Retinal ganglion cells (RGCs) play important roles in retinogenesis. They are required for normal retinal histogenesis and retinal cell number balance. Developmental RGC loss is typically characterized by initial retinal neuronal number imbalance and subsequent loss of retinal neurons. However, it is not clear whether loss of a specific non-RGC cell type in the RGC-depleted retina is due to reduced cell production or subsequent degeneration. Taking advantage of three knockout mice with varying degrees of RGC depletion, we re-examined bipolar cell production in these retinas from various aspects. Results show that generation of the cone bipolar cells is correlated with the existing number of RGCs. However, generation of the rod bipolar cells is unaffected by RGC shortage. Results report the first observation that RGCs selectively influence the genesis of subsequent retinal cell types. Our results show that, in the −/−Atoh7 retina, R-BPCs are generated in a normal number but degeneration become apparent after 1 month of age. Moreover, in the same retina, significantly fewer cone bipolar cells (C-BPCs) are generated, but their number remains stable throughout the adulthood. To investigate whether reduced C-BPC production is a cell autonomous or non-cell autonomous event, we further included two other retinas that had varying degree of RGC loss. Our results indicate that production of C-BPC is related to the existing number of RGCs, but production of R-BPC is independent from RGCs.The mammalian retina contains six major neuronal and one glial cell types. Among them, retinal ganglion cells (RGC), horizontal cells, amacrine cells and cone photoreceptors are born in the early phase of retinal neurogenesis and are regarded as embryonic or early cell types. Rod photoreceptors, bipolar cells and Müller glia are born in the late phase of retinal neurogenesis and are regarded as post-natal or late cell types. RGC, for its leading birth sequence, is believed to play an important developmental role in the formation of the remaining retinaAnimal use in these experiments was approved by the Animal Welfare Committee of the University of Texas Health Science Center at Houston in accordance with the guidelines established by the National Institutes of Health. In the process of euthanasia, animals at or younger than postnatal day 7 (P7) were anesthetized using hypothermia prior to decapitation to reduce pain and eliminate suffering. Animals at or older than P10 were euthanized through cervical dislocation by highly trained personnel to ensure no suffering in the process.Pou4f2 (AP/APBrn3b) and Atoh7 (LacZ/LacZMath5) mice were established as described previously −/−Pou4f2 and −/−Atoh7 in this study as we did not utilize their knock-in reporter genes. Double knockout of Atoh7 and Pou4f2  mice were generated previously in our laboratory −/−;Pou4f2−/−Atoh7 for the same reason. For simplicity, it is mentioned in the text as double knockout (DKO).Single knockouts of GFP/GFPAtoh7 and LacZ/LacZVsx1 mice were crossed to generate an −/−Atoh7;LacZ/LacZVsx1 line. The established line was then crossed with +/−Atoh7 mice to create −/−;Vsx1LacZ/+Atoh7 and +/−;Vsx1LacZ/+Atoh7 pups for examining C-BPC subpopulations using LacZVsx1 expression. Genotyping for LacZ/LacZVsx1 was conducted as described previously The −/−Pou4f2 and −/−Atoh7 retinas, were determined previously using various methods including 1,1′-dioactadecyl-3,3,3′,3′-tetramethylindocarbodyanine perchlorate (Dil) labeling by gene gun and retrograde labeling −/−Pou4f2 and −/−Atoh7 retinas respectively. These numbers are slightly higher than the rough estimations reported previously −/−;Pou4f2−/−Atoh7 DKO retina, DiI gunning yielded no RGCs and there was no optic nerve, making retrograde dye tracing impossible. Therefore, RGC number was estimated by comparing neurofilament light chain (NF-L) positive cells in the RGC layer in flat-mounted retinas with that of −/−Atoh7 retinas −/−;Pou4f2−/−Atoh7 retina had less than one fifth of the number of NF-L-positive axons that were observed in the −/−Atoh7 single mutant retina, which is corresponding to approximately less than 1% of normal RGC population. In this study, we designate the −/−;Pou4f2−/−Atoh7 (DKO) retina as having 1% of normal RGC number for simplicity.Numbers of RGCs in the The main sampling stage for estimating the number of proliferating cells in this study was postnatal day 4 (P4). This stage was selected for the best avoidance of overlapping production time of bipolar cells from other cell types. All the early cell types, RGCs, horizontal cells, cones, and amacrine cells, have ceased production at P4; while BPC production remains at its height and production of rod photoreceptors has dropped significantly Eyeballs were collected and fixed in 4% paraformaldehyde  in phosphate buffered saline (PBS) for 2 hr, washed 3 times with PBS-T, infiltrated in 15% sucrose overnight, and embedded in O.C.T. (Tissue-Tek 4583). Radial cryosections were collected at 30 µm thickness and only three most center sections in each retina were used. Center of the retina was judged by their largest circumference and presence of optic disc. Occasional obliquely sectioned retinas, as judged by shorter soma and abnormally thick nuclear layers, were discarded.Cryosections were blocked for 1 hr with 2% BSA 5% normal goat serum in PBS with 0.1% Triton X-100 (PBS-T). Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C. Samples were equilibrated to room temperature for 1 hr next morning, followed by 3× washing and 2 hr incubation with appropriate secondary antibody. For labeling proliferating cells at S-phase, BrdU (0.1 mg/g body weight) was intraperitoneally injected 2 hr prior to retina collection. For neuronal birthdating, BrdU (0.1 mg/g body weight) was injected twice in the selected postnatal date - one in the morning at approximately 10 am, the other in the afternoon at approximately 4 pm. Retinas were collected at P20. Primary antibodies and their working dilutions used in this study were as follows: mouse anti-Phospho Histone-3 , rat anti-Bromodeoxyuridine , sheep anti-Chx10 , rabbit anti-PKCα , rabbit anti-cleaved Caspase3 , mouse anti-Pax6 , and rabbit anti-β-gal . Nuclei were counter-labeled with TOPRO-3 or Propidium Iodide (PI).For flat-mount immunofluorescence of PH3 labeling, eyeballs were fixed with 4% PFA for 15 min, dissected into anterior and posterior halves, post-fixed for 1 hr, washed with PBS, blocked in 2% BSA, 5% normal goat serum in PBS-T for 1 hr at room temperature. Antibody labeling was processed the same as described for cryosections. Both the anterior  and posterior  halves of the retinas were mounted for confocal image collection.Images were captured using a Zeiss 510 confocal microscope. The laser power setting, gain and offset parameters were optimized in the initial experiments and remained unchanged for retinas from all genotypes. For retinal radial sections, images were taken from central and peripheral two distinct regions. The central region was defined by approximately 10–15% along the distance from the optic disc or from the center point when the retina does not have an optic disc. The peripheral region was collected as guided by the edge of the retina. The reason for sampling from two distinct regions of the retina is that, during retinogenesis, the central and peripheral portions of retina represent different developmental stages t-test was used for comparing data from only two genotypes. Statistical significance is defined as that p-value equal to or less than 0.05.Cell counts from six independent individual retinas, collected from 6 pups of at least 3 different litters , were averaged to represent data of one stage of each genotype. To compare the means among 4 genotypes at each time point, one-way ANOVA was performed with post hoc Tukey multiple comparisons. The independent samples −/−Atoh7 retina −/−Atoh7 retina was normal −/−Atoh7 mice, prompting the hypothesis that the apparent defective −/−Atoh7 retina undertook a degenerative path. Therefore, the discrepancy among different studies could have been results of honest reports on the continuously degrading retinas. To test whether there was a loss of R-BPCs in the −/−Atoh7 retinas after they reached maturation, we labeled retinas with PKCα and compared their numbers at the ages of 1-month, 2-month, 3-month and 8-month. Results showed that there was progressive R-BPC loss in the −/−Atoh7 retina  and retinas were collected at P20. BrdU co-labeling with Chx10 but not PKCα marked C-BPCs born at the injection day. BrdU co-labeling with Chx10 and PKCα marked the birth date of R-BPCs. Other BrdU positive cells lacking Chx10 or PKCα representing other late born cells  were counted but not presented because they were not related to the current study. Results showed that fewer C-BPCs (<50%) were generated at P1, P3, P5 in the −/−Atoh7 retina and no C-BPCs were generated in both −/−Atoh7 and wildtype retinas in the examined region at P7 (n = 3). In contrast to C-BPC birth, R-BPCs in the −/−Atoh7 retina were generated in relatively normal numbers if not at an increased level  and the −/−;Pou4f2−/−Atoh7 retina has more RGC loss (∼99%) compared to the −/−Atoh7 retina. We reason that proliferating cell numbers should reflect the existing RGC numbers if loss of RGCs, but not loss of genes, is the cause of reduced C-BPC production. For simplicity the −/−;Pou4f2−/−Atoh7 retina will be regarded as DKO herein.Neuronal production is often referred to as cell “birth” as neurons are specified at, or immediately preceding, their exit from the last cell cycle To cover the majority of the BPC production time window, retinas were collected at P0, P4, P7, and P10. Three center most cryosections, as judged by their largest circumference and presence of optic disc , were labeled with Phospho Histone-3 (PH3) and Bromodeoxyuridine (BrdU) antibodies to detect cells at M-phase and S-phase respectively. For BrdU labeling, BrdU was administered 2 hr prior to retina collection. The same antibodies were also used to label the flat-mounted retina for independent estimations. However, BrdU on flat-mounted retinas was later aborted because an objective counting was not achievable in our hands. To aid analyses, image data from the central retina and the peripheral retina were focused separately.−/−Pou4f2 nor −/−Atoh7 was significantly different to the wildtype at P0 but were both significantly lower than the wildtype at P4, the peak BPC production time. The DKO retina exhibited interesting proliferation dynamics in the sampling region: it was drastically lower than all other genotypes at P0, but became significantly higher than −/−Pou4f2 and −/−Atoh7 while remaining lower than the wildtype at P4  of BrdU-positive cells in the DKO retina may actually reflect the abnormally higher number of proliferating cells at a later stage. To avoid further confusion elicited by the unique cell proliferation dynamics in the central DKO retina, here we take only the PH3 data into consideration, which presents the accumulated proliferating cell number in the central retina as wildtype >−/−Pou4f2≈−/−Atoh7>DKO.In the central retina, the number of proliferating cells in neither pe at P4 . It appe−/−Pou4f2>−/−Atoh7>DKO. The accumulated proliferation of the whole retina was estimated by combining data from central and peripheral retinas of all examined stages: this result corresponds to the amount of existing RGCs in the retina showing wildtype >−/−Pou4f2>−/−Atoh7>DKO. Collectively, by examining retinas with varying degrees of RGC depletion, our results show that the amount of proliferating cells corresponds to the existing RGC number. Therefore, the observed low C-BPC production in the −/−Atoh7, and other RGC-depleted retinas is most likely a non-cell autonomous result of RGC-depletion but not a direct cell autonomous result of gene loss.Birth of BPCs begin prior to P0 despite peaking at ∼P3, P4. A retinal neurogenesis wave ripples from the center towards the periphery and development of peripheral retina is about 3 days behind the center of the retina −/−Atoh7 (5% RGC) and the DKO (1% RGC) retinas had a significantly lower BPC apoptotic rate compared to the wildtype. At P10, all RGC-depleted retinas had a significantly lower rate of BPC apoptosis compared to the wildtype. At P15, no Chx10-positive apoptotic cells were found in any retinal samples examined. The mean of the apoptotic rate showed a correlation between the existing RGC number and the BPC apoptotic rate. The retinas with the lowest RGC number displayed the lowest level of apoptotic BPCs  and the DKO retina (1% RGC), but they both had significantly fewer Chx10-positive cells than the −/−Pou4f2 retina (20% RGC). In the sampling area, there are only a handful of progenitor cells, which is negligible when compared to the number of bipolar cells at this stage. Therefore, differences in Chx10-positive cell numbers should represent differences in total bipolar cell numbers. Progenitors cease to exist in the sampling region prior to P7  are produced at a normal number but cone bipolar cells (C-BPCs) are produced at a lower number. The normally produced R-BPCs quickly degenerate between 1-month and 2-month of age, after which degeneration slowly proceeds. In contrast, the insufficiently produced C-BPCs are maintained at a stable number through out adulthood. By including two other RGC-depleted retinas, we further show that differential regulation of R-BPC and C-BPC production is a result of RGC loss and is unlikely a result of loss of Atoh7's cell autonomous function. We conclude that the number of C-BPC being born is related to the number of RGCs in the retina, but the total number of R-BPC being born is independent of RGCs.In this study, we re-examine the Pou4f2 is expressed after the final cell cycle and thus cannot be involved in any aspect of cell cycle regulation. Therefore, the reduction in cell proliferation observed in the −/−Pou4f2 retinas is more reasonably attributable to a secondary effect of the loss of RGCs. Atoh7 appears at S-phase of the last cell cycle before a cell is committed to become a neuron Pou4f2 is only expressed in RGCs and their precursors, but not in BPCs or their immediate precursors Atoh7 is downregulated at the time of BPC birth and it is not found in the cell lineage that give rise to BPCs −/−Pou4f2, −/−Atoh7, and DKO retinas are not directly related to gene loss. Third, reduction of cell proliferation and C-BPC production corresponds to the extent of RGC loss further strengthening our argument that reduced C-BPC production is the result of RGC shortage but not specific gene deletion.During retinogenesis, genetic networks are tightly intertwined. Knocking out a transcription factor often elicits a cascade of events and a series of phenotypes. Some phenotypes are related to the primary cell autonomous function of the missing factor that often leads to missing or aberrant cell types. Other secondary phenotypes may appear following the initially missing or over-produced cells. Therefore, using knockout mice to study gross cellular events in a given tissue presents a potential dilemma in deciphering the results. It is difficult to distinguish whether the observed phenotype is the primary effect of loss of the protein's cell autonomous function or the secondary effect elicited by the altered cell population. However, by comparing multiple knockout mice that have a similar alteration of cell populations, the primary cell autonomous and the secondary non-cell autonomous effects can be dissected. Here, we provide three arguments that suggest, rather than a direct effect of losing Atoh7 and/or Pou4f2 protein function, our observations of reduced cell proliferation and reduced C-BPC production are secondary effects caused by RGC loss. First, neither of these genes is directly involved in promoting cell cycles: −/−Atoh7 and −/−Pou4f2 retinas are cell autonomous consequences of the missing proteins. It has been shown in the −/−Atoh7 retina that amacrine cells, cone cells, and horizontal cells are increased −/−Atoh7-expressing cells transdifferentiate into other early retinal cell types at the expense of RGCs −/−Pou4f2-expressing cells become amacrine-like cells at the expense of RGCs −/−Pou4f2 and DKO retinas at P5 observed in this study  or loss of RGCs. R-BPCs do not form direct synapses with RGCs in the retinal circuitry. Instead, R-BPCs synapse with AII (A2) amacrine cells −/−Atoh7 retina Our results show that production of R-BPCs is not affected by either loss of genes (The results of this study provide the initial suggestion that production of C-BPC and R-BPC are differently regulated. However, our results do not predict possible molecular candidates that are involved in C-BPC and R-BPC fate decision. Emerging evidence shows that retinal progenitors are heterogeneous


The optimal cut-off value with the best prognostication effect for the hearing improvement at the fixed stage was an initial I/Clatency on affected-ear stimulation of 1.34  and an initial I/Clatency on healthy-ear stimulation of 0.76 , respectively. This study suggested that a dynamic process of central auditory plasticity can be induced by peripheral lesions. The hemispheric asymmetry at the initial stage bears an excellent prognostic potential for the treatment outcomes and hearing level at the fixed stage in ISSNHL. Our study demonstrated that such brain signature of central auditory plasticity in terms of both N100m latency and amplitude at defined time can serve as a prognostication predictor for ISSNHL. Further studies are needed to explore the long-term temporal scenario of auditory hemispheric asymmetry and to get better psychoacoustic correlates of pathological hemispheric asymmetry in ISSNHL.The longitudinal relationship between central plastic changes and clinical presentations of peripheral hearing impairment remains unknown. Previously, we reported a unique plastic pattern of “healthy-side dominance” in acute unilateral idiopathic sudden sensorineural hearing loss (ISSNHL). This study aimed to explore whether such hemispheric asymmetry bears any prognostic relevance to ISSNHL along the disease course. Using magnetoencephalography (MEG), inter-hemispheric differences in peak dipole amplitude and latency of N100m to monaural tones were evaluated in 21 controls and 21 ISSNHL patients at two stages: initial and fixed stage (1 month later). Dynamics/Prognostication of hemispheric asymmetry were assessed by the interplay between hearing level/hearing gain and ipsilateral/contralateral ratio (I/C) of N100m latency and amplitude. Healthy-side dominance of N100m amplitude was observed in ISSNHL initially. The pattern changed with disease process. There is a strong correlation between the hearing level at the fixed stage and initial I/C Functional imaging of brain reorganization and neurodynamics in response to central lesions provides essential information related to the prognosis of animals Idiopathic sudden sensorineural hearing loss (ISSNHL) is a good disease model for the study of the association between auditory neuroplasticity and clinical presentations. ISSNHL, a disease entity of unknown pathogenesis, is widely varied in the presenting signs and prognosis. Though it is possible that the neural deficit(s) lie at a higher level of the auditory pathway, the cochlea has generally been considered the most probable lesion site of ISSNHL. About one third to a half of affected persons achieve partial or complete recovery of hearing after appropriate interventions By choosing patients with mild-to-moderate hearing impairment, we have previously confirmed by means of MEG that acute unilateral ISSNHL can induce functional reorganization in terms of altered hemispheric asymmetry for sound processing in the central auditory pathway on either affected- or healthy-ear stimulation Our MEG findings are corroborated by a functional magnetic resonance imaging (fMRI) study in which loss of contralateral dominance on healthy-ear stimulation in patients with unilateral ISSNHL was reported We thus investigated whether or not the pattern of hemispheric asymmetry bears any prognostic effect with respect to the recovery of function in patients with acute unilateral ISSNHL by using MEG in this study. AEFs were assessed by measuring N100m in ISSNHL  and in normal hearing subjects (once only during the study). Ipsilateral/contralateral ratio was used to assess the degree of hemispheric asymmetry during follow-ups and determine the prognostic relevance to unilateral ISSNHL The study was in compliance with Declaration of Helsinki. Written informed consent was obtained from each participant with a protocol approved by the Institutional Ethics and Research Committee of Taipei Veterans General Hospital and Cheng Hsin General Hospital.Twenty-one right-handed, previously untreated adult patients with acute unilateral left (n = 11) or right (n = 10) ISSNHL  were studied. Initial MEG responses of thirteen patients had been reported in our preliminary one-time point studies Twenty-one right-handed healthy volunteers with normal hearing  served as control. Thirteen controls were involved in our previously published studies All participants underwent PTA exam to determine both air and bone conduction threshold, using test frequencies between 250 Hz to 8000 Hz. Controls had normal PTA results . A unilateral sensorineural hearing loss was confirmed in all ISSNHL patients, characterized as cochlea being the lesion site based on results of reduced distortion-product otoacoustic emissions (DPOAEs) and within-normal-limit interaural latency differences for auditory brainstem responses (ABRs) μV were rejected. MEG signals were sampled at 400 Hz and band-pass filtered at 0.03 to 100 Hz. About 90 artifact-free trials were averaged. Digital low-pass filtering at 30 Hz and high-pass filtering at 1 Hz was performed off-line. An equivalent current dipole (ECD) model consisting of bilateral sources was used to explain the MEG signals MEG measurements were done in a magnetically shielded room using a whole-head 306-channel neuromagnetometer . Subjects sat upright with eyes open during measurements. Tone bursts  were delivered monaurally via molded earpieces using the SoundProbe™ program on a Macintosh computer. The contralateral (i.e. non-stimulated) ear was plugged by using molded earpiece to minimize the ear-to-ear crosstalk. Affected and healthy ears were monaurally stimulated in separate sessions separated by two minutes of rest. Trials with electro-oculographic amplitudes exceeding 150 a and I/Cl for N100m dipole moment and latency, respectively), i.e., ratio of N100m in ipsilateral to that in contralateral hemisphere on unilateral-ear stimulation, and (2) I/C made by reactivity of same hemisphere to stimulus at two ears , i.e., ratio of N100m in one hemisphere on ipsilateral-ear stimulation to that in the same hemisphere on contralateral-ear stimulation. The prognostic relevance of the hemispheric asymmetry as expressed in the relationship between I/C and hearing level/hearing gain in ISSNHL was evaluated using Spearman' rank correlation. Differences of I/C among prognostic subgroups  were evaluated using both Kruskal-Wallis and Mann-Whitney U test. Receiver operating characteristic (ROC) curve was then used to determine the best cut-off value of I/C for the prognostication of hearing improvement at the fixed stage. Statistical significance was thresholded at P<0.05.The epoch analyzed ranged from 50 ms before to 350 ms after stimulation onset. The prestimulus interval was used as baseline. The time window for N100m was 70∼160 ms In all subjects, N100m dipole was identifiable over each hemisphere and was localized bilaterally on the superior temporal planum with an orientation centrifugal to the auditory cortex , and S3.as on healthy-ear stimulation (p = 0.046), initial I/Cls on healthy-ear stimulation (p = 0.027), and initial I/Cls on affected-ear stimulation (p = 0.02). Mann-Whitney U test furthermore showed that the differences existed between subgroups of complete and partial recovery , as well as those of partial and no recovery , but not those of complete and no recovery . The ROC curves in turn showed the best prediction effect of I/C for the hearing improvement at fixed stage: between subgroups of complete and partial recovery, the optimal cut-off value was an initial I/Cls on affected-ear stimulation at 1.34 ; between subgroups of partial and no recovery, the optimal cut-off value was an initial I/Cls on healthy-ear stimulation at 0.76 , a contralateral dominance of dipole moment was noted (p<0.001). A faster N100m response was also noted in the contralateral hemisphere (p<0.001). A subset analysis (n = 21) of peak N100m moment made according to the ear stimulated revealed a significant contralateral preponderance upon both left-ear (p<0.001) and right-ear stimulation (p = 0.002). Inter-hemispheric latency differences were significant for both left-ear (p<0.001) and right-ear stimulation (p = 0.002) on the subset level.On initial MEG exam of patients, the contralateral N100m was significantly shorter in response latency (p = 0.001) as compared to that of ipsilateral hemisphere (n = 42). Contralateral dominance was not observed (p = 0.062) on the pooled data from stimulation of both ears . However, a healthy-side dominance was observed when responses from hemispheres ipsilateral to the healthy ears were pooled and compared with those from hemispheres ipsilateral to the deaf ears, irrespective of the ear stimulated . No inter-hemispheric difference in latency was observed (p = 0.677).On MEG exam of patients at the fixed stage, the contralateral N100m was significantly shorter in response latency (p<0.001) as compared to that of ipsilateral hemisphere (n = 42). Contralateral dominance was observed (p = 0.015) on the pooled data from stimulation of both ears (n = 42). Neither evidence of “healthy-side dominance” (p = 0.142) nor inter-hemispheric difference in latency (p = 0.441) was observed when responses from hemispheres ipsilateral to the healthy ears were pooled and compared with those from hemispheres ipsilateral to the deaf ears, irrespective of the ear stimulated (n = 42).ls on affected-ear stimulation at 1.34 for the prognostication between subgroups of complete and partial recovery, and an initial I/Cls on healthy-ear stimulation at 0.76 for that between subgroups of partial and no recovery, respectively  was about the same to that of the N100m response in the contralateral hemisphere relative to healthy-ear stimulation in 1-mon MEG exam (64.8). This seems surprising given that the stimulus level used was constant. With a stimulus level of 75 dB SPL at 1000 Hz, the difference on hearing threshold translates into a pronounced disparity in sensation level (dB SL) of bilateral ears. Roughly, 65 dB HL translates to approximately 10 dB SL in the affected ear. For the healthy ear (no worse than 25 dB HL), the stimulus level is probably at least 50 dB SL. One reasonable explanation of this result should be the inner-ear hearing impairment itself. Previous study reported that inner-ear hearing loss revealed enhanced N100m response, and the amplitude of N100m response on affected-ear stimulation at 5∼10 dB SL was almost equal to that on healthy-ear stimulation at 50∼60 dB SL One major and novel finding in the current study is the prognostic relevance of hemispheric asymmetry in terms of ipsilateral/contralateral ratio for N100m responses. When ipsilateral/contralateral ratio were grouped according to the prognosis and analyzed, the ROC curves demonstrated that the cut-off value with best prediction effect of hearing improvement at the fixed stage was an initial I/Cls on either affected- or healthy-ear stimulation and I/Ca on affected-ear stimulation in ISSNHL might entail a better adaptation and compensational bi-hemispheric synergism of the auditory system to the abrupt hearing loss for the functional restitution after the insult. The observations in the current study suggested that the ipsilateral/contralateral ratio of both N100m latency and amplitude be a sensitive parameter to index for the subtle functional modulation/plasticity in ISSNHL of slight to moderate degree. Although efforts have been made to correlate various variables with the prognosis, no single biomarker was yet found to reliably prognosticate the eventual outcome and/or hearing level in ISSNHL The above-mentioned findings thus pinpoint a good prognostication of the outcome at defined time. Since the ipsilateral/contralateral ratio subserves an indicator of auditory plasticity The actual mechanisms of central auditory plasticity in the functional recovery of ISSNHL are currently unknown. Although the mean age of our controls is less than that of patients, the observations cannot be ascribed to the ageing effect or to the influence of dipole location In the present study, elapsed time for the initial MEG exam after disease onset ranged from 3 days to 3 weeks. It seemed that this could lead to an underestimate of the difference between findings at initial and fixed stages in patients who had late initial scan at 3 weeks, though the test interval between two stages was actually the same among patients. However, MEG results were significantly different between the two stages. The significantly higher initial ipsilateral/contralateral ratio on healthy-ear stimulation and the significantly lower initial ipsilateral/contralateral ratio on affected-ear stimulation, in comparison to that of controls on either-ear stimulation, yielded a pattern of healthy-side dominance in the acute stage of ISSNHL. Higher ipsilateral/contralateral ratio on healthy-ear stimulation and lower ipsilateral/contralateral ratio on affected-ear stimulation at initial MEG exam for patients were both replaced by values approaching ∼1.0 at the fixed stage, resulting in a relatively symmetrical pattern of N100m responses. This finding confirmed previous results showing a pattern of more equal activation over bilateral hemispheres on monaural stimulation in chronically unilateral deaf patients than those in acute stage and normal-hearing subjects One possible explanation of our finding is the involvement of neurochemical changes observed in the auditory system following peripheral hearing loss. Animal studies have revealed a down-regulation of both ipsilateral excitatory receptor expression/binding and contralateral inhibitory neurotransmitters synthesis with respect to the affected ear in the central auditory pathway In summary, the hemispheric asymmetry expression at initial stages bears the best prognostic potential for the treatment outcomes and/or the hearing levels at the fixed stage in acute unilateral ISSNHL of mild-to-moderate degree. In addition, this study suggested that a dynamic process of central auditory plasticity can be induced by peripheral lesions. Our study demonstrated that such brain signature of central auditory plasticity at defined time can serve as a prognostication predictor for ISSNHL. To strengthen the clinical application of our findings, studies using electroencephalography are required to verify the temporal changes in asymmetry expression within clinical settings since MEG is mainly a research tool.Figure S1Source waveforms at respective stages by ear stimulation in ISSNHL patients. Healthy-side dominance of N100m responses was observed initially . At the fixed stage , a relatively symmetrical pattern  of N100m responses was noted. Healthy, healthy-ear stimulation; Affected, affected-ear stimulation; Contra, hemisphere contralateral to the stimulated ear; Ipsi, hemisphere ipsilateral to the stimulated ear.(TIF)Click here for additional data file.Table S1Amplitude and latency of N100m dipole moment on initial MEG exam and 1 month after initial exam in controls. The excellent consistence of MEG makes it suitable for repeated follow-up measurements of auditory evoked responses, which is in line with experiences in our lab. To verify that the findings observed in our patients did not result from the differences due simply to the test-retest bias, we surveyed again the test-retest reliability for six additional controls with the same test-interval (i.e. one month) applied in ISSNHL patients. The results of evaluation for the reliability of repeated MEG exams in these normal hearing subjects showed no significant differences between test and retest over an interval of about one month in terms of peak dipole moment amplitude and peak latency.(DOC)Click here for additional data file.Table S2Relative position of N100m peak dipole at various stages in terms of Talairach coordinates . Relative position of N100m peak dipole between 2 repeated measurements in ISSNHL was expressed in terms of Talairach' nomenclature. Differences of N100m dipole location  at various stages were evaluated using Wilcoxon signed rank test. There are no significant differences between N100m source locations of two stages.(DOC)Click here for additional data file.Table S3Amplitude and latency of peak dipole moment for N100m.(DOC)Click here for additional data file.


Pyrenophora and few have investigated the genetic contributions of HGTs to fungi. We investigated HGT events in P. teres and P. tritici-repentis and discovered that both species harbored 14 HGT genes derived from bacteria and plants, including 12 HGT genes that occurred in both species. One gene coding a leucine-rich repeat protein was present in both species of Pyrenophora and it may have been transferred from a host plant. The transfer of genes from a host plant to pathogenic fungi has been reported rarely and we discovered the first evidence for this transfer in phytopathogenic Pyrenophora. Two HGTs in Pyrenophora underwent subsequent duplications. Some HGT genes had homologs in a few other fungi, indicating relatively ancient transfer events. Functional analyses indicated that half of the HGT genes encoded extracellular proteins and these may have facilitated the infection of plants by Pyrenophora via interference with plant defense-response and the degradation of plant cell walls. Some other HGT genes appeared to participate in carbohydrate metabolism. Together, these functions implied that HGTs may have led to highly efficient mechanisms of infection as well as the utilization of host carbohydrates. Evolutionary analyses indicated that HGT genes experienced amelioration, purifying selection, and accelerated evolution. These appeared to constitute adaptations to the background genome of the recipient. The discovery of multiple interkingdom HGTs in Pyrenophora, their significance to infection, and their adaptive evolution, provided valuable insights into the evolutionary significance of interkingdom HGTs from multiple donors.Many studies have reported horizontal gene transfer (HGT) events from eukaryotes, especially fungi. However, only a few investigations summarized multiple interkingdom HGTs involving important phytopathogenic species of  Pyrenophora includes almost two hundred species, some of which are important plant pathogens that cause significant losses to crop yields, such as P. teres, P. graminea, and P. tritici-repentis. Pyrenophora teres, a necrotrophic fungal pathogen, causes net-spot blotch on economically important agricultural crops such as barley (Hordeum vulgare) Pyrenophora graminea is the causal agent of barley stripe, which once caused significant crop yield losses in many areas of the world. Similarly, P. tritici-repentis is the agent of tan (or yellow) spot that mainly affects wheat (Triticum aestivum); it causes great losses by reducing photosynthesis. The published genomes of two fungal pathogens, P. teres and P. tritici-repentis, facilitate genetic investigations of their pathogenicity, virulence, and mechanisms of infection, both of which are important factors for combating the negative effects of these fungi.Fungal genus Horizontal gene transfer (HGT) involves the transfer of genetic information between distantly related organisms outside of normal  mating systems. HGT is an important driving force in genomic evolution because the recipient acquires novel genes that award new functions. For example, such genes can accelerate adaptation to new environments, broaden host range or change its diet, and even provide the ability to survive in previously lethal conditions Well-supported cases of HGT exist, especially in archaeal and eubacterial genomes Pyrenophora is notable for its recentness; an 11 kb region encoding the host-specific toxin ToxA is thought to have been transferred from Phaeosphaeria nodorum (anamorph Stagonospora nodorum) to the previously avirulent fungus P. tritici-repentis only about 70 years ago P. teres, suggesting a possible, recent HGT between P. tritici-repentis and P. teresPyrenophora.One case of HGT in Pyrenophora, it is necessary to identify potential interkingdom HGT events that may be important to the evolution of phytopathogenic species.Interkingdom HGT, especially bacterial–fungal HGT, plays an important role in the evolution of fungal metabolism, propagation, and pathogenicity P. teres and P. tritici-repentis. We comprehensively search for homologies and employ phylogenetic analyses. The analyses discover multiple ancient bacterial–fungal HGT events. Some bacterial–fungal HGT genes participate in metabolic pathways and in doing so they add important traits to the recipients. Other HGTs involving protein-coding genes associated with extracellular locations may advance virulence. A probable HGT gene coding a leucine-rich repeat protein in two species of Pyrenophora may originate from a host plant; this gene may play an important role in plant pathogenesis and host protein–protein interactions. Homology distributions of the HGT genes derived from bacteria and plants, taken together with their compositional characteristics and conservative synteny in fungal recipients, identify some ancient HGT events. Evolutionary analyses identify amelioration, purifying selection, and accelerated molecular evolution in these HGT genes, indicating adaption to new genomic backgrounds. Ours is the first thorough survey of HGT in species of Pyrenophora and their closely related species using analyses of whole genomes. It opens the first window to understanding the role interkingdom HGT plays in the evolution of these fungi.Herein, we explore HGT in a suite of taxa representing a variety of kingdoms ranging from viruses, bacteria, protists, plants, and animals to the fungi P. teres and P. tritici-repentis  associate with plants. Except for enophora . Thus, HCOG, GO, and KEGG analyses were used to predict the function of each HGT gene . UnfortuPyrenophora and their host plants. This is especially true for leucine-rich repeat protein, whose gene may be from the host plant itself.It is surprising that seven, about half of the 16 HGT genes have putative extracellular locations. Extracellular enzymes may relate to interactions between species of Pyrenophora may interfere with Tsn1–ToxA interactions. Unfortunately, the absence of EST data for leucine-rich repeat protein precludes analyses of variation in expression during the process of infection. Although we cannot demonstrate its function directly, the LRR domain, protein-binding function, and extracellular location indicate that leucine-rich repeat protein may facilitate interaction of the two species of Pyrenophora and their hosts. In some plants, this protein appears to function as receptor-like kinase or disease resistance protein. Faris et al. suggested that species of Pyrenophora may thrive by subverting the resistance mechanisms acquired by plants to combat other pathogens Genes with nucleotide binding sites (NBS), leucine-rich repeats (LRR) and/or serine/threonine protein kinase (S/TPK) domains often confer plant disease-resistance. Tsn1 has disease-resistance gene-like features, including S/TPK and NBS-LRR domains in host plants Pyrenophora. Xylanase is an important constituent of the plant's cell wall, and xylanase A can interact with it to promote the degradation of the structure. Species of Pyrenophora and other fungi acquired the gene encoding xylanase A from bacteria and they secrete the enzyme extracellularly. The phyletic distributions of the recipients indicates that all fungi with this gene associate with plants   . This diPyrenophora and donors include a diversity of bacteria and host plants. This is the first report of probable HGT from host plants to species of Pyrenophora. Seven HGT genes encode extracellular proteins that may drive the infection of host plants by species of Pyrenophora and closely related species. Some of these proteins likely interfere with plant defense-response. Other enzymes participate in the degradation of plant cell walls. Further, some intracellular enzymes encoded by HGT genes may participate in carbohydrate metabolism. Therefore, the HGT genes undoubtedly contribute to virulence and the utilization of their carbohydrates of host plants by species of Pyrenophora. Amelioration, purifying selection, and accelerated molecular evolution adapted HGT genes to new genomic backgrounds. Multiple interkingdom horizontal gene transfers may make significant contributions to the evolution of phytopathogenic Pyrenophora and their closely related species. Further investigations will lead to a more complete understanding of the biological functions of these HGT genes.Multiple interkingdom HGT occurs in species of P. teres and P. tritici-repentis, which have 33.58 and 37.36 million base pairs (Mbp) with 11,799 and 12,169 identified protein-coding genes, respectively The genomic sequences of http://www.ncbi.nlm.nih.gov/) along with the corresponding taxonomy (http://www.ncbi.nlm.nih.gov/taxonomy). This data contained a wide diversity of viral, eukaryotic, and prokaryotic taxa in the following groups: viruses, bacteria, fungi, protozoans, plants, and animals.The RefSeq and non-redundant (nr) sequences (2012.6.21) were downloaded from NCBI  but a wide taxonomic diversity in other groups  P. teres and P. tritici-repentis, we used a multistep, bioinformatic pipeline. Each genome of Pyrenophora was treated separately. We used BLAST to detect proteins present in a few fungal species only but widespread in other kingdoms −5. We aimed to detect recent HGT events involving species of Pyrenophora only as well as to identify ancient HGTs in Pyrenophora and other fungi. Therefore, proteins found in less than 10 fungal species were considered to be candidates. Next, candidate protein-sequences were compared to the non-fungi database using BLASTP with an e-value threshold of 1e−5. Proteins with over 20 species-hits in non-fungi were selected for further study To identify possible HGT events in P. teres and P. tritici-repentis we were calculated. We searched the transferred proteins in Pyrenophora against their own corresponding genome using a TBLASTN  to identify potential pseudogenes, those that could not translate intact to protein sequences.The amino acid (AA) lengths and number of introns of candidate HGT genes in the genomes of Pyrenophora. HGT genes present in a single genome of Pyrenophora were tested for being a product of DNA contamination from other organisms during genome-sequencing. When flanking sequences of HGT gene indicated vertical inheritance by being congruent with the species phylogeny, we assumed the putative HGT gene was located on the genome of Pyrenophora and physically linked to native genes We tested for possible artifacts and contamination in the two species of −5. The selected sets of significant homologous proteins were subjected to multiple alignment with ClustalW2 We reconstructed gene trees for all retained candidate HGT genes including their closest homologs. Homologs were identified by searching non-redundant databases by BLASTP with an e-value 1ePhylogenetic analyses were performed with two approaches: maximum likelihood (ML) and Bayesian inference (BI). We also employed distance-based neighbor-joining (NJ) trees, which were constructed by using the program Neighbor in Mega5 Pyrenophora, the transfer was assumed to have occurred in that specific lineage only. Candidate genes that had hits in two species of Pyrenophora indicated a HGT event in their common ancestor. When candidate HGT genes had homologs in several fungal species that formed a monophyletic group and then nested in non-fungal species, we assumed that the common ancestor of all fungal species received the HGT. Multiple HGT events from a similar donor with subsequent transfer among fungi was also possible. In this case, we analyzed the tree topology and investigated synteny of each HGT gene across both species of Pyrenophora and other fungi. If the homologs did not form a monophyletic group, the protein could have been transferred via multiple independent HGTs When a transferred gene occurred in a single species of We performed COG, GO, and KEGG analyses to infer function. KEGG analyses inferred that HGT proteins functioned within the metabolic network. For this analysis, we used KAAS (KEGG automated annotation server) to query metabolic pathways of HGT genes by BDH  orthology searches Pyrenophora were subjected to SignalP To investigate putative cellular locations of each HGT-encoded protein, all HGT genes in both species of −10.We compared HGT genes to those in the GenBank EST database to clarify the expression-pattern. We also searched the Taxonomically Broad EST eukaryotic database (TBestDB) P. teres, P. tritici-repentis, and species with highest identity to HGT genes in non-fungi groups were calculated using CodonW (http://codonw.sourceforge.net/). Four indices of codon usage bias included CAI (codon adaptation index), CBI (codon bias index), FOP , and ENC (effective number of codons).GC content, GC3s content, and codon usage of each gene in Pyrenophora and in their corresponding donor groups We conducted selection pressure analyses for each HGT gene. Using Codeml in PAML 4 Figure S1The phylogenetic trees of 16 types HGT genes in Pyrenophora species. Bayesian trees are shown; the ML trees and NJ trees exhibited substantially the same topologies. Nodal support values ≥50 shown (BI/ML/NJ). Asterisks (*) indicate support values <50. Pyrenophora sequences are indicated in red, while HGT gene sequences from other fungi are indicated in green.(PDF)Click here for additional data file.Figure S2Multiple alignment of amino acid sequences of enterochelin esterase-like enzyme in Pyrenophora.(DOC)Click here for additional data file.Figure S3GC and GC3s content of horizontally transferred genes in P. tritici-repentis and top-hit species in non-fungal groups. GC3s and GC content of P. tritici-repentis are the mean values of the complete coding sequence (CDS). Gene 1–14 refers to the genes coding leucine-rich repeat protein, methyltransferase MppJ, beta-galactosidase, UDP-glucosyltransferase, GCN5-related N-acetyltransferase, oxidoreductase, Gfo/Idh/MocA family, enterochelin esterase-like enzyme, N-acetylglucosaminyltransferase, succinylglutamate desuccinylase/aspartoacylase, 5-formyltetrahydrofolate cyclo-ligase, NmrA family protein, glcG protein, xylanase A, cyanophycinase, respectively.(TIF)Click here for additional data file.Figure S4The value of 4 index of codon bias: CAI, CBI, Fop and ENC of HGT genes, P. tritici-repentis and top-hit species in non-fungal groups. CAI value. (B) CBI value. (C) Fop value. (D) ENC value. The CAI, CBI, Fop and ENC value of P. tritici-repentis are the mean value of all the CDS. Gene 1–14 refers to the genes coding leucine rich repeat protein, methyltransferase MppJ, beta-galactosidase, UDP-glucosyltransferase, GCN5-related N-acetyltransferase, oxidoreductase, Gfo/Idh/MocA family, enterochelin esterase-like enzyme, N-acetylglucosaminyltransferase, succinylglutamate desuccinylase/aspartoacylase, 5-formyltetrahydrofolate cyclo-ligase, NmrA family protein, glcG protein, xylanase A, cyanophycinase, respectively.(TIF)Click here for additional data file.Table S1The amino acid (AA) lengths and number of introns of candidate HGT genes in the genomes of Pyrenophora teres and P. tritici-repentis.(DOC)Click here for additional data file.Table S2The synteny of HGT genes in Pyrenophora and closely related species. Upstream gene of HGT, HGT genes and downstream gene of HGT in P. teres were used to compare with P. tritici-repentis and other closely related fungal recipients. If homologs of these genes located with seriation in other fungi, this HGT gene can be regarded as good synteny in recipients.(XLS)Click here for additional data file.Table S3Lists of 93 fungi used in the analyses. Species with red font means that they associate with plants in their lifestyles.(XLS)Click here for additional data file.


Objectives. Vestibular hearing as an auditory sensitivity of the saccule in the human ear is revealed by cervical vestibular evoked myogenic potentials (cVEMPs). The range of the vestibular hearing lies in the low frequency. Also, the amplitude of an auditory brainstem response component depends on the amount of synchronized neural activity, and the auditory nerve fibers' responses have the best synchronization with the low frequency. Thus, the aim of this study was to investigate correlation between vestibular hearing using cVEMPs and neural synchronization via slow wave Auditory Brainstem Responses (sABR). Study Design. This case-control survey was consisted of twenty-two dizzy patients, compared to twenty healthy controls. Methods. Intervention comprised of Pure Tone Audiometry (PTA), Impedance acoustic metry (IA), Videonystagmography (VNG), fast wave ABR (fABR), sABR, and cVEMPs. Results. The affected ears of the dizzy patients had the abnormal findings of cVEMPs (insecure vestibular hearing) and the abnormal findings of sABR . Comparison of the cVEMPs at affected ears versus unaffected ears and the normal persons revealed significant differences (P < 0.05). Conclusion. Safe vestibular hearing was effective in the improvement of the neural synchronization. F0) frequency and exceeds that of the cochlea for low frequencies , where An = p13 − n23  and Ad  = p13 − n23 (the peak-to-peak amplitude in the affected ear). In bilateral case cVEMPs asymmetry ratio is not calculated. In the control group, this ratio was calculated using the peak-to-peak amplitudes for the right ear and left ear, respectively. The cVEMPs results for the control group were used as normative data. The normative values for latency and cVEMPs asymmetry ratio were calculated as mean ± two standard deviations [During cVEMPs recording patients were instructed to turn and hold their heads as far as possible toward the side contralateral to the stimulated ear . MoreoveThe subjects were tested without sedation, with noninverting electrode placed at the high forehead and inverting electrode on ipsilateral mastoid and ground electrode on contralateral. Electrode impedances were roughly equivalent and were <5 kilohms at the start of the test. Responses to 2000 stimuli were averaged, and each response (rate of 37/s) was replicated. Responses were filtered from 30 to 3000 Hz. The stimulus in our paradigm was a 2-0-2 tone burst , Blackman windowed. A response window of 25 ms was used when responses were recorded for all toneburst stimuli , 15. Thet-test for equality of means, Levene's test for equality of variances, and one-way ANOVA for continuous variables. A P-value of <0.05 was considered to indicate statistical significance.Data were analyzed by The dizzy patients presented with a total of forty-four ears . The affected ears consisted of benign paroxysmal positional vertigo (11 ears with BPPV = 25%), migraineurs (5 ears = %11.4), vestibular neuritis (2 ears = %4.4), psychogenic causes with the symptom of true vertigo during few hours after divorce, strife and death of father (5 women-5 ears: %11.4). Twenty-one patients were ipsilesional affected and one patient with BPPV was affected bilaterally.μv. The mean cVEMPs asymmetry ratio was 6.5 ± 10.2%, and the upper limit for this ratio (two standard deviations above the mean) was 26.9%. Testing of cVEMPs was done in both ears of each control subject (20 right and 20 left ears). The latency and the amplitude values of cVEMPs were detectable in all healthy persons (40 ears safe vestibular hearing). The mean latency values for p13 and n23 were 12.7 ± 1.0 and 20.1 ± 2.2 ms, respectively . TherefoP < 0.05 for both). The mean peak-to-peak amplitude in the affected ears was significantly lower than that in the control group (P < 0.05).The cVEMPs abnormalities (insecure vestibular hearing) included both decreased amplitudes and delayed latencies in twelve  and absent responses in eleven . In all dizzy patients, the cVEMPs asymmetry ratio findings indicated depressed response on the side with lower amplitude findings in a single ear only. The mean p13 and n23 latencies in the affected ears were both longer than the respective means in the control group . Also, tsABR was recordable bilaterally from all healthy persons (P < 0.05). Comparison of the sABR at affected ears versus unaffected ears and the normal persons revealed significant differences (P < 0.05). persons . It had sABR) and unaffected ears , respectively.The main outcome measures were differences in amplitudes, p13-n23 latencies of the cVEMPs between affected ears . Thus, safe vestibular hearing improved neural synchronization.Comparison of the cVEMPs at affected ears versus unaffected ears and the normal persons revealed significant differences (pitch [F0 = ∼100 Hz), women (F0 = ∼200 Hz), children (F0 = ∼400 Hz), [Pitch is the perceptual correlate of the fundamental frequency (F0) [pitch or voiced speech sounds and notes from musical instruments often consist of frequencies at integer multiples of F0. Such sounds, like a single violin note or a syllable in speech, are usually heard during neural synchronization [The range of vestibular hearing happens to coincide with the range of our voice pitch , which v400 Hz), –26. Pitccy (F0) , 26. Thenization , 27–29.F0 [F0 [Also, the auditory nerve fibers in the brainstem pathway are temporally precise, with better stimulus synchronization to F0 . Indeed,F0 [F0 , 24, 29.F0 [F0 , 30, 31.Also, the ABR is composed of several voltage deflections occurring within the first 15 ms after stimulus onset. These deflections (peaks and troughs) represent far-field synchronous activity produced by onset responses of neural elements and abrupt bends in the neural fiber tracts of the eighth nerve and the auditory brainstem pathway. The amplitude of an ABR component depends on the amount of synchronized neural activity and varies with level of stimulation. These factors affect the amount of neural activity generated, the degree of synchronization among neural elements activated, or both. The larger amplitudes of the waves in ABR may represent better auditory fibers that synchronized activation of fewer elements rather than activation of larger numbers of neural elements , 15, 32.Consequently, the major measures of the ABR are the latency and amplitude of its peaks and dependent on synchronization of stimulus onset. The neural synchronization or the neural conduction time in the brainstem pathway is responsible for the peak neural activity. Thus, a change in the transmission time of neural fiber activity results in poorer synchronization and delays in neural activation, leading to longer peak latencies of the combined activity of the neural fibers .Moreover, a number of specific brain areas may be activated by the vestibular hearing . And theFinally, we concluded that people with safe vestibular hearing have intact projections to cochlear nucleus, lateral lemniscus, and to inferior colliculus. sABR. On the other hand, the lower amplitude, rounded shape, and longer latency of sABR are the evidence for uncertain saccular projections in the brainstem pathway and insecure vestibular hearing. Then, safe vestibular hearing is effective in the improvement of the neural synchronization. These projections can increase the peaks of


The cremaster muscle in rats was spread over a plastic chamber and a gastric tube was placed into the stomach. Blood flow in the cremasteric artery was determined using a laser Doppler flowmeter, while blood pressure and heart rate were measured by the tail-cuff method. Red blood cell velocity in arterioles and blood flow in the artery were significantly increased 5 min after the administration of 10 mg/kg flavan-3-ols compared with distilled water treatment. The number of capillaries recruited in the cremaster muscle was also significantly increased 15 min after treatment. Microscopic observation confirmed that increased shear stress on endothelial cells was maintained during the measurement period. The mean arterial blood pressure and heart rate were also significantly elevated soon after administration and returned to baseline before the end of the observation period. Plasma nitrate and nitrite levels, and NO phosphorylation of aortic tissue were significantly increased at 60 min after administration of flavan-3-ols. According to these results, a single oral dose of flavan-3-ols elevates blood pressure and flow transiently, and these effects induce NO production through increased shear stress on endothelial cells.Several systematic reviews have reported that flow mediated dilatation (FMD) was significantly increased in subjects after ingestion of chocolate that contains flavan-3-ols; however, the mechanisms responsible for this effect are not clear. In this study, we evaluated the effects of a single oral dose of flavan-3-ols on the systemic circulation and microcirculation in the cremaster muscle using intravital video microscopy  Theobroma cocoa, is one of the ingredients in chocolate and cocoa. It is known to be rich in polyphenols, such as the flavan 3-ol monomers, (+)-catechin and (-)-epicatechin; and oligomers, such as the B-type flavan 3-ols linked by C4-C8 bonds Cocoa bean, the seed of In this study, we evaluated the acute effects of flavan-3-ols on the systemic circulation and microcirculation in skeletal muscle using intravital video microscopy under physiological conditions. We also examined changes in eNOS phosphorylation in aortic tissue and nitrate/nitrite levels in blood to elucidate the mechanisms of the acute response to flavan-3-ols.Urethane and Krebs- Ringer bicarbonate buffer were purchased from Sigma Chemicals . The flavan-3-ol fraction was provided by Meiji Co., Ltd , and the concentrations of polyphenols are shown in This study was approved by the Animal Care and Use Committee of the Shibaura Institute of Technology (Permit Number: 27-2956). All animals received humane care under the guidelines of this institution. Male Wistar rats weighting 200–250 g were obtained from Saitama Experimental Animal Supply . The rats were kept in a room with controlled lighting (12 h light and dark cycles at a regulated temperature of 23–25°C). The diet was certified diet obtained from the Oriental Yeast Co., Ltd., Tokyo, Japan.2 and 5% CO2 at 37°C. Thirty min after post-surgical equilibration period, a single unbranched arteriole with a resting inner diameter of 15–20 µm was selected from the microscopic images for measurement of red blood cell velocity and diameter of the vessels. After 15 min of baseline observation, 10 mg/kg of flavan- 3-ols or distilled water was administered orally to animals through the fixed gastric tube. The dosage of flavan-3-ols was determined according to a preliminary experiment (data not shown). The microcirculation was visualized by placing the chamber on a three-way movable stage, and the cremaster was transilluminated with a 150-W halogen light. The microcirculation was observed using an intravital microscope  equipped with a charge- coupled video camera . The images were displayed on a high-resolution television monitor at a final magnification of 1450× and stored for off-line analysis. Red blood cell (RBC) velocity and the number of newly-recruited capillaries were measured using a video image with 8-bit gray levels at a resolution of 512×512 pixels. RBC velocity was measured by monitoring the change in the position of a RBC over time in successive frames, and each velocity measurement was repeated 3 times. The number of newly-recruited capillaries was counted over an 846×307 µm area of the cremaster muscle throughout the observation period.Thirty two animals were fed a basal diet for 4 days and then allocated to two groups, with each group treated 4 ml/kg distilled water  or 10 mg/kg flavan-3-ol fraction , flavan-3-ols was dissolved in distilled water. Microscopic observation was carried out using eight rats then sacrificed under anesthesia, laser Doppler and tail cuff measurement was performed the other eight rats in each group. A gastric tube was inserted under urethan anesthesia . The cremaster muscle was exteriorized and carefully spread out on a dedicated plastic chamber with an optical port for transillumination; the surface was superfused with Krebs-Ringer bicarbonate buffer (pH 7.3–7.4) in an environment with 95% NA schematic diagram of the experimental apparatus is shown in Aortic tissue was homogenized in a microtube with lysis buffer  containing a protease inhibitor  and 0.2% SDS. Protein concentration was measured by the Bradford method. Protein (50 µg) was separated by SDS-PAGE using a 4–12% Bis-Tris gel and transferred onto a polyvinylidene difluoride membrane (Life Technology). The membrane was blocked with membrane-blocking reagent  for 1 hour. After blocking, the membrane was probed with a rabbit polyclonal primary antibody against phosphorylated eNOS  and an α-tublin antibody  for 2 hours. After the primary antibody reaction, the membrane was incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1∶100000) for 1 hour. Immunoreactivity was detected by chemiluminescence using the ECL Select™ Western Blotting Reagent . Fluorescence band images were analyzed using Just TLC (SWEDAY) analysis software. Values were normalized to those for α-tubulin.The plasma nitrate and nitrite concentrations were determined by using the Griess reaction Data are expressed as the means and standard deviations. Statistical analyses were performed using Student's t-test or Dunnett's test. P<0.05 was considered significant.Changes in the microcirculation of the cremaster muscle before and after oral administration of flavan-3-ols are shown in supplemental videos 1 (before treatment) and 2 (5 min after flavan-3-ols treatment). The marked elevation of RBC velocity in the arterioles can be seen in the video image, and this confirms that endothelial cells were exposed to severe shear stress. The average RBC velocity in the arterioles shortly after treatment with flavan-3-ols is shown in Changes of blood pressure and heart rate measured by the tail cuff method are shown in In the present study, we observed increased RBC velocity in the cremasteric arterioles  and blooSeveral previous studies was indicated that (-)-epicatechin in flavan-3-ols activated eNOS directly 2 for ATP production during exercise In this study, we found an increase in newly recruited capillaries in the cremaster muscle , and theIn conclusion, we found an increase in RBC velocity in arterioles and blood flow in artery in the cremaster muscle, along with transient elevation of blood pressure and heart rate after a single oral administration of flavan-3-ols. In this condition, the elevation of shear stress on endothelial cells was confirmed by microscopic observation. These results suggest that the mechanism of FMD elevation after acute chocolate administration was partly due to enhanced NO release from endothelial cells due to increased shear stress.Video S1Microcirculation in cremaster muscle of before administration of flavan-3-ols.(MPG)Click here for additional data file.Video S2Microcirculation in cremaster muscle of 5 min after administration of flavan-3-ols.(MPG)Click here for additional data file.


Schistosoma hematobium (squamous cell carcinoma of the bladder) and the liver flukes Opisthorchis and Chlonorchis (cholangiocarcinoma). A large number of parasites are known to colonize the gastrointestinal tract. We sought to review the evidence that implicates these parasites in gastrointestinal neoplasia. Schistosoma japonicum, which is endemic primarily in east Asia, has been shown in multiple studies to convey a mildly increased risk of colorectal adenocarcinoma. The data supporting a causative role for Schistosoma mansoni in colorectal or other neoplastic processes are less convincing, limited primarily to small case-control studies and case series. Reports of possible associations between other gastrointestinal parasites  and neoplasia may be found in the literature but are limited to individual cases. We conclude that, other than S. japonicum and to a lesser extent S. mansoni, there is little evidence of an association between gastrointestinal parasites and neoplasia.Several parasitic species are well known to have carcinogenic properties, namely;  Entamoeba histolytica, Giardia lamblia, Cryptosporidia, and Trypansoma cruzi. The multi cellular helminths are further divided into three groups, cestodes/tapeworms , nematodes/round worms , and trematodes/flukes .A wide variety of parasites are known to cause disease in the human gastrointestinal tract, including some species that are very prevalent over a large geographical area. Human parasites are traditionally divided into two broad groups, protozoa and helminths. The phylum protozoa includes a number of gastrointestinal parasites, with some notable members such as Spiroptera later developed stomach cancer. This work was later debunked on at least two levels. It was shown that the risk of “cancer” was only seen in a specific strain of mice, and later review actually challenged whether the lesions in question were truly carcinomatous in nature or just hyperplastic [S. hematobium and squamous cell carcinoma of the bladder and the liver flukes (Opisthorchis and Clonorchis) and cholangiocarcinoma have been well documented for decades. Thus, there is a body of evidence that links specific parasites to nongastrointestinal malignancies. Despite the fact that the gastrointestinal tract is the main site of infection for many parasites, there has, to our knowledge, been no review that explores the possible association between parasites of the gastrointestinal tract and subsequent neoplasia. There has long been scientific interest in exploring the possibility of infectious causes of cancer, including bacterial, viral, and parasitic causes. Mathematical modeling has estimated that approximately 16% of cancers throughout the world may be attributable to infection [rplastic . DespiteSchistosomiasis is a major worldwide health problem, with an estimated 600 million at-risk people across 74 countries and directly affecting approximately 200 million people . SchistoS. hematobium is endemic primarily in the Middle East, particularly in Egypt. There is a wealth of epidemiologic, experimental, and histopathologic data that link infection by S. hematobium with squamous cell carcinoma of the urinary bladder . Most t bladder , 6. The A damage , 7. Two te 1970s . IntestiS. japonicum and colorectal neoplasia has been debated for decades. The first reports on this topic began to appear in the English literature in the late 1950's [S. japonicum . Case riewed in –15. ManyS. japonicum prevalence. Most found a positive and significant correlation between cancers of the colon and rectum and mortality due to schistosomiasis, although all are to some extent confounded by difficulties in controlling for influences such as diet and other causes of colorectal cancer. In Xu and Su 1984 [S. japonicum in two provinces in eastern China with variable levels of schistosomiasis prevalence, including data over a three-year period (1977–1979). The prevalence of schistosomiasis in the studied areas ranged from 10% to 79%, as determined by skin testing and rectal biopsy. The investigators found a statistically significant positive correlation  between mortality due to colorectal cancer and that due to S. japonicum. In comparison, there was no statistical relationship between mortality due to S. japonicum and that due to lung cancer.A series of studies from China and Japan, published between 1983 and 2005, examined this topic from an epidemiological and geographical standpoint, which were reviewed by the International Agency for the Research of Cancer (IARC) in 1994 , 16–20.  Su 1984 , colorecS. japonicum and colorectal cancer [S. japonicum, as compared to 9.6% of those with nonmalignant conditions, a difference that was statistically significant. Interestingly, patients with malignant tumors in the 40–49-year-old age range were much more likely to show evidence of S. japonicum infection. In Xu and Su 1984 [S. japonicum infection, as compared to 14% of the control patients. From this data, the authors reported a statistically significant association between S. japonicum infection and colon cancer . Their results indicate a fraction of disease attributable to schistosomiasis of 24% for colon cancer. The investigators did not specifically compare colon and rectal cancers with regards to schistosomiasis.One of the first case-control studies to examine the association between l cancer  found th Su 1984 , the inv Su 1984 . Out of S. hematobium infection, this link was relatively simple to conclusively establish. In contrast, adenocarcinomas that arise in patients with S. japonicum infection have few overt histopathological differences from those not associated with schistosomiasis. However, a few studies have noted subtle features that distinguish S. japonicum-associated colorectal carcinomas from nonschistosomal-associated cancers. Cancers arising in the context of S. japonicum infection tend to occur in young patients, with 57% of S. japonicum-associated cancers being found in the 21–40-year-old range, as compared to 39% of nonschistosomal-associated colon cancers [S. japonicum-associated colorectal carcinomas with 165 nonschistosomal associated colorectal carcinomas, with the intent of identifying transitional mucosal changes that might distinguish schistosomal-associated colorectal cancers. Cancers associated with S. japonicum tended to be more often well differentiated than controls , while control cases were more frequently associated with adenomatous polyps. It was also noted in this study that cancers associated with S. japonicum tended to occur in younger patients (mean 40.3 years) than in nonschistosomal-associated cancers (mean 46.3), although no statistical significance was given. This group published a follow-up study in 1981 that examined a subset of the previously published group of S. japonicum-associated colorectal cancers [S. japonicum-associated colorectal cancers tended to have features similar to cancers arising in the context of inflammatory bowel disease, although this was not formally compared to a series of matched controls.Because squamous cell carcinoma of the bladder is rare in the absence of  cancers . One stu cancers  compared cancers . This stS. japonicum-associated colorectal cancers remains enigmatic, although, as discussed above, it appears that the chronic inflammatory response due to deposited schistosome ova plays an etiologic role [S. japonicum [S. japonicum . Another study attempted to identify characteristic mutations in p53 in schistosomal and nonschistosomal-associated rectal cancers [S. japonicum and twenty-two nonschistosomal rectal cancers were subjected to single strand conformational polymorphism testing for p53 mutations and subsequent sequencing. In schistosomal rectal cancers, thirteen p53 mutations were identified in ten cases, most of which were found to be base-pair substitutions, while thirteen mutations were found in nine nonschistosomal rectal cancers. A higher proportion of base-pair substitutions were found at CpG dinucleotides in S. japonicum-associated rectal cancers as compared to nonschistosomal rectal cancers, although this difference did not reach statistical significance. These findings led the authors to suggest that S. japonicum may release a directly genotoxic agent. In summary, the pathogenesis of S. japonicum-associated colorectal cancers may have some similarities to those seen arising in the setting of inflammatory bowel disease, but much remains unknown.The precise pathogenesis of gic role . This toaponicum . Two stuiewed in ), but nomes test . No evid cancers . Twenty- cancers . In thisS. hematobium. For instance, glutathione S-transferase gene deletions were shown to be significantly more common in patients with S. hematobium-associated squamous cell carcinomas of the bladder [S. hematobium-associated squamous cell carcinoma of the bladder [S. japonicum-associated colorectal cancer. In the approaching era of relatively inexpensive and widely available high throughput gene sequencing, it will be fascinating to ascertain whether there are host factors that predispose patients to the development of colorectal carcinoma due to schistosomiasis. A few studies have endeavored to explore potential host factors that could predispose patients to develop squamous cell carcinomas of the bladder in the context of  bladder . Similar bladder . To dateS. japonicum and colorectal cancer in 1994, as part of a monograph examining the carcinogenic risks to humans of schistosomes, liver flukes, and Helicobacter pylori [S. hematobium and species of Opisthorchis and Clonorchis were definitely carcinogenic to humans. In this monograph, the IARC concluded that S. japonicum is possibly carcinogenic to humans. Since this monograph was published, only a handful of additional studies have been published on this topic, although at least one additional case control study published in 2005 did find epidemiological evidence again implicating S. japonicum as a causative agent in colorectal carcinoma [The IARC formally reviewed the association between r pylori . The IARarcinoma .S. japonicum and colorectal malignancy, there is evidence that implicates this parasite in hepatocellular carcinogenesis. An epidemiological study of 907 patients with chronic liver disease from Japan found that 19% of patients with nonneoplastic chronic liver disease and 51% of patient with hepatocellular carcinoma showed evidence of infection by S. japonicum, a difference that was found to be statistically significant [S. japonicum, possibly biasing the data. In a mouse model, it was demonstrated that S. japonicum infected mice were at significantly increased risk of hepatocellular carcinoma. Out of 61 mice with hepatic infection by S. japonicum, 48 had single or multiple hepatocellular carcinomas [S. japonicum and then also given carcinogenic agents [In addition to the very probable link between nificant . In the rcinomas . Similarc agents .S. japonicum and cancers of other sites. A few studies have peripherally addressed a potential link between S. japonicum and cancers of the stomach and esophagus. Several case series of gastric cancer associated with S. japonicum infection have been published from both China and Japan . In oneiewed in , signifiar study , no posiS. japonicum, including gastrointestinal tumors. A case of a rectal carcinoid was described with evidence of S. japonicum in a 44-year-old Philippines-born female patient [S. japonicum eggs in the appendiceal wall [S. japonicum ova associated with nongastrointestinal tumors, such as squamous cell carcinoma of the skin [A few individual cases have been reported of other neoplasms with evidence of infection by  patient . In a seeal wall . A few cthe skin , malignathe skin , bronchothe skin , and brethe skin . Given tS. mansoni infection have been reported for decades, primarily in the Middle East [S. japonicum, there is a distinct lack of epidemiological evidence that links the geographical distribution of S. mansoni to cancers of any type, gastrointestinal or otherwise. In one series, a large group of patients in an area of endemic S. mansoni (Egypt) underwent lower endoscopy and biopsy. Of 638 patients with biopsies, 219 had histologic evidence of S. mansoni infection, while 26 had colorectal adenocarcinoma. However, only two patients in this series had evidence of both S. mansoni infection and colorectal adenocarcinoma, leading the authors to conclude that there was no causative role for S. mansoni in colorectal neoplasia [S. mansoni (Egypt). The authors found that patients with schistosomal-associated colorectal cancers were younger  and more often had multiple synchronous tumors . Pathologically, colorectal cancers associated with schistosomiasis were more often mucinous  and tended to present at high stage. Immunohistochemical staining revealed that significantly more schistosomal-associated colorectal cancers overexpressed p53 . The authors speculated that p53 alterations may act as an inciting event in schistosomal-associated neoplasia [Patients, both individually and in small series, with evidence of both colorectal adenocarcinoma and dle East , 41–43. eoplasia . In anoteoplasia .S. japonicum, a series of S. mansoni-associated colorectal cancers and nonschistosomal colorectal cancers were found to show no significant differences in p53 and c-myc immunohistochemical expression, but Bcl-2 was found to be significantly more likely to be expressed in S. mansoni-associated cancers [S. mansoni ova in 32% of resection specimens, similar to the reported prevalence in Egypt in the general population [S. mansoni ova were more commonly found in MSI-H colorectal cancers . S. mansoni ova were also more frequent among patients whose tumors showed KRAS mutations, although this was seen only in a small number of patients .As discussed in the above section on  cancers . A case pulation . This stS. japonicum, in 1994 the IARC formally reviewed the evidence for a causative link between S. mansoni infection and colorectal adenocarcinoma [S. mansoni as a carcinogenic agent in humans.Similar to arcinoma . The autS. mansoni ova have been reported as individual cases in a variety of other nongastrointestinal tumors, including prostatic adenocarcinoma [S. mansoni plays a causative role in the carcinogenesis of malignancies of other sites.arcinoma , ovarianarcinoma , uterinearcinoma , squamouarcinoma , and infarcinoma . Other tE. histolytica trophozoites [G. lamblia trophozoites but no cancer. A follow-up EUS-FNA a month later did identify adenocarcinoma, and a subsequent pancreaticoduodenectomy revealed a ductal adenocarcinoma [G. lamblia trophozoites. Subsequent distal pancreatectomy and omentectomy revealed a well to moderately differentiated ductal adenocarcoma with metastasis to the omentum [A number of reports in the literature discuss the wide variety of parasites that may opportunistically infect immunosuppressed patients (reviewed in ). Similahozoites . There ahozoites . Two womhozoites . The finhozoites . Colonichozoites , 59. Giaarcinoma . The sec omentum . Given tSpirocerca lupi. A large group of patients with Chagas disease (Trypansom cruzi) in South America were examined, and no increased risk of gastrointestinal neoplasia was noted [Echinococcus is not a gastrointestinal parasite, at least one published report found that Echinococcal hydatid cyst disease was significantly less prevalent than what would be expected given the prevalence in Turkey in a large group of cancer patients, leading the authors to conclude that this parasite may actually protect patients from neoplasia [In addition, a few investigators have specifically addressed this potential link in larger studies and reviews for a few selected gastrointestinal anatomic sites and parasitic species. Infectious causes of esophageal neoplasia were reviewed by Eslick  who concas noted . Althougeoplasia .S. japonicum infection of the colon and rectum conveys an increased risk for the development of adenocarcinoma. The molecular mechanisms that underlie this increased risk are not clear but are likely related to the long-term inflammatory response, similar to inflammatory bowel disease. The evidence linking S. mansoni infection and colorectal neoplasia is weaker, but at least one study does suggest that cancers arising in the setting of chronic S. mansoni infection have distinctive features, resembling cancers arising in the setting of inflammatory bowel disease [We have endeavored to review the literature that may demonstrate a causative role for parasites in gastrointestinal neoplasia, summarized in  disease .S. hematobium and the liver flukes, the increased risk of cancer is relatively low. It will require further very large epidemiologic studies to show a statistically increased risk of gastrointestinal neoplasia in parasites with low rates of prevalence, if one exists at all.Despite a scattering of case reports and small case series, there is no consistent or convincing evidence that links gastrointestinal neoplasia and any other parasite besides the schistosome species. Is there simply no significant relationship between most gastrointestinal parasites and neoplastic complications? Perhaps, but it is also possible that a weakly increased risk may be impossible to prove statistically in a parasite with low prevalence. Schistosomal infections are a very common parasitic infection in humans, resulting in a large infected population that facilitates epidemiological studies. Even in the well-studied carcinogenic parasites such as


Treacher Collins syndrome (TCS) is an inherited developmental disorder. More than 40% of individuals with TCS have conductive hearing loss attributed to external and middle ear anomalies. Mild cases of TCS often pass undiagnosed at birth or early childhood. The disease may be manifested as conductive hearing loss in teenagers and may resemble juvenile otosclerosis. Patients could suffer from slight facial variabilities including retrognathia (as in our case) and others, which point out to a possible middle ear anomaly. Surgical corrections of middle ear anomalies including TCS generally lead to poorer outcomes comparing with juvenile otosclerosis, which should be discussed with parents during preoperative counselling. Treacher Collins syndrome (TCS) is an inherited autosomal dominant disorder with a prevalence estimated at 1 in 40000–70000 of live births. About 60% of patients have the disorder as the result of a de novo gene mutation. In the vast majority of cases, full expressivity of the syndrome occurs, and TCS is clearly diagnosed at birth. However, patients with mild form of TCS may remain undiagnosed for a long time [A 15-year-old female patient was presented with a 3-year long right side hearing loss without dizziness or tinnitus. There was neither history of any trauma, previous ear surgery, or ear infection, nor was there any abnormality of the pinna, ear canal, or the tympanic membrane. However, mild retrognathia was noticed . A pure Middle ear abnormalities occur in approximately 20%–25% as a part of a malformation syndrome of which TCS is one of the most frequently diagnosed . TCS is Cremers and Teunissen noticed that specific syndromes are associated with characteristic middle ear malformations , 4. TheyThe most common HRCT findings in patients with TCS are narrow epitympanum, abnormalities of the ossicular chain, abnormal course of the facial nerve, and absence of mastoid pneumatization, which is a rather constant finding  . All of It might be challenging to distinguish between juvenile otosclerosis and congenital ossicular problem (as those attributed to TCS) during preoperative examination . There aVery important information for distinguishing between juvenile otosclerosis and minor congenital middle ear malformation is progression of the hearing loss. This is only detectible when the child undergoes regular examinations and cooperates well. Progressive hearing loss is typical for juvenile stapes otosclerosis, while nonprogressive hearing loss is typical for middle ear malformations . HoweverOther very useful information, which should point out to possible middle ear malformation, is the presence of even slight facial variability (in our case retrognathia). That is why even minor facial variabilities with conductive hearing loss should lead us to think of a middle ear anomaly and indicate HRCT as an important part of preoperative assessment. Suspicion of congenital middle ear anomaly is particularly important for giving appropriate information  to the parents during preoperative counseling, and hearing aid should be offered as an alternative option , 5. HoweThe technique of middle ear surgical reconstruction for combined middle ear anomalies depends on the level of the middle ear findings and in comparison with stapedotomy for juvenile otosclerosis generally leads to poorer outcomes, comparable to middle ear reconstruction for chronic ear disease , 5. In o