Acquisition of social dominance is important for social species including mice, for preferential access to foods and mates. Male mice establish social rank through agonistic behaviors, which are regulated by gonadal steroid hormone, testosterone, as its original form and aromatized form. It is well known that estrogen receptors (ERs), particularly ER α (ERα), mediate effects of aromatized testosterone, i.e., 17β-estradiol, but precise role played by ER β (ERβ) is still unclear. In the present study, we investigated effects of ERβ gene disruption on social rank establishment in male mice. Adult male ERβ knockout (βERKO) mice and their wild type (WT) littermates were paired based on genotype- and weight-matched manner and tested against each other repeatedly during 7 days experimental period. They underwent 4 trials of social interaction test in neutral cage (homogeneous set test) every other day. Along repeated trials, WT but not βERKO pairs showed a gradual increase of agonistic behaviors including aggression and tail rattling, and a gradual decrease of latency to social rank determination in tube test conducted after each trial of the social interaction test. Analysis of behavioral transition further suggested that WT winners in the tube test showed one-sided aggression during social interaction test suggesting WT pairs went through a process of social rank establishment. On the other hand, a dominant-subordinate relationship in βERKO pairs was not as apparent as that in WT pairs. Moreover, βERKO mice showed lower levels of aggressive behavior than WT mice in social interaction tests. These findings collectively suggest that ERβ may play a significant role in the establishment and maintenance of hierarchical social relationships among male mice. Individuals of social species often establish hierarchical social relationships with their conspecifics. Once their social rank is determined, dominant (higher rank) individuals can get preferential access to resources including food, territory and mates. It is known that group-housed male mice establish a social hierarchy both in the wild and in laboratory housing conditions. Their social order is determined through agonistic interactions, which include not only active aggressive behavior but also various behavioral responses to opponents’ aggression, such as fleeing, immobility and upright submissive posture  over 7 days. Social rank was also assessed with the use of the tube test, right after each trial of the social interaction test. In order to identify critical behavioral acts between paired mice for the determination of social rank, we also analyzed behavioral transition patterns and compared these between WT pairs and βERKO pairs.n = 24, WT: 10 pairs, n = 20) were used as experimental animals. They were obtained from a breeding colony maintained at the University of Tsukuba. Original breeding pairs were provided by Dr. KS Korach at the National Institute of Environmental Health Sciences  and completely backcrossed to C57BL/6J mice . Non-littermate mice from the same genotype and matched body weight (±3.5 g) were paired (homogeneous pair) and tested against each other throughout the experiment. After 1 week of individual housing, mice were trained for the tube test on two consecutive days. Starting on the next day, each pair underwent the social interaction test followed by the tube test on every other day  for a total of 4 trials . All behavioral tests were recorded using digital video cameras and scored by an experimenter unaware of the animals’ experimental group, using a digital event recorder program .Social interaction behaviors between the paired mice were assessed in a neutral testing cage (19 × 29 × 12 cm) for 15 min. All tests were done under red light illumination during the dark phase of the light/dark cycle. At first, the testing cage was divided into two compartments by inserting a black Plexiglas board (divider) at the middle of the cage and mice were habituated in each compartment for 5 min. At the beginning of the test, the divider was removed and social interaction behaviors were observed. The cumulative number and duration of aggression, fleeing, immobility, upright submissive posture, approach, sniffing, huddling and grooming were recorded. Aggression was defined as a series of behavioral interactions consisting of at least one of the following: chasing, boxing, wrestling, biting and offensive lateral attack, often accompanied by biting. The cumulative number of tail rattling was also recorded. These nine behavioral acts were classified into two groups for further analysis: agonistic behaviors  and prosocial behaviors . Sniffing and grooming were further categorized into face-targeted or body-targeted. Thereafter, ratio of the face-targeted was calculated and compared between the ranks determined by the tube test. Sniffing and grooming were combined for this analysis  was placed at the center of the testing arena (70 × 50 cm) surrounded by black wall (20 cm). Starting from 2 days before the first social interaction test, all mice were trained individually to run through the tube from one end to the other eight times per day for two consecutive days, as previously described  of the behavioral events. Among nine behavioral acts, upright submissive posture was excluded from this analysis because only limited mice showed this behavior. In monad transitions, actors of the two consecutive behavioral events were the same mouse. In dyad transitions, an actor of the first behavioral event (initiator) and that of the second behavioral event (responder) were different mice. For monad transitions, the probabilities of transitions were calculated and 8 kinetograms were constructed for each trial and genotype. For analysis of dyad transitions, eight behavioral events recorded in social interaction test was partially combined as follows; subordinate behaviors including fleeing and immobility, and prosocial behaviors including sniffing, grooming and huddling. Probabilities of dyad transitions were then calculated and 8 kinetograms were constructed for each test, genotype and rank (winner or loser in the tube test). Differences between tests, genotypes and ranks were analyzed qualitatively based on the diagrams. For dyad transitions, the number of all transitions, transitions initiated with approach, and transitions responded with subordinate behaviors were also counted and statistically analyzed.Post hoc power analyses for the main effects and their interaction of ANOVAs , repeated measurements of the main effects for genotype, trials and their interaction. s Cohen,  were cons Cohen, . GenotypF = 14.741, p < 0.001, d = 0.592, power (1-β) = 0.997; trial: F = 6.333, p < 0.001, d = 0.388, power (1-β) = 0.999; genotype × trial: F = 3.617, p = 0.015, d = 0.293, power (1-β) = 0.998). In WT mice, the number of agonistic behaviors in trials 3 and 4 were significantly higher than in trial 1 (p < 0.01), whereas no significant difference was observed between trials in βERKO mice. Moreover, βERKO mice showed a significantly lower number of agonistic behaviors, compared to WT pairs in trial 2 (p < 0.05), and trials 3 and 4 (p < 0.01), although there was no genotype difference in trial 1. βERKO mice also showed significantly a shorter overall cumulative duration of agonistic behaviors, compared to WT mice  = 8.629, p = 0.005, d = 0.453, power (1-β) = 0.903). Significant genotype difference was observed in trial 2 (p < 0.05), and trials 3 and 4 (p < 0.01), although there was no significant main effect of trial and interaction of genotype and trial . In contrast, both number and duration of prosocial behaviors were not different between genotypes and did not change over the four trials  = 9.143, p < 0.001, d = 0.733, power (1-β) = 0.999; genotype × day: F = 8.551, p = 0.007, d = 0.709, power (1-β) = 1.000; genotype: n.s.). As for the winner shift, there was a trend of a gradual decrease from trial 2 to trial 4 only in WT, but not in βERKO mice, although there were no statistically significant genotype differences  responded to a preceding behavioral event acted by the other mouse (initiator), were analyzed.p < 0.100; trial 3: p < 0.010; trial 4: p < 0.010). It should be noted that throughout the 4 trials, the numbers of dyad transitions initiated by winners and losers were roughly equal in both genotypes  was a predominant type of dyad transitions in trial 1 Figure . HoweverIn contrast to WT pairs, the most predominant transition was approach—approach in βERKO pairs in all four trials Figure . In triap = 0.016; βERKO, trial 1; p < 0.001, Binomial test). Consistent with these findings, losers of WT pairs showed a significantly higher number of subordinate behavior than winners in trial 2 . On the other hand, losers of βERKO pairs showed a significantly higher number of subordinate behaviors than winners in trial 1 .The number of transitions initiated by approach  followed by tube tests for social rank evaluation.Over four trials, WT pairs showed a gradual increase in agonistic behaviors, such as aggression and tail rattling in social interaction tests. In tube tests, a corresponding decrease of latency to loser ejection was observed. After an initial investigative period in trial 1, WT winners defeated the losers and acquired their dominance. Detailed analysis of behavioral transitions revealed that an asymmetry in behaviors of the winners and losers appeared in trial 2 in WT pairs—i.e., losers responded to winners’ one-sided attack with subordinate behaviors. Summarized kinetograms . In future studies, it is needed to investigate further whether the βERKO mice are able to establish hierarchical social relationships in larger groups since being in a pair and in a larger group induce different hormonal status in male mice (Williamson et al., MN, AÅ, and SO designed research. MN, AÅ and SS performed research and analyzed data; MN and SO wrote the article.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.


Bovine viral diarrhea virus (BVDV) affects cows, pigs, sheep, goats, and other ruminants, as well as some wild animals. BVDV causes considerable economic losses every year and many countries have developed programs aimed at the eradication of this disease. The genetic diversity of BVDV in diseased goats has never been described in southwestern China. Thus, in this study, we applied antigen-capture ELISA and RT-PCR to survey the infection rate of BVDV in diseased goats in this region. Our results demonstrated that the average BVDV infection rate in goats was 17.51%, with all positive samples indicating infection by BVDV-1 and not BVDV-2, BVDV-3, or Border disease virus. The molecular characteristics of the 5′-untranslated region (5′-UTR) of BVDV-1 were recognized as belonging predominantly to the BVDV-1a, 1b, 1c, 1m, and 1p subtypes. BVDV-1b and 1m were the most abundant subtypes identified in this region, similar to the BVDV epidemics in cattle in other regions of China. This is the first study that describes the genetic characterization of BVDV in sick goats from southwestern China and is important for future studies and control programs. Pestivirus and family Flaviviridae [ Pestivirus species, HoBi-like virus, was first identified in fetal bovine serum from Brazil and was related to BVDV at the antigenic and genetic levels. Thus, the viruses were referred to as “BVDV–3” [ Pestivirus species have been detected in Europe, North America, South America, and Asia [ Pestivirus (family Flaviviridae) was proposed, where all species were redesignated as Pestivirus A-Pestivirus K [Bovine viral diarrhea virus–1 (BVDV–1) and BVDV–2, together with classical swine fever virus and border disease virus (BDV), are currently classified as the genusivirus K .The natural host of BVDV is cattle though it is also capable of infecting sheep, goats, piglets, and other domestic and wild ruminants by persistently infected (PI) animals and transiently infected individuals . PI catt Pestivirus strains is usually based on the characteristics of partial sequences from the 5′–UTR, Npro or E2 regions of the genome. However, the 5′–UTR of Pestiviruses has the highest degree of sequence conservation and is therefore most frequently used for phylogenetic analysis. According to sequence comparison analyses of this region, there are 21 subtypes of BVDV–1(1a–1u) [BVDV has a single stranded positive sense RNA genome of 12.3–12.5 kb. The full-length viral genome is flanked at both ends by 5′ and 3′ untranslated regions (UTRs) and contains a single open reading frame that encodes a single polyprotein that is approximately 4000 amino acids. The genetic diversity of novel1(1a–1u) –9 and fo1(1a–1u) –12.A previous study based on a strain of BVDV–1 that infected goats in eastern China showed that a high proportion (29/236) of BVDV–positive goats came from eastern China . HoweverA total of 217 blood samples taken from diseased goats that presented with symptoms, such as diarrhea, respiratory tract infection, and mucositis, were collected between March 2013 and April 2016, from multiple goat farms in southwestern China.Sera were screened for BVDV using IDEXX BVD Ag Test , which can be applied for detection of genetically diverse BVDV strains, according to the manufacturer's instructions. The antigen-positive samples were then followed up with RT-PCR detection.μL aliquots of goat serum using the QIAamp Viral RNA Mini Kit , following the manufacturer's recommendations. Four different PCR assays for BVDV–1 [Viral RNA was extracted from 200 r BVDV–1 , BVDV–2 r BVDV–1 , BVDV–3 r BVDV–1 , and BDVr BVDV–1  were perhttp://www.ncbi.nlm.nih.gov/genbank) using the Basic Local Alignment Search Tool (BLAST). The sequences in this study were trimmed and analyzed with MEGA7.0 software [The 5′–UTR genomic sequences obtained were compared with the relevant sequences published in GenBank  in southwestern China.BVDV–1 was detected in 12 of 38 BVDV–positive samples by first round RT-PCR, which produced a 305 bp product that was not detected in the other 26 BVDV–1–positive samples. The sequences detected with the first round RT-PCR in the 12 BVDV–1 positive samples were deposited in GenBank under accession numbers MG323516–MG323527 .Twelve of those sequences were selected for the construction of a phylogenetic tree and were divided into five different subtypes. Five isolates  were clustered within BVDV–1b, one (DS1324) was typed as BVDV–1a, one (FS1573) as BVDV–1c, one (FS1426) as BVDV–1p, and four  were characterized as BVDV–1m. No BVDV–2, BVDV–3, or BDV was detected in field isolates by phylogenetic analysis.In this study, we identified 12 selected BVDV-1-positive samples that represented different flocks or geographical origins of blood samples collected between 2013 and 2016. Experimental or natural infection in goats has been previously confirmed . Goats iAmong the BVDV–1 types, the most frequent subtype was BVDV–1b (n=5) . Five BVThe 1b subtype is widely considered to be a major contributor to infection in Chinese cattle , 22. SimBVDV–1m is another major subtype in this region . Four BVOur positive samples, DS1324, FS1573, and FS1426, respectively, shared 87.9%, 97.5%, and 98.4% sequence identity with the reference strains BVDV–1a NADL (M31182), BVDV–1c Manasi (EU159702), Bega (AF049221), BVDV–1p TJ06 (GU120246), and TJ07 (GU120247) . These pIn summary, this study demonstrated that BVDV–1 was a common causative agent for infection of goats in southwestern China and that 17.51% of the detected samples from ill goats were BVDV–1 positive. However, we did not assess samples from the healthy goats and cannot determine the prevalence of BVDV in healthy goats. To further our understanding of the genetic diversity of BVDV strains detected, all antigen-positive samples were confirmed and characterized with RT-PCR as being infected by BVDV–1, but not BVDV–2, BVDV–3, or BDV. Twelve BVDV–1-positive samples were identified from these RT-PCR positive samples and were clustered into the six subtypes BVDV–1a, 1b, 1c, 1m, and 1p. Due to the possibility of cross infections of BVDV that might occur among cattle, piglets, and goats, much more emphasis should be placed on epidemiology surveys in these animals. This is the first description of the genetic diversity of BVDV–1-positive samples collected from goats in southwestern China.


This article contains supportive data related to a research article entitled “Annual variation of species richness and lorica oral diameter characteristics of tintinnids in a semi-enclosed bay of western Pacific”   Specifications TableValue of the data●This dataset contains raw and processed data of tintinnid assemblages, one of the important microzooplankton groups, at 4 sampling sites during a ten-year cycle in a semi-enclosed Bay in western Pacific.●The variation of plankton groups as well as phenology and be explored and analyzed using such statistical methods as analysis of variance, regression, factor analysis, cluster analysis, or structural equation modeling.●a, and other zooplankton groups, and microbial food loop are welcome.New collaborations about copepods, Chl 1Tintinnid samples were collected aboard R/V ‘Kejiao No.1’ in Jiaozhou Bay, Yellow sea, in the temperate western Pacific. Samplings were conducted at 4 sites  once a month from May 2003 to December 2012. A 30 L surface water was collected at each site by a large volume water sampler and then filtered slowly and gently through a net (mesh size 20 μm). The concentrated tintinnid samples (~150 ml) were fixed with formalin solution to 5% final concentration. Subsamples of 20 ml from well-mixed concentrated samples were pipetted into a sedimentation chamber and settled for 12–24 h Tintinnid species identifications were made on the basis of lorica morphology and dimensions according to literatures 22.1Favella, Leprotintinnus, Metacylis, Stenosemella, Tintinnidium, Tintinnopsis) and cosmopolitan  biogeographical types.A total of 26 species belonging to 9 genera were found in the 428 samples . Tintinn2.2−1, 2–1300 ind L−1, 0–1030 ind L−1, and 1–1102 ind L−1, respectively.The abundance of tintinnid assemblages were different in the four sampling sites . The ran2.3Tintinnopsis had more species (15) than other genera. The remaining genera were represented by 1 or 2 species. All the species have agglutinated lorica except 4 hyaline species: Amphorellopsis acuta, Eutintinnus lusus-undae, Favella sp. and Metacylis jorgensenii. All the 4 hyaline species occurred in July-September period. Hyaline forms herein occurred mainly in summer, as well as agglutinated forms peaked in summer.Species occurrence were showed in


Chl a, ash free dry weight (AFDW), and dry weight (DW) were measured to assess phototrophic and whole biofilm biomass development over time. From the analysis of photosynthetic parameter variation with light intensity, temperature and flow rate, it was possible to identify the set of experimental values favoring biofilm photosynthetic activity. Biomass increased over time, especially at the highest irradiances, where substrata were fastly colonized and mature biofilms developed at all temperatures and flow conditions tested.Photosynthetic performance of algal-bacterial biofilms from an Italian wastewater treatment plant was studied in a flow-lane photobioreactor at different irradiances, temperatures, and flow regime to evaluate the effects of these environmental parameters on biofilms’ functioning, in view of application of these communities in wastewater biological treatment. Pulse amplitude modulated fluorescence was used to estimate the effective quantum yield of PSII (ΔF/F Biological treatment is an important and integral part of wastewater treatment (WWT) procedures. Currently, the activated sludge process is the most commonly applied biological WWT technology. The activated sludge treatment utilizes dense bacterial suspended communities, forming biological flocs, to degrade organic material under aerobic conditions . AlthougAlthough the complex interactions between microalgae and bacteria in WWT are not yet fully elucidated, outputs of many studies conducted to date have suggested that microalgae, through photosynthesis, provide oxygen to heterotrophic bacteria to reduce chemical oxygen demand (COD). On the other side, bacteria release carbon dioxide and mineral nutrients from respiration and organic matter degradation which are efficiently converted into biomass by microalgae when exposed to solar light in outdoor systems ,4,11,12.Therefore, understanding the overall community photosynthesis and photoacclimation capability is pivotal to the exploitation of phototrophic microbial consortia in biological treatment of wastewaters. Different approaches have been applied to measure photosynthesis of aquatic phototrophic biofilms, such as oxygen microelectrodes, photosynthetic chambers in combination with oxygen probes, and pulse amplitude modulated (PAM) fluorimetry ,33,34,35Accordingly, the aim of this study was to investigate the photosynthetic behavior, biomass development, and production of phototrophic biofilms collected from an Italian WWT plant and cultured in a flow-lane incubator system at different abiotic conditions. Species composition, matrix characterization and nutrient removal capability of these autotrophic-heterotrophic cultured biofilms have been previously investigated ,38,39,403 sewage per day. Physico-chemical characteristics of the plant have been previously described [Biofilm samples were collected by scraping off the overflow system of the sedimentation tank (ST) of an Italian wastewater treatment plant (WWTP) located in Fiumicino . Taxonomic assessment of in situ consortia can be found in ,43,44,45escribed ,46.− and PO4− concentration was modified to approximate wastewater levels. Thereafter, biofilms were homogenized and frozen at −20 °C for 24 h to remove grazers [After sampling, ST biofilm biomass was subjected to a pre-treatment to obtain safe-handling and homogeneous inocula according to . Briefly grazers . To starThe resulting mixture was inoculated in a semi-continuous flow-lane incubator system (PBI) .PBI contained four separate light chambers (LCs) (120 × 10 cm each) a.Medium flowed above polycarbonate slides (76 × 25 × 1), used as substratum for biofilm growth, and was changed at regular intervals (twice per week). Flow rate was valve-regulated. “True light”  lamps were used as the light source. −2 s−1 (LC1), 60 µmol photon m−2 s−1 (LC2), 30 µmol photon m−2 s−1 (LC3), 15 µmol photon m−2 s−1 (LC4). Two temperatures (20 °C and 30 °C) and two flow conditions (25 L h−1 and 100 L h−1) were tested. These four incubator experiments were referred to as “Runs” (Biofilms were cultured at four photon flux densities (PFDs): 120 μmol photon ms “Runs” b. TransmBiofilms were sampled at three stages of development as function of biofilm light absorbance values : (1) Initial (in), when the average value of absorption was 10%; (2) Active (ac), when mean value was 50%; (3) Mature (ma), at 90–95%. Each run lasted ~30 days. On the last day, biofilm was collected without regard of light absorption and labelled as last sampling (ls). Photosynthetic parameters of cultured biofilms were assessed at each sampling stage using a miniaturized pulse amplitude modulated (Mini-PAM) fluorometer, coupled to WinControl Software  for computer control and data analysis. In the Mini-PAM, fluorescence was excited by pulses (3 μs width) of measuring red light from a light-emitting-diode (LED) with the peak excitation band at 650 nm. Fluorescence measurements were performed on light-acclimated biofilms to assess the effective quantum yield of charge separation in photosystem II PSII, either under the incubator light conditions or during the measurement of electron transport rate versus irradiance (ETR/I) curves. Three slides were sampled in the middle of light period (8 h ca.) at the initial stage of biofilm development, immersed in culture medium to avoid desiccation, inhibition of photosynthesis, and/or damage to photosystems, and positioned on a specifically designed grid so that biofilm surface could be virtually divided into 27 spots (8 × 8 mm each). The fiberoptic was kept at 10 mm above biofilm surface using a holder to obtain a homogeneous field of illumination c. After m’) of the light-acclimated biofilms was calculated according to [m’ is the maximum light-adapted fluorescence after the application of a single saturating light pulse  [m’ was measured on 27 spots of three slides (81 replicates) to take into account the heterogeneity of microalgal distribution. Thereafter, ETR/I curves were recorded on three spots on each slide providing nine replicates for each sampling. The position of the spots was chosen randomly at the initial sampling and then kept constant in the active and mature phase. ETR/I curves were measured by exposing the sample to eight increasing PFDs, up to 720 μmol photon m-2 s-1. Each period of actinic light lasted 10 s before the saturation pulse was applied to determine ΔF/Fm’. The internal halogen lamp (20 W) of the Mini-PAM served as light source for saturation pulse and for continuous actinic illumination. The photosynthetic electron transport rate (ETR) between PSII and PSI was calculated from the estimate of ΔF/Fm’ at different PFDs in actinic light according to the expression:PSII is the optical cross section of PSII. Since we could not measure a*PSII, relative ETR was calculated as ΔF/Fm’ ∙ PFD ∙ 0.5.The effective quantum yield of charge separation in PSII (ΔF/Frding to  the follm-2 s-1) ,50. ΔF/Fmax (1 -e −α I ETRmax) [max), the initial slope, i.e., photosynthetic efficiency (α) and the light saturation parameter (Ik = rel.ETRmax/α) were calculated.The ETR/I curves were fitted by means of an automatic spreadsheet based on linear regression for estimating ETR per light intensity and a chi-square minimization for the exponential function ETR = ETR ETRmax) . From tha concentration per unit area (mg m−2). Chlorophyll a was extracted overnight in 90% acetone and then quantified spectrophotometrically [Phototrophic biomass was assessed by determining chlorophyll trically .−2 d−1) by oven drying samples at 60 °C for 72 h. Samples were then burnt at 500 °C for 1 h to determine organic biomass (phototrophs + heterotrophs) as ash-free dry weight (AFDW) per unit area (mg m−2) [Biomass productivity was determined as dry weight (DW g m(mg m−2) .a and AFDW was assessed by applying the Spearman’s rho-statistic [To test the influence of the experimental conditions and their interactions on all biofilm photosynthetic parameters, the standard ANOVA provided by the Past software (v 3.2.5) was used in combination with the Tukey Honestly Significant Difference (HSD) test for multiple post-hoc testing or Mann–Whitney test. The effect of these environmental conditions on initial and latest phases of biofilm development was evaluated by repeated ANOVA. Correlation between Chl tatistic .Biofilm development in the microcosm was monitored through an online and automatic system that continuously recorded the light transmitted through biofilm thickness over experimental time, allowing the calculation of absorbance and the subsequent construction of growth curves ,55. Grow−2 s−1 (LC1) in all runs when biofilms were dominated by filamentous cyanobacteria [A sigmoidal trend was observed for biofilms grown at the higher irradiances (LC1 and LC2) in all runs, diversely from most of the cultures under lower irradiances (LC3 and LC4) that reached only the initial and active phase. Initial phase lasted between 7 (LC1 Run3) and 14 days (LC2 Run1) for high light cultures and between 18  and 30 days (LC4 Run4) for the low light ones. Irradiance appeared to influence biofilm development, in particular the duration of the initial phase, i.e., the higher the irradiance, the shorter resulted the lag phase. This is more evident in LC1 cultures, likely due to the dominance of unicellular green algae which have higher growth rates . The slobacteria  able to bacteria , allowinWhen observed at the macroscopic level, biofilm biomass accumulation on the slides was patchy at the initial phase of development reflecting the adhesion of the pioneer microorganisms during the colonization process . The folBiofilm detachment from the slides occurred in a number of cases, visible from the concomitant appearance of bare areas on the slides and biofilm flocs in the circulating medium, these representing the dispersion, i.e., the last phase of biofilms development. Beyond self-detachment, which occurs when losses, due for example to cell death, grazing, pathogens, etc., become equal to growth, other factors  may have an impact ,56. NutrResults of photosynthetic performance of light-acclimated cultures, obtained after PAM measurements, are reported in m’) represents the efficiency of the whole-chain photosynthetic electron transport of steady-state photosynthesis in the light [−2 s−1, 20 °C and 25 L h−1 (LC2ls Run1). Most yield values were included between 0.5 and 0.7, falling in the ranges reported for natural biofilms from different environments, thus exposed at various ambient light intensities [The effective quantum yield of photosynthesis  followed by a significant decrease from 60 to 120 μmol photon m−2 s−1 (p = 0.00001254). In Run2 a significant increase occurred from 15 to 120 μmol photon m−2 s−1   . −2 s−1 showed significantly higher yield than biofilms grown at the highest irradiance . This was also recorded at the runs’ end at 20 °C (Run1 LC2ls-Run2 LC2ls p = 0) while no significant difference was found between yields at different flow rates at 30 °C  than at all other conditions  than at almost all the other conditions (p = 1). In the last phase, 60 μmol photon m−2 s−1, 20 °C and 25 L h−1 (Run1 LC2) were the conditions providing higher yield values . Photoacclimation of cultured biofilms to different experimental irradiances was indicated also by variations in ETR/I curve parameters such as rel.ETRk, and α . Rel.ETRd α , reflecting the photoacclimation of the benthic phototrophic communities at increasing irradiance [max variations at both flow conditions (max of LC1ls cultures at 25 L h−1 at 20 °C (p = 0.0004123). As for the effect of flow rate, only few variations were significant both in samples at the initial phase and at the last run day  , Run2LC2-Run2LC3 (p = 0.03837) (p < 0.05 see −2 s−1 (p = 0.006375). Variation of α with temperature, likewise ΔF/Fm’, depends on the photochemical reactions that are temperature dependent (e.g enzymes of photophosporylation and electron transport and plastoquinone diffusion). As for the flow rate variation, similarly to temperature, no significant differences were observed in initial biofilms, at both temperatures. In cultures at the runs’ end at 20 °C, significant higher α values were obtained at low than high flow at LC2 (p = 0.002964), LC3 (p = 0.0001108), LC4 (p =1.894E-8). Regarding the interaction between light intensity, temperature, and flow rate, for biofilms in the initial phase it was not possible to find the conditions optimizing α, while for cultures sampled at the runs’ end two combinations were identified, 60 and 120 μmol photon m−2 s−1, 20 °C, 25 L h−1 (As for photosynthetic efficiency (α), it was included between 0.14 ± 0.02 (LC4 Run2) and 0.43 ± 0.01 (LC1ac Run1). The visual analysis of α values in relation to irradiance showed that in initial biofilms of Run1–3, α increased from 15 to 30 μmol photon m m−2 s−1 . Neverth.004918) . In biof0.03837) . At the 25 L h−1 .k ranged (−2 s−1 (LC1ac Run2). Most values of rel.ETRmax and Ik obtained matched those found for sub-tidal phototrophic biofilms from different geographical areas [k only in few cases . As for the flow rate, Ik did not have a significant variation in initial cultures while being significantly higher at 20 °C and lower at 30 °C at 100 L h−1 at almost all light conditions  considering that they were grown at the lowest light. This result suggests that the phototrophic community studied had the potential to acclimate to higher irradiances than those tested in the incubator cultures. Nevertheless, occurrence of photoinhibition in benthic communities during laboratory measurements and its relationship with the growth light regime is still quite controversial [LC4 cultures showed photoinhibition at lower but, nevertheless, quite moderate irradiance  [Chl ely, −2)  and for ely, −2) ,69,70. a trend, at 120 and 60 µmol photons m−2 s−1, Chl a concentration increased over time following biofilm development, up to approximately 120 mg m−2 in final cultures. This suggested that 60 µmol photons m−2 s−1 was sufficient for biofilm development and higher intensity did not appear to exert any additional effect. Nevertheless, at this irradiance a longer lag phase was required for biofilm growth than at 120 µmol photons m−2 s−1. This might have implications in the selection of the light condition for culturing microbial-algal consortia in a wastewater photobioreactor. Looking at Chl −2 (LC1ls Run4)  . This varcentage . A signiand AFDW , suggestand AFDW , indicata in mature communities. The raise of heterotrophs might be explained by the increased amount, in mature biofilms at high light intensity, of algal exudates that represent a high-quality organic substrate for bacteria [Values of ca. 140 were obtained in LC1 mature cultures at the highest light in Run3 and Run4, indicating a balance between the photo- and heterotrophic components ,74. Nevebacteria ,75,76. Wbacteria  observedbacteria ,40. Finabacteria  where va−2 d−1  and 2.10 ± 0.36 (LC1ma Run4) g DW m−2 d−1 . −2 d−1). The highest productivity values obtained here were higher than those attained with three different biofilm-forming cyanobacterial isolates grown in the same system [Chlorella vulgaris grown in a rotating algal bioreactor [Productivity was consistent with those reported  for mixee system  and withoreactor . Higher oreactor . This study provided new insights into photosynthetic activity of light-acclimated cultures, contributing to the knowledge of wastewater biofilm ecophysiology, in light of their exploitation in water bioremediation. The difficulty of studying these communities in nature, due to the complex interactions among several environmental factors, was overridden in the flow lane system, which allowed the investigation of key variables that influence photosynthetic performance.The tested light–dark cycle appeared to be suitable for community development and functioning.−2 s−1, 20 °C, 25 l−1. This has implications for biomass accumulation and nutrient uptake related to photosynthesis. Therefore, this set of values appears to be appropriate for the optimal operating of a WWT photobioreactor that purifies municipal sewage by means of mixed phototrophic biofilms.It was possible to identify light intensity, temperature, and flow value favoring photosynthetic activity, i.e., 60 µmol photons mFurther development would be in the cultivation of these microbial communities in wastewater effluents to evaluate the treatment efficiency at the set of conditions here identified.


To better investigate this aspect, we performed whole-cell patch-clamp recordings from mouse Type I hair cells or their associated calyx. We found that [K+] in the calyceal synaptic cleft is elevated at rest relative to the interstitial (extracellular) solution and can increase or decrease during hair cell depolarization or repolarization, respectively. The change in [K+] was primarily driven by GK,L, the low-voltage-activated, non-inactivating K+ conductance specifically expressed by Type I hair cells. Simple diffusion of K+ between the cleft and the extracellular compartment appeared substantially restricted by the calyx inner membrane, with the ion channels and active transporters playing a crucial role in regulating intercellular [K+]. Calyx recordings were consistent with K+ leaving the synaptic cleft through postsynaptic voltage-gated K+ channels involving KV1 and KV7 subunits. The above scenario is consistent with direct depolarization and hyperpolarization of the calyx membrane potential by intercellular K+.Vestibular organs of Amniotes contain two types of sensory cells, named Type I and Type II hair cells. While Type II hair cells are contacted by several small bouton nerve terminals, Type I hair cells receive a giant terminal, called a calyx, which encloses their basolateral membrane almost completely. Both hair cell types release glutamate, which depolarizes the afferent terminal by binding to AMPA post-synaptic receptors. However, there is evidence that non-vesicular signal transmission also occurs at the Type I hair cell-calyx synapse, possibly involving direct depolarization of the calyx by K Upon stchannels  triggerschannels . Howeverchannels , though + exiting the hair cell upon excitatory stimuli, accumulates rapidly in the synaptic cleft, thus directly depolarizing the pre- and postsynaptic membrane  compartment that extends over a long distance, it has been speculated that Kmembrane . Since tmulation .+ efflux from the hair cell produces a shift of the K+ current reversal potential (VrevK+), consistent with K+ accumulation in the intercellular compartment enclosed by the residual calyx  been achieved in mammalian vestibular epithelia, possibly because of the very thin and fragile neck region of the Type I hair cells. However, indirect information can be obtained by recording from Type I hair cells. Given that the basolateral membrane of Type I hair cells is completely enclosed in the afferent calyx, the patch pipette must be advanced through the calyx to reach it. Despite the calyx being pierced by the patch pipette, Kal calyx . Indeed,ir cells .VrevK+ (VrevK+ on K+ current elicited in Type I hair cells by investigating the accumulation factor (AF). The AF provides an indication of the “quality” of the residual calyx in terms of its residual ability to confine intercellular K+. Large AF values correlated with a depolarized resting membrane potential of the hair cell and a pronounced outward K+ current relaxation. We also found that intercellular [K+] increased or decreased depending on the size, kinetics and direction of K+ flow through GK,L, the low-voltage activated K+ conductance specifically expressed by Type I hair cells. Finally, since VrevK+ also depends on postsynaptic K+ channels, we recorded from in situ calyces. Our results are consistent with KV1 and KV7 channels being involved in the direct modulation of the postsynaptic membrane by intercellular K+.The reported shift of VrevK+  was highad libitum.All procedures for animal housing and experimentation were approved by the Ministero Italiano della Salute  and animal experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Mice (Swiss CD1 and C57BL/6N) from both sexes were obtained from Charles River  and from the Animal Care Facilities of the University of Pavia . In the UK, experiments were performed in accordance with Home Office regulations under the Animals (Scientific Procedures Act) 1986 and following approval by the University of Sheffield Ethical Review Committee. Mice were maintained under controlled light–dark cycles and received rodent pellets and water crista Type I hair cells were recorded either in situ or after enzymatic dissociation, as indicated in figure legends or text. A few recordings were also made in situ from mouse utricle Type I hair cells. All calyces were recorded in situ from the mouse crista or utricle. Data from utricle and canal hair cells were pooled. The location of the recorded cells was not assessed. Briefly, following anesthesia via inhalation with halothane  in Italy, and cervical dislocation in the UK, mice were decapitated and ampullae or utricles were surgically removed in chilled extracellular solution : NaCl 135, CaCl2 1.3, KCl 5.8, MgCl2 0.9, HEPES 10, glucose 5.6, NaH2PO4 0.7, Na-pyruvate 2. Vitamins  and amino acids  for Eagle’s minimum essential medium (MEM) were also added. The pH was adjusted to 7.4 with NaOH .The age of the mice ranged from postnatal day (PD) 7 to P47. Semicircular canal in situ recordings, following vestibular ganglia removal the cristae or the maculae were immobilized at the bottom of the recording chamber by mean of a weighted nylon mesh. Sensory epithelia were viewed by using an upright microscope equipped with differential interference contrast optics  and 60× water immersion objective. For hair cell dissociation, the mechanical-enzymatic treatment was the same as reported in For in situ and 44 isolated Type I hair cells with the K+-based intracellular solution (see below), from 17 in situ Type I hair cells with the Cs+-based intracellular solution (see below), and from 22 in situ calyces (20 with Intra_Cs+ and 2 with Intra_K+). In some experiments, the general outward rectifier K+ channels blockers tetraethylammonium  and 4-Aminopyridine , plus Cs+ which also blocks HCN channel (+ channel blockers was as follows (in mM): NaCl 110, CaCl2 1.3, CsCl 5.8, MgCl2 0.9, HEPES 10, glucose 5.6, NaH2PO4 0.7, TEACl 30, 4-AP 15. The pH was adjusted to 7.38 with HCl . In some experiments, CdCl2 0.1 mM (Sigma-Aldrich) was also added to the latter solution to block the Ca2+ current. All solutions were made freshly every morning and used in the course of the day.Recordings were obtained from 76 Type I hair cells  channel , were adThe amplifier’s filter bandwidth was generally set at 5 or 10 kHz. Digital sampling frequency was three to five times the analog bandwidth of the signal recorded. Current and voltage were measured and controlled through a DigiData 1322A or 1440 interface  connected to a computer running pClamp software.in situ were obtained after removal of visible calyx and tissue debris above the hair cell by using the patch pipette, and the ‘cleaning’ procedure was repeated with one (or more) patch-pipette until a GigaΩ-seal was performed. For dissociated hair cells, removal of the residual calyx by the patch pipette was not possible because during the procedure the hair cells became detached from the bottom of the Petri dish.Whole-cell recordings from Type I hair cells + currents. Sometimes, the seal was performed in ‘blind patch’ because the thickness of the preparation precluded visual identification of the thin calyx structure.For calyx recordings, after seal formation and suction, calyx identification was assessed by the presence of Na+-based intracellular solution : KCl 131, MgCl2 3, Na2-Phosphocreatine 10, Na2ATP 5, HEPES 5, EGTA 1, pH 7.2 with KOH, for a final osmolality of 293 mOsm/kg. In some experiments, K+ was omitted from the pipette solution, which contained : l-glutamic acid 110, CsCl 20, Na2-Phosphocreatine 10, MgCl2 3, Na2ATP 5, Hepes 5, EGTA 1, GTP 0.3, pH 7.28 with CsOH, for a final osmolality of 290 mOsm/kg). When filled with either intra-pipette solution, micropipettes had a resistance in the bath of 2–5 MΩ. All voltages in text and figures were corrected for the liquid junction potential (LJP) of −4 mV when using Intra_K+ or −11 mV when using Intra_Cs+, measured between electrode and bath solution . A few recordings, as noted in the figure legends, were obtained at body temperature . The patch-clamp amplifier was an Axopatch 200B  or Optopatch  amplifier. Patch pipettes were pulled from soda glass capillaries , fire-polished (in some cases) and partially coated with Sylgard  or surf wax . The micropipettes were filled with a Ksolution . All valVrest) was measured with the K+-based intracellular solution as the zero-current voltage in current-clamp mode.The cell resting membrane potential (Rm) was measured in voltage-clamp from the steady-state current elicited by a voltage step from −64 mV, or −61 mV, to −54 mV, or −51 mV, in Intra_K+ or Intra_Cs+, respectively. Since GK,L is fully active near −60 mV  is concerned, this current was only detected in a minority of Type I hair cells and, when present, it was very small (see Results). Series resistance (Rs) and cell membrane capacitance (Cm) were calculated off-line by the capacitive artifact elicited by a voltage step from −124 mV to −44 mV in Intra_K+, or −131 mV to −51 mV in Intra_Cs+. At these voltages IK,L and IK,v activated slowly enough to minimize overlap with the capacitive artefact. Moreover, a possible contamination by Ih, when present, should be minimal since with Intra_K+ and Extra_std it should reverse close to −40 mV (Cm and Rs measurement had a sampling rate of 100 kHz (an example is shown in the enclosed raw data file Neuroscience16226048Cm&Rs). Fit was performed from the average trace of 10 sweeps. We did not attempt on-line Rs compensation because GK,L is active up to −100 mV and repetitive voltage pulses more negative than −100 mV applied in close sequence damaged the cell. Moreover, given the residual calyx, Rs might also include a small contribution from the intercellular resistance. Therefore, we preferred to minimize Rs by keeping the pipette resistance as low as possible (tip diameters of about 2 μm) despite the greater difficulty in obtaining a gigaΩ-seal. We calculated an average Rs of 8.13 ± 5.34 MΩ (n = 120). Given an average Cm of 9.36 ± 6.12 pF (n = 119), the mean voltage-clamp time constant of the amplifier calculated by multiplying Cm and Rs for each cell was 58 ± 30 μs (n = 119). Although the clamp speed was reasonably good, depolarization above −30 mV elicited K+ currents of several nA in Type I hair cells, thus producing large voltage drops across the residual Rs (VRs). For the analysis of the relation between the quantity of K+ flowing through the cell membrane and tail current amplitude we did not correct for VRs since tail currents at −44 mV had a limited peak amplitude , producing a mean VRs of 5.19 ± 4.98 mV (n = 119). As far as the experiments with Intra_Cs+ are concerned, the amplitude of the currents, and therefore VRs, was much smaller than with Intra_K+  fired repetitively, indicative of inadequate space-clamp, Cm measurements were not considered. In the 4 remaining calyces, the mean Cm was 39.0 ± 18.1 pF (n = 4).For calyx recordings, + ions flowing during a given voltage step was calculated by integrating the macroscopic current tracing with Clampfit, which provided a quantity of charge ms−1 (Q).Analysis of traces and results were performed with Clampfit , Origin 6.1  and Microsoft Excel . The quantity of K+ (EK) was calculated according to the Nernst equation:The equilibrium potential for KVrev) of the mixed Cs+/K+ current  delivered from the conditioning voltage of −131 mV and considering a Vrev of −40 mV (see Results), with the following Boltzmann function:G(V) is conductance at voltage V, Gmin and Gmax are minimum and maximum chord conductances, V1/2 is voltage corresponding to half-maximal activation, and S is the voltage corresponding to an e-fold increase in G(V).NeuroscienceFig1 to 7, together with Origin files used for figures. All analyses performed can be found in NeuroscienceDatasheetExcel.All raw data cited in results can be found in folders t-test with or without Welch correction, was used for mean (median) comparison, as stated in the text. For parametric tests, the degrees of freedom and statistic’s values (t and F), in addition to the p value, are shown in the text. For non-parametric tests (Mann–Whitney), the statistic’s value U is provided in addition to the p value. Statistical relationship between two quantitative, continuous variables was estimated providing the Pearson’s correlation coefficient. Data are expressed as median and/or mean ± standard deviation (S.D.), or standard error (S.E.) when indicated; n = number of values.Statistical analysis was performed by Prism GraphPad 6.0 Software . Following Kolmogorov-Smirnov normality test, Mann–Whitney, or unpaired + in the pipette, 74 recordings were obtained from 74 different mice, while 46 from 20 mice, of which 16 provided 2 recordings each, 1 provided 3 recordings, and 2 provided 4 recordings each. Therefore, the contribution from nested data was largely diluted in the data pool. Moreover, AF measurements obtained from cells of the same animal were not clustered . As far as data used for AF statistical analyses is concerned, they all came from different mice except for 2 cells (in 10) coming from the same animal for the low-AF group, for which two cells the average value was taken. Therefore, we did not perform a multilayer analysis. Finally, as far as recordings with Intra_Cs+ are concerned, all averaged values are from different animals.In a few cases, recordings were obtained from distinct cells from the same animal, as follows. With Intra_K+ conductance GK,L, which activates at about −100 mV, is almost fully active at −60 mV and shows negligible inactivation during hundreds ms depolarizing steps , which activates for hyperpolarization below −60 mV  shift of the K+ current reversal potential (VrevK+) towards depolarized voltages, which has been attributed to K+ accumulation in the residual calyceal synaptic cleft  at −44 mV (red arrow in conds). Note that, in the absence of intercellular K+ accumulation, the outward Ii_tails should increase with Vcond depolarization, consistent with the increase of the K+ conductance and the driving force. When substantial intercellular K+ accumulation occurs, the outward Ii_tails will decrease with Vcond depolarization, because of the increase in K+ exit, until eventually reversing. The reversal of Ii_tails is associated with a relaxation of the outward K+ current during Vcond (black arrow in VrevK+ (see below). Smaller effects are produced in the presence of a reduced shift in VrevK+  exiting the hair cell in response to different Vconds . Q at a single Vcond (same cell as cond: −4 mV). The Ii_tail/Q relationships for the current responses of + ions theoretically required to reverse Ii_tail at −44 mV. The progressively steeper Ii_tail/Q relationship  compared to the latter ones  – (t(13.17) = 9.11, p < 0.0001; F = 4.07, p = 0.048; unpaired t-test with Welch correction). The smaller peak outward K+ current amplitude seen in high-AF than in low-AF cells was not necessarily due to a smaller GK,L amplitude, as it could be also explained by a smaller driving force for K+ to exit because of substantial intercellular K+ accumulation. Consistent with this hypothesis, AF correlated with a significantly more depolarized Vrest of the cells. Vrest was −74.4 ± 2.9 mV  in low-AF cells vs. −70.1 ± 3.5 mV  in high-AF cells – (t(16) = 2.76, p = 0.014; F = 1.44, p = 0.6446; unpaired t-test). Since a small Ih was only detected in one low-AF cells and one high-AF cells (not shown), and Rm between −124 mV and −114 mV was not significantly different between cells with a low-AF cells  and a high-AF  –  = 45, p = 0.7245; Mann–Whitney Test), the more depolarized Vrest in high-AF cells was not attributable to Ih or to “leaky” hair cells. Therefore, the above results indicates that in high-AF cell responses, intercellular K+ accumulation already occurs at rest.Paradoxically, the outward K+ current amplitude between low- and high-AF cells  – (t(12.85) = 10.62, p < 0.0001; F = 11.05, p = 0.0014; unpaired t-test with Welch correction).The difference in the steady-state outward KAF cells D was eve+ current during step depolarization in high-AF cells compared to low-AF cells. For example, at −31 ± 6 mV the outward current in low-AF cells relaxed to 96 ± 3% of the peak current , while it relaxed to 64 ± 14 % at −32 ± 4 mV in high-AF cells  − (t(10.08) = 7.32, p < 0.0001; F = 16.63, p = 0.0003; unpaired t-test with Welch correction). This result is consistent with outward K+ current relaxation in high-AF cells mainly produced by progressive intercellular K+ accumulation during Vcond. The role of voltage-dependent inactivation should instead be minimal since even in low-AF cells the small outward current relaxation was associated to a shift of VrevK+ towards depolarized voltages . Since GK,L does not inactivate nor deactivate significantly in the range of the hair cells receptor potential  in high-AF cell compared to 101.41 ms  in low-AF cells – (t(17) = 0.5986, p = 0.5573; ; p = 0.5839; Unpaired t-test). However, the difference was not statistically significant, possibly because the gating of GK,L is affected not only by membrane voltage but also by K+ since it is slowed down by an increase of external K+ concentration . Different from most voltage-gated K+ conductances, GK,L is significantly permeable to Cs+ . However, GK,L is less permeable to Cs+ than K+, causing the macroscopic current to reverse at significantly more depolarized voltages  than with Intra_K+ . This depolarized Vrev is well consistent with the estimated Vrev of −39 mV in our experimental condition, as calculated by Eq. +] in the pipette solution of 88 mM and a total [K+] in the extracellular solution of 5.8 mM, and given the reported permeability ratio of Cs+ to K+ of 0.31 .The hypothesis that e to Cs+ . In the  of 0.31 . However of 0.15 , which iVrev in Intra_Cs+, the current was inward in the voltage-activation range of GK,L . This inward current should be almost exclusively carried by K+ through GK,L since Na+ does not permeate GK,L  is absent in postnatal mouse Type I hair cells (2+ current (ICa) expressed by mouse Type I hair cells activates positive to −60 mV and is very small  confirmed the absence of INa and the presence of a very small ICa , −91 mV , −81 mV , −71 mV  and −61 mV  gave a reversal potential of +8 mV, which was considered to be mostly leak current. Assuming that leakage did not change before and after perfusion of TEA, 4-AP and Cs+, its contribution to the control current at −61 mV  was therefore 11%.On average, the steady-state inward current at −61 mV was −0.28 ± 0.22nA  after leak current subtraction.The inset in the bottom panel of + also blocked the outward Cs+ currents elicited by voltage steps less negative than Vrev (n = 3) to −18 ± 7 pA (n = 3) − see inset in Perfusion of TEA, 4-AP and Cshan Vrev A. For ex+ we analysed the activation kinetics of GK,L at voltages negative to −61 mV to avoid overlap with GK,v, which activates positive to −50 mV  Ih are not activated, inward current relaxation can only be explained by a progressive leftward (towards more negative voltages) shift of Vrev as produced by removal of intercellular K+. The degree of inward current relaxation, measured as the difference between the peak and the steady-state inward current amplitude  of the initial peak value. In the same cells, voltages less negative than Vrev elicited outward currents showing variable degree of relaxation  of the initial peak value. Outward current relaxation is consistent with intercellular Cs+ accumulation in a similar way as K+ (R): 0.31; p: 0.42) was found between the percentage of inward current relaxation at −61 mV and of outward current relaxation at −21 mV by linear regression of data, possibly because inward current relaxation overlaps to GK,L activation time course which at hyperpolarized voltages is very slow  was inward, consistent with intercellular Cs+ accumulation. However, following long-lasting inward currents . The mean Ii_tail/Vcond relation, obtained from three cells by using the voltage protocol illustrated above, is shown in To further test for intercellular Kmulation ; Fig. 2.GK,L is responsible for intercellular K+ accumulation or clearance depending upon hair cell depolarization or hyperpolarization, respectively. It should be noted that, depending upon VrevK+, GK,v is also contributing to K+ flux into the cleft during hair cell depolarization above −40 mV, and back into the hair cell during hair cell hyperpolarization before it deactivates.Taken as a whole, the above experiments show that + currents evoked by calyx depolarization could produce a shift of VrevK+ toward less negative voltages . Analogous space-clamp problems have been reported with a K+-based intracellular solution in whole mount vestibular preparations , depolarization above −71 mV elicited a volley of rapid transient inward currents, whose frequency increased with depolarization A. Since arations . In 10 ot −81 mV B, indicanditions .Fig. 5Redissociated rodent vestibular calyces a single transient Na+ current was elicited by depolarization above −60 mV ; 17623021 (P15); 17629002 (P16); 17630019 (P17). The presence of INa is consistent with cell identification as a calyx since in the mouse, different to the rat . An example of calyx response with a single Na+ action current is shown in hold of −71 mV, a small sustained inward current (−64 pA) was present. On average, the sustained inward current at −71 mV was −132 pA .In order to avoid the problem of poor clamp, we restricted our analysis to those calyces that, like dissociated calyces, showed a single transient Na the rat , INa is re birth . Moreoven = 4). For depolarization above Vrev, the outward current increased linearly  to −109 ± 134 pA (n = 4) and the peak outward current at 9 mV decreased from 3427 ± 1552 pA (n = 4) to 1298 ± 0.602 (n = 4). The outward current blocked by TEA + 4-AP + Cs+, obtained by subtracting the residual current after block from the control current, appeared near −40 mV and increased monotonically with depolarization  found at the gerbil vestibular calyx terminal , which blocks all voltage-gated Ca2+ channels at sub-millimolar concentration (n = 2) to 158 ± 40 pA (n = 2)  A. Fig. 7te , despite the partial removal of the calyx by the patch pipette  current will produce an almost synchronous change of IK,L amplitude and presumably of intercellular K+ concentration. Recording from the calyx while mechanically stimulating the associated Type I hair cell in early postnatal (<P9) excised saccule preparations, + exit through GK,L seems a good candidate.Intercellular Kugh GK,L , Fig. 4,membrane , which sin vitro  might be+ transporters not precluding changes in intercellular K+, though their regulatory function remains to be determined.In summary, the above studies are consistent with active KIn situ recordings using slice preparations from the gerbil crista have reported the presence of a non-inactivating K+ current which was sensitive to dendrotoxin-K, suggesting the contribution of Kv1.1 and/or Kv1.2 channel subunits, and a slowly-inactivating K+ current sensitive to margatoxin, indicating the contribution of Kv1.3 and Kv1.6 channel subunits  are not involved. Ephaptic transmission requires a high intercellular resistance (Ri) and an extended apposition of pre- and postsynaptic membranes, such that current flowing through Ri produces an extracellular potential drop that instantaneously affects the activity of pre- and postsynaptic voltage-gated channels  decrease of intercellular K+ content, thereby increasing the driving force for K+ to exit from the calyx into the cleft through KV1 and KV7 channels expressed at the calyx inner membrane, thus hyperpolarizing the calyx.Several lines of evidence support the hypothesis that intercellular Kal cleft . Immunolmembrane , providinterpart , demonstw −60 mV . Given acordings , the intin vivo due to the presumably larger MET current through fully functional MET channels and the relatively low endolymphatic Ca2+ concentration (20 μM; in vivo Vrest might be even more depolarized than found here.As a final consideration, it should be mentioned that the zero-current potential measured with the hair cell in artificial perilymph is likely to be less depolarized than n 20 μM; . Therefo


Early exposure to inflammation in childhood is increasingly recognized as one of the major factors that hinder millions of children worldwide from meeting their full developmental potential. The current study examined the association between systemic inflammation and children’s neural responses to facial stimuli and explored if this activity mediated the relation between inflammation and cognitive outcomes.Two separate cohorts of children living in an urban slum in Dhaka, Bangladesh who are at high-risk for sustained inflammation were recruited in this study. The concentration of C-reactive protein (CRP) in blood samples served as our index of inflammation. Blood samples were collected once at 18 weeks for the younger (infant) cohort (N = 125) and at 6, 18, 40, 53, and 104 weeks for the older (toddler) cohort (N = 120). Event-related potentials (ERPs) were also recorded separately for the two cohorts: at 6 months for the younger cohort (N = 48) and at 36 months for the older cohort (N = 93), using a face-oddball paradigm in which standard and oddball faces were presented. Cognitive outcomes were evaluated with Mullen Scales of Early Learning (MSEL) at 27 months for the younger cohort (N = 98) and with Wechsler Preschool and Primary Scale of Intelligence (WPPSI) at 48 months for the older cohort (N = 124).For the older toddler cohort, the P400 and Nc amplitude differences between the two types of stimuli were found to be associated with the frequency of elevated CRP such that more episodes of elevated CRP corresponded to smaller P400 and Nc differences between the two conditions. In addition, the P400 and Nc differences were both found to mediate the relation between inflammation and performance IQ scores. For the younger infant cohort, the participants showed differentiated N290 response to the two types of stimuli, but no association between the ERP response and CRP concentration was found.These findings suggest that chronic systemic inflammation has a long-term impact on children’s brain functioning and cognitive development. The neural circuitries associated with social attention and recognition memory of faces may be potential pathways by which inflammation exerts its effect on cognitive development. •Chronic inflammation is assoicated with ERP responses to faces in Bangladeshi children.•Children more frequently having elevated CRP level show smaller ERP difference between familiar and novel faces.•The ERP difference is associated with future IQ.•The ERP activation mediates the relation between inflammation and future IQ. Systemic inflammation is commonly accompanied by infections as the result of the release of pro-inflammatory cytokines and proteins . The impThere are different pathways by which systemic inflammation can communicate with the brain. Systemic inflammation is characterized by increased inflammatory cytokines , IL-6), proteins ) and other immunologically active peptides generated at the site of infection . These pConnections between early-life inflammation and neurodevelopmental impairments in children are increasingly inferred from behavioral studies . For insFace perception and social attention are fundamental skills for children to master in order to learn and interact effectively in a social world. The investigation of how early-life inflammation may impact the neural correlates of these cognitive processes is critical for understanding the effect of early-life inflammation on child cognitive development. The face-oddball paradigm in which frequent  and oddball  faces are presented to participants has been widely employed to examine the neural correlates of social attention and recognition memory of faces in children . In partThe objective of the current study was to investigate the effect of early-life inflammation on brain functioning and to explore how brain functioning mediates the relation between inflammation and cognitive outcomes. The concentration of CRP in peripheral blood samples was measured as the biomarker of systemic inflammation at 18 weeks for the infant cohort and at 5 consecutive times during the first two years of life for the toddler cohort. Children’s brain functioning was assessed by measuring differences in the brain’s electrical activity (ERPs) in the face-oddball task. For the infant cohort, we evaluated their cognitive outcome at 27 months of age using Mullen Scales of Early Learning (MSEL) . For the22.1The infant cohort consisted of 130 (56M/74F) participants who were recruited from the “Cryptosporidium Burden” (CRYPTO) study and had their blood sample collected at 18-weeks of age, height measured at 4.5  and 6 months, ERP tested at 6 months  and cognitive assessment at 27 months . The toddler cohort consisted of 130 (72M/58F) participants who were recruited from the “Performance of Rotavirus and Oral Polio Vaccines in Developing Countries” (PROVIDE) study had their blood samples collected at 6, 18, 40, 53, and 104 weeks, height measured at 21 , 30  and 36  months ERP tested at 36 months  and cognitive assessment at 48 months . Participants’ HAZ scores were calculated based on World Health Organization (WHO) standards for each time point and then averaged across the time points. All infants and toddlers were born ≥ 34 gestational weeks. These participants had no known history of neurological abnormalities, traumatic brain injury, genetic disorders, or visual or auditory impairments.The two cohorts of participants were recruited from an impoverished neighborhood (urban slum) in Dhaka, Bangladesh. Children living there are often exposed to air and water pollution, significant poverty and unsanitary living conditions. These environmental hazards have been associated with various kinds of health issues including growth faltering, enteric diseases and inflammation-related brain injury , 2017. TEthical approval for the study was obtained from research review and ethics review committees at the International Centre for Diarrheal Disease Research, Bangladesh and Institutional Review Boards at Boston Children’s Hospital and were in accordance with local guidelines and regulations. We have collected written consent forms from all families who participated in the study.2.2Continuous EEG was recorded while the participants were seated on a caregiver’s lap approximately 65 cm from the presentation screen. The face-oddball task is illustrated in 2.3EEG was recorded from a 128-channel HydroCel Geodesic Sensor Net (HGSN) that was connected to a NetAmps 300 amplifier . Channel impedances were kept at or below 100 kΩ and signals were sampled at 500 Hz. EEG recordings were filtered with a 0.3–30 Hz finite impulse response (FIR) band-pass filter and segmented from 100 ms before stimulus onset as the baseline to 900 ms following stimulus onset. The segmented epochs were inspected for artifacts automatically in ERPLAB  using boForty-eight of the original 130 infants had usable ERP data. Among the 82 excluded infants, 22 did not participate in the EEG portion of the study, one infant’s data were not collected correctly due to an experimenter error, and 59 did not have enough ERP trials during data collection or after artifact rejection for at least one condition. There were 93 out of the original 130 toddlers having usable ERP data. One participant did not participate in the EEG section, 3 participants’ data were rejected because of experimenter error or equipment failure, and 33 participants had not enough ERP trials for at least one condition.The electrodes were combined into different electrode clusters covering the commonly studied scalp regions in previous examination of the N170/N290, P400, and Nc components in child face processing . The “Fr2.4The concentration of CRP in peripheral blood samples was measured as the index of inflammation. The blood sample for CRP assessment was collected at 18 weeks for the infant cohort and at 6, 18, 40, 53, and 104 weeks for the older toddler cohort. We evaluated the chronic (persistent) inflammation burden for the 36-month-old cohort by calculating the cumulative number of times the child experienced elevated CRP level. CRP elevation was defined as the child’s CRP concentration (mg/L) being at the top 50% in the group at the measurement time . As a reFor the 6-month-old cohort, the CRP concentration at 18 weeks was log transformed to create a continuous and normally distributed measure for 125 infants who had usable blood samples. We were not able to evaluate the level of chronic inflammation for this younger cohort because of the only CRP measurement before the ERP test.2.5The cognitive outcome of the infant cohort was assessed with Mullen Scales of Early Learning (MSEL)  at 27 mop = .979. CRP concentration was slightly higher for infants with than without MSEL data, but the difference was not significant either, t = 1.68, 95%CI = [-0.091 1.122], p = .095.Given a large portion of infants ended up with no usable ERP or MSEL data, CRP concentration was compared between infants with and without usable data to rule out the possibility that infants with higher CRP concentration were more likely to have low-quality ERP and behavioral data. No difference in CRP concentration was found between infants with and without usable ERP data, t = 0.027, 95%CI = [-0.536 0.557], The cognitive outcome of the toddler cohort was assessed with Wechesler Preschool and Primary Scalre of Intelligence (WPPSI-III)  at 48 mo2.6p < .05) main or interaction effects emerged, post hoc comparisons were conducted with a Bonferroni correction.Repeated-measures analyses of variance (ANOVAs) were performed to test whether the participants would show different ERP responses to frequent and infrequent faces on the group level. The ERP amplitudes for the N290, P400, and Nc components were analyzed as the dependent variables separately in three ANOVAs, which included age (2: 6 and 36 months) as a between-subjects independent variable and stimulus condition (2: frequent and infrequent) as a within-subjects independent variable. Greenhouse-Geisser corrections were applied when the assumption of sphericity was violated. When significant (Multiple linear regression was performed to test the associations between inflammation and ERP response (the difference between frequent and infrequent conditions for the three components), with the factor of HAZ being included as an independent variable (covariate). Regression was also performed to test the associations of ERP response and inflammation with cognitive outcomes . Repeated-measures ANOVAs and regression analyses were run in IBM SPSS Statistics .X2 (p > .05), CFI > 0.95, SRMR < 0.08, and RMSEA < 0.06. Indirect effects were estimated using bootstrapping across 10,000 draws with bias-corrected confidence intervals.Longitudinal path analysis was finally conducted with Mplus (version 7.4) to test the hypothesized indirect pathways by which inflammation could affect children’s cognitive outcomes. One pathway is through the ERP response, i.e., to test whether ERP response mediates the relation between inflammation and cognitive outcomes. The other pathway is through HAZ and ERP reponse, i.e., to test whether inflammation is associated with malnutrition, which in turn may impact ERP response and cognitive outcomes. Only participants with ERP data were included in the analysis. Missing values for WPPSI scores (N = 5) for the toddler cohort were handled using full-information maximum likelihood (FIML) estimation with robust standard errors. Model fit was evaluated based on a non-significant 33.1For the infant cohort, the mean/median of the CRP concentration at 18 weeks were 4.379/1.335 mg/L (SD = 8.605). For the toddler cohort, the mean/median of the CRP concentration at 6, 18, 40, 53, and 104 weeks were 0.423/.097 (SD = 1.083), 2.727/.549 (SD = 6.393), 5.354/1.712 (SD = 10.240), 3.831/1.106 (SD = 7.903), and 6.213/1.593 (SD = 8.605) mg/L. The frequency of CRP elevation varies between 0 and 5: 0 (N = 8), 1 (N = 19), 2 (N = 35), 3 (N = 34), 4 (N = 28), and 5 (N = 6).3.23.2.1p < .0001, ηp2 = 0.113. The N290 amplitude for the frequent condition was greater (more negative) than that for the infrequent condition, Mdiff = −1.868, 95% CI [-2.746 -.990]. There was no interaction between age and stimulus condition  = 17.694, ondition .Fig. 2Th3.2.2p = .002, ηp2 = 0.066. Post-hoc comparisons with Bonferroni adjustment showed that the effect of stimulus condition was only significant for the 36 months old cohort, such that the P400 amplitude for the frequent condition was significantly smaller (less positive) than that for the infrequent condition, Mdiff = −3.971, 95% CI [-5.052 -.2890]  = 9.796,  -.2890] . There w3.2.3p = .007, ηp2 = 0.051. Post-hoc comparisons showed that the Nc amplitude for the frequent condition was smaller (less negative) than that for the infrequent condition only for the 36 months old cohort, Mdiff = 2.571, 95% CI [1.799 3.342]  = 7.430, 9 3.342] . No effe3.3We further tested the association between inflammation and the ERP amplitude difference between the two conditions.3.3.1Multiple linear regression analysis was performed to examine the associations of ERP responses with CRP concentration at 18 weeks and the average HAZ score between 4.5 and 6 months, separately for the N290, P400 and Nc components. The analyses revealed no effect of CRP concentration or HAZ on the ERP response for all three components.3.3.2p = .148, R2 = 0.042, and neither frequency of CRP elevation  or average HAZ score from 21 to 36 months significantly predicted the N290 difference between the two conditions.The multiple linear regression analysis for the older cohort showed different results for three ERP components. The overall regression model for the N290 component was not significant F = 1.951, p = .003, R2 = 0.119. The frequency of CRP elevation but not HAZ was significantly associated with the P400 difference between the two conditions, β = −0.337, p = .002, such that more episodes of elevated CRP corresponds to smaller P400 difference between the two conditions  = 6.089, nditions A. In Fignditions B.Fig. 3Lp = .05, R2 = 0.064. Only the frequency of CRP elevation was a significant predictor of the Nc difference between the two conditions, β = −.250, p = .021, such that more episodes of elevated CRP corresponds to smaller Nc difference between the two conditions  = 3.075, nditions , Fig. 4.3.4p = .025, β = −1.341, R2 = 0.201] and FSIQ  at 48 months. No significiant association was found between the CRP concentration at 18 weeks and MSEL composite or visual reception scores at 27 months.Linear regression analyses were conducted to determine the assoication between inflammation and future cognitive outcomes. Our results showed that for the toddler cohort, the frequency of CRP elevation negatively predicted children’s PIQ  and Nc  difference both positively predicted the PIQ but not FSIQ scores at 48 months, such that the greater the difference the higher the PIQ  = 5.652,  the PIQ C and D. 3.52(1) = 0.074, p = .791; CFI = 1.00; SRMR = 0.008; RMSEA = 0.000; the “Nc model”: χ2(1) = 2.109, P = .146; CFI = 0.956; SRMR = 0.049; RMSEA = 0.109.Path analysis was run for the toddler not the infant cohort given the absence of associations between the factors for the latter. The indirect effect of inflammation on PIQ through the ERP responses was tested in the mediation model. The indirect effect of inflammation on PIQ through HAZ and ERP responses was not tested because there was no association between HAZ and ERP responses revealed by the regression analyses. The mediation model  was run The path analysis for the P400 component revealed that the frequency of CRP elevation was negatively associated with the P400 difference, which in turn was positively associated with PIQ. The mediation effect of the P400 response on the relation between frequency of CRP elevation and PIQ was significant: 95% confidence interval (CI) of the estimate = [-0.163 -.017] . The ana4The goal of the current study was to understand the long-term consequences of chronic systemic inflammation during infancy on the development of brain function in early childhood. We examined two groups of children living in an impoverished neighborhood in Dhaka, Bangladesh who are at high-risk for infectious diseases and chronic inflammation from early childhood. We measured an inflammatory marker – CRP concentration – along with ERP responses to familiar and novel (or “frequently and infrequently presented”) faces and cognitive scores at specific ages. As expected, the frequency of CRP elevation was negatively associated with the P400 and Nc differences between frequent and infrequent faces for the toddler cohort, such that more episodes of elevated CRP corresponded to smaller P400 and Nc differences. Children in the toddler cohort showing greater ERP evidence of discrimination between the two types of stimuli were found to have higher PIQ scores at 48 months. In addition, the ERP responses mediated the relation between the frequency of CRP elevation and PIQ scores; in other words, there was an indirect effect of chronic inflammation on PIQ through the ERP responses. In contrast to our hypothesis, the younger infant cohort whose CRP concentration was measured once at 18 weeks did not show an association between CRP concentration and ERP differences between conditions or a correlation between ERP responses and cognitive outcomes.4.1In the current study we provide the first empirical evidence for the association between chronic inflammation and children’s brain functioning measured with EEG-based tools – the ERP. The P400 and Nc components have been widely used as neural indices of stimulus processing and sustained attention in children , and theChildren with <2 times of CRP elevation showed significant difference in the P400 and Nc components between the infrequent and frequent faces, and such difference reflects the ability to discriminate the infrequent from the frequent stimuli by the brain . This fip = .055. The N290 component is regarded as the precursor of the adult N170 face-sensitive component  scores at four years of age. This finding provides converging evidence for effects of early-life inflammation on neurocognitive development that have been reported previously . A recen4.3In contrast to our hypothesis, the infant cohort did not show an association of inflammation with their ERP responses or cognitive outcomes. It is possible that we were unable to detect an association between inflammation and brain activity at 6 months of age, as such an association may take time to manifest itself , or our ERP measure lacks the sensitivity to detect the derailed development of the neural circuits underlying social attention due to inflammation early in life. The non-significant association between inflammation and MSEL scores could be due to that tasks of MSEL are too demanding  for infants growing up in such low-resource settings who may be delayed in development in various cognitive domains. In addition, the only measurement of CRP level at 18 weeks for the infant cohort may not be able to account for the effect of chronic inflammation during the first few months of life. It is valuable to follow this younger cohort and measure their brain activity, cognitive performance and CRP concentration on additional time points to justify these explanations.One limitation of the current study is limited control of other potential covariates in addition to stunting due to the (relatively) small sample size. These children are growing up in an impoverished neighbourhood who are likely to suffer from other adverse experiences as well, such as poverty or reduced caregiving quality . Future 5The current study demonstrates that chronic systemic inflammation during infancy has a long-term impact on children’s brain functioning and cognitive development. The neural circuitry associated with face processing and sustained attention are likely to be affected by early-life inflammation and be potential pathways by which inflammation derails cognitive development in relative domains. The measure of ERP with EEG recordings is shown to be a useful tool to study brain functioning in low-resource settings. Children growing up in these low-resource settings are at high-risk for inflammation-related health issues from early childhood, and thus immune-based therapies may be critical to prevent developmental delays.


In this paper, we first prove an integral majorization theorem related to integral inequalities for functions defined on rectangles. We then apply the result to establish some new integral inequalities for functions defined on rectangles. The results obtained are generalizations of weighted Favard’s inequality, which also provide a generalization of the results given by Maligranda et al. (J. Math. Anal. Appl. 190:248–262,  The notion arises in a variety of contexts, and it can be made precise in a number of ways. In remarkably many cases, the appropriate statement is that x is majorized by y (or y majorizes x). Namely, for two n-tuples x is said to be majorized by y , 3533, 3Letbe a continuous convex function on the intervalI, and letandbe two n-tuples such thatIfxis majorized byy, thenn-tuples x and y can be found in [n-convex functions; see [The inequality asserted by Theorem found in . Generalfound in  and Niezfound in . In receons; see , 41–45 aRecently, it has come to our attention that certain integral majorization theorems, we begin with recalling some relevant results. In 1947, Fuchs [Letbe continuous and increasing functions, and letbe a function of bounded variation. (i)Ifandthen for every continuous convex functionϕ, we have(ii)Ifthen for every continuous increasing convex functionϕ, we haveIn 1995, Maligranda, Pečarić, and Persson  establisLetwbe a weight function, and letfandgbe positive integrable functions onSuppose thatis a convex function and thatand(i)Iffis a decreasing function onthen(ii)Ifgis an increasing function onthenIn 1933, Favard  proved tLet Φ be a nonnegative continuous concave function onnot identically zero, and let ϒ be a convex function onwhereThenAs a consequence of Theorem Let Φ be a concave nonnegative function onIfthenMaligranda, Pečarić, and Persson  gave the(i)Let Φ be a positive increasing concave function onand let ϒ be a convex function onwhereThen(ii)Let Φ be a positive decreasing concave function onand let ϒ be a convex function onwhereThenFor further results related to generalizations, extensions, and refinements of the integral inequalities of majorization type, we refer the reader to , 46–48.In this paper, we extend majorization and Favard inequalities from functions defined on intervals to functions defined on rectangles. The results presented in this paper generalize the results of Maligranda, Pečarić, and Persson .In this section, we introduce some notions and lemmas.A functiondefined on a convex subset Ω ofis said to be convex iffor allandϕ is convex, thenIn this paper, convex functions considered are supposed to be twice differentiable. It is well know that if the function In the literature, there are many generalizations of convex functions in different directions. One of them is coordinate convex functions introduced by Dragomir .Let us consider a bidimensional intervalA functionis said to be coordinate convex if the partial functionsdefined asanddefined asare convex where defined for allandEvery convex functionis coordinate convex.Letvbe a weight function on(i)Ifhis a decreasing function onthen(ii)Ifhis an increasing function onthenIn this section, we establish some majorization integral inequalities for functions defined on rectangles. The following theorem is a generalization of Theorem Letwandpbe positive continuous functions onandrespectively, and letf, gandh, kbe positive differentiable functions onandrespectively. Suppose thatis a convex function and thatand(i)Ifgandkare decreasing functions onandrespectively, then(ii)Iffandhare increasing functions onandrespectively, then(i)Since Put Integrating both sides of inequality  gives\dBy Fubini’s theorem we haveUsing integration by parts, we obtainϕ is convex on ϕ is coordinate convex on k and g are decreasing, so that Since Combining , 3.6), , 3.6), aequality .(ii)Inequality  can be p □Letwandube positive continuous functions onandrespectively, and letf, gandk, lbe positive differentiable functions onandrespectively. Suppose thatis a convex function. (i)Letandbe decreasing functions onandrespectively. Iffandlare increasing functions onandrespectively, thenIfgandkare decreasing functions onandrespectively, then the reverse inequality of (holds.(ii)Letandbe increasing functions onandrespectively. Ifgandkare increasing functions onandrespectively, thenIffandlare decreasing functions onandrespectively, then the reverse inequality of (holds.(i)Using Lemma /g(t) in , we obtaAlso, putting /k(s) in  gives3.From  and 3.1 we deducAdditionally, it is easy to observe thatBy relations , 3.13),,3.13), , and 3., using iequality .Using the majorization relations , 3.13),,3.13), , and 3. and ineq(ii)If we perform an interchange equality  follows equality . The revequality  can be dequality  by using □As an application of Theorem Letwandube positive continuous functions onandrespectively, and letfandlbe positive differentiable functions onandrespectively. Suppose thatis a convex function. (i)Letandbe decreasing functions onandrespectively. Iffandlare increasing functions onandrespectively, then(ii)Letandbe increasing function onandrespectively. Iffandlare decreasing functions onandrespectively, thenNote the simple fact that if equality  and chooequality . Applyinequality  and chooequality . □Letwandube positive continuous functions onandrespectively, and letfandlbe positive differentiable functions onandrespectively. Suppose thatis a convex function. (i)Letandbe decreasing functions onandrespectively. Iffandlare increasing functions onandrespectively, thenwhereand(ii)Letandbe increasing functions onandrespectively. Iffandlare decreasing functions onandrespectively, thenwhereandSubstituting sides of , we getequality , leads tequality .Similarly, we can deduce inequality  by substsides of . □Letwandube positive continuous functions onandrespectively. Suppose thatis a convex function. (i)Iffandlare positive increasing concave functions onandrespectively, thenwhereand(ii)Iffandlare positive decreasing concave functions onandrespectively, thenwhereand(i)By the first part of Corollary Consider the functiont givesDifferentiation of f is a positive concave function on It follows that equality .(ii)f is positive concave function on Similarly, we haveequality  follows  □Obviously, the results of Corollary =ϒ(x) in  and (4.6=ϒ(x) in . Further=ϒ(x) in  by takin


We describe a wireless microsystem for gastrointestinal manometry that couples a microfabricated capacitive transducer to a dual-axis inductor, forming a resonant inductor-capacitor (LC) sensor within an ingestible 3D printed biocompatible capsule measuring ø 12 mm × 24 mm. An inductively coupled external telemetry unit wirelessly monitors the pressure dependent resonant frequency of the LC sensor, eliminating the need for integrated power sources within the ingested capsule. In vitro tests in saline show pressure response of −0.6 kHz/mmHg, interrogation distance up to 6 cm, and resolution up to 0.8 mmHg. In vivo functionality is validated with gastrointestinal pressure monitoring in a canine beagle over a 26-hour period. Many industries can benefit from miniaturized wireless pressure monitoring, but it is perhaps the biomedical field which may have the most compelling needs . IntraocThe gastrointestinal (GI) tract is dependent on regular contractions to ensure consistent digestion and passage of food. Abnormalities in these contractions and the resulting pressure waves can result in, or result from, a number of motility disorders associated with nausea, abdominal pain, and vomiting ; detectiTM , which images the intestinal tract but lacks physical sensors [TM , which records pressure, offering >100 mmHg pressure range, 10 mmHg pressure resolution, and 1 Hz readout bandwidth [Another diagnosis regime employs pill-shaped ingestible devices, typically containing sensors, readout and data transmission electronics, and batteries . Commerc sensors , and theandwidth . Ongoingandwidth . While tTM HF System  was utilized to measure pulmonary artery pressure to monitor for congestive heart failure; it provides 320 mmHg pressure range, 1 mmHg resolution, and 200 Hz readout bandwidth [A widely employed passive telemetry method utilizes the phase response of an inductor-capacitor resonant circuit (LC sensor), in which the resonant peak is altered by capacitively sensed pressure . Prior wandwidth .Our effort assesses an alternate method of diagnosing motility disorders that eliminates the need to ingest radiological or battery materials while simultaneously reducing sensor pill complexity, and potentially reducing the cost for this single-use disposable device. It realizes an ingestible capsule containing a passive LC sensor and inductively coupled external telemetry unit with control software for monitoring gastric pressure , focusinROC, ROR, and PSC, SR are the parasitic capacitance and resistance of the readout coil and sensor, SensC is the pressure transducer, and M is the mutual inductance between the inductors ROL and SL. Relevant design equations are summarized in inZ, of the external readout coil is calculated from ROV and ROI, the complex input voltage and current (1). Hence, the frequency dependent phase of the input impedance, Zin(f)ϕ, is the difference between the phase components of ROV and ROI (2). At the resonant frequency of the LC sensor, 0f, this phase is reduced relative to higher and lower frequencies. The size of this “phase-dip”, ΔPDϕ, is indicative of the level of coupling, k, between the external coil and LC sensor, and is also affected by the quality factor, Q, of the LC sensor. The ΔPDϕ at 0f can be calculated using (3), where Q is calculated using (4) [k is calculated using (5) [0f, is given by (6).A lumped model for the sensing system is shown in sing (4)  and k issing (5) . The LC Q and k affect ΔPDϕ, Q also impacts the frequency span over which the phase-dip is evident. As the frequency span of the phase-dip increases, the minimum pressure resolvable by the system declines due to a degradation in the accuracy with which 0f can be extracted through curve fitting of discrete data. The degradation of Q is primarily caused by series resistance, SR, of the LC sensor. The AC resistance of the coil, S,CoilR, can be estimated using equations that account for the skin-depth effect [S,CoilR can be further increased as much as 5-fold due to the proximity effect with closely spaced (S) inductor windings [Although both h effect . RS,Coilwindings . The equk, is affected by many parameters; if the readout coil is much larger than the sensor and well aligned to it, coupling is approximated by (5), where 0µ is the permeability of free space, n and r are the turns and radius of each coil, and z is the coil separation . Available equations [SL and ROL. Using (3)–(6), coil design can be refined to maximize performance within the physical boundaries of the gastric manometry application. Coil coupling, quations  can be uROr, of ≤7.5 cm (to permit ease of use during in vivo canine testing) and its self-resonant frequency, 0,ROf, was constrained to be at least double of the LC sensor 0f to prevent interference. Maximum coupling, k, at a given interrogation distance, z, can be achieved when ROr is equal to z. The maximum ROr of 7.5 cm was chosen to maximize the allowed value of z. To prevent 0,ROf from decreasing below 25 MHz, a maximum ROL of 20 µH was set, assuming that the parasitic capacitance of the readout coil, ROC, was 2 pF.In this work, the planar external readout coil was constrained to a physical radius, Physical size constraints for the LC sensor footprint were set by the capsule interior volume at ø 10 mm × 20 mm. The sensor coil utilized a dual-axis design to improve coupling in non-ideal alignment scenarios, as sensor orientation cannot be determined once deployed. These z-axis and x-axis coils were connected in series to the pressure transducer, as shown in 0C/C ratio, which enhanced LC sensor response (0/∆P∆f). It was designed to maximize capacitance change over 0–300 mmHg of applied gauge pressure , it reduced the ESR to <20 Ω, nearly doubling LC sensor Q.The capacitive pressure transducer was fabricated on a dielectric substrate with features made from dielectric layers to enabled low offset and parasitic capacitances. This permitted an increase in Δal tract ) using tPDϕ, Q, and LC sensor response were calculated for all combinations of coil parameters within the boundary conditions. Due to the compromises associated with the LC sensor design, no one solution offers an optimal choice of maximum ΔPDϕ, Q, and LC sensor response. For example, increasing LC sensor Q  will negatively impact LC sensor response (0/∆P∆f), as 0∆f is inversely proportional to the offset capacitance, as shown in (6). A figure of merit was created by multiplying the ΔPDϕ, Q, and 0/∆P∆f to identify a design which balances all three parameters. The final design and measured device values are given in The ΔThe LC sensor support structure and housing were fabricated with 3D printing using a ProJet MultiJet printer and M3 Crystal, a biocompatible, acrylic-based resin . The supg = 500 nm), and diaphragm diameter (ø 200 µm). The upper electrode (200 nm) was deposited by sputtering, followed by an initial diaphragm layer of PECVD silicon nitride and silicon dioxide (2.3 µm). Etchant access slits were opened and the α-Si was removed with XeF2. The diaphragm was sealed with PECVD dielectric (2.0 µm) and atomic layer deposition of Al2O3 (100 nm), h = 4.6 μm. Contact pads were opened with a dry etch and conventional dicing was used for singulation. Optical images are shown in A surface micromachining process was utilized to fabricate the capacitive pressure transducer . It bega0f, with sufficient readout bandwidth (≥2 Hz) and calculate the corresponding pressure with adequate resolution (≤5 mmHg). The input impedance phase (inϕZ) between the ROV and ROI was measured using a phase-to-voltage IC  [ROV) and digitized the output of the AD8302. To meet the readout bandwidth and resolution requirements, a balance was calculated between the interrogated frequency range (IFR), the number of discrete interrogated frequencies , and the interrogation time at each frequency  . The reqssf, up to 12.5 ms is available at each frequency. System non-idealities such as frequency switching, data transfer, and curve fitting cause additional delays. Therefore, a total available averaging time of 5 ms is assumed, permitting up to 5 × 104 samples at 107 samples per second. When averaged, this can reduce uncorrelated white RMS noise by more than 200-fold [Sample averaging was utilized to reduce noise and improve the minimum resolvable pressure. To permit a 2 Hz readout bandwidth at the necessary IFR and 200-fold . The capacitive transducer and antenna inductor coils were independently characterized, then assembled in the LC sensor and tested in vitro in conductive saline to mimic deployment conditions. The same characterized device was then successfully deployed and interrogated in vivo in the GI tract of a canine model. The pressure transducer provided an offset capacitance of 10 pF and response of 1.8 fF/mmHg over an applied gauge pressure of 0–300 mmHg. Simulation parameters were fitted and found to be within the range of expected variation, given in 0f as a function of applied pressure at z = 2 cm and z = 6 cm, shown in 0f data points. The theoretical model and experimentally measured size of the phase-dip, ΔPDϕ, and in vitro system resolution as a function of interrogation distance, z, are shown in PDϕ is a function of z, resolution could be determined through ΔPDϕ when z is unknown, such as during in vivo interrogation. The predicted ΔPDϕ with an almost ideal capacitive transducer (ESR = 5 Ω) is also given, presenting a maximum z of ≈8 cm for the same noise floor limit (ΔPDϕ ≈ 0.15°). However, sensor Q would increase with a reduced ESR, permitting 0f to be more accurately measured, further reducing the minimum detectable signal and thus increasing the maximum interrogation range. Passive interrogation of the LC sensor used the system described in Canis familiaris) weighing ≈10 kilograms. Beagles are commonly used for bioequivalence studies of ingestible medication. The LC sensor was administered via mouth and radiographs taken prior to each measurement to verify LC sensor location, as seen in z = 2 cm. Sensor interrogation was completed immediately after ingestion (0f was measured and, using the in vitro characterizations relating 0f to applied pressure (PD∆ϕ to resolution (In vivo testing of the characterized LC sensor was performed on a beagle canine specimen (ngestion e and aftngestion f; the sepressure b and ∆ϕPsolution c, the co0f or response. After 26 h, the canine specimen ingested part of its bedding, causing it to regurgitate both the cloth and LC sensor, ending the in vivo experiment. The recovered LC sensor was re-tested and found to be functional, with no physical damage or change in A passive wireless pressure sensing system was evaluated for gastric manometry in order to monitor GI tract pressure and aid in the diagnosis of motility disorders. The sensor was packaged into a biocompatible 3D printed ingestible capsule measuring ø 12 mm × 24 mm, utilizing inductive coupling telemetry to passively monitor pressure. A surface micromachined capacitive pressure transducer was coupled to an 11.0 µH dual-axis inductor coil forming the resonant LC sensor. It provided a response of −0.6 kHz/mmHg over applied pressures of 0–262 mmHg with a real-time readout bandwidth of 2 Hz, maximum interrogation distance of 6 cm, and pressure resolution of up to 0.8 mmHg in vitro. The sensor was successfully deployed and interrogated in vivo, demonstrating functionality in a canine GI tract over a 26-hour residence period. While in vivo testing was performed with a small planar coil to permit ease of use during in vivo canine testing, a larger coil belted around or draped on the torso is envisioned. Performance may be improved by reducing resistive losses of the capacitive transducer, which limited interrogation distance and resolution, as well as improving interrogation hardware to reduce switching delays in order to improve readout bandwidth and reduce noise. In general, a completely passive sensing system rivaling active GI tract sensors could be realized, increasing safety and reducing cost.