–SnO2/rGO sensor was explored. This work provides guidance for an acetylene sensor based on Ag–SnO2/rGO ternary nanocomposites.In this work, an acetylene gas sensor, based on Ag–SnO


Parasympathetic innervation of meninges and ability of carbachol, acetylcholine (ACh) receptor (AChR) agonist, to induce headaches suggests contribution of cholinergic mechanisms to primary headaches. However, neurochemical mechanisms of cholinergic regulation of peripheral nociception in meninges, origin place for headache, are almost unknown.Using electrophysiology, calcium imaging, immunohistochemistry, and staining of meningeal mast cells, we studied effects of cholinergic agents on peripheral nociception in rat hemiskulls and isolated trigeminal neurons.d-tubocurarine but was insensitive to the TRPA1 antagonist HC-300033. Carbachol but not nicotine induced massive degranulation of meningeal mast cells known to release multiple pro-nociceptive mediators. Enzymes terminating ACh action, acetylcholinesterase (AChE) and butyrylcholinesterase, were revealed in perivascular meningeal nerves. The inhibitor of AChE neostigmine did not change the firing per se but induced nociceptive activity, sensitive to d-tubocurarine, after pretreatment of meninges with the migraine mediator CGRP. This observation suggested the pro-nociceptive action of endogenous ACh in meninges. Both nicotine and carbachol induced intracellular Ca2+ transients in trigeminal neurons partially overlapping with expression of capsaicin-sensitive TRPV1 receptors.Both ACh and carbachol significantly increased nociceptive firing in peripheral terminals of meningeal trigeminal nerves recorded by local suction electrode. Strong nociceptive firing was also induced by nicotine, implying essential role of nicotinic AChRs in control of excitability of trigeminal nerve endings. Nociceptive firing induced by carbachol was reduced by muscarinic antagonist atropine, whereas the action of nicotine was prevented by the nicotinic blocker Trigeminal nerve terminals in meninges, as well as dural mast cells and trigeminal ganglion neurons express a repertoire of pro-nociceptive nicotinic and muscarinic AChRs, which could be activated by the ACh released from parasympathetic nerves. These receptors represent a potential target for novel therapeutic interventions in trigeminal pain and probably in migraine. In migraine pathophysiology, much attention was traditionally paid to the role of trigeminal innervation of meninges, likely representing a triggering zone for migraine pain –3. Peripvia released acetylcholine (ACh), with somatic trigeminal nerves located around essential meningeal vessels  can innervate and interact,  vessels . Moreove vessels , 7.in vivo pain models  . In contn models –17. One n models , 19.It is widely accepted nowadays that the key role in migraine pathology belongs to the neuropeptide CGRP, which is released in venous blood flow during migraine attack . Carbachex vivo rat hemiskull preparation with preserved innervation, we showed that the main AChR agonists, ACh, carbachol, and nicotine, all activated nociceptive firing in meningeal nerves and that carbachol (but not nicotine) induced degranulation of dural mast cells. Ca2+ imaging allowed us to confirm the pro-nociceptive action of nicotine and carbachol at the level of single trigeminal neurons.In the current project, using Wistar rats of both sexes from the Animal House of the University of Eastern Finland were used in this study. P9–P13 rats were used for trigeminal cell culture preparation and mast cell degranulation assay, whereas adult (P35–P36) rats were used for electrophysiology and immunohistochemistry.2/95% O2) artificial cerebrospinal fluid (ACSF) containing (in millimolars): NaCl 120, KCl 3, CaCl2 2, MgCl2 1, NaH2PO4 1, NaHCO3 25, and glucose 10, pH 7.3 (6–7 ml/min). The nervus spinosus, which is a part of the mandibular branch of the trigeminal nerve  was used for recordings. Signals were visualized and analyzed with the WinEDR V3.4.6 software . Spontaneous and drug-induced action potentials generated in the distal parts of the transected nervus spinosus were recorded at room temperature. Spike frequencies (number of spikes per every 2 min) were estimated and analyzed using OriginPro 9.1 software . For control values, the frequency of spikes during 10 min prior to drug application was taken.Preparation of male rat (P35–P36) hemiskulls was performed as published earlier , 24. BriIsolated male rat dura mater preparations were fixed during 60 min in 2% p-formaldehyde  and rinsed three times for 30 min in the phosphate buffer . Then, the preparations were consecutively incubated for 30 min in 0.3% Triton X-100 (Sigma-Aldrich) for 15 min in a solution containing 5% goat serum (Sigma-Aldrich), 1% bovine serum albumin , and 0.3% Triton X-100 and for 15 min in a solution of 1% BSA and 0.3% Triton X-100 (solution A). These solutions were based on PBS.Next, preparations were incubated for 15 h at 4°C in the solution A containing goat polyclonal antibody to AChE , or mouse monoclonal antibody to BuChE  and rabbit polyclonal antibodies to the neurofilament NF-H . The preparations were rinsed three times in the solution A and then incubated for 1 h at room temperature with the appropriate secondary antibodies conjugated to Alexa Fluor 488 or 647  diluted 1:1,000 in the solution A.After washing in PBS, the preparations were exposed to PBS solution diluted by glycerol (1:1) and used for microscopy  with a laser scanning confocal microscope Zeiss LSM 510 Meta .2, 2.1 MgCl2 (pH adjusted to 7.4 with NaOH), whereas others were filled with BSS containing 50 µM carbachol or 100 µM nicotine. Then, the hemiskulls were left in 4% PFA solution for at least 4 h. After that meninges were dissected carefully and placed on a microscope slide, where they were stained with 0.1% toluidine blue , containing (in millimolars) 152 NaCl, 5 KCl, 10 HEPES, 10 glucose, 2.6 CaCline blue . Images ine blue , 26. To ine blue . Then, tl-lysine  and were cultured in F12 medium  at 37°C, 5% CO2 for 48 h prior to an experimental treatment.Trigeminal ganglion (TG) cell cultures were prepared as described previously . Briefly2, 1 MgCl2 (pH adjusted to 7.4) followed by loading with 2 µM fluo-3 acetoxymethyl ester  at 37°C for 45 min in BS. After 20 min postincubation, dishes were transferred to TILL Photonics imaging system  and were constantly perfused (at 1.2 ml/min) with BS. The setup was equipped by fast perfusion system , which allowed rapid (exchange time ~30 ms) application of various compounds. Cells were viewed via Olympus IX-70  microscope with a specific filter of 488 nm wavelength using 10× objective. Images were collected using CCD camera  at sampling frequency set to 2 fps. Cells were further characterized by their responsiveness to a brief application of high potassium (50 mM KCl with compensated osmolarity) as a marker for neurons.Primary TG cells kept in culture for 2 days were rinsed with basic solution (BS) containing (in millimolar) 152 NaCl, 2.5 KCl, 10 HEPES, 10 glucose, 2 CaClpost hoc test was used. Mast cell degranulation data were analyzed using two-tailed Mann–Whitney U test with GraphPad Prism software . Two-tailed Student’s t-test [OriginPro 9.1 software ] was applied to evaluate the significance of increase in basal meningeal firing after 1 µM CGRP pre-application. Results are expressed as mean of data ± SE unless otherwise stated. In all tests, p < 0.05 was taken as significant.Nociceptive firing was statistically analyzed using OriginPro 9.1 software  and SigmaPlot 11.0 . One-way repeated measures ANOVA with Tukey To study if cholinergic agents can stimulate or inhibit meningeal nociceptive signaling, we applied various AChR agonists directly to the rat hemiskull preparation with preserved dura mater innervation. To this end, we recorded spontaneous nociceptive firing of meningeal trigeminal nerves in basal conditions, then the preparation was superfused with a ligand solution, and changes in spike frequency were monitored.inset). On average, the frequency of spikes was increased by 2.5-fold by 10 min of 250 µM ACh application  are also expressed by nociceptive sensory neurons to modulate the nociceptive signaling –37. Durin = 7 hemiskulls), although spare labeling was observed across the dura mater. Figures To study a role of endogenous meningeal cholinergic system in rat dura mater, we explored the expression and localization of AChE and BuChE, two key enzymes implicated in cholinergic signaling by termination of ACh action. The cholinesterase-positive nerve fibers were found mainly in perivascular regions  versus 385 ± 75 spikes in control . Importantly, co-application of 50 µM d-tubocurarine (non-selective nAChR antagonist) prevented the facilitatory pro-nociceptive action of neostigmine  did not change the nociceptive firing n = 4), whereas it was largely enhanced up to 62 ± 5% after 50 µM carbachol treatment . In females, there was 19 ± 5% of degranulation in control (n = 4), and the large enhancement of degranulation up to 61 ± 8% was observed after 50 µM carbachol exposure .As degranulation of mast cells has been recently suggested as one of potential triggers of migraine , 41, we n = 4) and 20 ± 4% after application of 100 µM nicotine . Likewise, in females, these values were 24 ± 8% for vehicle (n = 4) and 24 ± 5% for nicotine treatment .In contrast to carbachol, the concentrations of nicotine, which induced the nociceptive firing, did not promote degranulation of mast cells Figure D suggestThus, the pro-nociceptive action of nicotine was clearly mast cells independent, whereas the action of carbachol could potentially be, at least partly, mediated by products released from degranulated mast cells.2+ signals in neurons isolated from TG. Application of 50 mM KCl at the end of protocol was used to identify neurons , and the experimental protocol was approved by the Animal Care and Use Committee of the University of Eastern Finland (license EKS-004-2014). All efforts were made to minimize the number of animals used and their suffering.Design of the study: IS, EN, and RG. Conducting the study and analysis: IS, NM, PA, and LN. Manuscript preparation: IS, NM, and RG.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.


MAPT), progranulin (GRN) and chromosome 9 open reading frame 72 (C9orf72) being the most common genetic causes. No other risk factors for FTD had been identified until a genome-wide association study of patients with the most common pathological form of FTD, frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), showed an association with single nucleotide polymorphisms (SNPs) in a region encoding transmembrane protein 106b (TMEM106b).GRN mutations (who have FTLD-TDP pathology).GRN cohorts, in those with C9orf72 expansions  and in one undifferentiated clinical FTD cohort.GRN cohort.Frontotemporal dementia (FTD) is a neurodegenerative disease associated with impaired behaviour, language and motor function. Around a third of FTD is familial, with mutations in microtubule-associated protein tau (GRN (n=10), C9orf72 (n=17), dual GRN/C9orf72 (n=1), TBK1 (n=1) and SQSTM1 (n=2) mutations) and those with clinical syndromes associated with FTLD-TDP (FTD-MND and svPPA). Genotyping of the rs1990622 polymorphism in the total FTD cohort showed a frequency of AA 77 , AG 90  and GG 31 , and in the TDP-43 subset of AA 33 (43.4%), AG 34 (44.7%) and GG 9 (11.8%). The frequency within a UK control population of 5020 people from the Wellcome Trust Case Control Consortium was AA 1637 (32.6%), AG 2485 (49.5%) and GG 898 (17.9%). Using a homozygous protective allele model, there was no difference in the TDP-43 subset  or the total FTD cohort  compared with the control group, nor did allelic tests show significant differences between patients and controls. However, a homozygous risk allele model in the TDP-43 subset gave Fisher’s exact test, p=0.049 compared with the control group, and in the total FTD cohort gave Fisher’s exact test, p=0.076 compared with the control group.The UCL FTD DNA cohort database was searched for those with volumetric MRI. Data were available from 198 patients with an FTD spectrum disorder: 87 with behavioural variant FTD (bvFTD), 46 with non-fluent variant primary progressive aphasia (nfvPPA), 43 with semantic variant PPA (svPPA), 6 with a primary progressive aphasia (PPA) syndrome not meeting criteria for any of the other PPA syndromes , 5 with FTD associated with motor neurone disease (FTD-MND), 7 with corticobasal syndrome (CBS) and 4 with progressive supranuclear palsy (PSP). Within this cohort, 76 individuals had definite or likely TDP-43 pathology: this group included cases with genetic mutations known to cause TDP-43 pathology  for the insula, cingulate, frontal, temporal, parietal and occipital lobes in each hemisphere were generated from volumetric T1 MRI using a previously described methodology.In the total FTD cohort, significantly lower volumes were present in the AA  group compared with the AG/GG group in the frontal (p=0.009), temporal (p=0.029), cingulate (p=0.014) and insula (p=0.018) cortices .As previous studies have shown an association of the rs1990622 polymorphism with decreased volumes in the left hemisphere,In the TDP-43 subset, although similar or larger absolute differences in volume were seen in the AA group compared with the AG/GG group, a significant difference was only seen between the groups in the frontal lobe (p=0.044), with a trend to a difference in the cingulate (p=0.085) and insula (p=0.084) cortices. As with the clinical cohort, this was driven by an effect within the left hemisphere, with significant differences seen between groups in the frontal (p=0.013) and parietal (p=0.037) cortices, and a trend to a difference in the cingulate (p=0.082) and insula (p=0.072) .In a cohort of patients clinically diagnosed with FTD and also in a subset of patients with likely TDP-43 pathology, we show that homozygosity for the TMEM106B risk allele is associated with reduced grey matter volume in key cortical regions implicated in FTD. Consistent with previous research showing an effect of the TMEM106B risk allele on left hemisphere structures within the general population, decreased grey matter volumes were found in the left but not right hemisphere in both the total cohort and the TDP-43 subset.GRN or C9orf72, as well as those without.Despite some studies showing no effect, overall, previous studies have shown that the effects of TMEM106B variants are seen in cases of FTD with FTLD-TDP (but not FTLD-tau), including those with mutations in TMEM106B is a glycoprotein critical for normal lysosomal function.


Laparoscopic totally extraperitoneal (TEP) hernia repair is a technically demanding procedure. Recent studies have identified BMI as an independent factor for technical difficulty in the learning period. To analyze the effect of overweight and obesity on the technical difficulties of TEP. Prospective study on patients who underwent a symptomatic inguinal hernia by means of the TEP technique. Were analyzed gender, BMI, previous surgery, hernia type, operative time and complications. Technical difficulty was defined by operative time, major complications and recurrence. Patients were classified into four groups: 1) underweight, if less than 18,5 kg/m²; 2) normal range if BMI between 18,5 and 24,9 kg/m²; 3) overweight if BMI between 25-29,9 kg/m²; and 4) obese if BMI≥30 kg/m². The cohort had a total of 190 patients, 185 men and 5 women. BMI values ranged from 16-36 kg/m² (average 26 kg/m²). Average operating time was 55.4 min in bilateral hernia (15-150) and 37.8 min in unilateral (13-150). Time of surgery was statistically correlated with increased BMI in the first 93 patients (p=0.049). High BMI and prolonged operative time are undoubtedly correlated. However, this relationship may be statistically significant only in the learning period. Although several clinical features can influence surgical time, upon reaching an experienced level, surgeons appear to easily handle the challenges. The World Health Organization estimates a total of 700 millions obese and 2.3 billion overweight adults by 2015. Besides directly linked to several deseases including hypertension, diabetes mellitus and cardiovascular diseases,,,3,18, 23,25,28.Body mass index (BMI), which is calculated by dividing weight in kilograms by height in meters squared, is the most accepted measurement of obesity. BMI lower than 18,5 kg/m² is considered underweight; between 18,5 and 24,99 kg/m² is the normal range; exceeding 30 kg/m² is defined as obese. Several studies have demonstrated the effects of high BMI on surgical procedures, postoperative complications and anesthetics risks13, 21 and provides less postoperative pain and fast recoveryEndoscopic approach is widely accepted technique for hernioplasties among obeseThe aim of the present study was to analyze the effect of overweight and obesity on the technical difficulty of TEP performed by a single experienced surgeon.After Ethics Committee approval, a prospective study of patients who underwent a symptomatic inguinal hernia by means of the TEP technique between May 2009 and May 2014 was performed. Medical records from patients operated by a single senior surgeon were analyzed in terms of previous surgery, BMI, type of hernia, operative time and complications. Technical difficulty was defined by prolonged operative time and major complications. All patients signed an informed consent form.Patients were classified into four groups: 1) underweight, if BMI less than 18.5 kg/m²; 2) normal range, if BMI between 18.5 and 24.99 kg/m²; 3) overweight, if BMI between 25 and 29.99 kg/m²; and 4) obese, if BMI≥30 kg/m².,Variables were analyzed by Sperman’s correlation. Statistically significant values were defined as p<0.05Among a total of 238 patients identified on the period of study, sufficient data was obtained from 190.There were 185 men (97.4%) and five women (2.6%). Average age was 52 years, average BMI was 26 and average time of surgery was 44 min . Types aAverage operative time was 55.4 min per bilateral hernia (15-150) and 37.8 min per unilateral (13-150). Among patients with BMI≤24.99 kg/m², 36 had bilateral hernia; 25 at least one previous surgery; and four postoperative complications, including two conversion to open surgery, one haematoma, one cord edema and one hernia recurrence. Average operative time in this group was 41.6 min (16-120). Overweight patients presented 35 bilateral hernias; 32 had previous surgery; and six had complications: three conversions, one haematoma, one hematuria and one seroma. Average operative time was 43.8 min (13-150). Among 22 obese, nine had bilateral hernia; 10 underwent a previous surgery; and no complications were seen. Average time of surgery was 51.9 min (20-130).Distribution according to BMI is shown in According to ASA classification, 40% were classified as ASA 1, 55.8% as ASA 2 and 4.2% as ASA 3. Four patients (2.1%) stayed longer than 12 h in hospital.In group A, time of surgery was statistically correlated with increased BMI in the first 93 patients (p=0.049). Among patients who underwent unilateral hernioplasty (group B) p value was 0.42. Best p value was obtained from the first 14 patients (p=0.083). Patients who underwent bilateral surgery (Group C) had the best p-value obtained from the first 42 patients p<0.001, .,Among this cohort of 190 patients, BMI values ranged from 16-36 kg/m² (average 25.8 kg/m²) and there was a prevalence of men (97%), similar to other hernia studies ,This series demonstrates a significant correlation between BMI and operative time, suggesting that higher BMI is related to more technical difficulty. Not only anatomical factors, but also the higher prevalence of obesity-related comorbidity  can make surgical procedures in obese patients more difficult. Akagi et al. presented a statistically significant correlation between BMI and technical difficulty during laparoscopic anterior resection15, 26. Estimated number of surgeries ranges from 30-60 in literature4,15, 26. Arriving at an experienced level, it seems that surgeons can deal with challenges proficiently. Nevertheless, the correlation was statistically significant only in the first 14 patients who underwent a unilateral hernia  and in the first 42 patients who underwent a bilateral hernia repair . It suggests an influence of a learning curve on technical procedure. Due to unfamiliar pelvic anatomy and limited working space, TEP hernioplasty requires time to achieve excellence. This gap is more affected by patients’ clinical characteristics and anatomical aspects. Previous studies have affirmed that BMI may be a significant factor influencing surgical difficulty only in the learning period,,,Comparing bilateral and unilateral hernias, the first obviously demands longer operative time and has worse postoperative complication according to Jacob et. al.Difficulty associated with BMI in the learning period can help young surgeons to select hernia patients in order to overcome the learning period easily. Since this study included a series of cases of a single surgeon, further studies gathering young surgeons could be helpful to better evaluate the correlation between high BMI and operative time. High BMI and longer operative time are undoubtedly related. However, this correlation may be statistical significant only in the learning period. Although several clinical characteristics may influence operative time, when arriving at an experienced level surgeons seem to deal with challenges easily.