Rett syndrome is a severe pediatric neurological disorder caused by loss of function mutations within the gene encoding methyl CpG-binding protein 2 (MeCP2). Although MeCP2 is expressed near ubiquitously, the primary pathophysiology of Rett syndrome stems from impairments of nervous system function. One alteration within different regions of the MeCP2-deficient brain is the presence of hyper-excitable network responses. In the hippocampus, such responses exist despite there being an overall decrease in spontaneous excitatory drive within the network. In this study, we generated and used mathematical, neuronal network models to resolve this apparent paradox. We did this by taking advantage of previous mathematical modelling insights that indicated that decreased excitatory fluctuations, but not mean excitatory drive, more critically explain observed changes in hippocampal network oscillations from MeCP2-null mouse slices. Importantly, reduced excitatory fluctuations could also bring about hyper-excitable responses in our network models. Therefore, these results indicate that diminished excitatory fluctuations may be responsible for the hyper-excitable state of MeCP2-deficient hippocampal circuitry. Rett syndrome is an X-linked genetic disorder that largely affects females, and stems from mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2) in vivo investigations, mouse models of Rett syndrome have been developed that recapitulate many of the cardinal features seen in Rett syndrome patients To facilitate One step towards obtaining a better understanding of how network dynamics influence Rett syndrome phenotypes is to use a ‘reduced microcircuit’ strategy to identify how synaptic alterations in a defined structure affect its network activities. The hippocampal formation is one neural structure that has commonly been the focus of such investigations. Previously, Zhang and colleagues in vitro electrophysiological assays. None of the mutant animals displayed complete immobility or a moribund appearance at the time of sacrifice, although each subject did display impairments in hind limb elevation reflex indicating the presence of Rett-like symptoms All animal experimentation was conducted in accordance with the guidelines of the Canadian Council of Animal Care, and thoroughly reviewed and approved before implementation by the Toronto General and Western animal care committee (Protocols 882.6 and 1321.9). All surgeries were performed under general anesthesia, and every effort was made to minimize pain in the experimental subjects. Hippocampal slices (thickness 0.5 mm) were prepared as described previously Extracellular recordings were conducted using a dual channel amplifier  as described previously The basic set of differential equations governing both the model inhibitory interneurons and pyramidal cells Excitatory, pyramidal cell model: We use Inhibitory, interneuron cell model: We use a reduced version of Synaptic model: The synaptic current Population model representation - local field potential (LFP) analogue: In our simulations, we represent the experimentally observed LFP modulations due to SPAs . We use the Euler algorithm for numerical integration. Integration time step for each simulation ranges from 0.01 to 0.02 ms.In the results here, we first illustrate and describe the experimental findings of Spontaneous population activities produced from thick slice preparations of hippocampus are illustrated in Comparing What mechanism(s) could cause the observed changes in these hippocampal population activities in MeCP2-null samples? Zhang et al. In this section, we show, via a simulation approach, that decreased excitatory fluctuations are sufficient to account for all the observations by In It is clear from the four LFP model examples  to a bursting-like mode  as the excitatory-excitatory synaptic strength values  are affected in a mouse model of Rett syndrome. Three principle observations emerge from our work: 1) changes in mean excitatory drive within the hippocampal network are not necessarily a critical component of these network alterations, 2) decreased excitatory fluctuations can be a critical factor in changes in hippocampal network oscillations in Rett syndrome, and 3) these fluctuations in excitatory levels can also explain hyper-excitable responses in MeCP2-deficient hippocampal networks. For the first time, we have brought together observations, some of them seemingly paradoxical , under the unifying framework of excitatory fluctuations. We found that decreasing excitatory fluctuations is sufficient to account for all of the observations in Our model results indicating that oscillatory activity could be dramatically influenced by fluctuations led to the question of whether the fluctuation component could also explain hyper-excitable responses seen in MeCP2-deficient hippocampal networks. For this to be examined, we took advantage of previous excitatory network models expressing population bursts While it might seem paradoxical that reduced excitatory fluctuations can lead to network hyper-excitable responses , our simIdeally, one would like to have a single model network which incorporates both excitatory and inhibitory populations, and be able to recapitulate all the aspects of neuronal activities in MeCP2-null slice samples. We did not pursue this approach in this work, however. Instead, we teased apart the network into inhibitory , excitatIn the network models employed, two features are represented: excitatory drive (ance see  and reprIn conclusion, the work presented here indicates that decreased excitatory fluctuations, and not just the level of excitatory drive, can be instrumental in ascribing the functional properties of a network system. For Rett syndrome, where network hyper-excitable responses co-exist with attenuated spontaneous excitatory drive, our network models allowed us to dissect out how such seemingly paradoxical states could co-exist. Our results surprisingly show that attenuated excitatory fluctuations can in fact lead to a robust decrease in inhibitory population activities and simultaneously allow for excitatory network hyper-excitable responses to manifest. This has clear therapeutic implications for MeCP2-deficient systems, as it suggests that an increase in excitatory fluctuations, and not necessarily changes in tonic excitatory levels, would restore the normal dynamics of the network and decrease hyper-excitable responses.


Biliary complications have great importance for liver transplant recipients because of affecting long-term prognosis. In rare situations, an internal hernia of the Roux-en-Y loop cause graft injury. A 42-year-old woman with a history of living donor liver transplantation 6 years ago presented with prolonged graft injury during the past 6 months. She suddenly developed ileus of the small bowel with internal hernia through the defect of the mesentery around the Roux-en-Y limb of the hepaticojejunostomy. Emergent surgery was performed to reduce the hernia and volvulus; also the mesenteric rent was closed with interrupted suture of silk. Internal hernia of the small bowel after liver transplantation is rare but causes graft injury due to associated biliary complications and rapid deterioration of patient's condition. Liver transplantation has become an accepted therapeutic procedure for patients with end-stage liver disease, and its outcome has become satisfactory , 2. HoweA 42-year-old woman presented to her local emergency room with symptoms of episodic nausea and sudden abdominal pain. She had undergone living donor liver transplantation with biliary reconstruction as hepaticojejunostomy for primary sclerosing cholangitis at the age of 36 year, and was under tacrolimus immunosuppression. During the past 6 months, her ductal enzyme levels were significantly elevated, and cholestasis was suspected. She was admitted to our institute a few weeks prior to this episode for close examination. During the hospitalization, she needed to undergo percutaneous liver biopsy and transhepatic biliary drainage. The drainage exudates looked like diluted bile, and the amount was about 1000 mL/day. The biopsy specimen revealed chronic cholestasis with fibrosis. Signs of chronic rejection were not detected in the specimen.Abdominal computed tomography (CT) images revealed ascites and markedly dilated small bowel loops filled with small intestinal juice. Additionally, the small intestine was evident through the orifice of the internal hernia . These fAt the time of admission, she had diffuse abdominal guarding and rigidity with increased bowel sounds. Her laboratory data revealed leukocytosis and liver injury; therefore, she underwent emergent laparotomy.During the operation, the patient was found to have internal herniation and volvulus of the small bowel through the defect of the mesentery around the Roux-en-Y limb of the hepaticojejunostomy . The herLiver transplantation has become an accepted therapeutic procedure for patients with end-stage liver disease, and its outcome has become satisfactory , 2. FollThere have been detailed reports of 11 cases (including the current case) of internal hernia related to the hepaticojejunostomy after liver transplantation  6–9]. I–9. I6–9]Among the 11 cases, 4 patients received a right lobe graft, and 2 patients received a left lobe graft. In other 5 patients, graft type was not specified in the reports. The time of onset of internal hernia after liver transplantation varied from 13 days to 11 years. Computed tomography was performed in 8 patients, and the correct diagnosis was obtained in 7 patients. In spite of emergent surgery, 1 patient died. Thus, in patients having abdominal pain and a history of liver transplantation with hepaticojejunostomy, emergent CT is important for prompt and correct diagnosis.Once an internal hernia without strangulation develops, cholestasis can occur occasionally due to outflow obstruction of the efferent loop. Stenosis of the hepaticojejunostomy and intrahepatic bile duct can result from the inflammatory change caused by the repetitive cholangitis . ElevatiIn order to prevent internal hernia in transplant recipients, the most important and essential technical point is to close the defects with nonabsorbable suture materials . AbsorbaFor liver transplantation recipients, stasis of enteric fluid due to internal hernia can cause biliary complications and graft injury. This complication might be rare but can be avoided by careful mesenteric defect closure with nonabsorbable suture materials when performing Roux-en-Y hepaticojejunostomy or duct-to-duct biliary reconstruction.


Acorus calamus rhizoma, Cannabis sativa fructus, Chamomilla matricaria, Ginkgo biloba, Salvia officinalis, Sweet bee venom, Fritillaria cirrhosae bulbus, and the herbal combinations Bu Yang Huan Wu, modified Bu Yang Huan Wu plus Liuwei Di Huang, modified Chai Hu Long Gu Mu Li Wan, Geranii herba plus Aconiti lateralis praeparata radix , Niu Che Sen Qi Wan (Goshajinkigan), Gui Zhi Jia Shu Fu Tang (Keishikajutsubuto), Huang Qi Wu Wu Tang (Ogikeishigomotsuto), and Shao Yao Gan Cao Tang (Shakuyakukanzoto). The knowledge of mechanism of action is still limited, the quality of clinical trials needs further improvement, and studies have not yielded enough evidence to establish a standard practice, but a lot of promising substances have been identified. While CIPN has multiple mechanisms of neuronal degeneration, a combination of herbs or substances might deal with multiple targets for the aim of neuroprotection or neuroregeneration in CIPN.Chemotherapy-induced neuropathy (CIPN) has a relevant impact on the quality of life of cancer patients. There are no curative conventional treatments, so further options have to be investigated. We conducted a systematic review in English and Chinese language databases to illuminate the role of medical herbs. 26 relevant studies on 5 single herbs, one extract, one receptor-agonist, and 8 combinations of herbs were identified focusing on the single herbs  Several chemotherapeutic drugs are known to be neurotoxic and can lead to chemotherapy-induced peripheral neuropathy (CIPN). It is one of the main dose-limiting toxicities in oncologic treatments and a potential reason to terminate or suspend chemotherapy, in some cases leading to disease progression . CIPN inThe development of CIPN is usually dependent on the cumulative dose and symptoms may progressively aggravate . After sSome differences concerning the mechanisms of CIPN are described for different therapeutic drugs. In platinum compounds the dorsal root ganglia are the main parts of the nervous systems that are injured  and apopThe taxanes paxlitaxel and docetaxel are antitubulins and mainly damage the soma of the sensory neurons and the nerve axons. This is induced by an interference with the axonal transport, which is caused by enhancement of microtubule polymerization . MicroglVinca alkaloids  prevent tubulin polymerization from soluble dimers into microtubules . By affeEpothilones  are like the taxanes antitubulins, so there might be similar mechanisms of peripheral neurotoxicity. They damage the ganglion soma cells and peripheral axons through disruption of microtubules of the mitotic spindle and interfere with the axonal transport in the neurons  and can κB pathway can lead to disarrangement of the neurotrophin network -methanone) and the cannabilactone AM1714 -6H-benzo[c]chromene-6-one))—had been tested for their dose related suppression of established paclitaxel-evoked mechanical allodynia in a rat model. (R)-AM1241, but not (S)-AM1241, suppressed paclitaxel evoked mechanical allodynia, and AM1714 induced a modest antinociceptive effect. So the authors suggested that cannabinoid CB2 receptors may be important therapeutic targets for the treatment of chemotherapy-evoked neuropathy [Cannabis sativa or Cannabis extracts have not been clinically explicitly tested for CIPN but for other clinical conditions like neuropathic pain in HIV [Two structurally distinct cannabinoid CB2 agonists—the aminoalkylindole -n in HIV . Multipln in HIV –82.Matricaria chamomilla seem to have an antispasmodic effect and main components such as α-bisabolol or chamazulene have anti-inflammatory effects [Matricaria chamomilla extract-treated mice had a significant reduction of cisplatin-induced peripheral pain [Matricaria chamomilla hydroalcoholic extract was able to decrease cisplatin-induced pain and inflammation better than morphine [This is a commonly used herb in western as well as in eastern phytopharmacological tradition. Flavonoids from  effects . En-In-D effects . In a moral pain . Matricamorphine .Ginkgo biloba alcoholic extract in cisplatin-induced peripheral neuropathy in mice [Ginkgo biloba extract prevented reduction in NCV. In another study a Ginkgo biloba extract prevented some functional and morphological deteriorations induced by cisplatin, antagonizing the decrease in the number of migrating cells and in the length of outgrowing axons [Ginkgo biloba extract, but no specification of the extraction method was provided in the published abstract. The researchers found that 11 of the 17 patients developed a grade 1 peripheral neuropathy (PN) after the first cycle of oxaliplatin. Five of six patients who received Ginkgo biloba after the second cycle of oxaliplatin reported decreased intensity and duration of sensory PN. No Ginkgo biloba related side effects have been observed. The data suggested that Ginkgo biloba extract appears to attenuate the intensity and duration of acute dysesthesias caused by oxaliplatin and may yield synergistic antitumor activity [The popular herb from the maidenhair tree that has shown some promising effects as an neuroprotectant. The most unique components of the extracts are the terpene trilactones, that is, ginkgolides and bilobalide . In vitr in mice . Developng axons . Marshalactivity .Salvia species and their isolated constituents possess significant antioxidant activity in enzyme-dependent and enzyme-independent systems [β-induced toxicity [salvia species and their isolated constituents showed anti-inflammatory properties [Salvia officinalis extract can have anti-inflammatory and also antinociceptive effects on chemical behavioral models of nociception in mice that involve an opioid mechanism [Salvia officinalis hydroalcoholic extract on vincristine-induced PN in mice in comparison with morphine with a decrease of pain response, suggesting that Salvia officinalis extract could be useful in the treatment of vincristine-induced peripheral neuropathic pain [ systems –115. Thetoxicity . In addioperties , 118. Saechanism . An animhic pain .The venom of honey bees with its active peptide Melittin has been tested for injection into the acupuncture point Zusanli (ST 36) for its effect on CIPN in animal models. It showed to alleviate thermal hyperalgesia and mechanical allodynia. The results indicated an association with the activation of the LC noradrenergic system and with a reduction in spinal pNR1 , 121.Sweet bee venom. A total of 11 eligible consecutive CIPN patients were treated for 3 weeks and observed for another 3 weeks. A significant intraindividual improvement was found for pain and neuropathy scales [In a first case series this was tested in 5 patients in a 1-week course of treatment, which showed no side effects and gave evidence of clinical improvement . Anothery scales . Fritillaria bulbus, was evaluated in mice for its analgesic activities in murine models of inflammatory and neuropathic pain. It was shown that oral administration of hydroalcoholic extracted verticinone could significantly inhibit acetic acid-induced writhing response in a dose-dependent manner superior to acetylsalicyl acid. In the rat model of paclitaxel induced neuropathic pain, in contrast to the declined analgesic effect of morphine after repeated administration with the same dose, a relatively constant analgesic effect of verticinone was observed, so verticinone is expected to become a potentially novel sedative-analgesic agent without producing tolerance [Verticinone, an isosteroidal alkaloid isolated from olerance .Bu Yang Huan Wu is a classical combination of Chinese herbs, first mentioned in Wang Qing-Ren's Yi Lin Gai Cuo (Correcting the Errors in the Field of Medicine) published in 1830 [Astragalus membranaceus radix, Angelica sinensis radix, Prunus persicae semen, Paeoniae rubra radix, Ligustici chuanxiong rhizoma, Lumbricus terrestris, Spatholobi caulis, Curcuma radix, Chaenomeles lagenaria fructus, and Achyranthes bidentatae radix.  in 1830 . This ren = 44, control group n = 40) after the treatment of oxaliplatin and showed reduced development of CIPN in the treatment group tested by standardized clinical tests [The decoction has been used in a randomized Chinese study of 84 patients  [The remaining dregs of decoction were additionally used for washing the lower extremities. The treatment was used on 32 patients with existing CIPN following different chemotherapies and compared with 32 patients who were treated orally with vitamin B1 2500 ug plus by intramuscular injection with vitamin B1 100 mg. Herbal treatment was found to be significantly more effective to vitamin treatment ( < 0.05) . Chai Hu Long Gu Mu Li Wan is a traditional recipe derived from the Shang Han Lun [ Han Lun . A modifPseudostellaria heterophylla, Pinelliae rhizoma, Glycyrrhizae radix, Scutellaria baicalensis radix, Bupleuri radix, Fossilia ossis mastodi, Ostreae concha, Rubia cordifolia radix, Scutellariae barbatae herba, and Fritillariae thunbergii bulbi. The external washing was done with Astragali membranaceus radix, Angelica sinensis radix, Paeoniae rubra radix, Lumbricus terrestris, Ligustici chuanxiong rhizoma, Prunus persicae semen, and Carthami flos.The oral combination of herbs consists of P < 0.05) [The incidence of CIPN was almost half as high in the patients treated additionally with Chinese herbs as evaluated by clinical testing ( < 0.05) .Geranii herba and Aconiti radix extract has been shown to be effective in a rat model of oxaliplatin evoked neuropathy. Mechanical allodynia and thermal hyperalgesia were alleviated. NGF was increased, substance P decreased in the group treated with Geranii herba and Aconiti radix extract additionally to oxaliplatin compared to oxaliplatin alone [External application of a combination of in alone .Geranii herba plus Aconiti radix granule can relieve neuropathy and improve the quality of life. Unfortunately the authors did not provide data in the published abstract, which species of Geranii herba or Aconiti radix they used [In the following randomized clinical study 58 patients with CIPN from oxaliplatin, taxol, or capecitabine were assigned prospectively in a controlled randomized trial: 30 patients were assigned to the study group and 28 were used as a control. The clinical study revealed that symptoms of pain, paraesthesia, and swelling were relieved after one week of therapy and it was concluded that hey used .Jisheng Fang, written by Yan Yonghe, a little known but highly regarded physician of the Song Dynasty, published in 1253 [Rehmannia viride radix, Achyranthis bidentatae radix, Corni fructus, Dioscorea opposita rhizoma, Plantaginis semen, Alismatis rhizoma, Moutan cortex, Cinnamomi cortex, Aconiti lateralis praeparata tuber, and Poria alba) [This formula derives from the ia alba) . GJG hasia alba) , 128.GJG was tested for its effect on CIPN in animal studies. In a rat model of oxaliplatin-induced neuropathy repeated administration of GJG prevented the oxaliplatin-induced cold hyperalgesia but not mechanical allodynia and axonal degeneration of the rat sciatic nerve. A single administration of GJG reduced both cold hyperalgesia and mechanical allodynia after the development of neuropathy. GJG did not affect the antitumour effect of oxaliplatin on the tumour cells or mice implanted with tumour cells .GJG was also tested in a rat model for paclitaxel-induced peripheral neuropathy, but as with oxaliplatin no regeneration was found in histological examination . NeverthGJG has been widely used to treat symptoms like numbness, vibration sensation, cold sensation, and limb pain associated with diabetic neuropathy .P < 0.01, log-rank test). The incidence of grade 3 PN after 10 courses of chemotherapy was 0% in the GJG group and 12% in the control group, and after 20 courses was 33% in the GJG group and 75% in the control group [2+/Mg+, and two control groups: FOLFOX without additional therapy and FOLFOX plus Ca2+/Mg+. When a cumulative dose of oxaliplatin exceeded 500 mg/m2, the incidence of PN was 91% in the FOLFOX without additional therapy group, 100% in the FOLFOX group with additional Ca2+/Mg+ therapy, and 79% in the FOLFOX plus GJG plus Ca2+/Mg+ therapy group and 50% in the GJG plus FOLFOX group. The cumulative oxaliplatin dose when 50% of patients developed neurotoxicity was 765 mg/m2 in the GJG plus FOLFOX and 340 mg/m2 in the FOLFOX plus GJG plus Ca2+/Mg+ group, respectively, and 255 mg/m2 in both control groups. The authors concluded that concomitant administration of GJG reduced the neurotoxicity of oxaliplatin without having a negative influence on the response rate [It has been shown to prevent oxaliplatin-induced peripheral neuropathy in a FOLFOX-regimen (FOL: Folinic acid (leucovorin), F: Fluorouracil (5-FU), OX: Oxaliplatin (Eloxatin)) in clinical studies , 100. Innse rate , so for nse rate .GJG was also tested for paclitaxel-induced neuropathy in breast and gynecological cancer. A retrospective study on 82 patients found that GJG was possibly effective for the treatment and the prevention of peripheral neuropathy and seemed to be more effective when administered from the beginning of chemotherapy using paclitaxel .National Cancer Institute-Common Toxicity criteria) grade 3 of neurotoxicity developed in 2 patients (14.3%) after 6 weeks in the vitamin B12, whereas no neurotoxicity was observed in the vitamin B12/GJG group. The change in the frequency of abnormal current perception threshold (CPT) ratio was significantly lower in the VB12/GJG group in comparison to VitB12 alone (P < 0.05), which suggests that GJG inhibits the progression of PN [In a prospective study in paclitaxel/carboplatin treated patients with ovarian or endometrial cancer, patients were randomly divided into two groups. 14 patients received vitamin B12 and 15 patients vitamin B12 plus GJG. The observation period was 6 weeks following treatment initiation. A NCI-CTCAE . It contains Cinamomi cortex, Aconiti lateralis praeparata tuber, Zingiberis rhizoma, Jujubae fructus, Glycyrrhizae radix, and Atracylodis macrocephalae rhizoma.This formula has its basis from the Aconiti lateralis praeparata tuber was used as a granule for 11 patients with metastatic colorectal cancer receiving FOLFOX in a noncontrolled study. Reduction of neuropathy was observed in 5 cases after chemotherapy (45.5%) [This herbal combination with an increased dose of  (45.5%) . Keishikajutsubuto was not possible due to hot flashes or gastric discomfort. The remaining 12 patients showed a VAS improvement rate of 76.5 ± 27.7% (mean ± standard deviation) [The same herbal granule was also used in a study on postherpetic neuralgia and was found to be effective. In three of 15 patients in this noncontrolled trial continuation of the treatment with viation) .Jin Gui Yao Lue  [Ogikeishigomotsuto, containing Astragali membranaceus radix, Cinnamomi cortex, Paeonia alba radix, Jujubae fructus, and Zingiberis rhizoma. It has been used in individual cases for neuropathic pain due to ANCA-associated vasculitis [This classical combination derives from Chamber) . In Kampsculitis .In single case report the granule showed a positive effect on neuropathic pain and it allowed the continuation of the suspended chemotherapy with oxaliplatin . Shang Han Lun [Shakuyakukanzoto and it is a herbal granule of Paeonia alba radix and Glycyrrhizae radix. It is used to relieve menstrual pain and muscle spasm as well as muscle pain due to the chemotherapeutic agents paclitaxel and carboplatin [This classical formula derives from the  Han Lun . In Kampboplatin –136 and boplatin .Shakuyakukanzoto significantly relieved the allodynia and hyperalgesia induced by paclitaxel [Shakuyakukanzoto has also been tested for preventing neurotoxic side effects of FOLFOX and the effect was retrospectively compared to the treatment with GJG (see n = 20) or Shakuyakukanzoto (n = 24). The response was 50.0% in the GJG and 65% in the Shakuyakkanzoto group. The authors concluded that both recipes are able to reduce the FOLFOX-induced neurotoxicity [This was supported by animal studies in a mouse model of paclitaxel-induced pain. clitaxel . Shakuya GJG see . 44 patitoxicity .Capsicum. Topical creams with capsaicin are used to treat peripheral neuropathic pain. Following application to the skin capsaicin causes enhanced sensitivity, followed by a period with reduced sensitivity and, after repeated applications, persistent desensitisation. Capsaicin is the active component of chili peppers, which are plants belonging to the genus Topical capsaicin is used to treat postherpetic neuralgia and HIV-neuropathy and has been found to be effective in multiple trials. There are risks of epidermal innervation upon repeated application over long periods . Aconiti lateralis praeparata radix is a herb used in many recipes for neuropathy like GJG  and in the second model by selective L5 spinal nerve transection. The extracts of Moutan cortex and Coicis semen dose dependently alleviated the PGF2alpha-induced allodynia. The increase in NADPH diaphorase activity in the spinal cord associated with neuropathic pain was also blocked by these extracts. These results suggest that Moutan cortex and Coicis semen are substances effective in the treatment of neuropathic pain [hic pain .Nigella sativa and one main compound thymoquinone were beneficial on histopathological changes of sciatic nerves in streptozotocin (STZ) induced diabetic rats. Evaluation of the tissues in the diabetic animals showed fewer morphologic alterations, and myelin breakdown decreased significantly after treatment with Nigella sativa and thymoquinone. The ultrastructural features of axons also showed improvement [rovement .Ocimum sanctum was investigated in sciatic nerve transection induced peripheral neuropathy in rats. Axonal degeneration was assessed histopathologically. Paw pressure, Von Frey Hair, tail cold-hyperalgesia, and motor in-coordination tests were performed to assess the in vivo extent of neuropathy. Biochemical estimations of thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH), and total calcium levels were also performed. Ocimum sanctum attenuated axonal degeneration, rise in TBARS, total calcium, and decrease in GSH levels in a dose-dependent manner. In vivo reduction of nociceptive threshold and motor in-coordination was found. The authors concluded that anti-oxidant and calcium attenuating actions may be responsible for the amelioration [ioration .Teucrium polium. The treated rats exhibited a lower nociceptive score as compared to untreated diabetic rats. The results may suggest a therapeutic potential of Teucrium polium for the treatment of hyperalgesia [In an another animal study STZ-induced diabetic rats received intraperitoneal injection of this extract of ralgesia .Phyllanthus amarus has been shown to be effective in a neuropathic model of nociception. The antiallodynic effects seemed not to be associated with the impairment of motor coordination or with the development of tolerance. Apart from its anti-inflammatory actions, which are probably linked to the presence of lignans, another as yet unidentified active principle(s) present in the hexanic extract of Phyllanthus amarus produces pronounced anti-allodynia [A hexanic extract from llodynia . Sesbania sesban was tested in STZ-induced diabetic rats. The treated group showed an increased tail flick latency significantly when compared with pregabalin and reduced superoxide anion and total calcium levels which gave evidence of neuroprotective effects [ An aqueous extract of  effects .In spite of intense research in the last decades, no conventional pharmacological substance has been established as a sufficient and safe treatment of CIPN-induced neuroprotection and regeneration , 65–67. One main problem of doing research on herbs and understanding the mechanisms of their action is the fact that herbs contain a number of active compounds and by tradition, especially in Asian herbal therapy, combinations of multiple herbs are common. Classical research mainly focusing on a single active compound has been done often without regard to historical knowledge of the therapeutic utility of the plant source . This doSalvia officinalis seems to protect neurons against toxicity [Ginkgo biloba as a single herb shows some evidence of preventing neuronal degeneration and inducing neural regeneration in CIPN [The positive effect of a single herb on neuroprotection or regeneration was not found in any clinical study and rarely found in animal studies. Only the flavonoid apigenin from toxicity  and Gink in CIPN –111.Acorus calamus rhizoma due to its antioxidative, anti-inflammatory, and calcium inhibitory actions [Matricaria chamomilla have an antispasmodic and an anti-inflammatory effect [Salvia officinalis is probably working due to its antioxidant activity and anti-inflammatory properties [Fritillaria bulbus might be effective due to its isosteroidal alkaloid verticinone [Cannabis sativa is effective in multiple and central pain syndromes [Cannabis agonists had been shown to be effective in CIPN in a rat model could be a hint that Cannabis sativa might be a promising substance for pain in CIPN in the future. Mainly in animal studies only a few single herbs have been tested for treatment of CIPN improving neuropathic pain. This effect might be induced by  actions . Flavonoy effect , 84. Saloperties –115 and operties . Fritillticinone . While Cyndromes –78, the Ginkgo biloba extract has been positively tested in a small retrospective clinical study in humans [Sweet bee venom had shown positive results by injection into acupuncture points in a small clinical case series [Only n humans  and Sweee series , 90. Tope series .Moutan cortex and Coicis semen have been tested positively in two different neuropathic pain models. Both herbs dose dependently alleviated the PGF2alpha-induced allodynia and blocked NADPH diaphorase activity in the spinal cord associated with neuropathic pain [There are a few more single herbs tested in animal models for the treatment of neuropathic pain in other conditions than CIPN. hic pain . Nigella sativa, Ocimum sanctum, Teucrium polium, Phyllanthus amarus, and Sesbania sesban also have been tested in single studies to ameliorate neuropathic pain [hic pain –145.Aconiti lateralis praeparata radix in a mouse model of CIPN had analgesic effects by inhibition of spinal astrocytic activation [Aconiti lateralis praeparata radix had a dose-dependent effect on allodynia and thermal hyperalgesia which was induced via spinal kappa-opioid receptor mechanisms [tivation  and in achanisms , which cchanisms . Active chanisms –155.Aconiti lateralis praeparata radix is often used for several types of persistent pain while it is rarely used as a single herb for CIPN. But interestingly Aconiti lateralis praeparata radix is part of many recipes used for CIPN as GJG, Shakuyakukanzoto, Keishikajutsubuto, and Geranii herba plus Aconiti radix combination  [GJG is the best tested herbal recipe for CIPN. But not all details of its mechanism have been clearly identified and for some ingredients the effects are unknown. It is considered that the analgesic effect is exerted through the improvement of peripheral nocireceptor sensitivity, vasodilation, and peripheral circulation by the promotion of NO production due to the effects of eceptors . Anotheror (NGF) , which cor (NGF) . One furor (NGF) .Even though the full recipe was clinically positively tested in a couple of clinical trials, most of them were uncontrolled or retrospective analyses –102, 130Shakuyakukanzoto, and both were found to be effective in the treatment of CIPN, but there was no negative control group [Shakuyakukanzoto was analyzed in a mouse model [Shakuyakukanzoto contains only two herbs, Paeonia alba radix and Glycyrrhizae radix, the future evaluation of its role for treating CIPN might be easier than for other herbal recipes. While neuronal apoptosis can be triggered by oxidative stress and mitochondrial dysfunction, substances with an antioxidative potency are possible candidates for treatment of CIPN [Paeonia alba radix has been positively tested as an antioxidant in a nonneuronal cell model might be relevant [Paeonia alba radix was used in another recipe , so at least something is known about its possible mechanisms of effect.The herbal recipe d trials , 93  radix, Astragalus membranaceus radix, and Glycyrrhizae radix. The most used herbs of herbal recipes in clinical trials on CIPN are Astragalus membranaceus radix is supporting the Qi, which means in western terms supporting the general energy level of the body, which is usually reduced in CIPN by activating the vegetative nervous system. Rehmannia praeparata radix is basically supporting the Yin, which in western terms reflects the structural damage of tissue, in the case of CIPN the peripheral nerves. Paeonia alba radix and Paeonia rubra radix promote the flow of the Xue, which in western terminology has its correlate in enhancing the microcirculation which might be reduced in CIPN; promotion of the perfusion might enhance the regeneration. In Chinese medicine experience Aconiti lateralis praeparata radix supports in Chinese medicine theory the Yang. A typical symptom of Yang deficiency is ice-coldness, which is prominent in the extremities in some cases of CIPN. Glycyrrhizae radix supports the fluids and has a balancing effect on the whole recipe.So in spite of using different terms, Chinese medicine theory describes physical reactions of the body that can be explained by western physiology and has found its proof by experimental studies , 159–164So using these herbs has as rational foundation on the basis of Chinese medical theory as well as from experimental studies, but unfortunately still little is known about the complex physiological mechanisms of herbal combinations, the interactions of the substances, and the mechanisms of action of many other substances used in herbal medicine. The challenge for future research is bringing historical knowledge and modern scientific analysis together. Due to this limited knowledge on the mechanisms of the action of herbs, we additionally collected data on herbs that are a putative treatment option for CIPN. While oxidative stress and mitochondrial dysfunction promote CIPN, substances with an antioxidative potency are possible candidates for the treatment of CIPN. So in the list below we list herbal antioxidants tested in neuronal cell or disease models. Additionally we added herbs for enhancement of nerve growth as putative treatment options for CIPN. NGF exhibits potent biological activities such as preventing neuronal death, promoting neurite outgrowth, and supporting synapse formation , which hPutative Herbs or Herbal Compounds for the Treatment of CIPNHerbal Antioxidants Tested in Neuronal Cell or Disease Models. Puerarin (from Pueraria lobata radix) [Lycium barbarum) [Magnolia officinalis) [Ginkgo biloba) [Huperzia serrata) [Polygonum multiflorum) [Tripterygium wilfordii Hook) [Salvia miltiorrhiza) [Salvia miltiorrhizae) [Gastrodia elata rhizoma [Astragalus membranaceus radix) [Ligusticum wallichi) [Scutellaria baicalensis radix Georgi) [Uncaria rhynchophylla [Dioscorea opposita radix) [a radix) , 166, Ica radix) , a fractarbarum) , Ginsenoarbarum) , honokiocinalis) , Ginkgol biloba) , huperziserrata) , Ginsengserrata) , Notoginserrata) , 3,5,4′-iflorum) –176, celii Hook) , Salvianorrhiza) , 179, tarrhizae) , Gastrod rhizoma , 182, Ass radix) , Tetrameallichi) , 184, Ziallichi) , Baicale Georgi) , Uncariahophylla , 6,7-diha radix) .Herbs or Herbal Compounds That Enhance Nerve Growth. Cistanches herba [Huperzia serrata [Astragalus membranaceus radix [Gentiana rigescens radix [Rehmanniae praeparata radix [Paeoniflorin of Paeonia alba radix [es herba , 188, Hu serrata , 190, a  serrata . Astragaus radix , Gentisins radix , Rehmannta radix , Paeonifba radix .Research with the aim of proving the benefits of promising substances or herbal combinations has to describe the mechanism of action for single herbs and single components, investigate the interactions of combinations of substances, and analyse the promoting effects of combinations.From this viewpoint, research in this field has not very much progressed, but if research does not provide these data, herbal combinations will not find acceptance in mainstream treatments in non-Asian countries in Europe, North America, or Australia. This challenge could be taken if more international cooperation of interested research groups would be organized. Experimental and clinical studies have not yielded enough evidence to establish a standard practice for the treatment of CIPN, but from this literature review, a lot of promising substances, mainly Chinese medical herbs with possible effect in CIPN or a putative influence on mechanisms of CIPN, have been identified in the last years. The knowledge of the mechanisms of action is still limited and the quality of the clinical trials needs further improvement. In the future not only the mechanisms of action for single herbs and single components have to be described, but interactions of combinations of substances as well as interactions with chemotherapy have to be investigated and analysed in depth. While CIPN has multiple possible mechanisms of neuronal degeneration, a combination of components might be a promising opportunity focusing on multiple targets of degeneration or activating regeneration.


Disrupted white matter integrity and abnormal cortical thickness are widely reported in the pathophysiology of obsessive-compulsive disorder (OCD). However, the relationship between alterations in white matter connectivity and cortical thickness in OCD is unclear. In addition, the heritability of this relationship is poorly understood. To investigate the relationship of white matter microstructure with cortical thickness, we measure fractional anisotropy (FA) of white matter in 30 OCD patients, 19 unaffected siblings and 30 matched healthy controls. Then, we take those regions of significantly altered FA in OCD patients compared with healthy controls to perform fiber tracking. Next, we calculate the fiber quantity in the same tracts. Lastly, we compare cortical thickness in the target regions of those tracts. Patients with OCD exhibited decreased FA in cingulum, arcuate fibers near the superior parietal lobule, inferior longitudinal fasciculus near the right superior temporal gyrus and uncinate fasciculus. Siblings showed reduced FA in arcuate fibers near the superior parietal lobule and anterior limb of internal capsule. Significant reductions in both fiber quantities and cortical thickness in OCD patients and their unaffected siblings were also observed in the projected brain areas when using the arcuate fibers near the left superior parietal lobule as the starting points. Reduced FA in the left superior parietal lobule was observed not only in patients with OCD but also in their unaffected siblings. Originated from the superior parietal lobule, the number of fibers was also found to be decreased and the corresponding cortical regions were thinner relative to controls. The linkage between disrupted white matter integrity and the abnormal cortical thickness may be a vulnerability marker for OCD. OCD is frequently familial, and first-degree relatives of individuals with OCD have a fivefold increased risk of developing the disorder Obsessive-compulsive disorder (OCD) is a chronically debilitating psychiatric disorder with a prevalance of approximately 2% et al. reported that patients with OCD showed increased grey matter in the left posterior orbitofrontal cortex, anterior insula, bilateral parahippocampal gyrus, and right fusiform gyrus, and decreased grey matter in the left anterior cingulate gyrus and medial frontal gyrus, compared with healthy controls Alterations in brain structures as well as disrupted connectivity in cortico-striato-thalamo-cortical circuitry have been implicated as pathophysiological mechanisms of OCD et al. reported that patients with OCD had significantly lower fractional anisotropy (FA), an indicator of the white matter microstructural integrity, in the bilateral anterior cingulate gyrus, parietal lobe, right posterior cingulate gyrus, and left lingual gyrus using VBM In addition to findings of atypical grey matter volumes and cortical thickness, there is increasing evidence of structural aberrations in white matter within OCD's classical neuroanatomical circuits et al. found that patients with OCD and their unaffected first-degree relatives both had reduced FA in the right inferior parietal lobe and increased FA in the right medial frontal lobe, indicating that these white matter abnormalities may be an endophenotype for OCD et al. found a significant linkage between disrupted white matter  and reduced cortical thickness  in patients with schizophrenia Of note, structural changes in grey and white matter have also been detected in high-risk subjects, which indicates that morphometric abnormalities may exist prior to illness onset and might even increase the risk for the subsequent emergence of clinical symptoms Due to their high risk of developing OCD, unaffected siblings of patients with the disorder can provide rich genetic information for OCD research, thus helping disentangle the state and trait markers of the illness. Consequently, an important step towards investigating how combined alterations of white matter and cortical thickness may serve as a putative biological marker is to examine these alterations in the unaffected siblings of individuals with OCD.The goal of the present study is to investigate two hypotheses: (1) whether there exists a potential linkage between disruptions in white matter connectivity and alterations in cortical thickness in patients with OCD; and (2) whether similar abnormalities are evident in unaffected siblings, thus representing a potential biomarker of increased risk for OCD. Specifically, in order to detect the correlation between abnormal white matter and abnormal cortical thickness, brain regions with significant FA differences between individuals with OCD and healthy controls were used as starting points for fiber tracking. Cortical thickness in regions implicated by these tracts, as well as the number of fibers connecting cortical regions, can be compared between groups of individuals with OCD, their siblings, and healthy controls. We expect that disrupted white matter integrity should go along with reduced cortical thickness in individuals with OCD, and, given the high heritability of OCD, similar abnormalities will be present in their unaffected siblings.A total of 30 adult outpatient subjects with a DSM-IV diagnosis of OCD, 19 unaffected full siblings, and 30 healthy subjects were included in this study see .Unaffected siblings and healthy subjects were interviewed by the SCID-I/NP All procedures were approved by the ethics committee of Guangzhou Psychiatry Hospital, China, and written informed consent was obtained from all participants.Obsessive-compulsive (OC) symptom severity was evaluated using the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) 2 and one without diffusion gradient (b = 0) image. Images were acquired parallel to the anterior-posterior commissure. A high-resolution T1-weighted anatomical image was acquired by using a 3-dimensional fast spoiled gradient recalled (FSPGR) sequence with 128 contiguous slices . Magnetic field uniform was reached before each scanning. All the images were visually inspected for clinical abnormalities and common artifacts by a radiologist (C.Z. S.) prior to analysis.After clinical interview and assessment, participants underwent MRI examination. The MRI data were acquired on a Signa HDe 1.5-T GE scanner  equipped with an 8-channel phased-array head coil at the First Affiliated Hospital of Jinan University. Diffusion-weighted images were obtained using echo-planar imaging . Diffusion-sensitizing gradient encoding was applied in 25 different directions with a diffusion-weighing factor of b = 1000 s/mmhttp://www.fmrib.ox.ac.uk/fsl) http://www.fil.ion.ucl.ac.uk.spm), including the following steps: a) each subject's b0 image was coregistered to T1 image, and the same coregistration parameters were applied to the FA map; b) each subject's T1 image was then nonlinearly normalized to the SPM T1 template, and the same normalization parameters were applied to the respective aligned FA maps; c) The final normalized FA map was further smoothed with a 6-mm full-width at half-maximum (FWHM) isotropic Gaussian kernel. A white matter mask was defined by binarizing the SPM white matter template to restrict the search volume for analysis during the corrections for multiple comparisons. FA maps of all subjects were then put into a general linear model (GLM) to remove the effects of age, gender, and mean FA of whole brain. Statistical analyses were conducted in SPM8. Statistical significance of FA between groups was finally evaluated by two-sample t-test, and the significance level was set at p<0.05, corrected for multiple comparisons , with a minimum cluster size of 200 voxels.First, the DTI data were corrected for motion and eddy current effects by applying an affine alignment of the diffusion-weighted images to the b0 image using the FSL software package (http://www.paraview.org/), and the number of fiber tracts passing through each ROI was calculated. The obtained ROI-based fiber count was compared between each pair of groups after regressing out the effects of age, gender, and total fiber count of each subject.Regions with significantly differed FA between OCD patients and healthy controls were selected as regions of interest (ROIs) in MNI template space. We then mapped each ROI back to the native space of each subject, and use it as the starting point for streamline fiber tracking by using ExploreDTI toolbox Individual T1 MRI images were processed using the FreeSurfer software package In order to examine the relationship between clinical variables and FA values, cortical thickness, and the number of fibers in OCD patient group, we extracted the mean value of the corresponding morphometric measures from each ROI. A linear regression was carried out with all morphometric measures entered simultaneously as predictors for each clinical variable . Age, gender and IQ were entered as covariates in this regression.Demographic and clinical data are shown in Group comparisons showed a pattern of reduced FA in patients with OCD compared with healthy controls, which includes the cingulum, arcuate fibers near the superior parietal lobule, inferior longitudinal fasciculus (ILF) near the right superior temporal gyrus and uncinate fasciculus. Relative to the unaffected siblings, patients with OCD exhibited decreased FA in the cingulum and superior longitudinal fasciculus (SLF), while no increased FA was found. Compared with healthy controls, unaffected siblings showed the reduced FA in arcuate fibers near the superior parietal lobule and anterior limb of internal capsule. Patients with OCD and their unaffected siblings both showed the decreased FA in arcuate fibers near the superior parietal lobule. The present study was aimed to investigate the potential vulnerability markers for OCD, which means that these markers should exist in both OCD patients and their siblings OCD = 0.034, uncorrected; PSiblings = 0.046, uncorrected). Patients with OCD also exhibited lower fiber counts in corresponding cortical regions compared to healthy controls using the ILF near right superior temporal gyrus as a starting point . Compared with healthy controls, patients with OCD and their siblings both showed significantly decreased fiber count within the high-connectivity cortical regions measured using the arcuate fibers near superior parietal lobule as a starting point  and the ILF near right superior temporal gyrus  as starting points. NO areas of increased cortical thickness associated with OCD were found. Compared with healthy controls, unaffected siblings were similarly characterized by significantly less cortical thickness in the left superior parietal lobule . No significant differences in cortical thickness were found between patients with OCD and their unaffected siblings. Cortical thickness data are presented in Our findings revealed that greater obsession subtotal scores on the Y-BOCS were correlated significantly with lower FA in the arcuate fibers near superior parietal lobule . The value of projected cortical thickness using superior parietal lobule as starting point had a significant association with the total scores of OCI-R . None of the other morphometric measures were significantly associated with these clinical variables . The relationship between whole-brain FA values and clinical variables and also the relationship between cortical thickness and clinical variables are shown in To the best of our knowledge, the current study is the first to investigate the combined relationship between white matter microstructure and cortical thickness in patients with OCD and their unaffected siblings. We found that patients with OCD exhibited reduced FA in the cingulum, arcuate fibers near the superior parietal lobule, the ILF near right superior temporal gyrus and uncinate fasciculus, compared with healthy controls. Similarly, siblings exhibited lower FA in the arcuate fibers near left superior parietal lobule than that observed in the healthy controls. Interestingly, relative to healthy controls, patients with OCD showed not only decreased fiber quantities, originated from the left superior parietal lobule seed, but also significantly thinner cortical thickness within corresponding cortical regions. Importantly, unaffected siblings exhibited similar fiber counts and cortical thickness abnormalities as patients with OCD. Overall, the current study indicates that the disruption of white matter in the left superior parietal lobule is accompanied by the reduced fiber quantities and cortical thickness within projection regions in both patients with OCD and their unaffected siblings, suggesting possible hereditary risk factors for OCD.Our findings provide evidence of atypical structural neural circuitry in individuals with OCD. Specifically, we found decreased FA in the cingulum, arcuate fibers near the superior parietal lobule, the ILF near right superior temporal gyrus and uncinate fasciculus in patients with OCD, compared with healthy controls. These fiber bundles directly connect the brain regions within the cortico-striato-thalamo-cortical circuitry. For instance, the uncinate fasciculus is a prominent white matter tract connecting the OFC with the limbic system, such as hippocampus and amygdala in the temporal lobe The left parietal lobe is associated with motor attention. Rushworth et al. found that subjects with impairment in left parietal lobe had a difficulty in shifting the focus of motor attention from one movement in a sequence to the text, while not in the right parietal lobe et al. found that patients with OCD exhibited increased connectivity within the fronto-parietal network (FPN) compared with matched controls at the resting state Functionally, Stern Although functional connectivity is not a direct proxy for anatomic connectivity, it is possible that anatomic connectivity constrains neural activity fluctuations. Actually, coupled fluctuations were observed between many regions that possess anatomic connections Importantly, abnormal white matter near the superior parietal lobule and decreased cortical thickness was also found in unaffected siblings, suggesting a possible endophenotype for OCD. Previous studies have separately provided evidence that white matter The current study has several potential limitations. First, all patients with OCD were being treated with psychotropic medications at the time of MRI examination, and the potential effect of these medications on FA, cortical thickness, and behavioral assessments cannot yet be determined. However, drug-naïve patients with OCD have been found to exhibit similar brain differences to those observed in the individuals with OCD and a history of drug treatment In summary, our data provides substantial evidence that disrupted white matter microstructure was associated with decreased cortical thickness in corresponding cortical regions in patients with OCD and their unaffected siblings when using the white matter near superior parietal lobule as a starting point for tractography, suggesting that this feature may be an endophenotype representing increased genetic risk for OCD.Figure S1Regions showing significant cortical thickness difference between patients with OCD, their unaffected siblings, and healthy controls.(ZIP)Click here for additional data file.Figure S2The regression analysis of whole-brain FA and clinical variables in OCD patient group.(ZIP)Click here for additional data file.Figure S3The regression analysis of cortical thickness and clinical variables in OCD patient group.(ZIP)Click here for additional data file.Table S1Treatment details of OCD patients.(ZIP)Click here for additional data file.Table S2Inter-correlations between the anatomical measurements. The correlation coefficients between each pair of mean FA, fiber-quantity (FQ) and mean cortical-thickness (CT) values from each of the 5 ROIs are shown below.(ZIP)Click here for additional data file.


Drosophila BHD model through genomic deletion of the FLCN gene (−DBHD). The DBHD mutant larvae grew slowly and stopped development before pupation, displaying various characteristics of malnutrition. We found the growth delay was sensitive to the nutrient supplies. It became more severe upon restrictions of the dietary yeast; while high levels of yeast significantly restored the normal growth, but not viability. We further demonstrated that leucine was able to substitute for yeast to provide similar rescues. Moreover, the human FLCN could partially rescue the −DBHD phenotypes, indicating the two genes are involved in certain common mechanisms. Our work provides a new animal model of the BHD syndrome and suggests that modulation of the local nutrient condition might be a potential treatment of the BHD lesions.Folliculin (FLCN) is a conserved tumor suppressor gene whose loss is associated with the human Birt-Hogg-Dubé (BHD) syndrome. However, its molecular functions remain largely unknown. In this work, we generated a  The BHD syndrome is a rare genetic disorder that is clinically characterized by frequent lung cysts, benign hair follicle tumors, and a high risk to develop kidney cancers. Inactivation of the folliculin (FLCN) gene is the genetic basis of BHD syndrome. FLCN is present in a wide range of organisms, from the single-cell yeast to human, indicating it may regulate certain basic cellular processes. However, its biological functions are still not clear −) − mouse tissues − mice stopped development at very early embryonic stages with severely disorganized structures Several FLCN mutant cell lines and animal models have been developed to unravel its functions. One discovery from these works is the intriguing relations between FLCN and the mechanistic target of rapamycin (mTOR), a highly conserved nutrient sensor among eukaryotes whose mutations have been found in certain human diseases including cancer Drosophila provides an ideal model system to study the genotype-phenotype relationships. Its genome contains a single FLCN homologue gene (DBHD). Using an RNAi-mediated gene knockdown assay, people uncovered a role of DBHD in the male germline stem cell maintenance and suggested the dysregulated stem cell homeostasis might be a potential mechanism of the BHD tumorigenesis DBHD knockout allele (−DBHD). The DBHD mutant larvae displayed various features of malnutrition, including growth retardation, small body size and larval lethality. The growth defects, but not the lethality, could be significantly rescued by dietary yeast or the branched-chain amino acid of leucine. We further demonstrated that the rescue effect is likely a consequence of elevated dTOR signaling, because a specific dTOR signaling suppressor, rapamycin, could reverse the rescues of −DBHD mutants by yeast or leucine. Moreover, the human FLCN could partially rescue the −DBHD mutants, indicating at least some molecular functions of the two homologous genes are conserved. Our work provides a novel animal model of the BHD syndrome and suggests that modulation of the local nutrient conditions deserves further investigations for treatment of BHD.DBHD targeting construct, two genomic fragments from both sides of the DBHD locus, about 5 kb each, were amplified and inserted into the NotI and AscI cloning sites of the pW25 vector TCTTTTTAGGCGCGCCTACACTTGGGCCTCC and TGACCAGGCGCGCCCATTCACTGAAATACCAG (with AscI site); CAATCCGCGGCCGCTTTCACTGATAAAAACGAG and GACTATGCGGCCGCAATCATTGATGAGGGGTTG (with NotI site). The screening procedure was performed according the protocol described before To make the DBHD-res, a genomic fragment covering the complete DBHD locus till the adjacent gene (CG14829) was amplified from the Drosophila genomic DNA. PCR amplification primers: GCACTCTAGACCACAGGTAATGAACAG and GCTGTCTAGACTGGATTCGGCATC. EGFP tag was then fused in frame with either terminus of the DBHD transcription unit by fusion-PCR. The human FLCN cDNA (a kind gift of Laura S. Schmidt) was amplified by PCR and cloned into the pUAST vector to make the UAS-hFLCN transgene. To generate a polyclonal DBHD antibody, the EcoRI-XhoI fragment of DBHD cDNA, which encodes the N-terminal 88 aa from N2 to L89, was amplified by PCR and inserted into the expression vector pGEX4T1. This construct was transfected into bacteria BL21 for expressing GST-fusion protein. The purified GST-fusion protein was injected into rabbit to generate antiserum against DBHD. On SDS-PAGE, it recognizes a major band at about 55 kDa, the same size as the predicted DBHD protein.To make the DBHD mutant screening experiment, we used the following stocks: y w; 70FLP, 70I-SceI (BL#6934) and 1118; 70FLPw (BL#6938). The following flies were used to generate mosaic clones: −hsp-flp; RRT80B ubi-GFP/FRT80B DBHD and −eyeless-flp; FRT80B ubi-GFP/FRT80B DBHD. We crossed the following flies to rescue −DBHD with hFLCN: −/TM3, Sbhsp-Gal4, DBHD and −/TM3, SbUAS-hFLCN; DBHD. The normal food recipe used in our lab: 8% sugar, 10% corn flour, 1.5% baker’s yeast, 1% agar, 0.4% Propionic Acid and 0.1% Nipagin. Chemicals used in the feeding experiments: 3-methyl adenine (Invitrogen); Rapamycin (LC Laboratories). Leucine, arginine, glutamine, tryptophan, cholesterol, and riboflavin were all bought from Sigma. For the free amino acids analysis, the third instar larvae were rinsed with 70% ethanol and allowed to be air-dried. More than 55 larvae were homogenized followed by sonication. Proteins were precipitated by sulfosalicyclic acid. After centrifugation, the supernatant were passed through 0.22 µm filter and analyzed using the Hitachi automatic amino acid analyzer L-8900.In the The following primary antibodies were used: rabbit anti GFP ; rabbit anti PH3 . Mouse anti Armadillo (1∶200) and Prospero (1∶200) were from DSHB. Guinea pig anti-Dpn (1∶1000) was a gift from Jim Skeath. ApopTag-Red assay kit was from Millipore. The fluorescent secondary antibodies, LysoTracker, and Click-iT EdU assay kit were all bought from Invitrogen.DBHD function, we used the homologous recombination strategy to delete the DBHD genomic sequence within the germ cells from living animals . The DBHD gene encodes a 460-amino-acid protein, spanning 1712 base pairs (bp) on the left arm of chromosome 3 with three exons. The targeting construct contains a 4.8 kb and a 5 kb of genomic fragments flanking the DBHD transcription unit (DBHD including sequences encoding the first 400 amino acids and the 5′ untranslated region (5′ UTR) will be replaced with a white marker gene. This should give rise to a DBHD null allele . One copy of DBHD-res could rescue the mutants to healthy adults without any obvious abnormalities compared with the heterozygotes. These results revealed that we obtained a clean null allele of DBHD.We performed several experiments to check the mutation. We first did PCR analysis and confirmed the targeting effects on both arms as predicted (DBHD mutants (hereafter referred to as −/−DBHD) never survived to adults. To trace the developmental progresses, we combined the −DBHD mutant allele with a GFP-marked balancer chromosome . Firstly, we wanted to check if the mutation had any dominant effects. We compared the developmental profiles of the heterozygotes  and another strain containing an isogenized wild-type third chromosome (recovered from single 1118w fly) with the same balancer chromosome . Embryos were collected within three hours and allowed to develop in stable environment . Under such conditions, the heterozygotes behaved similarly with the wild-type controls  by developing into healthy adults at around the same time point  hatched normally . The −/−DBHD larvae could survive for a prolonged period of time (up to three weeks). Eventually, all mutants died as small larvae. We separately cultured the mutants and the heterozygotes in different food vials. Each vial contained no more than ten newly hatched larvae. This should provide each individual with sufficient food and space, and minimize the potential toxicities brought by the crowds or heterozygotes. The −/−DBHD larvae still showed the same growth retardation phenotype, which could rule out the weakness in intraspecific competition as a causal mechanism.Based on the statistical analysis and direct check under fluorescence microscope, we found that the Drosophila zygotic mutants can survive the embryogenesis because of the large amounts of maternal gene products deposited in the eggs. To exclude the potential maternal contributions, we generated −/−DBHD germline clones by removing the DBHD products at the beginning of egg formation −/−DBHD germline clones showed no difference with the zygotic mutants in development: they successively passed through the embryo stage and died before pupation. We conclude that DBHD plays essential roles in the larval stages.Some Drosophila larvae feed continuously to increase their body mass dramatically before the onset of pupation. The larval imaginal and endoreplicative cells make the main contributions to this change. The imaginal cells, including those in the imaginal discs, the gonads and the brain, go through active mitosis to increase the cell number. In contrast, the endoreplicative cells from the gut, salivary gland and fat body, increase the DNA polyploidy and cell volume without further cell divisions After hatching from eggs, the −/−DBHD larvae had tiny or even no visible imaginal discs. The brains were also reduced in size .Next, we checked their growth responses to nutrient food. We picked no more than twenty newly hatch larvae, put them on culture medium with different concentrations of yeast and analyzed their developmental profiles Table 1.DBHD had no gross influences on cell fate specifications  and amino acids. The larval growth will be delayed if any of these components is limited nutrient Table 2.orphosis . We conc−/−DBHD larvae fed normally. Next, we checked their abilities of food digestion and amino acid absorption. The third instar larvae were homogenized. The concentrations of 16 free amino acids from the whole extracts (including those in the haemolymph and cytoplasm) were measured using the automatic amino acid analyzer . With either normal food or yeast paste, no significant differences were observed between the heterozygotes and the −/−DBHD larvae . We generated a UAS-hFLCN transgenic fly and expressed the human FLCN gene under the control of hsp-Gal4 driver. The newly hatched larvae fed with normal food were heated for 30 minutes at 37°C, twice a day, to ubiquitously induce the expression of hFLCN. No rescued −/−DBHD flies eclosed. However, some −/−DBHD larvae expressing hsp/hFLCN (about one third) could develop into pupae with no clear defects of the cuticles . The transcriptions were under the control of native DBHD promoter and the upstream sequence till the adjacent gene. Either transgene could fully rescue the −/−DBHD animals into healthy adults. Therefore, the EGFP signal should be able to monitor the essential localizations of DBHD proteins. Both transgenes showed the same EGFP expression patterns, suggesting the DBHD proteins functioned in full length throughout their lifetime.We made two DBHD-res; −/−DBHD) to check the expression of DBHD-res, so that there were no endogenous DBHD proteins. The EGFP is broadly present in larval phases, including the imaginal discs, fat bodies, central nervous system (CNS) and midguts (Deadpan (dpn) DBHD-res in most, if not all neuroblasts in the larval CNS . These results suggest that DBHD is not required cell-autonomously in these tissues.The Drosophila and mammals. An interesting discovery from our fly model is that the growth defects of DBHD mutant larvae could be substantially rescued by nutrient. It is, therefore, of great interest to investigate if modulation of the local nutrient conditions is beneficial for the treatment of BHD lesions in mammalian systems.In summary, we developed a new animal model of the BHD syndrome and demonstrated that some functions of FLCN are conserved in −/−DBHD animals. DBHD either functions in parallel with, or is directly involved in the leucine-mediated mechanisms. Because the normal food can support the growth of heterzygotes, but not −/−DBHD, we speculate that leucine must play roles other than protein synthesis. So far, the best known role of leucine as a signaling factor is to activate mTOR dTOR by rapamycin reversed the rescue effects of leucine or yeast.It is not clear why nutrient (particularly leucine) could rescue the growth defects of DBHD is involved in dTOR signaling, as it does in mammals, we propose here one mechanism that DBHD functions to sequester amino acids within cellular organelles to activate dTOR in some Drosophila tissues. DBHD does not encode a typical membrane protein and DBHD-res is mainly expressed in the cytoplasm of several cell types , they eventually stop development before eclosion.Unlike mice, DBHD controls the growth of the imaginal disc cells in a non-cell-autonomous manner, which does not support a classical anti-tumor function. Our work suggests that at least in the Drosophila imaginal discs, DBHD controls growth through some neuronal/hormonal mechanisms. Consistently, DBHD-res is expressed in various endocrine cells or tissues, including the gut epithelia, fat body, brain, and ring glands (Drosophila insulin-like peptides (DILPs). Flies missing these products will develop the starvation-like phenotypes. At present, we are analyzing the functions of DBHD in these places.It is surprising that g glands Figure 6


Microchimerism is the presence of cells from one individual in another genetically distinct individual. Pregnancy is the main cause of natural microchimerism through transplacental bidirectional cell trafficking between mother and fetus. The consequences of pregnancy-related microchimerism are under active investigation. However, many authors have suggested a close relationship linking fetal microchimerism and the development of autoimmune diseases. It has been more than ten years now since the demonstration of the presence of a significant high number of fetal microchimeric cells residing in thyroid glands from operated patients with Graves' disease. This intrathyroidal fetal microchimerism is an attractive candidate mechanism for the modulation of Graves' disease in pregnancy and the postpartum period. Microchimerism is defined by the presence of alien cells within an individual tissue with genetically different background . Microch Contrary to previous expectations, the placental trophoblastic physical barrier effect is not a perfect cutoff system. A certain cell leakage is present between mother and fetus during gestation, and this transplacental cell trafficking is a two-way process. Fetal cells movement into maternal circulation starts very early during pregnancy. Circulating fetal cells have been found in maternal blood as soon as the fourth week of gestation , whereas So far, only the human leukocyte antigen (HLA) compatibility between mother and fetus has been identified as a factor influencing the persistence of microchimeric cells. Microchimerism and chronic graft versus host disease resemble each other. Both diseases share many clinical and pathological features with autoimmune diseases. Since autoimmune disease are more frequent in childbearing age females, it has been hypothesized that fetal microchimerism may be involved in their etiology. For a successful pregnancy, the maternal immune system must not overreact to the fetus. Dramatic changes throughout gestation make possible maternal tolerance of the fetus and permit fetal cells to move into maternal circulation and settle in maternal tissues. As a result, maternal tolerance allows the persistence of fetal microchimerism . Intrathyroidal fetal microchimerism has been reported using different techniques. PCR-based analysis identifies Y-chromosomal and merely demonstrates the presence of male cells in maternal tissues , 12. Oth According to cellular characterization, fetal microchimeric cells could cover a wide spectrum of action. Experimental data support a variety of important hypotheses concerning their biological implications. The types of cells crossing the placenta into the mother include both immune cells and cytokeratin-positive epithelial cells. Such cells have been identified as hematopoietic progenitor cells, nucleated erythrocytes, trophoblast cells, and leukocytes . Therefopathogenic, beneficial, and neutral microchimerism [pathogenic microchimerism initially suggested by Nelson [beneficial microchimerism, where persistent fetal cells may have a beneficial effect as a new source of progenitor cells potentially capable to contribute to maternal tissue repair processes. The third possibility could be neutral microchimerism, where fetal cells may act as innocent bystanders playing no role in biology at all. We have previously proposed a three-role division for fetal microchimerism, which covers himerism . The cony Nelson , hypothe In line with our proposed microchimeric fetal cells varied effects, Fugazzola et al.  have rec For a successful pregnancy, the maternal immune system must not overreact to the fetus. The mechanisms through which the immune systems between mother and fetus interact to induce and maintain this immune tolerance are not fully understood, but several changes have been described. Trophoblast cells serve both as physical barrier as well as immune modulator by expressing several molecules and secreting specific cytokines. This involves the expression of Fas-ligand, cytokines, indoleamine 2,3-dioxygenase, immune-modulating sex steroids, and HLA-G. Fas-ligand is involved in the removal of maternal T-cell clones that react to fetal antigens. In animal models, indoleamine 2,3-dioxygenase catabolizes tryptophan from maternal immune cells in the placental area, which is essential for a successful pregnancy in murine models, whereas HLA-G, exclusively expressed on trophoblast cells, inhibits natural killer cell-mediated cellular immunity . These changes also include a reduction of T-regulatory cells activity and affect maternal T-helper (Th) cell differentiation. Placental immune modulation promotes suppression of Th1 (cellular), whereas relative enhancement of Th2  immunity occur. Thus placental immune suppression helps establish fetal microchimerism. Immune tolerance to fetal implant allows pregnant woman to accept fetal circulating cells . Consequ This complex modulation of the maternal immune system by pregnancy is also reflected by the variation of circulating levels of autoantibodies. Furthermore, there is compelling evidences that circulating thyroid autoantibodies are predictors for the increased risk of pregnancy complications like miscarriage, breech presentation, or prematurity . Probabl At the same time, it has been hypothesized that pregnancy-related immune changes have important positive effects for the fetal immune system. There is evidence suggesting that maternal immune cells instruct fetal cells how to balance the requirement for self-defense on one hand and the need for immunologic tolerance on the other. There is still much to understand about tolerogenic versus immunogenic forms of microchimerism, both of which have been reported . Taken together, these different observations a question may be raised whether the sequence of events in autoimmunity is at least partly due to alloimmunity rather than autoimmunity.It is well recognized the highest prevalence of autoimmune diseases in childbearing-age women than in men. It is also well known that autoimmune diseases have a profound influence on pregnancy outcome , 25–27.  Fetal cells have been found not just in peripheral blood but also within a variety of damaged tissues, where the autoimmune reaction is taking place , 28–31.  On the other hand, it should be emphasized that a positive effect of pregnancy on autoimmune disease is generally observed. As previously mentioned, the placenta induces immune suppression and so lessens autoimmune activity. Actually, the amelioration of autoimmune clinical manifestations along gestation is a usual clinical observation. Experimental data suggest that, despite the relative enhancement of Th2 reaction, both arms of the immune response (Th1 and Th2) are globally reduced during pregnancy. The observation is supported by the increase in T-regulatory cells observed in pregnancy and because autoantibodies greatly decrease during pregnancy .Assuming that part of microchimeric fetal cells could be progenitor cells of the fetal immune system, the pathogenic effect of fetal microchimeric cells in autoimmune diseases can be perpetuated. These cells could survive in bone marrow or move to maternal different organs, where they could proliferate, differentiate, and activate. The activation of fetal immature T cells, monocytes, macrophages, and natural killer cells and the production of inflammatory cytokines and chemokines are believed to initiate then autoimmune diseases , 34. Alt Finally, this potential association is also supported by the observation that, albeit with few exceptions, the resetting to normal immune status in the postpartum period is usually coincidental with a clinical exacerbation of many autoimmune diseases. Several evidences have shown that female subjects with autoimmune thyroid diseases frequently have microchimeric fetal cells residing within their thyroid glands , 11, 12. Davies' laboratory has extensively investigated the influence of pregnancy in autoimmune thyroid diseases, including the relationship between fetal microchimerism and autoimmune thyroid diseases , 35. The Davies' group, going forward in the study of fetal microchimerism as a cause involved in the development of autoimmune thyroid disease, used their experimental autoimmune thyroiditis model using sex-determining region Y (SRY) gene as the marker of presence of male microchimeric cells within maternal thyroids. The results demonstrated the presence of fetal cells in 46% of Tg-immunized pregnant mice, whereas few male fetal cells were detected in only 20% of controls or nonimmunized pregnant mice. Subsequent studies of cell characterization revealed the immune origin of the cells that accumulate within the thyroid of mice with experimental autoimmune thyroiditis during pregnancy and early postpartum . These f As aforementioned, several clinical studies have found male cells in thyroid samples of women previously diagnosed of Hashimoto's thyroiditis –14, 37. Pregnancy-related factors have a strong influence in Graves' disease . Normall In the postpartum period as the pregnancy-associated immune-privileged state disappears, a relapse, exacerbation, or new onset of Graves' disease may occur. This outbreak normally happens between 4–12 months after delivery . In factIt was also Davies' group who in 2002 first demonstrated that intrathyroidal fetal microchimerism was common and profound in female patients with Graves' disease . Renné e Davies' group research was conducted in a sample of 27 thyroid glands from patients with a past medical history of Graves' disease . The inv5, with a median of 37/105 male/female cells. SRY gene was searched in 10 thyroid adenoma specimens (6 in paraffin and 4 frozen). None of the 6 paraffin-embedded thyroid adenoma was positive for the SRY gene analysis, whereas 1 out of 4 frozen samples with thyroid nodules showed male cells. Authors speculate that the greater detection of the SRY gene in frozen female thyroid tissues was probably due to DNA fragmentation in the paraffin-derived samples. Unexpectedly, many of the patients with male cell-positive thyroids had no history of earlier male pregnancies at the time of surgery. However, as the authors stated in the discussion, this did not necessarily exclude the possibility of undetected first trimester pregnancies because it has been demonstrated that fetal microchimerism can be established in the first month of pregnancy [ A subsequent analysis was focused on thyroid samples from females previously diagnosed of Graves' and in a group of thyroid adenomas as control. The storage of the thyroid samples had been diversed. Twenty glands had been prepared in paraffin blocks, whereas the remaining seven were frozen. Interestingly, SRY gene was found in only 20% of paraffin embedded tissues, while 86% of the frozen samples were positive for the Y chromosome gene. In the former group, the ratio of male to female cells ranges from 14 to 295 by 10regnancy , 46.in situ hybridization for X chromosome and Y chromosome specific staining from paraffin-embedded thyroid specimen taken at surgery. The results showed that 23 out of 49 thyroids (47%) were positive for Y-chromosome-specific staining. These authors found no Y-chromosomes-positive thyrocytes. The proportion of Y-chromosome-positive thyroid section was highest in Hashimoto's thyroiditis , lower in Graves' disease , and infrequent in follicular adenomas  [Renné et al. study compared three entities: Graves' disease, Hashimoto's thyroiditis, and nodular or diffuse follicular adenomas from women whose childbirth history was positive for sons . These i/9; 22%) . The res/9; 22%) , 13.A summary of these findings is presented in  Those dissenting from the appraisal of the relation between autoimmune thyroid disease and microchimerism mainly base on data from epidemiological studies. An Australian study over a large community-based female population  showed no association between parity and presence of thyroid autoantibodies or thyroid dysfunction. Hence, the authors suggested a lesser role of fetal microchimerism in autoimmune thyroid diseases . A year  Currently, there is a large body of evidence showing that maternal thyroid gland is a significant organ where a variety of fetal cells settle and persist for decades. These foreign immune cells may be activated after delivery once placental immune suppression is over. This remains an attractive hypothesis for the postpartum increase of prevalence of Graves' disease.


However, identification and functional validation of chromatin-interacting RNAs are technically challenging with respect to distinguishing RNAs that bind chromatin modifying machinery from artifacts.Long non-coding RNAs (ncRNAs) are increasingly recognized as important regulators of genomic processes and cell specification. Many IncRNAs are hypothesized to localize to and regulate chromatin, functionally impacting epigenetic state through interactions with chromatin-modifying proteins’. Numerous IncRNAs have been reported to play a role in the activity or recruitment of the epigenetic factors the Polycomb group (PcG) proteins to genomic sitesWe developed a method to enrich for bonafide ncRNA-PcG interactions on the DNA. Chromatin, the substrate of the PcG proteins, was purified away from the cytoplasm and soluble fraction of the nucleus in crosslinked HeLa cells. The PcG component Bmi1, and a stable FLAG-Bmi1 were then specifically immunoprecipitated from the chromatin extract. Bmi1-associated ncRNAs were biochemically isolated and further studied with the aim of validating and understanding the roles of individual IncRNAs in PcG biology. Cross-validated Bmi1-interacting candidates were compared with immunoprecipitations against replicate Bmi1 samples, additional PcG components, and other chromatin protein controls .We have identified strong candidates at known IncRNA sites that are expressed from loci with distinct chromatin signatures. These noncoding RNAs are specifically and reproducibly enriched across replicates. For comparison, no mRNA exons are enriched in both FLAG-Bmi1 and Bmi1 pulldowns. Since mRNA exons are usually enriched in canonical RNA immunoprecipitations, but are considered a major source of noise, this protocol effectively eliminates a large number of false positives. Validation of individual candidates is underway, including siRNA screening for candidates which affect Bmi1 localization (ChIP) or expression of Bmi1 targets (RNA-seq).This technology offers an advantage over current RNA immunoprecipitation techniques by enriching the input for ncRNA-chromatin interactions, cross-validating results and employing stringent wash conditions and controls. Application of this technology to a variety of chromatin proteins yields distinct spectra of candidate IncRNA-chromatin interactions, and will enable future identification of IncRNAs that coordinate with proteins to direct chromatin state.