Treacher Collins syndrome  is characterized by deafness, hypoplasia of facial bones , antimongoloid slant of palpebral fissures, coloboma of the lower lid and bilateral anomalies of the auricle. Hypoplasia of the facial bones may be the first indicator of the disorder. We present a case report of Treacher Collins syndrome with their extraoral findings, intraoral findings and their treatment plan. We have also included the various etiological factors, clinical diagnostic aids, and multidisciplinary team approach. TCS is characterized by deafness, hypoplasia of facial bones , antimongoloid slant of palpebral fissures, coloboma of the lower lid and bilateral anomalies of auricle. It is a condition in which the cheek bones and jawbones are underdeveloped.2Treacher Collins syndrome (TCS), also called Treacher Collins-Franceschetti syndrome or mandibulofacial dysostosis, is an autosomal dominant disorder affecting the development of structures derived from the first and second brachial arches during early embryonic development.A 9-year-old boy reported to the department of pedodontics and preventive dentistry, AJ institute of dental sciences, Kuntikana, Mangalore; complaining of retained deciduous teeth in the upper anterior region. The patient’s mother also complained of a symmetry of his face and deafness .On general examination, the patient was of short stature (115 cm), low weight (18 kg) and pallor was observed. On both the lower limbs, the 2nd toe was missing and webbing of the 3rd and 4th toes was seen .Ophthalmologicalfindings: Bilateral corneal opacity with adherent leucoma. On examination, the fundus was bilaterally hypermetropic with macular dystrophy.ENT findings: Conductive hearing loss, large and everted ears, narrow nasal cavity, soft palate drooping downward  .2The only gene currently known to be associated with TCS is TCOF1 which is mapped to chromosome 5 q31.3-q33.3, encoding a serine/alanine-rich protein, called ‘treacle’.3 There is no preference among genders or races and it consists of autosomal dominant trait of variable expressiveness. Its phenotypical expression probably results from bilateral congenital malformation involving the first and second brachial arches.45Thomson was the first to refer to this syndrome in 1846. In 1900, Dr E Treacher Collins, a British ophthalmologist, described two children who had very small cheek bones and notches in their lower eyelids. Therefore, the condition gets its name from him. For unaffected parents with one child with TCS, the chance of giving birth to a second child with the condition is negligible. Adults with TCS have a 50% chance of passing the condition to the offspring. When a parent with TCS passes on the genes, the children may be affected in varying degrees. The degree may be the same as the parent, milder or more severe. There are two possible ways that TCS develops. First, TCS can develop as a new mutation. This means that both parents pass on normal genes to their child. The second way that TCS develops is by inheriting it from one of the parents.67Neural crest cells migrate over extensive distances to the periphery of the face giving rise to most of the cartilage, bone, connective and peripheral tissues in the head. Most disorders of craniofacial development are thought to be caused by defects in the formation, proliferation, migration and/or differentiation of cranial neural crest cells and TCS is no exception. Hence, abnormal neural crest migration, ectopic cell death and inappropriate differentiation have all been hypothesized as underlying causes of TCS. Therefore, focuses on recent advances in our understanding of the basic etiology, pathogenesis and emerging prospects for prevention are required.These are the diagnostic features of TCS:Eyes I.  Antimongoloid slant of the palpebral fissures Colobomata and hypoplasia of the lower lids and lateral canthi Partial absence of eyelid cilia Hypertelorism.Ears External ear anomalies External auditory canal abnormalities Middle ear cavity ossicular deformities Conductive hearing loss results from variable degrees of hypoplasia of the external auditory canals and ossicles of the middle ears.Nose/mouth Respiratory compromise in severely affected patients as a result of the following two factors: Presence of maxillary hypoplasia, which tends to constrict the nasal passages and results in a degree of choanal stenosis or atresia. Presence of mandibular micrognathia and a retro-positioned tongue obstructing the oropharyngeal and hypopharyngeal spaces. Nasal deformity Microstomia Cleft palate with or without cleft lip High-arched palate Malocclusion Open bite.Facial bone malformation―the most characteristic findings are as follows: Hypoplasia of the malar bones Often with clefting through the arches. Limited formation of the residual zygomatic complex. Orbits Hypoplastic lateral aspects of the orbits Dysplastic inferior lateral orbit. Maxilla and mandible Characteristically hypoplastic Variable effects on the temporomandibular joints Anterior open bite A steep occlusal plane.138 Sleep apnea and sudden infant death syndrome. Nager’s acrofacial dysostosis Miller acrofacial dysostosis1 Oculoauriculovertebral spectrum. Radiographs and CT for evaluation of craniofacial abnormality Audiological evaluation for hearing impairmentDNA diagnosis: Direct sequencing of the coding and flanking intronic regions of TCOF1 defects mutations in about 90 to 95% of patients.1 Two-dimensional and preferably three-dimensional sonography Polyhydramnios Demonstration of characteristic facies of TCS, like downward slanting palpebral fissures, microgna-thia, abnormal appearance of the nose with narrow nostrils, cleft lip/palate, low set dysplastic ears and abnormal fetal swallowing. Amniocentesis or CVS To detect TCOF1 The disease causing allele of an affected individual must be identified before prenatal testing can be performed1 The presence of a TCOF1 mutation detected by prenatal diagnosis does not predict the specific malformation or severity of the disease.9 Depending on the severity of the TCS, the patient may need some or all of the following procedures: A conductive hearing aid, correction of the cleft palate, repair of the sidewall and floor of the eye socket, repair of cheek bones, repair of eyelid notches, correction of the undeveloped jaw and chin, surgery to correct the beak-like nose, reconstruction of the ear.10The current approach for TCS’s clinical deformities seeks functional and esthetical corrections as well as psychosocial support. Multidisciplinary approach, including otorhino-laryngologists, craniofacial surgeons, ophthalmologists, speech therapists, psychologists and pediatric dentist, is the most appropriate way to manage these patients. In addition to anatomical and physiological anomalies, TCS patients carry a social stigma because of its severe facial deformities.467The major problem in anesthesia lies on the maintenance of a free airway and intubation. During postoperative period, pharyngeal and laryngeal edema may develop even after pharyngoplasty. Cases of sleep apnea, respiratory distress and even sudden death have been reported.2Since the children with TCS often have problems with their teeth and jaw, the pediatric dentist will care for these specialized problems. There are circumstances in which teeth are absent or a patient may be unable to open the mouth. This makes the care of their teeth quite difficult; therefore, the special skills of a pediatric dentist are needed.TCS is an autosomal dominant disorder of craniofacial development which presents with unusual clinical features associated with abnormalities of structures derived from the first and second brachial arches, including antimongoloid slant of palpebral fissures, colobomas of the lower eyelid, eyelash malformations, deafness, hypoplasia of facial bones  and malar and mandibular defects. Management of TCS needs a multidisciplinary approach. The treatment plan is made to meet the individual patients need, considering the growth patterns, function and psychological development.


Chlorella ellipsoidea) was investigated. The reported 96-h LC50 values for the studied invertebrate species ranged from 72 to 962 μg/L. There was no significant difference between the sensitivity of aquatic invertebrate species from tropical and temperate regions. EC50 values for C. ellipsoidea, with and without pH buffer, were significantly different. The findings of this study can be used to support site-specific water quality criteria and environmental risk assessment for TCS in tropical regions. However, further chronic and semi-field experiments with TCS could potentially enable a refined assessment of direct and indirect effects on tropical aquatic communities and further explore functional endpoints of tropical ecosystems.The antimicrobial triclosan (TCS) has been detected in household wastewaters (untreated and treated) and receiving environments across the globe. The toxic effects of TCS on temperate standard aquatic test organisms have been widely reported with microalgae being the most sensitive. However, environmental differences between tropical and temperate regions may have selected different trait compositions between these two regions, which in turn may lead to a difference in species sensitivity. Therefore, additional information is required to better characterize risks to organisms in tropics and ensure biodiversity in these regions is not adversely impacted. This study aims to supplement existing TCS toxicity data with five aquatic invertebrates found in tropics and to compare the sensitivity between aquatic invertebrate species from tropical and temperate regions. In addition, the effect of pH on the toxicity of neutral and ionized forms of TCS to microalgae (The online version of this article (doi:10.1007/s11356-016-7302-0) contains supplementary material, which is available to authorized users. Ampelisca abdita and Chironomus plumosus, respectively phenol, TCS), an antimicrobial agent used in some personal care products, has been detected in household wastewater discharge and receiving environments including regions with poor connectivity to wastewater treatment facilities , Anostraca (Branchinella thailandensis), Oligochaeta (Tubifex tubifex), Trichoptera (Leptocerus sp.), and Decapoda (Macrobrachium lanchesteri). Details of the test organisms, toxicity test endpoints, and test conditions are provided in Table Baetis sp. and Leptocerus sp.) also occur in temperate regions, but unfortunately, due to a lack of identification keys, we could not evaluate whether we have tested a uniquely tropical species or one that has a cosmopolitan distribution and one species tested (T. tubifex) is cosmopolitan. However, these tests differed with a temperate test in two aspects. Firstly, these tests were performed under a tropical condition (the test temperature was around 29 °C), and the higher test temperature may increase the bioavailability of chemicals causing the species tested in tropics to be more sensitive , was purchased as Irgasan from Sigma-Aldrich. TCS stock solutions were made by dissolving TCS in acetone, and test mediums were prepared by diluting the stock solutions in a filtered water. To spike the stock solutions, the volume of acetone added in the test medium never exceeded 0.1 mL/L. Blank controls and solvent controls were performed in three replicates for each endpoint observation. The studied endpoints were immobility for 24-h test of B. thailandensis nauplii and mortality for other invertebrate species. Immobility of the tested nauplii was considered when no movement was observed after three-time repeated tactile stimulation with a laboratory needle. The wait period to confirm the immobility of nauplii was 3 s between each tactile stimulation. The observations of immobility and mortality of the nauplii were done under a microscope. The effects of TCS on the studied endpoints were recorded every 24 h throughout the 96-h exposure periods except for the B. thailandensis where the effect on mortality was assessed every 48 h during the medium renewal which was performed every other day  was assessed. Laboratory axenic cultures of C. ellipsoidea were obtained from the Inland Feed Research Institute, Department of Fisheries, Ministry of Agriculture and Cooperatives, Thailand. The culture was inspected for contamination on a weekly basis, and only log-phase algae were used for the toxicity tests. Two experiments were conducted in accordance with OECD guideline 201 (OECD 2C(CH2OH)3, ≥99.8 % Purity, was purchased from UNIVAR. A preliminary test was also performed to assess the effect of Tris buffer on the growth of algae prior performing the actual tests. The tests were conducted with an application of ultraviolet (UV) covers on the lights to avoid photolysis of TCS. Both tests were performed in 250-mL glass Erlenmeyer flasks containing 100 mL of test medium, modified BG-11  within the 72-h test period. Therefore, the initial cell density of the algal test with Tris buffer was adjusted to 5 × 104 cell/mL following the OECD guidelines and Roberts et al.  of methanol in Milli-Q water. If the sample contained a lot of suspension, it was re-dissolved in 1-mL methanol and filtered through 0.22 μm organic syringe filter. The known volume filtrate was subsequently transferred to another 2-mL amber vial and dried with nitrogen gas and finally re-dissolved in a solution of 10 % (v/v) of methanol in Milli-Q water prior to HPLC analysis.To verify the exposure concentrations, 3-mL sample of test media was taken from one replicate of the lowest and highest test concentrations at the beginning and at the end of each test. Two-milliliter subsamples of the test media from the invertebrate tests were transferred to 2-mL amber vials and allowed to settle for an hour in the dark, then 0.5-mL supernatant was taken for TCS determination by high-performance liquid chromatography (HPLC). The samples from algae tests required a use of liquid-liquid extraction method to remove microalgae cells from the samples. The aqueous TCS cannot be directly filtered through any filtered material as TCS can bind or adsorb into it. Therefore, the samples needed to be prepared in an organic solvent before the filtration. Firstly, the samples were extracted three times by dichloromethane . Secondly, the extracts were transferred into amber vials and then dried with nitrogen gas. Then, if the sample was relatively clean, it was re-dissolved in a mixed solution of 10 % (v/v) were used as the mobile phase. The injection volume was 100 μL and the flow rate was set at 1 mL/min. The UV wavelength for TCS detection was 205 nm. The limit of quantitation for TCS was 5 μg/L. The recovery of TCS analyses ranged from ca. 85–95 %.TCS was analyzed on the HPLC, Agilent 1260 Infinity series equipped with an UV detector. A Zorbax Eclipse Plus-C18 column  was used for the separation. The column temperature was set at 30 °C. Acetonitrile and 0.1 % acetic acid solution  were derived after a number of different exposure periods as described in Table http://cfpub.epa.gov/ecotox/). The data selection followed those outlined by Maltby et al. , T. tubifex (259 μg/L), Leptocerus sp. (760 μg/L), and M. lanchesteri (962 μg/L). In addition, this study demonstrated that a 72-h exposure time was sufficient to test these invertebrates. The 72-h LC50 values of Baetis sp., T. tubifex, Leptocerus sp., and M. lanchesteri were 96, 266, 866, and 1005 μg/L, respectively, which was not significantly different from the 96-h LC50 values. B. thailandensis test was only evaluated at 48-h intervals, and its 48-h LC50 (130 μg/L) was not significantly different from the 96-h LC50. The LC50 values at different exposure times for each species are given in the Baetis sp. shows that TCS is relatively toxic to Ephemeroptera. For TCS, no previous toxicity tests with this taxon have been conducted. However, based on acute toxicity data for other chemicals like insecticides, Ephemeroptera seem to be among the most sensitive aquatic invertebrates   to TCS, with a 96-h LC50 of 100 μg/L, as the test was performed with algal feeding. However, for the fairy shrimp nauplii, the 24-h EC50 and 24-h LC50 were 402 and 522 μg/L, respectively. These results are in accordance with a previous study of Kim et al.  than reported by Wang et al. (Limnodrilus hoffmeisteri (96-h LC50 of 2046 μg/L). This could be because the body size of T. tubifex is much smaller than L. hoffmeisteri, but probably is the effect of a larger fraction of neutral TCS present in the T. tubifex test as it was performed at pH 7.0 ± 0.2 while the L. hoffmeisteri test was performed at pH 8.0 ± 0.2 . Both data sets passed the Anderson-Darling goodness of fit test (α = 0.05) indicating that the studied toxicity datasets satisfied the normally distributed conditions. The results of two-sample Kolmogorov-Smirnov tests did not reveal significant difference between the sensitivity distributions of tropical and temperate aquatic invertebrate species . The SSD curve of tropical and temperate aquatic invertebrate species is shown in Fig. The selected data Table  for the As TCS concentrations have been detected in the tropical and temperate environments, the risk of TCS to the aquatic organisms should be evaluated. The risk estimation of TCS to the aquatic environment studied by Lyndall et al.  was perfBaetis sp., was the most sensitive and Decapoda, M. lanchesteri, was the least sensitive. This study provides useful information for environmental risk assessments of TCS in regions that have tropical aquatic environments. We demonstrate that there was no significant difference between the sensitivity distributions for acute toxicity to TCS for both tropical and temperate aquatic invertebrate species. This implies that the aquatic toxicity data used to support environmental risk assessment of TCS in temperate regions can potentially be extrapolated to support risk assessments in tropical regions. Due to its potential constant discharge via household wastewaters in tropical regions, additional chronic and semi-field experiments with TCS could help further evaluate direct and indirect effects of TCS on tropical aquatic communities and explore impacts on functional endpoints of tropical ecosystems. Such data would help evaluate whether temperate water quality criteria can routinely be used to protect all aquatic ecosystems across the globe. Finally, we confirm the importance of the test design concerning media pH used to assess the toxicity of TCS and highlight that existing risk assessments that rely on historic microalgal toxicity data, which fail to account for differences in pH and/or photolysis during toxicity tests, should be interpreted with caution.This study supplements existing surface water risk assessments for TCS and provides new insights into its toxicity to a range of aquatic invertebrate species in the tropics. Of the species tested, we demonstrate that Ephemeroptera, ESM 1(DOCX 22.8 kb)


A) receptor . In α1β2γ2 GABAA receptors there are five subunit interfaces: two β+/α- interfaces, and one of each α+/β-, α+/γ-, and γ+/β-; where the sidedness of the subunits is designated + and -. Figure A key target for the intravenous anesthetics propofol and etomidate is the γ-aminobutyric acid type A  has indicated presence of binding sites for etomidate and propofol [2N265I mutation, we individually altered one of the two sites, and were able to dissect the functional contribution of the two β+/α- interfaces. Etomidate acted almost exclusively at the β+/α- interface flanked by γ and β subunits with a minor contribution of the β+/α- interface flanked by α and γ subunits [Introduction of the βpropofol . SeveralA receptors were identified that excert their action exclusively through the β+/α- interfaces. Similar to etomidate they predominantly act at the β+/α- interface flanked by γ and β subunits, but in contrast to etomidate they do not act at the γ+/β- subunit interface. Both substances induced loss of righting reflex in Xenopus laevis tadpoles with potencies similar to propofol [The importance of the corresponding binding pockets for anesthetic action is underlined by the fact that recently two new allosteric modulators of GABApropofol  and reprA receptors harbor modulatory sites for these anesthetics in three of the five-subunit interfaces. The anesthetics etomidate and propofol use a different sub-set each. Pentobarbital seems to use at least one additional interface [1β2γ2 GABAA receptor by these popular intravenous anesthetics.In summary, we have demonstrated that etomidate exerts its function predominantly through the γβ+/α-β and γ+/β- subunit interfaces while propofol acts predominantly at the γβ+/α-β, αβ+/α-γ and γ+/β- subunit interfaces. GABAnterface . Taken t


Ex vivo exposure of the organ of Corti from mice exposed to As significantly decreased the number of auditory neurons and fibers. Thus, our combined study showed that oral exposure to As caused hearing loss in young people and young mice.There is no information on the association between oral exposure to arsenic (As) and hearing loss in humans or mice. In this combined epidemiological study and experimental study, the association of oral exposure to As with hearing loss in people aged 12–29 years and young mice was examined. Subjects in the exposure group (n = 48), who were drinking tube well water contaminated with As, showed significantly higher risks of hearing loss at 4 kHz , 8 kHz  and 12 kHz  than did subjects in the control group (n = 29). We next performed an experiment in which young mice were exposed to As via drinking water at 22.5 mg/L, which is a much greater concentration than that in human studies. The exposure group showed hearing loss and accumulation of As in inner ears.  About 20 million people in Bangladesh are exposed to As by drinking well water in which As levels are above the national standard of 50 µg/L4. A previous study using univariate analysis showed that 10-year-old children in an As-polluted area had hearing losses at frequencies of 125, 250 and 8,000 Hz5. Thus, exposure to As is a potential risk for hearing loss in children. However, there has been no epidemiological study with multivariate analysis showing an association between oral exposure to As and hearing loss in young people aged 10–30 years.Contamination of arsenic (As) in drinking well water has been reported worldwide6. A previous study showed that intraperitoneal injection of As at 200 mg/L into guniea pigs for 2 months resulted in morphological impairments of the inner ears7. Thus, it is possible that exposure to As induces hearing loss in experimental animals. However, there is no direct evidence about whether oral exposure to As via drinking water causes hearing loss in mice determined by auditory brainstem response (ABR). It is also unclear whether oral exposure to As via drinking water modulates the As level in inner ears of mice.Inner ears, which are important organs for hearing, contain the organ of Corti with inner hair cells (IHCs) and outer hair cells (OHCs) and spiral ganglion neurons (SGNs)In this study, we examined the association of oral exposure to As with hearing loss in young people and young mice. For this purpose, we compared hearing levels of young people aged 12–29 years in a control group drinking tap water and an exposure group drinking tube well water contaminated with As in Bangladesh using a multivariate analysis adjusted for age, sex, body mass index (BMI) and smoking. We compared hearing levels by ABR and accumulation levels of As in inner ears of wild-type mice exposed to As via drinking water. We then tried to clarify the etiology of As-mediated hearing loss.The basic characteristics of the subjects are shown in Table 12. We categorized hearing levels with the mean values  of hearing thresholds in young people in this study. The multivariate analysis showed significantly higher risks of hearing loss at 4 kHz , 8 kHz  and 12 kHz  in the exposure group than those in the control group . Before exposure to As, hearing levels in the control group  and exposure group  were comparable Fig. . After e13. Correspondingly, our epidemiological data also showed significant correlations between As levels in hair and hearing loss at 4 kHz , 8 kHz  and 12 kHz  in humans  is about 100-times higher than the maximum level (221 µg/L) of As detected in tube well water in this study. On the other hand, our epidemiological study showed that the duration of drinking tube well water was also associated with hearing loss at 4 kHz , 8 kHz  and 12 kHz   Figure , suggestex vivo study, exposure of the organ of Corti to As at 0.3 µg/mL, which was detected in inner ears of mice orally exposed to As in vivo, significantly decreased the number of auditory fibers and SGNs but not hair cells. Thus, our results indicate the possibility that 0.3 µg/g of As that accumulated in inner ears caused a decrease in the numbers of the auditory nerve fibers and SGNs in vivo. In previous studies, aquaporins (AQPs) including AQP9 were shown to mediate cellular uptake of As in mammalian cells21. AQP9 was shown to be expressed in SGNs but not hair cells in the organ of Corti22. Therefore, it is possible that As accumulated in SGNs via AQPs caused a decrease in the numbers of auditory nerve fibers and SGNs. Previous studies also showed that As-mediated neurotoxicity was involved in apoptosis and demyelination of peripheral neurons including dorsal root ganglion (DRG) neurons24. As-mediated neurotoxicity was also shown to be involved in increased levels of oxidative stress markers including heme oxiygenase 1 (HO-1) and decreased levels of procaspases 3 and 12 in DRG explants25. Further study is needed to determine whether in vivo exposure to As induces oxidative stress, apoptosis and demyelination in auditory neurons and to elucidate the molecular mechanism by which hair cells were not affected by exposure to As in this study.In our In conclusion, our combined study suggests that exposure to As via drinking water causes hearing loss in young people and young mice. Hearing loss in young people has a negative impact on the quality of life. Therefore, further study is needed to determine the correlation between hearing loss and oral exposure to As via drinking water in order to prevent As-mediated hearing loss in humans.2). In this study, subjects were defined as being underweight if their BMI was less than 18.5 kg/m2, being of normal weight if BMI was 18.5–24.9 kg/m2 and being overweight if BMI was higher than 25 kg/m2 as set by the WHO26. We measured hearing thresholds at 1, 4, 8 and 12 kHz in the subjects by pure tone audiometry (PTA)27. For each frequency, the sound pressure level was increased from 0 dB to 90 dB in 5-dB steps until the subject showed a response. The epidemiological study was approved by Nagoya University International Bioethics Committee following the regulations of the Japanese government  and the Faculty of Biological Science, University of Dhaka (Ref. no. 5509/Bio.Sc).We performed a survey using self-administered questionnaire regarding age, smoking, sex, weight and height for 77 healthy subjects aged 12–29 years in Bangladesh. Informed consent in written form was obtained from all of the participants in the study. Subjects in the control group and subjects in the exposure group were drinking tap water and tube well water contaminated with As, respectively. Subjects with a drinking habit or a habit of using earphones to listen a music did not participate in this study. In addition, another ethnic group or race was not included as subjects in this study. In brief, BMI was calculated as weight (kg) divided by the square of height (m2) purchased from Sigma-Aldrich was dissolved in distilled water. We newly prepared drinking water containing As and changed the drinking water every week. We gave distilled water to the non-exposure group. We started the exposure experiment at 1 month of age. ABR measurements (AD Instruments Pty. Ltd.) were performed as described previously28. Tone burst stimuli were measured 5 dB-stepwise from 0 dB SPL to 90 dB SPL. The threshold was judged by the appearance of the lowest level of I-IV waves of ABR. Data are presented as means ± SD. The experimental study was approved by the Institutional Animal Care and Use Committee in Nagoya University  and followed the Japanese Government Regulations for Animal Experiments.Hairless mice having a C57BL/6 J mice background were purchased from Hoshino Laboratory Animal, Inc. The mice were maintained under specific pathogen-free (SPF) conditions at a fixed temperature (23 ± 2°C) and a 12-h light/dark cycle. Sodium arsenite  H2O2 was added and the samples were immediately incubated at 80°C for 3 hours. After cooling, the samples were diluted by Milli-Q water. For urinary samples, a 15 ml tube was filled with 4 ml of a urine sample followed by addition of 1 ml HNO3, and the tube was left overnight. The samples were incubated at 80°C for 24–72 hours until the solution became clear. After centrifuging for 1 min, the samples were adjusted with Milli-Q water up to 5 ml, and the As levels were measured by ICP-MS. Urinary As levels were normalized for dilution by specific gravity (SG) adjustment using the following formula27:target is a population mean SG. In this study, the target SG used was 1.012 for subjects. The levels of As in inner ears were determined by the same protocol as that described above.We collected biological samples  and performed measurements by ICP-MS (Agilent 7500cx) as described previouslyex vivo culture of the organ of Corti29. In brief, after dissection of the organs of Corti from C57BL6/J mice at postnatal days 4–6, the cochleas were placed in HBSS buffer. The cochlears were dissected and the organs of Corti containing IHC, OHC, auditory nerve fibers (ANFs) and SGNs were placed in a 6-well dish. D-MEM (low glucose) medium containing 10% FBS and 10 µg/mL of ampicillin was added to the dish. A 0.3 mg/mL stock solution of As was diluted in the culture medium to a final concentration of 0.3 µg/mL. The organ of Corti was placed in an incubator . On the next day (day 1), we removed the medium and added a fresh medium for the control group and exposure group (0.3 µg/mL of As). On day 2 and day 3, the medium was removed and the organ of Corti was washed three times with 0.1 M PBS. Specimens were fixed with 10% formalin for 1 hour and rinsed with PBS. To detect SGNs and auditory fibers, specimens were immunolabeled with a solution containing 1 µL of rabbit anti-neurofilament 200 kD  primary antibody, 40 µL of 10% Triton X-100, 6 µL of normal goat serum and 153 µL of PBS overnight at 4°C. On the second day, specimens were washed with PBS and immersed in a solution containing 1 µL of a secondary antibody, 20 µL of 10% Triton X-100, 6 µL of normal goat serum and 173 µL of PBS for 2 hours at room temperature. To evaluate hair cells, we labeled specimens with fluorescein-phalloidin . The specimens were immersed in a solution containing 1 µL of fluorescein-pholloidin (6.6 µM stock solution) and 199 µL of PBS. After washing with PBS, the specimens were mounted on glass sides, covered with antifade reagent, and observed with Super-resolution/confocal Microscopy (Carl Zeiss Microscopy LSM880-ELYRA PS.1). Hair cells were counted in the width (100 µm) of the field of view of the of microscope at 40X. The ANFs (length > 50 µm) and SGNs were counted in the field (100 µm × 100 µm) of view of the microscope at 40X. Ten specimens were examined for each group.We partially followed the previous method of 27. Since the Shapiro-Wilks test showed that As levels in biological samples and hearing thresholds for PTA and ABR of all frequencies were non-normal distributions, the Mann-Whitney U test to evaluate statistical differences of As levels and hearing levels between two groups. For multivariate analysis, we performed binary logistic regression analysis with adjustments for age8, sex9, BMI12 and smoking11 as confounding factors. In this study, values of p < 0.05 were considered statistically significant.All statistical analyses were performed by JMP Pro  as described previouslySupplementary Information


GJB2) gene are the most common cause of hearing loss. Of these, the p.V37I variant of GJB2 has a high allele frequency (up to 10%) in East Asians. Characterization of the phenotypic spectrum associated with p.V37I, as well as the role of this variant in the onset of hearing loss could have a remarkable effect on future diagnostic strategies. Here, we performed a pedigree analysis of unrelated families exhibiting various hearing phenotypes, and then conducted a meta-analysis to comprehensively assess the association between the p.V37I and the risk of hearing loss. Pedigree analyses showed that both homozygous p.V37I variants, as well as compound heterozygous p.V37I with other GJB2 pathogenic variants, contributed to various phenotypes of hearing loss. Meanwhile, meta-analysis demonstrated that, compared with those in the wild type group, both p.V37I homozygotes and compound heterozygous p.V37I variants were at significantly higher risk of developing hearing loss . Conversely, heterozygous p.V37I variants alone did not increase the risk of hearing loss. Given the high allele carriage rate of p.V37I (up to 10%) within the general population, our work not only provides information that might influence future genetic screening policies, but also offers insight into clinical risk evaluation and genetic counseling regarding hearing loss.Pathogenic variants in the gap junction protein beta-2 ( To date, greater than 300 variants within GJB2 have been found to be associated with HL (http://www.hgmd.cf.ac.uk/ac/), including c.35delG, c.235delC, and c.176_191del16.Hearing loss (HL) is the most sensory defect that affects 1-3 in every 1,000 newborns worldwide, and half of these cases are attributed to genetic factors [GJB2 has a high allele frequency (up to 10%) among East Asian populations [GJB2 pathogenic variants, indicates that p.V37I likely increases the risk of HL, particularly for mild-to-moderate cases [In particular, the p.V37I (c.109G>A) variant of ulations –6. This ulations . Howeverte cases –15. Intete cases .Given the high allelic frequency of the p.V37I variant (up to 10%), it is estimated that greater than five million East Asians suffer from HL due to homozygous or compound heterozygous carriage of this allele . It is tGJB2 gene. Pedigree analyses revealed that p.V37I was transmitted from heterozygous parents to their children, who suffered HL if he/she inherited two affected alleles (p.V37I homozygotes or compound heterozygous p.V37I variants). Meanwhile, siblings that inherited one affected allele retained normal hearing. These analyses strongly suggest that p.V37I increases the risk of HL through an autosomal recessive inheritance pattern.Seven families carrying the p.V37I variant were included for pedigree analyses Table . There wTo further determine the detrimental effects of p.V37I, we performed a meta-analysis. A flow chart of the literature search is shown in heterogeneityP < 0.001; 2I = 74.9%). Moreover, compared with individuals with wild type, p.V37I homozygotes , but not p.V37I heterozygotes , had a significantly higher risk (7.14-fold greater) of developing HL. Similar results were obtained using the recessive model . Sensitivity analyses demonstrated that our results were quite stable (P > 0.10).As shown in Table e stable  and no oheterogeneityP = 0.060; 2I = 44.9%). These results were strongly supported by sensitivity analyses (P = 0.152).Furthermore, we evaluated the association between compound heterozygous p.V37I variants on HL risk. Ten eligible studies comprising of 6,762 cases and 4,211 controls were included  were screened in our pedigree analysis. Second, the number of studies regarding compound heterozygous p.V37I variants evaluated herein was insufficient to further explore the concrete effects of particular compound variant types on HL risk. Third, although both our study and previous reports found that p.V37I is associated with various HL phenotypes, the specific mechanism by which this variant promotes HL, such as interactions between p.V37I and other genetic or environmental factors, remains unclear. Further studies are therefore needed to address these issues.The major strengths of our work were the variable phenotypic spectrum associated with p.V37I and that a large population  was used to evaluate the association between this variant and HL risk. However, our results should be interpreted with caution. First, only commonly known HL-related variants . For very young participants, auditory brainstem responses (ABR) and/or auditory steady-state responses (ASSR) were recorded. Degree of hearing loss was estimated as the average hearing levels at 0.5, 1.0, 2.0, and 4.0 kHz for the better ear. Severity of hearing loss was categorized as normal (<25 dB), mild (26-40 dB), moderate (41–70 dB), severe (71–95 dB), or profound ( >95 dB). In addition, we defined sudden deafness as over 30 dB of sensorineural hearing loss involving at least three frequencies occurring less than 72 hours .GJB2, SLC26A4, 12S rRNA, and GJB3, were polymerase chain reaction (PCR) amplified and then subjected to bidirectional sequencing with an ABI 3500 DNA sequencer (Applied Biosystems). The primers and PCR conditions used for these analyses are described in detail in Genomic DNA was extracted from anticoagulant peripheral blood by the QIAamp DNA blood mini kit . For mutation screening, the entire coding region and flanking sequences of four commonly mutated genes, GJB2 OR connexin 26”, “polymorphism OR variant OR mutation”, “hearing loss OR deafness”, and “case-control OR cohort OR population”. Only English language articles were included. The inclusion criteria were: (i) studies that investigated the association between p.V37I and HL under a case-control or cohort design; (ii) studies that reported genotypes or allelic data for p.V37I to estimate OR and 95%CI. In cases of overlapping populations, the study with the largest sample size was included. We used OR as the effect measure and combined data using a random-effects model. Hardy-Weinberg equilibrium (HWE) in the controls was checked by χ2 test. Meanwhile, Cochran χ2 test and 2I values were used to evaluate the heterogeneity between studies. Sensitivity analyses and publication bias assessment were also conducted. All statistical analyses were performed using Stata 12.1 software , and P ≤ 0.05 was considered significant for all tests.We further performed a meta-analysis to determine the detrimental effects of p.V37I on sporadic HL, according to the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . A struc


L/A scores worsened by 57% and 13%/14%, respectively. Using the hearing aid without CR treatment for 5 months no change in tinnitus symptoms was observed. However, after three months of CR treatment delivered through the hearing aids, the THI and VASL/A scores were reduced by 70% and 32%/32%, respectively.Primary tinnitus has a severe negative influence on the quality of life of a substantial portion of the general population. When acoustic coordinated reset (CR) neuromodulation stimuli are delivered for several hours per day over several weeks a clinically significant symptom reduction in patients with primary tonal tinnitus has been reported by several clinical sites. Here, we reported the first case where CR neuromodulation was delivered through a hearing aid. A 52-year-old man with chronic primary tonal tinnitus was previously considered untreatable with sound therapy. He initially received the classic CR treatment protocol with signals delivered with the separate proprietary device with his hearing aids removed during treatment. He was subsequently treated with the therapy being deployed through a set of contemporary hearing aids. After 5 months of classic CR treatment with the separate custom device, the THI and VAS Primary (subjective) tinnitus is an idiopathic symptom that may or may not be associated with sensorineural hearing loss . The preSensorineural hearing loss, especially that associated with noise exposure and aging, can result in a reduction in the number of functioning sensory hair cells in the cochlea. Although sensory hair cell loss can lead to a sensorineural hearing loss, it is highly unlikely that the hair cell loss itself is the source of the tinnitus. It is much more likely that the deafferentation process can trigger abnormal neuronal synchrony in the central nervous system and, if persistent, can lead to an abnormal upregulation of synaptic connectivity to other brain regions resulting in chronic primary tinnitus –10. ChroAcoustic coordinated reset (CR) therapy is a sound-based therapy supported by neuromodulation principals –23. UtilIn these studies, the daily therapy signals were delivered by a custom device (T30 CR neurostimulator) connected to earphones by cables, an arrangement that is not ideal. The use of the therapy devices requires that hearing aid patients remove their hearing aids during therapy so the benefits of the hearing aids are removed during the therapy, an important consideration because a large percentage of patients with chronic primary tinnitus also have a degree of hearing loss that warrants hearing aids and the use of hearing aids in tinnitus patients itself can reduce tinnitus symptoms –31. FurtWe report results obtained by the delivery of acoustic CR therapy via hearing aids and results obtained by delivery via a separate proprietary sound system in a single hard to treat patient.One male patient (52 years) with a diagnosis of idiopathic chronic primary bilateral tinnitus participated in this case study. His tinnitus symptoms started in 2005 at the age of 41 years. He presented bilateral, sensorineural, high frequency hearing loss with a pure-tone average of 49 dB HL in both ears .The patient wore hearing aids for 8 months prior to initial treatment with acoustic CR neuromodulation, relies on them for communication ability, and reported that they provide no benefits regarding tinnitus symptoms. The CR neuromodulation therapy was implemented for 19 weeks in 2012 with the standard acoustic stimuli delivered via a commercially available tinnitus device . The tinnitus therapy required removal of his hearing aids during the therapy. Subsequently (2015-2016), he was treated for 12 weeks with the same neuromodulation therapy device  but connected to an external “streamer”  that was a component of commercially available hearing aids , The streamer had a sampling rate of 22 kHz (stereo) and an audio bandwidth from 20 Hz to 10 kHz (Datasheet Phonak ComPilot), while the hearing aid (Phonak Audeo V50) had a bandwidth from 100 Hz to 9.2 kHz. These bandwidth limitations, well known for conventional hearing aid signal processing , may havNo further otolaryngology or audiology related issues were diagnosed while undergoing the two therapy phases and no pretreatments for tinnitus were reported except for the hearing aids that were fitted in July 2015. All treatments and hearing aid fittings were conducted by one of the authors .The standard acoustic CR neuromodulation therapy signals were used in both therapy periods and consisted of four pure tones determined by the pitch matched tinnitus frequency. The frequencies of the four therapy tones, two below and two above the tinnitus frequency, were a percentage of the tinnitus frequency, namely, from 77% to 140%, presented sequentially in a randomized fashion at approximately 5 dB above the individual threshold at that frequency. The daily stimulation duration was 4–6 hours [L on a scale from 0 to 100) and for tinnitus annoyance  at baseline and after treatment. Please note that the sensitivity of the THI score has been challenged by Tyler et al. [Tinnitus outcome measures used were the Tinnitus Handicap Inventory (THI) scores and the visual analog scale scores for tinnitus loudness , VASL (90 points), and VASA (80 points) scores worsened by 57%, 13%, and 14%, respectively, In the first treatment phase (June 2012–November 2012) the measured tinnitus pitch fluctuated substantially with a mean pitch of 8040 Hz and standard deviation of 1400 Hz. During this treatment phase, tinnitus pitch matching was performed using a bracketing method, similar to the well-known Vernon-method . Namely,Another follow-up visit in February 2013 showed no change in tinnitus symptoms and therapy was stopped. In July 2015 hearing aids were fitted . The hearing aid was set up using a recent audiogram applying a national acoustic laboratory nonlinear prescription formula (NAL NL1) . The patL was 92 points, and for VASA was 92 points. During two follow-up visits the therapy signal was readjusted to the slightly varying tinnitus frequency. After three months of therapy a large improvement of tinnitus symptoms was observed; namely, the THI score was reduced by 70% to 12 points, the VASL score was reduced by 32% to 63 points, and the VASA score was reduced by 32% to 63 points, For the second treatment phase, the T30 CR device was connected via direct cable to a hearing aid streamer  that wirelessly transmitted the therapy signals to hearing aids . The streamer sends an external audio signal  through an external sound input to hearing aids that provide higher amplification customized to the patients hearing loss. The hearing aids (Phonak Audeo V50) were fitted with the same NAL NL1 prescription formula as used for the initial hearing aid . This was done in order to ensure comparative amplification with the patient's original Oticon hearing aids used during phase one of treatment. Because the hearing loss was greater for high frequencies, in particular for frequencies above 6 kHz, the patient was not able to hear the highest stimulation tone, resulting in use of only the first three CR tones. Normally, this could cause the exclusion of the patient from treatment but was accepted for this case report. The tinnitus pitch was quite stable during the second treatment phase with a lower mean pitch of 6260 Hz and a much smaller standard deviation of 150 Hz. During this treatment phase, a newly developed automated and patented pitch matching method was utilized that integrated bracketing, similarity grading, a two-alternative forced-choice (2AFC) method, and fine-tuning into a single protocol of four distinct stages shown to improve pitch matching accuracy . Namely,L, VASA, and TQ scores. The magnitude of the hearing loss and the variable pitch matching results may have contributed to the unsuccessful initial acoustic CR neuromodulation treatment attempt. The application of acoustic CR neuromodulation through the hearing aids was well accepted and no side effects were observed throughout the intervention.During application of acoustic CR neuromodulation via a hearing aid a significant reduction in tinnitus symptoms was observed as documented by substantial decreases in VASAt the time of writing the majority of hearing instrument manufacturers provide aids with sound therapy features that can be utilized for a number of different therapeutic purposes and may serve to reduce the distress associated with tinnitus perception. Examples of these features include sound signals that are intended to be conducive to relaxation ; alternaResults obtained during this case report are encouraging from several clinical and technical standpoints. First, this approach is also applicable for tinnitus patients with sufficient hearing loss to warrant the use of hearing aids. Second, this approach allows simultaneously tinnitus therapy and hearing aid benefits. Third, this approach allows improved acoustic CR neuromodulation therapy signal control because of the inherent further amplification offered by the hearing aids used to treat the existing hearing loss. Fourth, this approach likely will result in higher compliance with the therapy regime because the primary device already is being worn. Fifth, the continued use of the hearing aid beyond acoustic CR neuromodulation therapy might help to stabilize the obtained therapeutic effects. Finally, this approach will allow easy implementation of additional CR therapy should the tinnitus return or worsen in the future.There are some limitations with this approach. First, current hearing aids have a restricted frequency range that will limit the delivery of high frequency stimulation tones with high enough levels in cases of high frequency tinnitus. Second, the available data did not fully explain why the patient did not respond to CR therapy without hearing aid support in the first phase of the experiment. This might have occurred because of inadequate and imprecise pitch matching see  or becauIn a single case, we were able to show that acoustic CR neuromodulation can be implemented effectively through contemporary hearing aids to allow the simultaneous benefits of hearing aid use and therapy delivery. This approach appeared to not negatively influence normal hearing aid functionality, increase the environments in which therapy can be delivered, and potentially increase likelihood of compliance with the tinnitus therapy regimen. Since only one case has been investigated so far, the results are clearly not yet conclusive. This case report encourages a more comprehensive investigation of the integration of acoustic CR therapy delivery with hearing aids including understanding the effects of nonlinear amplification, harmonic intermodulation, and temporal distortions inherent in contemporary hearing aids.


Visual working memory (VWM) allows us to temporarily maintain and manipulate visual information in order to solve a task. The study of the brain mechanisms underlying this function began more than a half century ago, with Scoville and Milner's  seminal Riley and Constantinidis contribute a thorough review of relevant primate studies, and provide compelling fresh evidence for it. They also survey a number of alternative models of VWM and conclude that each one can only mediate a limited range of memory-dependent behaviors. They also provide a detailed account of the tissue characteristics that make the prefrontal cortex (PFC) uniquely specialized to support this function.The classic model of VWM posits that persistent delay activity in the prefrontal cortex is both sufficient and necessary to mediate visual working memory. Boschin and Buckley, who enhance it by offering an account of the functions of the frontopolar cortex (FPC) from a series of pioneering lesion and behavioral studies in the non-human primate. Specifically, they suggest that the FPC supports the exploration and evaluation of relative values of novel alternatives, some of which may turn out to be distractors, while the dorsolateral PFC maintains, manipulates, and selects relevant information, rules and strategies for the task at hand. Mansouri et al. review the role of VWM in executive control functions with an emphasis on abstract features, and representations of errors and conflicts in order to make adaptive behavioral adjustments. They note that primate performance in a Wisconsin Card Sorting Task analog is disrupted after lesions of the dorsolateral PFC, orbitofrontal cortex, but also of anterior cingulate cortex. Tsutsui et al. offer an integration of findings on visuospatial WM from two animal models: primates and rodents. Both lesion and single unit studies, together with anatomical patterns of fronto-parietal connectivity indicate that the dorsolateral PFC in the macaque is analogous in function to the medial PFC in the rat.Further support for the classic model is provided by per se  correlated within individuals both in behavior and in brain activity , there were no significant brain-behavior correlations in capacity. Both of these empirical findings open up a lot of questions for future work.Let us now turn to the works in this Research Topic that examined VWM from a developmental perspective. In 2004, we presented a summary of what was then known about the early development of VWM in humans . Optical imaging (fNIRS) studies reported by Wilcox and Biondi provided converging evidence. The occipital cortex  was involved during all events when infants had to maintain object representations in VWM. In addition to this, the anterior temporal cortex was selectively activated when infants maintained two distinct objects in VWM.Two empirical studies in this Research Topic that tested human infants found VWM-related activity in the ventral visual stream. Prior EEG studies have demonstrated that gamma-band power in the temporal cortex increases during periods while infants are maintaining an object representation in VWM  has extensive connections with both frontal and temporal areas. Reynolds and Romano reviews the existing literature in infants, and conclude that while the role of sustained attention in long-term memory encoding has been well understood (see e.g., the now-classic works of Richards, Fitch et al. that “future research should aim to examine relations between attention and working memory in infancy and early childhood using both psychophysiological and neural measures.”Two articles in our Research Topic have examined the complex interactions between visual attention and memory during development. Shimi et al. investigated the magnitude of the N2pc in 10-year-old children and adults, and found that this neural signature of visual attention during the encoding phase of the task was related to their behavioral performance during the later recognition phase. This brain-behavior relationship was demonstrated on the individual level as well: children with large attentional cue benefits and high VWM capacity elicited an adult-like ERP response following attentional selection of the to-be-encoded item, whereas children with low VWM capacity did not.We know more about attention-VWM interactions in older children, thanks to, among others, the EEG/ERP studies of Scerif and her colleagues. In summary, this Research Topic includes nine up-to-date literature reviews and seven novel empirical studies approaching the neural mechanisms underlying visual working memory from human developmental, neuroimaging, and non-human mammalian perspectives. Together, they describe a common brain network that involves the fronto-parietal control system, various processing stages of the ventral visual stream, and the medial temporal lobe—with some differences in the weights and functions of the different structures. This extensive network seems to function in early infancy, and new multi-level approaches will help elucidate the details of the developmental trajectories.The two authors contributed equally to the preparation of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cold hypersensitivity in the hands and feet (CHHF) is a common symptom in Korea and patients with CHHF complain of coldness in the hands and feet in an environment that is not considered cold by unaffected people. In traditional East Asian medicine, CHHF is believed to be accompanied by various diseases and symptoms, and is considered a symptom that needs active treatment. CHHF is used for pattern identification in the cold pattern, yang deficiency, and constitution. This study aimed to examine the differences in frequencies of chronic diseases with respect to the presence of CHHF.n = 1909) and non-CHHF groups (n = 3017) according to the CHHF survey. The differences in frequencies of 18 diseases were analysed using chi-square tests, and the odds ratios (ORs) for each disease according to CHHF status were examined via logistic regression with adjustment for age, sex, and body mass index (BMI).Disease history, CHHF, body measurements, and blood test survey data from 6149 patients collected by 25 medical institutes in Korea were obtained from the Korean Medicine Data Center. The participants were divided into CHHF (Based on chi-square test results, the CHHF group showed a higher frequency of the following diseases: anaemia, hypotension, chronic gastritis, reflux oesophagitis, chronic rhinitis, dysmenorrhoea, and gastroduodenal ulcer. Diseases found in lower frequencies were as follows: hypertension, diabetes mellitus, impaired fasting glucose, dyslipidaemia, stroke, fatty liver, and angina pectoris. In addition, from the logistic regression with adjustment for age, sex, and BMI, the CHHF group showed a lower OR in diabetes mellitus and dyslipidaemia than the non-CHHF group, but a higher OR in degenerative arthritis, chronic gastritis, gastroduodenal ulcer, reflux oesophagitis, and chronic rhinitis.This study showed that CHHF is associated with chronic disease. Further large-scale prospective studies are needed to validate these associations. Cold hypersensitivity in the hands and feet (CHHF) is a sensation of coldness in the hands and feet in an environment not considered cold by unaffected people or a having a heightened cold sensation in a relatively low temperature area. This symptom is relatively common in Korea, especially in women , 2.Although the exact mechanism of CHHF is still unclear, it is associated with a hypersensitive vasoconstrictor response , 4 and aIn TEAM, CHHF is important for identifying yang deficiency and the cold pattern. Furthermore, the book, Internal Classic, by Huangdi introduced the concept of the “spleen and four extremities”, and suggested that the function of the spleen was to control the four limbs and that dysfunction in the limbs may indicate splenic problems , 8. KoreStudies have examined the association between CHHF and diseases such as functional dyspepsia , orthostHowever, the concepts behind “hie” and CHHF are not entirely the same; previous studies conducted in Korea had limitations regarding disease, sex, and age of the participants, while a few studies included only a small sample size, thus lacking statistical power , 13, 14.This cross-sectional study was conducted based on Korean medical data of 6149 patients collected from 25 Korean medical institutes in Korea between November 2006 and August 2013 and stored at the Korean Medicine Data Center (KDC) . Among tn = 1909), and those who responded “warm” or “intermediate” to either question were classified in the non-CHHF group (n = 3017). Those who responded, “I don’t know” to either of the two questions (n = 964) were excluded from the study . According to the Seventh Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC-7), hypertension was defined as a systolic blood pressure (sBP) higher than 140 mmHg or diastolic blood pressure (dBP) higher than 90 mmHg, or a diagnosis of hypertension with blood pressure managed by medication.Hypotension: Generally, hypotension is not defined as a disease and has no standard for diagnosis, but there are claims that hypotension accompanies CHHF , 13. ThuIFG and DM: Blood analysis was performed using an ADVIA 1800 Auto Analyzer . Fasting blood sugar (FBS) was measured after 12 h of fasting. IFG was defined as an FBS between 100 mg/dL and 125 mg/dL, and DM was defined as an FBS higher than 126 mg/dL or a diagnosis of DM with medication for lowering blood sugar, according to the American Diabetes Association.Dyslipidaemia: According to the National Cholesterol Education Program Adults Treatment Panel III, dyslipidaemia was defined as a total cholesterol (TC) higher than 240 mg/dL, low-density lipoprotein (LDL) higher than 160 mg/dL, high-density lipoprotein (HDL) lower than 40 mg/dL, triglycerides (TG) higher than 200 mg/dL, or a diagnosis of dyslipidaemia with medication.Anaemia: Haemoglobin (Hb) levels were measured using ADIVA2120i . According to a standard established by the World Health Organization [Dysmenorrhoea: Dysmenorrhoea was defined as lower abdominal, back, or pelvic pain occurring just before or during menstruation . HoweverChronic diseases that could be confirmed from the disease history survey of KDC data were degenerative arthritis, rheumatoid arthritis, angina, stroke, chronic gastritis, gastroduodenal ulcer, reflux oesophagitis, fatty liver, hepatitis, asthma, chronic rhinitis, and chronic sinusitis. The presence of dysmenorrhoea was confirmed by a menstrual pain scale that is often used in Korean medical clinics . ChronicP < 0.05. All statistical analyses were conducted using SPSS 21.0 for Windows .Demographic characteristics of the participants are presented as frequencies and percentages or mean and standard deviation, and analysed using the chi-square test. A comparison of the disease frequency according to CHHF status was presented as frequency and percentage and analysed using the chi-square test. In addition, in order to obtain the odds ratios (OR) and 95% confidence intervals (CIs) for CHHF in each disease, logistic regression with adjustment for sex, BMI, and age was performed. Participants with missing data regarding disease history, blood test results, and body measurement were excluded from the analysis for that respective disease. Furthermore, diabetic participants were excluded from the analysis of IFG, and dysmenorrhoea was analysed only in women aged 19–45 years who still experienced menstruation. The statistical significance level was set at The total number of participants was 4926, including 1698 men (34.5%) and 3228 women (65.5%). There were 3017 participants in the non-CHHF group  and 1909 participants in the CHHF group . Age, height, weight, BMI, sBP, dBP, FBS, TG, TC, LDL, and Hb were significantly higher in the non-CHHF group than the CHHF group, and HDL was significantly higher in the CHHF group than the non-CHHF group (Table P < 0.001), as well as angina pectoris (P = 0.007). Compared to that in the non-CHHF group, the CHHF group showed a higher frequency of anaemia, hypotension, chronic gastritis, reflux oesophagitis, and chronic rhinitis (P < 0.001), as well as dysmenorrhoea (P < 0.01) and gastroduodenal ulcer (P < 0.05). There was no significant difference in degenerative arthritis, rheumatoid arthritis, asthma, and chronic sinusitis between the two groups  in TEAM. In TEAM, pattern identification is an important factor to consider during disease diagnosis when prescribing herbal medicine, so it is important that both be identified . MoreovePrevious studies on CHHF were focused on a single disease or on the association between other symptoms and CHHF. Furthermore, studies on “hie” (cold disorder), which is a concept similar to CHHF in Japan, examined the association between cold disorder and various symptoms, but did not focus on its association with other diseases , 15. UnlWhen participant age, sex, and BMI were adjusted, the CHHF group showed ORs higher than 1 in degenerative arthritis, chronic gastritis, gastroduodenal ulcer, reflux oesophagitis, and chronic rhinitis compared to those in the non-CHHF group. This result is in line with a previous report showing a higher frequency of functional dyspepsia in the CHHF group  and a reIn our study, DM and dyslipidaemia had ORs lower than 1 in the CHHF group compared to those in the non-CHHF group when adjustments were made for age, sex, and BMI. This is contradictory to the results of a previous study showing that diabetic peripheral neuropathy, Type-I DM, and atherosclerosis could be the causes of CHHF , 10, 37.High ORs for dysmenorrhoea and hypotension were observed in the CHHF group, similar to the results of a previous study, showing that CHHF and the cold pattern are common in patients with dysmenorrhoea, and that the frequency of CHHF is high in orthostatic hypotension –15, 40.CHHF is a commonly observed symptom in East Asian countries including Korea and presumed to occur because of a genetic disposition and imbalance of the autonomic nerves triggering impaired peripheral circulation , 3, 41. This study has a few limitations. First, because it was a cross-sectional study, a causal relationship cannot be examined. Thus, it is unknown if our results were the effects of disease, cause of disease, or caused by the treatment of disease. Second, bias may have existed because most disease data were obtained via surveys and thus dependent on patient memory, and patients underwent only one round of examinations and measurements. Dysmenorrhoea was not classified into primary and secondary types, and the validity and reliability of the questionnaire used in this study have not been verified. In addition, we confirmed whether the participants had CHHF or not based on the sensations they experienced in the last six months. However, considering that thermal sensations are affected by ambient temperature, it is necessary to maintain a constant temperature inside the measurement room of each institution . UnfortuProspective studies with accurate disease diagnoses, which also allow the exploration of a causal relationship, should be conducted in the future.In this study, we found that CHHF was associated with DM, dyslipidaemia, degenerative arthritis, chronic gastritis, gastroduodenal ulcer, reflux oesophagitis, and chronic rhinitis. Our results allow a better understand of CHHF pathogenesis.


Drosophila grooming sequence that is induced by simultaneous activation of different sensory pathways that each elicit a distinct movement . Here, we confirm this model using transgenic expression to identify and optogenetically activate sensory neurons that elicit specific grooming movements. Simultaneous activation of different sensory pathways elicits a grooming sequence that resembles the naturally induced sequence. Moreover, the sequence proceeds after the sensory excitation is terminated, indicating that a persistent trace of this excitation induces the next grooming movement once the previous one is performed. This reveals a mechanism whereby parallel sensory inputs can be integrated and stored to elicit a delayed and sequential grooming response.A central model that describes how behavioral sequences are produced features a neural architecture that readies different movements simultaneously, and a mechanism where prioritized suppression between the movements determines their sequential performance. We previously described a model whereby suppression drives a  The hundreds of bristles on the compound eyes each contains the dendrite of a sensory neuron, which also projects an axon into an afferent tract that enters the CNS in the subesophageal zone (SEZ)  . In contne (SEZ) . We testne (SEZ) .Split GAL4 (spGAL4) technique to restrict expression to only the sensory neurons (DNA binding domain (DBD) and the transcriptional activation domain (AD), are expressed in the same cells. We generated spGAL4 flies that were anticipated to target the wing sensory neurons by expressing the DBD in the pattern of R31H10-GAL4 and the AD in the pattern of R30B01-GAL4 , where they follow diverse paths locally, with some further ascending to the SEZ in the brain  that expressed both in the eye bristle mechanosensory neurons and the same three categories of sensory neurons on the wings and halteres that were expressed in the R30B01-AD ∩ R31H10-DBD combination . SimultaThe goal of this work was to test the prediction of the model of hierarchical suppression that simultaneous activation of sensory neurons on different body parts elicits a prioritized grooming response. Two lines of evidence led us to this prediction. The first was based on our previous finding that coating the body of the fly in dust elicits grooming that prioritizes head over posterior body grooming . The secOne aim of this work was to identify sensory neurons that can induce grooming behavior. The bristles are canonically thought to be involved in insect grooming based on evidence that their tactile stimulation on different body parts induces site directed grooming responses . Here, wOne outstanding question is whether the sensory neurons have a direct role in establishing hierarchical suppression. We previously proposed two mechanisms of hierarchical suppression . One is The use of optogenetics to activate sensory neurons reveals new insights into the neural mechanisms that drive grooming behavior. One striking example is evidence of a persistent trace of those sensory neurons that were optogenetically activated (next Discussion section). Other data in Although suppression of eye by wing grooming is predicted by the model, it is unclear why the flies transition to wing grooming during the optogenetic stimulus . SimilarFuture studies will address how changing the relative activation levels between grooming movements results in a shift of suppression such that later movements suppress earlier ones. This could be tested by extending the fiber optic-directed optogenetic approach used in this study , such thDrosophila grooming movements have mechanism(s) for maintaining excitability. This was originally proposed from a study identifying a mechanosensory circuit that elicits persistent grooming of the antennae  (UAS-Channelrhodopsin-2 (UAS-CsChrimson (attP18)  , 13xLexA(pJFRC7) , UAS-Chadopsin-2 , and 20x(attP18) . GAL4, s2. The light was directed towards the dorsal posterior region, away from the anterior body, to activate sensory neurons primarily on the wings . ChR2 was used for these experiments because the intensity of the blue light used for its activation is attenuated by the cuticle of the fly . Therefohe wings . Alternahe wings . The LED2), R30B01-GAL4 (0.066 mW/mm2), and R81E10-GAL4 (0.077 mW/mm2). The light power used for each LexA and spGAL4 line was: VT17251-LexA (0.382 mW/mm2), R30B01-AD ∩ R31H10-DBD and R31H10-AD ∩ R34E03-DBD (0.135 mW/mm2). The red light frequency was 5 Hz (0.1 s on/off) for 5 s, followed by 30 or 60 s intervals where the red light was off. The experiment consisted of a total of three photostimulation periods with 30 or 60 s intervals between each stimulation. The experiment was recorded for manual annotation of the grooming movements performed.The camera and behavioral setups used for recording freely moving flies with optogentic activation were described previously . Flies wThe recorded grooming movements of flies were manually annotated as described previously . For theDissection and staining of the CNS was performed using a previously reported protocol . The heaUAS-mCD8::GFP (JFRC7) or 13xLexAop-myr::GFP (pJFRC19). The progeny were anesthetized using CO2, decapitated, dipped in 70% ethanol, transferred to PBS, and each body part was dissected as described below. The unfixed body parts were imaged immediately in PBS or Vectashield . We used both PBS and Vectashield and did not notice a difference in the cell morphology or expression pattern when using either reagent. The use of Vectashield had the advantage of resulting in fewer air bubbles between the coverslip and sample.Dissection of the different body parts and imaging of the PNS expression patterns of the different GAL4, LexA, and Split GAL4 lines were performed as follows. The lines were crossed to 20xHead: Flies were decapitated using a standard razor blade. Heads were then placed ‘face up’ on a slide in a small well that was made by stacking six reinforcement labels  and filled with PBS or Vectashield. A cover slip was then placed over the well. Abdomen: The abdomen was severed from the rest of the body just posterior to the scutellum. Abdomens were then placed on a slide in a well created as described above. The abdomens were placed either ventral or dorsal side up so that each side could be imaged. Notum: A scalpel was used to slice longitudinally between the legs and the dorsal side of the notum. The notum was imaged in the same well preparation described above. Wing: A scalpel was used to remove the left wing from the body of the fly. To ensure that the entire wing was obtained, part of the body wall was also cut with the wing. The wing was then placed on a drop of Vectashield and then covered with a coverslip. Leg: The left prothoracic leg was dissected in the same way as the wing. All body parts were imaged using a Zeiss 710 confocal microscope using 10x and 20x air objectives. Native GFP fluorescence was imaged using an excitation wavelength of 488 nm, whereas autofluorescence from cuticle was imaged using 568 nm. Body parts from at least three flies were imaged from separate crosses on different days. In some cases, the body parts were imaged at 20x and then stitched using a FIJI plugin , is a member of our Board of Reviewing Editors, and the evaluation has been overseen by K VijayRaghavan as the Senior Editor.Thank you for submitting your article "Simultaneous activation of parallel sensory pathways promotes a grooming sequence in The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:eLife in 2014, which established the grooming behavior of Drosophila as a very productive one for the study of sequential behaviors at the cellular level, using all the genetic tricks available. In that paper the authors proposed a model of hierarchical suppression where simultaneous activation of sensory neurons on different body parts (dusting the whole fly) elicits a prioritized grooming response, roughly anterior to posterior, with eye grooming dominant over wing grooming, for example. In this advance, the authors use Gal4 and spGal4 drivers to begin the identification of the sensory neurons involved in grooming. Whole body short duration optogenetic stimulation of using general Gal4 drivers for sensory neurons gives rise to sequencing with eye grooming not giving way to posterior body grooming until the activating light is turned off, but light stimulation limited to head or body give rise to eye grooming or body grooming respectively. Thus a persistent state of activation of posterior body grooming is revealed and the model is supported. They identify with spGal4 drivers and optogenetically activate specific sensory neurons that elicit different specific grooming movements, eye grooming and wing grooming. The goal is to activate them optogenetically together and test their effects on grooming behavior and in particular on its sequencing. The ultimate experiments, once limited identified populations of eye and the wing sensory neurons are under optogenetic control, was to simultaneously activate them and show that eye input remains dominant but sequencing persists. This is a significant advance in technique and it provides strong evidence for the hierarchical suppression model, which is of wide interest. The tools and reagents are now in place to explore directly how suppression is accomplished by sensory neurons. The work is carefully done with appropriate controls and characterization of Gal4 and spGal4 lines. The data is analyzed in an appropriate manner. The lines characterized will be very useful for fly workers who wish to pursue experiments in which limited classes of sensory neurons are to be optogenetically activated. The work is of interest to those who study the neuronal bases of natural behaviors and behavior sequences.This is an interesting advance on a seminal paper in Essential revisions:1) The experiment of 2) Experiments could, in principle, be done using the activation of mechanosensors in the abdomen, playing them against activation of the head and/or thoracic mechanosensors. The authors should discuss whether there is a reason that such experiments were not done.Reviewer #1:1) Paragraph two subsection “Competition between eye and wing sensory neurons elicits prioritized grooming” and 2) Paragraph three of subsection “Simultaneous excitation of sensory neurons across the body induces a grooming sequence” and Reviewer #2:eLife paper that proposes a suppression hierarchy that drives sequential grooming behavior by adult fruit flies. This study nicely tests the basic assumption of the model proposed in the previous study-that activation of mechanosensory neurons all over the fly's body activates pattern generators that arouses grooming behavior to all parts of the body, with the location of the grooming sequenced in an anterior-to-posterior pattern. The experiments are well presented, appropriate, and the results are convincing. The conclusions are fully justified and nicely qualified where appropriate.This is an excellent follow-up study to this group's 2014 Their basic technique  provides a strong test of the hypothesized mechanism. The results are convincingly in line with the hypothesis and, additionally, provide the added information that even short-lasting activation of a set of mechanosensors produces a long-lasting bout of grooming the appropriate area, a finding that should lead to interesting extensions of this work in the future.Two possible extensions of the research that should be addressed:1) The same kinds of experiments could, in principle, be done using the activation of mechanosensors in the abdomen, playing them against activation of the head and/or thoracic mechanosensors. The authors should discuss whether there is a reason that such experiments were not done.2) Have the authors tried weakly stimulating head mechanosensors and strongly stimulating the thoracic ones? Such an experiment would test whether there is a weak back-to-front inhibition of grooming behavior in addition to the clearly strong front-to-back inhibition. If they have such data, it would be good to show it. If not, they might mention this as a possible future experiment.Reviewer #3:Drosophila. Overall, I find the experimental data compelling, the writing is clear, and I have no major experimental concerns.This paper represents an important addition to the previous studies by this group looking at the emergence of grooming sequences in  Essential revisions:1) The experiment of We agree with the reviewers that the result shown in In the “Interestingly, the transition of eye to wing grooming occurred within the stimulus period, suggesting that eye grooming became inhibited by wing grooming during stimulus. This may reveal a prediction of the model of hierarchical suppression that later movements in the sequence can suppress earlier ones (see Discussion). However, the prioritized suppression came from eye grooming, as any ongoing grooming of the wings terminated and all flies groomed their eyes with each red light stimulus .”The following text has been added as a new Discussion section. This section also addresses the requested extension of reviewer 2: “Have the authors tried weakly stimulating head mechanosensors and strongly stimulating the thoracic ones?”“Using optogenetic control of sensory neurons to further probe the neural mechanisms of hierarchical suppression. The use of optogenetics to activate sensory neurons reveals new insights into the neural mechanisms that drive grooming behavior. […] In effect, changing the illumination levels on the different body parts could mimic the loss of dust as flies clean the different parts of their bodies.”2) Experiments could, in principle, be done using the activation of mechanosensors in the abdomen, playing them against activation of the head and/or thoracic mechanosensors. The authors should discuss whether there is a reason that such experiments were not done.We agree with the reviewer that this would be a very interesting set of experiments to do. However, we first need to identify transgenic driver lines (e.g. Split GAL4 lines) that specifically target two populations of neurons on the abdomen and head or thorax. This is a laborious, and often unsuccessful process that involves screening though many different combinations of Split GAL4 halves (i.e. AD and DBDs), searching for those that express exclusively in both populations of sensory neurons. It has been particularly difficult to identify lines that express in bristle mechanosensory neurons on specific body parts, which are likely the primary sensory neurons whose activation elicits abdominal and thoracic grooming. The main problem seems to be that the lines often express in mechanosensory bristles on several different body parts. The eye bristle driver that we identified and described in this manuscript seems to be an exception to that rule. Although we had success in identifying a Split GAL4 driver line that expresses in eye and wing/haltere mechanosensory neurons, we have yet to identify a driver that expresses exclusively in the abdomen and head or thorax. This is the reason why we have not tried this experiment yet.Reviewer #1:1) Paragraph two subsection “Competition between eye and wing sensory neurons elicits prioritized grooming” and We addressed this reviewer’s concern in the essential revisions section above.2) Paragraph three of subsection “Simultaneous excitation of sensory neurons across the body induces a grooming sequence” and This is an interesting question. The model of hierarchical suppression predicts that optogenetic activation of the sensory neurons at shorter interstimulus intervals than the 60 seconds shown in Reviewer #2:[…] Two possible extensions of the research that should be addressed:1) The same kinds of experiments could, in principle, be done using the activation of mechanosensors in the abdomen, playing them against activation of the head and/or thoracic mechanosensors. The authors should discuss whether there is a reason that such experiments were not done.We addressed this reviewer’s concern in the essential revisions section above.2) Have the authors tried weakly stimulating head mechanosensors and strongly stimulating the thoracic ones? Such an experiment would test whether there is a weak back-to-front inhibition of grooming behavior in addition to the clearly strong front-to-back inhibition. If they have such data, it would be good to show it. If not, they might mention this as a possible future experiment.We agree that this would be an excellent experiment to try. However, we do not yet have the necessary hardware to rigorously do this experiment. We are constructing an apparatus that uses two lasers to illuminate two body parts as the same time with different light powers. This will allow us to adjust the relative optogenetic light power on the two parts and test whether weak stimulation of the anterior body with strong stimulation of a posterior part results in the prioritization of posterior grooming and the suppression of anterior grooming.We realized with this reviewer’s comment that this would be a good point to mention in the manuscript, and added a paragraph to the new Discussion section called: “Optogenetic control of sensory neurons to further probe the neural mechanisms of hierarchical suppression.” The entire Discussion section is also shown above. The paragraph that is directly relevant to this reviewer’s comment follows:“Future studies will address how changing the relative activation levels between grooming movements results in a shift of suppression such that later movements suppress earlier ones. […] In effect, changing the illumination levels on the different body parts could mimic the loss of dust as flies clean the different parts of their bodies.”


Based on a deterministic continuous model of cell populations dynamics in the colonic crypt and in colorectal cancer, we propose four combinations of feedback mechanisms in the differentiations from stem cells (SCs) to transit cells (TCs) and then to differentiated cells (DCs), the four combinations include the double linear (LL), the linear and saturating (LS), the saturating and linear (SL), and the double saturating (SS) feedbacks, respectively. The relative fluctuations of the population of SCs, TCs, and DCs around equilibrium states with four feedback mechanisms are studied by using the Langevin method. With the increasing of net growth rate of TCs, it is found that the Fano factors of TCs and DCs go to a peak in a transient phase, and then increase again to infinity in the cases of LS and SS feedbacks. The “up-down-up” characteristic on the Fano factor  demonstrates that there exists a transient phase between the normal and cancerous phases, our novel findings suggest that the mathematical model with LS or SS feedback might be better to elucidate the dynamics of a normal and abnormal (cancerous) phases. Some researchers pointed out that the other factors, such as microenvironmental influences and abnormal regulation of signal pathway, can also induce the uncontrolled growth and the tumor initiation78910112et al. revealed that the numbers of actual functional stem cells in intestinal crypts (5–7 in each crypt) and adenomas (9 per gland) are small through continuous clonal labelinget al.On the other hand, various experimental evidences suggested that the number of SCs in a normal colonic crypt is often very limited13141516171415In this paper, based on the deterministic model, we firstly use a Hill function form to represent a general feedback mechanism (including the linear and the saturating), then there are four combinations of feedback mechanisms in the cell populations dynamics, i.e. the LL, LS, SL, SS feedbacks. Secondly, in order to study the relative fluctuations in the populations of SCs, TCs, and DCs, we derive the formulae of Fano factor, covariance and susceptibility of cell populations around equilibrium states by using the Langevin method212223252627A general deterministic continuous model, which is based on the three compartments assumption in a single crypt2In order to make the system structurally stable, the differentiations of both SCs and TCs are regulated by two alternative forms of feedback which maintain homeostasis in the crypt, respectively. Here we use a Hill function form to represent two possible feedback mechanisms (linear or saturating):i = 0 and 1, N0 and N1 are the population numbers of SCs and TCs, respectively, k0 and k1 represent the rates of population response to change, while m0 and m1 are feedback parameters. In the case of saturating feedback, there is a saturating value α2 + k0/m0 (or β2 + k1/m1) of the differentiation rate of SCs (or TCs). Therefore, there are four possible combinations (listed in m0 = 0 and m1 = 0), LS feedback (m0 = 0 and m1 = 0.01), SL feedback (m0 = 0.1 and m1 = 0), and SS feedback (m0 = 0.1 and m1 = 0.01).where In the deterministic description, the population dynamics in the crypt can be given by following ordinary differentiated equations (ODEs)23which give the steady statewithN2 is the number of DCs, and α = α3 − α2 − α1 and β = β3 − β2 − β1, which denote the net (per capita) growth rates of SCs and TCs, respectively. Combining that α1 + α2 + α3 = 1 and β1 + β2 + β3 = 1, the net growth rates (α and β) can be determined by the linear stability analysis: 0 < α < k0/m0 and −1 < β < k1/m1. So the k0/m0 and k1/m1 are the critical threshold values which discriminate the non-cancerous and cancerous states. When one of the net growth rates goes beyond its own threshold value, the cell population grows uncontrolled. In this paper, the values of the parameters β2 = 0.3, γ = 0.323, k0 = 0.1 and k1 = 0.01 are determined2and In the stochastic description, the dynamics of the three compartments are given by the following stochastic equations:ξi(t)  are random variables. Here, the statistical properties of the random variables ξi(t) around the steady state are derived by using two assumptionsξi(t) are zero, but the autocorrelation functions of ξi(t) relate with the system’s intrinsic characters  and can be described by following equationswhere ξi(t) take the formsand the cross-correlations between ξ0(t) is determined by the SCs’s net growth rate α, differentiation rate α2, and its steady state value ξ1(t), the cross-correlation between ξ0(t) and ξ1(t), and the cross-correlation between ξ1(t) and ξ2(t) have connection with the feedback mechanism on SCs or TCs. The autocorrelation of ξ2(t) is dependent of DCs’s steady state value and the removal rate. There is no cross-correlation between ξ0(t) and ξ2(t).From the above whereThe corresponding Fokker-Planck equation of the 3132According to the definition of the Fano factor , it is used to characterize the relative size of fluctuation and can be calculated through the Fokker-Planck equation. So, we can get the formulae of Fano factors of SCs, TCs, and DCs, respectivelyσ00, σ11, σ22) of three compartments and the covariance , which can describe the correlations between fluctuations of the cell population, are given in Part 2 of where the variances , which means the steady value of the system. Here, we consider the effects of the net growth rates, so z = α, β. According to this definition, the susceptibilities to the two net growth rates are derived in Part 3 of where α and β). Next, the differences on the mutational processes, steady state values, and the diversities of the fluctuation effects on Fano factors due to the net growth rates (α and β) of SCs and TCs in a colonic crypt with four feedback mechanisms are discussed in the range of physiologically reasonable parameter values2Although all the four feedbacks can maintain the homeostasis of the population in the colonic crypt , the mutα or β, and the mutations can give a selective advantage to the cell2N0(0) = 1.0, N1(0) = 1.0, N2(0) = 1.0, and α = 0.286, β = 0.432, α2 = 0.3, β2 = 0.3 was chosen with the four cases of feedbacks. As time goes on, the processes go with the same sequence of mutations in all the four feedbacks except the last mutation in which α = 1.1, β = 0.697  on the steady state values, and the cell population as a function of α or β with four feedbacks is shown by β, with the increasing of α, the stationary population of SCs is small and linearly increased with LL and LS feedbacks (α of SCs is close to the saturating threshold (k0/m0 = 1) with SL and SS feedback, the population of the crypt is rapidly increased and the tumor is more likely to be initiated. According to m0 = 0), i.e. LL and LS feedbacks, the stationary population of SCs is α/k0 which is small because of k0 = 0.1 and is proportional to α(0 ≤ α ≤ 1); when the feedback on the SCs is saturating (m0 = 0.1), i.e. SL and SS feedbacks, the stationary population of SCs is α close to the saturating threshold (k0/m0 = 1).From eedbacks , but it eedbacks . At the eedbacks , and is eedbacks , but unceedbacks . It can α, with the increasing of β, the stationary population of SCs is a constant , the whole population of the crypt is still controlled and limited with LL and SL feedbacks (β ≪ 0), transient (β ~ 0), and cancerous (β ≫ 0)For a given value of tant see  and smaltant see , the staeedbacks . Howevereedbacks . Above rα (α are illustrated by β (β = 0.432), it is obvious that the three Fano factors  with SL and SS feedbacks are quite different with LL and LS feedbacks. With the increasing of α, the Fano factor of SCs goes down rapidly for small values of α with four feedbacks, then reaches 1 for large values of α with LL and LS feedbacks, but goes up quickly again to infinity with SL and SS feedbacks when α is close to 1    ; the Fane k0/m0) . These ery small  and the oes down . Due to  F1, F2) . When α infinity , then thinfinity .β with determined α are shown by β   with the LS or SS feedback, the numbers of TCs and DCs increase uncontrolled to infinity (2122232526273637Fano factors of TCs and DCs with four feedbacks as a function of by β see  is a coneedbacks . When β  (β ~ 0) , which minfinity  and the infinity . This “uinfinity  which coβ with different α under LS and SS feedbacks respectively. For the LS feedback, with the increasing of α, the intensity of relative fluctuations goes down in the normal (β ≪ 0) and transient (β ~ 0) phases and is almost same in the cancerous phase (β ≫ 0 or β > 0.5) (F1 and F2) is decreasing with the increasing of α under the LS feedback , the intensity of relative fluctuations goes down with the increasing of α in the normal and transient phases and vice vesa  . This isfeedback , but wheice vesa . In the ing of β , which mα or β on the covariance and susceptibilities are compared and provided in Part 4 and 5 of In addition, the effects of the net growth rate The aim of this paper is mainly to compare the effects of the relative fluctuations of the cell population in a colonic crypt around the steady state with four feedbacks. There are mainly three methods to study the fluctuations of biochemical reaction systems. The first one is to solve the probability distribution for all the molecular components through the chemical master equationComparing the Langevin method of Swainα and β are compared with the same and different volumes of parameter Ω in α and β except about 0 ≤ α < 0.1 and −1 ≤ β < −0.4. Because the number of the populations of SCs is very small with low values of net growth rates of α and β with four feedbacks  of SCs (or TCs). Our theoretical results show that the stationary populations of TCs and DCs exhibit an approximately threshold behavior as a function of the net growth rate of TCs for the four feedback mechanisms. The reproductions of TCs and DCs can be classified into three phases: normal, transient, and cancerous. The LL feedback which cannot produce the uncontrolled growth is not suitable for the cancerous phase with increasing of the net growth rate of SCs or TCs. It is interesting that there is a so-called van der Waals loop in the Fano factors of TCs and DCs populations under the LS and SS feedback mechanisms, which can be considered as an evidence of phase transition, and this transient phase may be corresponded to the adenomas state before CRC tumor initiation252627The formulae of Fano factors, covariances, and susceptibilities are derived with the general model. Although the dynamics of cell populations of SCs, TCs, and DCs can reach a steady state with the four feedbacks, which is reasonable in the range of physiologically parameter values, the steady state values, and the effects of relative fluctuations on Fano factors, covariances, and susceptibilities obtained by using of Langevin methodα increases. However, above theoretical results and the other results23183637This three-compartment model discussed in this paper is a general model in normal tissue differentiation or the development of other tumors initiation. Thus, it is important to know which one is the best of these four feedback mechanisms for modeling the dynamics of a normal and abnormal (CRC) colonic crypt or other tumors initiation? Due to the difficulty of identifying cancer stem cells (CSCs), some researchers believed that the quantity of CSCs is small in tumors, just like the number of SCs in the normal tissues. A recent research4142How to cite this article: Qiu, K. et al. A van der Waals-like Transition Between Normal and Cancerous Phases in Cell Populations Dynamics of Colorectal Cancer. Sci. Rep.6, 36620; doi: 10.1038/srep36620 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.