Bombus terrestris before and after an artificial hibernation period of 16 weeks. Deep sequencing of 16S ribosomal RNA gene amplicons and clustering of sequence reads into operational taxonomic units (OTUs) at a similarity threshold of 97% revealed several bacterial taxa that are known to be strongly associated with corbiculate bees. Bacterial community composition after hibernation compared to before hibernation was characterized by higher OTU richness and evenness, with decreased levels of the core bacteria Gilliamella  and Snodgrassella , and increased relative abundance of non-core bacteria, including several psychrophilic and psychrotrophic taxa.Many reptiles, amphibians, mammals, and insects practice some form of hibernation during which their metabolic rate is drastically reduced. This allows them to conserve energy and survive the harsh winter conditions with little or no food. While it can be expected that a reduction in host metabolism has a substantial influence on the gut microbial community, little is known about the effects of hibernation on the composition of the microbial gut community, especially for insects. In this study, we assessed and compared the bacterial gut community composition within the midgut and ileum of indoor-reared queens of  Microorganisms are found virtually everywhere and provide numerous benefits to the environment and life on Earth ,2. SymbiIctidomys tridecemlineatus), hibernation increases the relative abundance of Bacteroidetes and Verrucomicrobia, which are particular species that are able to survive on host-derived substrates such as mucins. On the other hand, the relative abundance of Firmicutes that presumably rely on the presence of dietary polysaccharides is reduced [Ursus arctos), hibernation also leads to an increased relative abundance of Bacteroidetes, though not of Verrucomicrobia, and a decrease in Firmicutes and Actinobacteria [Many animals, including mammals, reptiles, and amphibians, hibernate when food becomes scarce, allowing them to conserve energy and survive the harsh and food-limited winter conditions . While e reduced ,15. In lbacteria .To date, little or nothing is known about the effect of hibernation on the gut microbiome of cold-blooded animals such as insects. Many insects have adapted to cold temperatures by entering diapause, which, similar to hibernation in higher animals, is a state of low metabolic activity that is associated with arrested development and increased resistance to environmental stress . A largeBombus terrestris) (n = 15) with that of their active counterparts (n = 15) using next-generation sequencing of bacterial 16S ribosomal RNA (rRNA) gene amplicons. In nature, bumblebee queens hibernate by digging themselves into the soil, from which they emerge again the following spring to form a new colony [In this study, we compared the gut microbiome from midgut to ileum in hibernating queens of buff-tailed bumblebees . Both food sources were refreshed weekly. Once the first daughter queen pupae were present, the full nest  was transferred to a bigger nest box to allow the sufficient production of new queens in a bigger space. Next, for each nest, daughter queens that had reached the age of three days were collected and put together in a separate nest box, and kept under the same conditions as mentioned above until they were six days old. This step ensured that the new queens were transferred to mating cages at the right age, without compromising the chances of getting contacts with nestmates. A timespan of three days between eclosion and separation of the queens from their nestmates has been shown to be long enough to establish stable gut microbiota through horizontal transmission, via feces secretion and social contacts [n = 15, randomly taken from the produced queens), bees from the hibernating treatment were individually placed in cardboard match boxes and subjected to an artificial hibernation period of 16 weeks at 3 °C without food and light. Immediately thereafter, 15 queens that survived hibernation  were dissected using the methodology described below.Following mating and a subsequent artificial hibernation period of three months (>80% relative humidity (RH) and 3 °C, no light) at a commercial bumblebee rearing facility , nine contacts ,22,23. SFollowing rinsing with 70% ethanol, each specimen  was pinned to a polyacrylamide gel plate and immersed in sterile Ringer’s solution. Next, the abdomen was opened by pulling the third segment outward to expose the intestines, and the midgut and ileum were collected into a vial containing one mL of a 40% glycerol solution and homogenized by using zirconia beads and a Fast-Prep24 Instrument . The dissections were conducted with the help of a binocular microscope . After every dissection, Ringer’s solution was replaced, and the gel plate was sterilized with 70% ethanol. Samples were preserved at −80 °C.−1). The reaction was initiated by denaturation at 94 °C for 120 s, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 59 °C for 45 s, and elongation at 72 °C for 45 s, followed by a final elongation at 72 °C for 10 min. Amplicons were then purified using Agencourt AMPure XP magnetic beads  according to the manufacturer’s instructions. Following quantification of the purified products using a Qubit High Sensitivity Fluorometer kit  amplicons were combined at equimolar concentrations into an amplicon library. Subsequently, the library was subjected to an ethanol precipitation and loaded on an agarose gel. Next, the band of the expected size (c. 250 bp) was excised, and the DNA was purified again, this time using the QIAquick Gel Extraction Kit . Finally, the DNA concentration was measured again, and the library was diluted to two nM and sequenced (together with a number of other samples) at the Center for Medical Genetics  using an Illumina MiSeq sequencer with a v2 500-cycle reagent kit .In order to extract DNA from the gut homogenates, the gut was crushed in a 170-µL lysozyme solution (100 mg/mL), and DNA was extracted according to Meeus et al. . A negatBombus bumblebees), identifications were refined by available information in the literature and GenBank [Sequences were received as a demultiplexed FASTQ file. Paired-end reads were merged using USEARCH (v10.0.240) to form consensus sequences  and trun GenBank ,32. Raw Nosema bombi  and the trypanosome Crithidia bombi . Previous research has shown that infection by these pathogens may be related to the gut community composition [N. bombi and C. bombi, respectively [Quantitative real-time PCR (qPCR) was used to estimate total bacterial abundance in the gut samples, as well as assess the presence and abundance of two widespread bumblebee pathogens, i.e., the microsporidian parasite position ,31,33. Tposition  were useectively . qPCR amS) was determined for each specimen by counting the number of observed OTUs, and bacterial diversity was approximated by the Shannon diversity index (H) and Pielou’s evenness (J = H/ln(S)). Shannon diversity was exponentially transformed (exp(H)), by which the variable behaves in a linear manner, in contrast to the non-transformed Shannon diversity [H), and evenness of each group of bees was determined as the average of the richness, exp(H), and evenness of the 15 bee samples, respectively, and compared between treatments by a simple t-test. Non-metric multidimensional scaling (NMDS) was used to visualize the level of similarity in community composition between the different samples based on Bray–Curtis similarities . Further, using the same distance matrix, UPGMA (unweighted pair group method with arithmetic mean) clustering was used to generate a dendrogram. Permutational multivariate analysis of variance (PERMANOVA) using the anosim function in the vegan package was performed to test for significant differences in gut microbial community composition between individuals before and after hibernation. Furthermore, β-diversity was calculated using the weighted UniFrac distance metric, and significant differences across both bee groups were again evaluated using PERMANOVA. Finally, an indicator species analysis (ISA) was performed using the Indicspecies package (v1.7-1) in R [For each sample, a rarefaction curve was constructed using the Vegan package (v2.4-6) for R . Additioiversity . The ric-1) in R  to ident-1) in R .8 and 2 × 108 gene copies before and after hibernation, respectively). None of the specimens investigated was found to be infected by the bumblebee pathogens N. bombi or C. bombi.High-throughput 16S rRNA gene sequencing and subsequent bioinformatics analysis yielded a dataset of 698,172 sequences  that could be classified into 674 bacterial OTUs . In genep < 0.05) higher for the bees after hibernation compared to before hibernation . More specifically, hibernation significantly increased the relative abundance of Acidobacteria (from 1.1% to 6.3%), Bacteroidetes (from 5.5% to 16.2%), and Firmicutes (from 8.5% to 15.7%), while a relative abundance of Proteobacteria was reduced (from 84.2% to 54.8%), especially due to lowered levels of Snodgrassella and Gilliamella  attributed to hibernating bumblebees. These were OTU5 , OTU7 , OTU9 , OTU10 , OTU11 , OTU13 , OTU18 , OTU 21 , and OTU 22 . No OTUs were significantly associated with the before hibernation treatment  and GilGamma-1” ). LikewiLacto-2” ) and LacLacto-1” ) were con = 30). All of them were found in the hibernating queens (accounting for 71.5% of the sequences); a subset of 13 OTUs was found before hibernation (90.0% of sequences)  and Gilliamella  (Snodgrassella OTU and 8.8% for Gilliamella). Besides Snodgrassella and Gilliamella, queens after hibernation mainly contained OTU5 , OTU10 , OTU7 , OTU9 (Buttiauxella sp. (Gamma-E1); 5.1%), OTU13 , and OTU11 , many of which were an indicator of OTU for hibernation and were not or are only sporadically detected in active queens  . As can ; 33.8%) . In conte queens .B. terrestris, which is one of the most common bumblebees in Europe, revealed several bacterial taxa that are known to be associated with corbiculate bees. Snodgrassella, Gilliamella, and Lactobacillus have been described as the core gut bacteria of Apis [B. terrestris [Snodgrassella  and Gilliamella  both occurred in the gut of every specimen investigated. Additionally, lactobacilli were found in almost every sample (27 out of 30 specimens). Specifically, the bee-specific lactobacilli L. bombi  and L. bombicola  were frequently found, i.e., in 19 and 18 of the 30 queens investigated, respectively. In addition, OTU194 corresponding to Lactobacillus apis  was fouBifidobacteriaceae  in the majority of B. terrestris workers investigated (22 out of 24 samples), bifidobacteria were only found in half of the specimens investigated here, with the bee-associated phylotype Bifido-3 , and another OTU corresponding to Bifidobacterium commune . While B. animalis is commonly found in the animal intestinal environment, B. commune has only recently been described as a novel species inhabiting the bumblebee gut [8 and 109 bacterial cells, respectively [Snodgrassella, Gilliamella, and the lactobacilli Firm-4/Lacto-2 and Firm-5/Lacto-1, the rectum is dominated by lactobacilli and bifidobacteria [In contrast to Meeus et al. , who fouagulans; ) as the ebee gut . Most prebee gut ,32,39. Pectively ,44. Furtbacteria ,44. TherAltogether, these results indicate that the gut microbiome of bumblebees is similar to that of honeybees and stingless bees, which are also social and contain gut communities dominated by some of the same core bacterial species as those found in bumblebees . PhylogeOur results further indicate that the hibernation of bumblebee queens leads to significantly altered gut communities that are characterized by a different bacterial community structure, higher overall diversity, and higher evenness. Hibernation significantly increased the relative abundance of Bacteroidetes (+10.7%) and Firmicutes (+7.2%), while the relative abundance of Proteobacteria drastically decreased (−29.4%). This is partially consistent with previous findings in hibernating mammals, where an increase in the relative abundance of Bacteroidetes was found, but in contrast to our results, there was also a reduction of Firmicutes ,12. In cSnodgrassella and Gilliamella, which were abundantly present before hibernation, perform worse during hibernation , thereby facilitating the growth of several other bacteria that are better adapted to thrive under the harsh conditions of hibernation . Indeed, hibernating bumblebees contained a wide diversity of bacteria, including several facultative psychrophilic (cold-loving) and psychrotrophic (cold-tolerant) bacteria [Acinetobacter, Buttiauxella, Chryseobacterium, Hafnia, Psychrobacter, and Pseudomonas . Therefore, non-hibernating queens of the same age as those investigated after a 16-week hibernation period could not be included as a control group. It also has to be noted that our experiments were performed using commercially reared bumblebees that were forced to undergo hibernation under controlled experimental conditions. We specifically used indoor-reared specimens to exclude genetic and external factors as much as possible. It remains to be investigated whether the same effects are observed in wild populations that face less sterile conditions in a fluctuating, more complex environment. Previous research has shown higher gut microbiome diversity in wild B. terrestris workers with more non-core bacteria compared to indoor-reared specimens [Due to practical limitations, we were not able to control for the factor of time (age), which also may have had an effect on gut microbiota as seen in honeybee queens . Bombus pecimens . TherefoGilliamella and Snodgrassella, and increased relative abundance of non-core bacteria, including several psychrophilic and psychrotrophic taxa. Most probably this can be explained because these core bacteria perform worse during hibernation, thereby facilitating the growth of other bacteria that are better adapted to thrive under the harsh conditions of hibernation.In conclusion, our study showed that the bacterial community composition of bumblebee queens after hibernation compared to before hibernation was characterized by higher OTU richness and evenness. Hibernation led to decreased levels of the core bacteria


Epilepsy is a common clinical syndrome characterized by sudden and recurrent attacks and temporary central nervous system dysfunction caused by excessive discharge of neurons in the brain. Amber, a fossilized organic substance formed by the resins of conifers and leguminous plants, was prescribed to tranquilize the mind in China. In this paper, the antiepileptic effect of amber was evaluated by a pentylenetetrazole (PTZ)-induced epileptic model. An untargeted metabolomics approach was applied to investigate metabolic changes in the epileptic model, which was based on HILIC-UHPLC-MS/MS multivariate statistical analysis and metabolism network analysis. The outcome of this study suggested that 35 endogenous metabolites showed marked perturbations. Moreover, four metabolism pathways were mainly involved in epilepsy. After treatment by amber, the endogenous metabolites had a marked tendency to revert back to the situation of the control group which was consistent with phenobarbital. This study characterized the pentylenetetrazole-induced epileptic model and provided new evidence for the sedative effect of amber. Epilepsy is a common clinical syndrome characterized by sudden and recurrent attacks and temporary central nervous system dysfunction caused by excessive discharge of neurons in the brain. This can result from a variety of endogenous or exogenous factors, often occurring months or years after a sudden injury . A seizu2+-signal transduction inhibitors [2+-signal transduction is involved in an allergy, 3-cler-oden-15-oic acid from Dominican amber can inhibit Ca2+-influx [Traditional Chinese medicine (TCM) has accumulated ambient experiences in treating epilepsy and mineral Chinese Materia Medica  has a long history of use for mind-tranquilizing with significant effects . Amber ihibitors ,18. When+-influx ,20. Howe1H-NMR spectroscopy [Metabolomics is a top–down systems biology approach which is the systematic study of the special chemical fingerprints left by specific cellular processes and the overall study of their small-molecule metabolites . Analytitroscopy ,23 and mtroscopy ,25,26 , and the Racine score of epilepsy reached 4 or 5. As shown in p < 0.01, Under the intervention of amber, the incubation period was significantly prolonged, and the level of seizures was reduced as well  for different groups are shown in + and ESI− modes, are shown in + mode and ESI− mode are shown in +) and 4B (ESI−). The QC samples are clustered, indicating good reproducibility of the data. Representative HILIC-UHPLC-ORBITRAP-MS cortex metabolic profiles from a control and a PTZ-treated animal, in ESIPCA analysis in positive and negative TIC was used to evaluate the PTZ-induced epileptic model. In the positive ion mode, a model with two principal components was obtained  while three principal components were obtained in negative mode . PCA scores showed that all samples in the positive ion mode were distributed in the 95% confidence interval ellipse while one sample was distributed out of the ellipse in negative mode. As shown in the PCA scores scatter plot, significant separation between the three groups in the unsupervised mode could be observed, indicating the difference in the metabolites between the different groups.n = 200) on the left are lower than the points on the right  model  showing significant changes in the model group compared to the control group were taken as candidate biomarkers [In this study, loading S-plots  and the omarkers . For thoPathway analysis of the discriminating metabolites was performed with MetaboAnalyst 4.0, a web-based tool for pathway analysis and visualization of metabolomics . As showThe variation of phosphorylcholine, choline, acetylcholine, phosphatidylcholine (PC) and lysophosphatidylcholine (LysoPC) in the cortex could potentially indicate that the glycerophospholipid metabolism was disrupted and played a major role in seizures. Increased choline may reflect myelin breakdown, increased cell density, or gliosis, which may indicate Alzheimer’s disease or epilepsy. Phosphatidylcholine is the main component of the cell membrane and usually exists on the surface of the ectoplasmic membrane. Excitotoxic events enhance the hydrolysis of phosphatidylcholine in the brain, which was evidenced caused by a concomitant increase in the levels of choline and free fatty acids .The variation of gamma-aminobutyric acid (GABA) in the cortex could potentially reflect that the metabolism of alanine, aspartate and glutamate is disrupted, which plays a major role in seizures. GABA, a key inhibitory neurotransmitter, is synthesized through the decarboxylation of glutamate via alanine, aspartate and glutamate metabolism . StudiesAcetonitrile, methanol, alcohol, acetone and formic acid (HPLC grade) were purchased from Merck Company Inc. ; HPLC grade water was produced by a Direct-Q3 Ultrapure Water System from Millipore . Other reagents and chemicals were of analytical grade.Amber samples were purchased from Shaanxi Science Pharmaceutical Co. Ltd. They were identified by Dr. Hui Yan . Pentylenetetrazol (PTZ), Phenobarbital (PB), and sodium chloride injections were from Macklin , Shanxi Yunpeng Pharmaceutical Co. Ltd. , and Chenxin Pharmaceutical Co. Ltd , respectively.Specific Pathogen-Free (SPF) male ICR mice were purchased from the Experimental Animal Center of Qinglongshan . All the mice were kept in the Specific Pathogen Free Center of Nanjing University of Chinese Medicine, Nanjing, China. All animal studies were in accordance with the guidelines of the Animal Ethics Committee of Nanjing University of Chinese Medicine (201810A016).After an initial acclimation period of 7 days in cages, 40 mice were randomly allocated into 4 groups: Control, model , PB , and amber .After the intraperitoneal injection of pentylenetetrazole, observation of the seizures lasted for 30 min, and the severity, latency and duration of seizures were recorded. The degree of epileptic behavior was divided into 6 grades according to the Racine standard of neurology: Level 0, no response; Level I, ear and facial twitch; Level II, myoclonus, but no upright position; Level III, myoclonus, with axial position; Level IV, systemic tonic-clonic seizure; and Level V, systemic tonic-clonic seizure and loss of postural control .The mice were sacrificed after the observation. Three brains of each group were taken and immersed in 4% paraformaldehyde, and paraffin sections of coronal plane were used for Nissl staining. The rest of the brain was divided into cortex and hippocampus, and kept in liquid nitrogen. After thawing in the fridge at 4 °C, 40 mg of the cortex sample was precisely weighed and used for the following process: 160 μL of extract solution, vortexed for 1 min, sonicated for 5 min in an ice bath, centrifuged for 10 min under 13,000 rpm at 4 °C, and then 100 μL of supernatant was collected and concentrated to dry. The extract was then reconstituted with 40 μL of mobile phase, vortexed for 1 min, sonicated for 5 min in ice bath and centrifuged for 10 min under 13,000 rpm at 4 °C. The supernatant was then finally collected for analysis. Quality control (QC) samples were prepared by pooling aliquots (2 μL) of each sample.m/z 100 to 1000 in profile mode. External calibration was carried out with a standard LTQ calibration mixture . The following settings were used for MS detection: vaporizer temperature, 280 °C; sheath and auxiliary gases, 35 and 15 (arbitrary units); spray voltage, 3.5 kV(ESI+), 2.5 kV(ESI−); capillary temperature, 350 °C; capillary voltage, 10 V; tube-lens voltage, 120 V; maximum injection time, 1000 ms; maximum number of ions collected for each scan, 5 × 105; mass resolution, 30,000; MS/MS mode, high-energy induced dissociation (HCD); Collision gas, N2.An LTQ–Orbitrap Velos pro-mass spectrometer (Thermo Scientific) equipped with an ESI source was set to collect data from For LC separation, UHPLC Dionex Ultimate 3000  and an ACQUITYTM UPLC BEH Amide column  were used. Water and acetonitrile modified with 5 mM ammonium formate, 5 mM ammonium acetate, and 0.1% formic acid were used as mobile phase A and B, respectively. The column was eluted with a program as follows: The percentage of B was decreased from 95% to 55% at the first 13 min, and then held for 2 min. The flow rate and injection volume were set at 0.4 mL/min and 2 μL, respectively.The experimental data were analyzed by R and Compound Discoverer 2.1 software , including peak extraction, peak alignment, background deduction and compound identification. After removing exogenous component interference, the extracted ion fragment peak area was normalized by SIMCA 14.1 software , and multivariate statistical analysis was conducted after standardization. The mean-centering method and pareto-scaling method were used to transform the data, and the importance of low-abundance ions was increased, while the noise was not obviously amplified. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used for classification. The s-plot was generated to discover the significant components between groups as potential markers.The potential endogenous biomarkers were identified based on accurate molecular mass, MS/MS fragments, and retention behavior by searching online databases. The potential markers were identified within 5 ppm. Moreover, the MS/MS spectrum match was searched in the METLIN database. In this study, the Compound Discoverer  was used to search KEGG, HMDB and LIPID MAPS database. Metabolism pathway analysis was performed with MetaboAnalyst 4.0 , a web-based tool for pathway analysis and visualization metabolomics.Metabolomics, as a systematic method, could systematically identify, quantify or reveal the metabolites of diseases, provide a basis for the diagnosis, biomarkers and/or monitoring tools of diseases, and provide potential targets for the treatment and prevention of diseases. In this study, the metabolic changes and potential biomarkers of epileptic models were studied by using the metabolomics method of LC-MS technology and metabolic network analysis. After intervention of amber, the incubation period was significantly prolonged, and the level of seizures was reduced as well. The damage to the cortex and hippocampal neuron cells was reversed, the fluctuating composition metabolites had a marked tendency to revert back to the control group which was consistent with phenobarbital. This study characterized the PTZ-induced epileptic model and provided new evidences for amber sedative effect. Our work enhanced not only the understanding of the pathology of epilepsy, but also revealed that amber could effectively inhibit seizures with a similar mechanism to that of phenobarbital.


This study examined the effects of different intravaginal device types used for estrous cycle management in sheep, and the timing of their insertion, on vaginal features  and fertility under field conditions.Salmonella spp. and Staphylococcus aureus. On the other hand, independently of the length of the treatment, only around 15–20% of the sheep treated with CIDRs evidenced vaginal discharge (p < 0.00005 when compared to sponge groups), and such discharge was scarce, clear, and showed no changes in vaginal pH and microbiota when compared to control sheep. Fertility yields were associated with vaginal features, being higher in both short-term treatments (75%) and the long-term CIDR-based treatment (70%) than in the long-term sponge-based treatment (45%).Induction and synchronization of estrus and ovulation in sheep is based on intravaginal progestagen-impregnated polyurethane sponges or progesterone-loaded silicon-based devices (CIDR), in either short- (6–7 days) or long-term (12–14 days) protocols. Bearing in mind that the use of intravaginal sponges in long-term protocols has been related to the presence of vaginitis at removal, we compared the effects of sponges and CIDRs, maintained during either 7 or 14 days, on vaginal features  and fertility under field conditions. Almost all the ewes treated with intravaginal sponges showed vaginal discharge at device withdrawal, which was purulent and/or bloody in around 15% and 80% of the females treated for 7 and 14 days, respectively. The vaginal pH and microbiota changed in both groups when compared to control sheep, especially in ewes treated for 14 days, which showed a pH value around 8 and a higher incidence of  Induction and synchronization of estrus and ovulation for reproductive management of sheep are traditionally based on the intravaginal insertion of progestagen-impregnated polyurethane sponges for 12–14 days, followed by the intramuscular injection of equine chorionic gonadotrophin (eCG) at sponge removal , which m® , which has been recently authorized for commercialization in the European Union. The CIDR is a Y-shaped silicon-based device and, due to its design and composition, which allows the drainage of any secretion, the rates of device loss, adherence and vaginitis are much lower than for sponges [A possible alternative for minimizing the physical effects of the intravaginal sponge is the use of a different intravaginal device, named CIDR sponges . HoweverIn recent years, regardless of the intravaginal device used, the protocols for induction and synchronization of ovulations are evolving to shorter treatments, around 6–7 days of treatment with the intravaginal device ,9,10. ThHence, bearing in mind the future availability of CIDR in the European Union and looking for a complete picture of the scenario, the objective of the present experiment was to compare vaginal features  and fertility among non-treated control ewes and ewes treated with different devices (sponge or CIDR) and treatment lengths (14 or 7 days). Our hypothesis was that the type of intravaginal device and the duration of the treatment would affect physical characteristics and microbiota of vaginal mucus, also affecting fertility afterwards.The study was performed during the breeding season (March) on a single commercial farm , according to the Spanish Policy for Animal Protection RD53/2013, which complies with the European Union Directive 2010/63/UE about the protection of animals used for research. The study protocol was reviewed by the Committee of Ethics in Animal Research of the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and no ethical implications were observed.n = 20 each one), based on the intravaginal device type  and treatment length  used for synchronization of ovarian activity, estrus and ovulation. Hence, 40 sheep were treated with one progestagen-impregnated sponge  for either fourteen  or seven days , while 40 sheep were treated with one progesterone-loaded CIDR  for either fourteen  or seven days , and 20 ewes remained as untreated controls (group CON-0). At intravaginal device removal, groups SP and CIDR received one intramuscular injection of 400 IU of eCG , while short-term treatments (groups SP-7 and CIDR-7) also received an intramuscular injection of 5 mg of prostaglandin F2α . The experimental group consisted of 100 Lacaune multiparous sheep (2 to 5 years old), housed indoors with outdoor access. All the animals were monitored for adequate health status and specific pathogens, and clinical examinations were performed daily during the period of insertion of the devices by the farm’s veterinarian; no signs of disease were found in any of the animals. The experimental group remained together but, for experimental purposes, it was divided into five equal subgroups  were scored , performed at Days 38 and 67 after device withdrawal for assessing induced and return estrus, and confirmed at lambing.Staphylococcus aureus was determined by using Baird Parker agar and incubation at 37 °C for 24 h. Typical colonies were confirmed by coagulase test [Escherichia coli and Klebsiella spp. was determined using violet red bile glucose agar plates, which were incubated at 37 °C for 48 h [E. coli isolation, and to MacConkey agar for Klebsiella spp. isolation, incubating at 37 °C for 48 h [Aeromonas spp., incubating at 37 °C for 48 h [Shigella spp. and Salmonella spp. were cultured on xylose lysine deoxicholate agar (XLD agar) and incubated at 37 °C for 24 h [Swabs were used to plate samples on different selective media , in order to determine the presence or absence of several bacteria. The presence of ase test . The prefor 48 h . Typicalfor 48 h . MacConkfor 48 h , while Sfor 24 h . In all ® 22.0 . The effects of the intravaginal device type  and the treatment length  on vaginal features at device removal  and subsequent fertility were assessed by analyses of variance (ANOVA). Statistical analysis of results expressed as percentages was performed after arcsine transformation of the values for each individual percentage. All results are expressed as mean ± S.E.M. and the statistical significance was accepted at p < 0.05, with p < 0.1 being considered a trend.Statistical analysis was performed using SPSSp < 0.00005 for the comparisons among SP and CIDR groups). The discharge was scarce, clear and mucous in females treated with CIDR; conversely, 10.5 and 83.3% of the females in the groups SP-7 and SP-14, respectively, showed purulent and/or bloody vaginal discharges .At removal of the intravaginal device, almost all ewes in the groups SP-7 and SP-14  showed vaginal discharge ; a percep > 0.818). Conversely, the value for pH increased in both SP groups, with significant differences when assessing the effect of duration of the treatment. Hence, SP-7, with a pH of 7.2 ± 0.1, had a trend for higher values than CON-0 (p = 0.07), CIDR-7 (p < 0.05) and CIDR-14 (p = 0.05), while the differences were highly significant when assessing the SP-14 group, with a pH of 8.0 ± 0.2, against SP-7 (p < 0.001) and CON-0, CIDR-7 and CIDR-14 .Assessment of vaginal pH at device removal also showed significant differences among treatments. The values were similar, around 6.8–6.9, in the controls CON-0 and the CIDR groups , and the Gram positive (G+) S. aureus (68.4% of the ewes). Assessment of the animals with intravaginal treatments showed similar prevalence for E. coli. However, all the treated groups showed appearance of the G– Salmonella spp. , although differences only showed a trend for SP groups when compared to CON-0 (p = 0.07). The SP-14 group also showed an increase in the appearance of S. aureus, Shigella spp. and the G– Klebsiella spp. ; however, there was only statistical significance in the case of S. aureus (p < 0.05). Finally, the CIDR groups showed a significantly decreased presence of Aeromonas spp. .The prevailing bacteria  in the Cp < 0.05 when compared with all the other groups). Moreover, it was detected that 15% of the sheep in the group SP-14 resulted pregnant after displaying a short estrous cycle in response to the treatment; a finding which was not identified in the other groups. As shown in p < 0.05 when compared with all the other groups).Assessment of fertility at induced estrus also showed significant differences among treatments . PregnanThe results of the present study indicate that both the type of intravaginal device (sponge or CIDR) and the duration of the progestative treatment (14 or 7 days) may affect characteristics of vaginal mucus discharge, pH and bacteriology, and fertility.Almost all the ewes treated with intravaginal sponges showed vaginal discharge at device withdrawal. The characteristics of such vaginal discharge were different depending on the duration of the sponge insertion. Around 80% of the sheep treated with classical long-term treatments (14-day) showed purulent and/or bloody vaginal discharges. These results reinforce previous studies reporting that long-term progestogen treatments are associated with soaking of the polyurethane sponge, retention of vaginal secretions and abnormal discharges at sponge withdrawal . HemorrhThe assessment of vaginal discharges at device removal showed that only around 15–20% of the sheep treated with CIDRs had a discharge. In all the cases, such discharges were scarce, clear and mucous. Hence, in agreement with previous studies , the desSalmonella spp. and especially Staphylococcus aureus. Both Salmonella spp. and S. aureus have been previously related to aerobic vaginitis causing increased inflammatory immune response [S. aureus is considered an inhabitant of vulva and vagina and identified as the most common causal agent of purulent vaginitis in ewes [E. coli, probably of fecal source, which are opportunistic agents of bacterial vaginitis [Previous studies have also shown that abnormal vaginal discharges after long-term progestogen treatments are the result of inflammatory and infectious processes caused by changes in the vaginal microbiota ,6. Althoresponse ,18 and, response ,20. Part in ewes , which iaginitis .Aeromonas spp. This is a surprising finding and we have actually not found a possible explanation for this change in the vaginal microbiota of sheep treated with CIDRs. A possible hypothesis may be based in the facts that Aeromonas spp. are ubiquitous in water and usually are associated with infections by exposure to contaminated water, while exposition to progesterone may decrease the water content of vaginal mucus by approximately 90% [On the other hand, in the present study, we have to highlight that the groups treated with CIDRs, either for 7 or 14 days, showed a significantly decreased presence of tely 90% .A possible option to avoid vaginitis, and further changes in vaginal features and fertility, in ewes treated with polyurethane sponges would be the use of an adequate prophylactic management that can include the disinfection of the vulvar area and all the material used for the application of the devices, and even the use of local and/or systemic antibiotics ,23. HoweThe changes in the vaginal secretion and microbiota found in sponge-treated sheep may be related to changes in the vaginal pH at device removal. Controls and ewes treated with CIDRs, either with short- or long-term protocols, showed near-neutral pH similarly to that previously described for non-treated sheep . The neuOur results for fertility are consistent with data for vaginal discharge characteristics. Fertility yields were similar among sheep treated with CIDR, in both short- and long-term protocols, and sheep treated with sponges in short-term protocols, in agreement with previous studies ,27,28. TS. aureus and a decrease in fertility. On the other hand, only around 15–20% of the sheep treated with CIDRs, independently of the length of the treatment, evidenced vaginal discharge , and both CIDR groups showed a vaginal pH and microbiota similar to control sheep.Almost all the ewes treated with intravaginal sponges showed vaginal discharge at device withdrawal, which was purulent and/or bloody in around 80% of the females treated for 14 days and around 15% of the females treated for 7 days. The vaginal pH and microbiota changed in both groups when compared to control ewes, especially in the long-term protocol, where pH was around 8 and the ewes showed a higher incidence of


A diet of precooked noodles (PN), as widely produced and consumed in Asia, was used to simulate an actual food context. Assays were conducted with male Wistar rats (n = 80); the rats were divided into five groups and fed with either PN (control), PN supplemented with medium-dose KGM (MK), precooked low-dose KGM-supplemented noodles (LKD), precooked medium-dose KGM-supplemented noodles (MKD) or precooked high-dose KGM supplemented noodles (HKD). The time-dependent changes in blood glucose and the sensitivity to insulin after intragastric administration were determined to evaluate the postprandial glycemic response. The activity of intestinal Na+-K+-ATPase and the levels of gut hormones including motilin, cholecystokin, GLP-1, and orexin were also determined to provide insights into the function of gastrointestinal motion and after-meal hormonal feedback in each group. The noodles-based KGM showed much more efficacy in sustaining glucose homeostasis compared with KGM supplemented in a diet of noodles, indicating there might be potential long-term health outcomes of satiety and energy balance using noodles-based KGM. The postprandial glycemia was largely moderated by LKD and MKD. Despite the significant reduction in the production of glucose, MKD caused insensitivity to insulin–blood glucose regulation and a rapid gut negative feedback following a severe blood glucose fluctuation. In conclusion, the health-promoting benefits of KGM supplements on glycemic response highly depend on the type of matrix and the dose of KGM.This study was conducted to investigate the hypothesis that the beneficial metabolic effects of dietary fiber, konjac glucomannan (KGM), related with  Konjac glucomannan is a soluble dietary fiber, which has been proven to be effective in lowering postprandial glycemic response after intake as a food supplement.This study investigated whether konjac glucomannan in a noodles-based food matrix has a beneficial effect on metabolism related with digestion.KGM inhibited in vitro enzymatic hydrolysis of wheat starch and wheat gluten nearly dose-dependently.KGM slowed the rise of postprandial blood glucose and increased satiety by affecting intestinal metabolism of rats.The glycemic index (GI) is associated with the postprandial blood glucose response of ingestion of a food with available carbohydrates. Intake of refined carbohydrates will raise postprandial blood glucose rapidly and even presents a risk factor associated with diseases including diabetes. Commonly eaten white noodles are readily digestible and their GI is really high (70 and above), whereas low-GI foods can control the release of glycosylated hemoglobin and fructosamine which lead to more stable postprandial blood glucose .in vivo postprandial blood glucose response. Accumulating evidence suggested that certain food contexts, through a variety of mechanisms, might alter the pattern of intestinal glucose uptake. The digestibility of dietary fiber extruded rice starch is remarkably lower than that of natural rice, and the GI decreases when the dietary fiber content is >6%  insulin radioimmunoassay kit (KJEIA0041D), motilin radioimmunoassay kit (KDEIA0087), and cholecystokinin radioimmunoassay kit (KDEIA0091) were purchased from Kangjia Hongyuan Biotechnology Co., Ltd . Ultramicro assay kit for Na+-K+-ATPase (A016-1) was purchased from Nanjing Jiancheng Biology Engineering Institute . Ghrelin ELISA kit (EK-031-31), glucagon-like peptide-1 ELISA kit (EK-028-11), and orexin A radioimmunoassay kit (EKE-003-30) were purchased from Phoenix Pharmaceuticals Inc. (USA). All the other chemicals and reagents were of analytical grade.Konjac glucomannan (KGM) was kindly provided by Shiyan Huaxianzi Konjac Products Co., Ltd. . The contents of moisture, ash, glucomannan, crude protein, and fat were 3.7 g/100 g, 0.8 g/100 g, 87.8 g/100 g, 0.6 g/100 g, and 0.1 g/100 g, respectively. Wheat flour  was used throughout the experiment, and the contents of moisture, ash, crude protein, starch, fat, and total dietary fiber were 12.83 g/100 g, 0.89 g/100 g, 11.2 g/100 g, 73.5 g/100 g, 1.6 g/100 g, and 1.21 g/100 g, respectively. The contents of moisture, ash, crude protein, amylose, and amylopectin in starch were 11.5 g/100 g, 0.89 g/100 g, <0.3 g/100 g, 17.78 g/100 g, and 66.1 g/100 g, respectively. The contents of moisture, ash, and protein in gluten were 6.34 g/100 g, 0.89 g/100 g, and 87.2 g/100 g respectively. Glucose determination kit (Ld 60201) was purchased from Laibang Biotechnology Co., Ltd . Iodine  were purchased from Huafukang Biotech  and raised in a specific pathogen-free (SPF) environment with free access to feed and water. After a week of adjustable feeding, 80 rats were randomly divided into five groups: Control, MK, LKD, MKD, and HKD. Rats were fasted for 12 h before orbital sinus blood sampling, and these samples were immediately freezed. Treated noodles containing different dose of KGM were suspended into water (0.1 g/mL), and the rats received 2 mL of the suspensions per 100 g of weight by gavage. After 15, 30, 60, and 120 min of gavage treatment, 4 rats were randomly selected and their blood samples were taken by puncturing the orbital sinus while rats were under diethyl-ether anesthesia. Anticoagulant (disodium hydrochloride solution) was added to the plasma, and the rest of the plasma was placed at −80°C for isolating serum.The blood glucose was determined by glucose determination kit; 10 μL of serum was mixed with 1 mL of R1 reagent. The absorbance at 505 nm was recorded to calculate the blood glucose content.125I) insulin radioimmunoassay kit; 200 μL of serum was mixed with the same volume of marker and antibody. The mixture was incubated at 4°C for 18 h; 1,000 μL of separating reagent was added and thoroughly mixed, followed by incubation in 4°C for 15 min and centrifugation at 3,600 rpm for 20 min. The supernatant was abandoned, and the cpm value of the sediment was measured to calculate the insulin level.Insulin level was determined using iodine  was determined by radioimmunoassay kit; 300 μL of plasma was thoroughly mixed with 100 μL of antiserum. After incubation at 4°C for 24 h, 100 μL of Ghrelin concentration was determined according to the Protocol for Catalog # EK-031-31 Ghrelin  EIA Kit  with no modification.GLP-1 concentration was determined according to the Protocol for Catalog # EK-028-11 GLP-1 -36-AmideOrexin A was determined according to the Orexin A  Extraction Free EIA Kit Protocol  with no modification.The results were statistically analyzed using SPSS . Analysis of variance (ANOVA) was used to determine significant differences between the results, and Duncan’s test was used to compare the means with a significance difference at the level of 0.05.in vitro digestibility of 1% KGM was significantly higher than those of the other samples. The differences in the inhibitory capacity on starch digestibility proved that the concentration of KGM is important for enzymatic hydrolysis, which could be explained in two aspects: firstly, in such a heterogeneous system, KGM molecules tended to be attached to the surface of starch particles, forming a barrier-like layer on the periphery, which blocked the contacts between enzymes and starch, especially the binding sites of enzymes and starch molecules. Besides, as the proportion of KGM increases, the thickening effects of KGM might limit mass transfer and further inhibit starch swelling . The enzymolysis of control began to stabilize after 150 min. However, the enzymolysis of 1% KGM and 5% KGM groups was not complete until 180 min, while the enzymolysis of 3% KGM group was still in progress. There is no significant difference in the digestibility of protein for either control or KGM groups before 120 min. However, the rate of wheat gluten digestion in the control group was strongly up-regulated at the stage of trypsin enzymolysis. To some extent, the degradation of gluten protein by trypsin leads to the appearance of pores, partial network destruction, and the increase in pore size, which leads to the increase in digestion rate when entering the digestion stage of trypsin. A highly entangled network structure of indigestible KGM could be formed around the gluten , but there was no significant difference between these two groups at 60 and 120 min after feeding, suggesting how KGM was incorporated in a diet might be also important for functioning.The secretion of insulin is closely related to the level of blood glucose response, and both of them can be used to indicate the extent of digestion and absorption of food. As shown in Fluctuations in blood glucose level impact the secretion of insulin, and the blood glucose level is regulated by insulin in turn. Insulin is the only known hormone in the body that lowers the blood glucose level. The sensitivity of the body to insulin is another important factor in stabilizing postprandial blood glucose. in vitro experiments have shown that in a KGM-thickened system, starch enzymolysis can be reduced, the efficacy of soluble dietary fibers to attenuate the blood glucose level in complex mixing conditions such as the small intestine may not be simply attributed to changes in intestinal digestive viscosity , indicating that the type of matrix where KGM was incorporated is critical to regulate the activity of Na+-K+-ATPase. It was demonstrated that the reduction of nutrient absorption capacity of KGM is related to its matrix. The activity of Na+-K+-ATPase would be lowered to accommodate these changes in the amount of small molecule nutrients needed to be transferred. Partial degradation of KGM in colon could produce short-chain fatty acids, decrease intestinal pH, and promote the passive regulation of Na+-K+-ATPase (Na+-ATPase .in vivo study of dietary from different food matrixes at the same concentration showed that intake of dietary fibers coming from less-processed foods resulted in higher plasma CCK level, that is, β-glucan from oat bran was more capable than that from oat flour and oatmeal to enhance the CCK level (Cholecystokinin is the most extensively studied gastrointestinal satiety hormone secreted by the I-cells that line the intestinal tract. The main role of CCK is to promote the secretion of various digestive enzymes from the pancreatic acinus and enhance the release of GLP-1 to reduce food intake . In Fig.CK level .Ghrelin, a gastrointestinal hormone synthesized and secreted primarily from the fundic mucosa of the stomach, appears to be an orexigenic signal which would increase in state of hunger or food deprivation. GLP-1 is a neuroendocrine polypeptide synthesized and secreted by enteroendocrine ‘L’ cells located within the ileum of the small intestine and colon. The biological activities of GLP-1 include stimulating the secretion and synthesis of insulin and enhancing insulin sensitivity, which are important for maintaining homeostasis in human body . Figure Orexin is a neuropeptide substance, which plays a physiological role in regulating appetite, glycolipid metabolism, and energy utilization through complex central and peripheral tissues . At 30 mIn vitro enzymatic hydrolysis of wheat starch and wheat gluten was nearly dose-dependent on KGM concentration, but the interaction between KGM and two wheat components certainly interferes the hydrolysis probably by its thickening and steric hindrance effects. In vivo assays could reveal a correlation between blood glucose elevation, insulin secretion and gastrointestinal hormone release under such a precooked noodles-based KGM system besides the digestive enzymes-induced enzymatic reaction. This study indicated that the beneficial metabolic effects of KGM related with digestion were not similar within different food matrixes. There are wild fluctuations of glucose in the blood of MK. This can eventually lead to low insulin sensitivity and increased risk of obesity and diabetes. Even at the same dose of KGM, MKD was more desirable in quickly satisfying the appetite of rats, enhancing satiety and promoting the effect of insulin to maintain stable blood glucose. HKD group tended to maintain satiety at the time when the digestion process has been basically completed. In conclusion, metabolism is extremely complicated and varies with the type of food matrix. In the analysis of the impacts of KGM on digestion, it is necessary to consider the combined effects of food pattern on various indicators including enzymatic reaction, gastrointestinal motility, and hormones. Feeding of KGM incorporated in staple foods like noodles is better than the supplementation in reducing food intake and weight control.


This paper focuses on extending drug release duration from contact lenses by incorporating catanionic aggregates. The aggregates consist of a long-chain cationic surfactant, i.e., cetalkonium chloride (CKC), and an oppositely charged anti-inflammatory amphiphilic drug. We studied three non-steroidal anti-inflammatory (NSAID) drugs with different octanol–water partition coefficients; diclofenac sodium (DFNa), flurbiprofen sodium (FBNa), and naproxen sodium (NPNa). Confirmation of catanionic aggregate formation in solution was determined by steady and dynamic shear rheology measurements. We observed the increased viscosity, shear thinning, and viscoelastic behavior characteristic of wormlike micelles; the rheological data are reasonably well described using a Maxwellian fluid model with a single relaxation time. In vitro release experiments demonstrated that the extension in the drug release time is dependent on the ability of a drug to form viscoelastic catanionic aggregates. Such aggregates retard the diffusive transport of drug molecules from the contact lenses. Our study revealed that the release kinetics depends on the CKC concentration and the alkyl chain length of the cationic surfactant. We demonstrated that more hydrophobic drugs such as diclofenac sodium show a more extended release than less hydrophobic drugs such as naproxen sodium. Ocular drug delivery is a challenging research field due to the presence of anatomical and physiological barriers that affect the drug bioavailability following multiple routes of administration, including topical, systemic, and injectable . Most ocE hydrophobic coating [In order to address the limitations of eye drops, researchers have explored the use of therapeutic contact lenses ,6. When  coating ,18,19,20 coating . A limit coating  and the  coating .The use of catanionic aggregates formed from drug compounds with oppositely-charged surfactants has received particular attention for drug delivery applications ,31,32,33Researchers have used micelles made of non-ionic surfactants in contact lenses to extend the delivery time of hydrophobic drugs such as cyclosporine A ,36,37, dIn this paper, we report the preparation of aggregates composed of a cationic surfactant and an anionic drug for the purpose of extending drug delivery from poly-hydroxy-ethyl-methacrylate (pHEMA) contact lenses. We study non-steroidal anti-inflammatory drugs (NSAIDs), a class of drugs that is used for ocular inflammation and pain relief . Due to iso-butrylonitrile (AIBN), benzyl-dimethyl-octyl-ammonium chloride (C8), and Dulbecco’s phosphate buffer saline (PBS) were purchased from Millipore-Sigma . Flurbiprofen sodium (FBNa) reference standard was purchased directly from the United States Pharmacopeia . All chemicals were used as received.Diclofenac sodium (DFNa), naproxen sodium (NPNa), Brij 97 (B97), cetalkonium chloride (CKC), 2-hydroxyethyl methacrylate (HEMA), ethylene glycol dimethacrylate (EGDMA), azobis-We obtained homogeneous surfactant solutions by mixing 2 g of Brij 97 in 10 mL of deionized water with 0, 125, 250, 500, 750, or 1000 mg of CKC. The amounts of CKC correspond to concentrations of 0, 25, 50, 100, 150, and 200 mM CKC, respectively. Because CKC has limited water solubility, a non-ionic surfactant was used to increase its aqueous solubility. We stirred each solution at 800 rpm at room temperature until the CKC was completely dissolved. Next, 50 mg of drug (DFNa or NPNa) was dissolved with 5 mL of the prepared surfactant solution. For FBNa, 25 mg was used instead of 50 mg. Drugs were dissolved for 20 min at 600 rpm at room temperature.Steady and dynamic shear rate rheological experiments were performed on an AR2000 controlled stress rheometer  using cone-and-plate geometry . Dynamic frequency spectra were conducted in the linear viscoelastic regime of the solutions, as determined from dynamic stress sweep measurements.We fabricated the contact lenses by thermally curing the monomer solutions in polypropylene lens molds. The HEMA monomer (2 mL) was mixed with 4.5 mg AIBN as thermal polymerization initiator, 7.5 μL EGDMA as crosslinker, and 1.5 g drug–surfactant solution. This solution was stirred at 300 rpm at room temperature for 10 min and three drops (~75 μL) of the mixed solution was poured into the contact lens mold . After curing at 70 °C for 24 h and demolding, the contact lenses were thoroughly washed in deionized water for 1 h to remove unreacted monomers.Each contact lens was immersed in 3 mL of PBS at pH 7.4 and room temperature. At predetermined time intervals, 1 mL aliquots were pipetted out and replaced by 1 mL fresh PBS. The drug concentration was determined using a ultraviolet (UV)-visible spectrophotometer  at wavelengths of 276 nm for DFNa, 248 nm for FBNa, and 271 nm for NPNa. Control contact lenses  were tested in the in vitro release experiments to ensure that there were no interfering absorbance readings.To determine the total amount of drug in the contact lenses, we immersed each lens in 3 mL of ethanol for 24 h to extract all of the drug. The total amount of drug per each lens was quantified using a UV-visible spectrophotometer (Varian Cary 50 Bio) with pre-established calibration curves. After the extraction, lenses were discarded and not used in subsequent experiments.The optical transparency was measured using a UV-visible spectrophotometer  from 380 to 740 nm wavelength range. Contact lenses were hydrated overnight by soaking in PBS at pH 7.4, and they were placed in a 24-well plate filled with 1 mL of PBS to measure transmittance.The water content of contact lenses was conducted by immersion of each contact lens in 2 mL of deionized water at room temperature for 24 h. The contact lens was removed from the solution, gently blotted to remove all the liquid from the surface, and wet lens weight was recorded. Dry weights were measured after removing the contact lenses from the hydrating solution and letting them dry at room temperature overnight. The following equation was used to calculate the % water content of contact lenses:−1. For concentrations of 25 mM CKC or higher, shear thinning behavior with a plateau in the viscosity at low shear rates is observed for all systems. Furthermore, unlike CKC, C8 is unable to aggregate with DFNa even at high concentrations, as demonstrated by its relatively low solution viscosity. As shown in In addition, from −1. However, 100 mM and 200 mM CKC solutions with NPNa exhibit a very weak shear thinning behavior. From −1, to 1 Pa.s at a shear rate of 100 s−1. For DFNa at 100 mM CKC  with no drug, no shear thinning behavior is observed, as shown in P) of 4.75, while NPNa has a log P of 3.39 , the response is elastic, whereas for time scales longer than Rt , the response is viscous. This time-dependent viscoelastic response has been reported to be typical of wormlike micelles [The dynamic rheological response of catanionic aggregates is presented in ion time . At timemicelles ,48.G′) and viscous (G″) moduli are described by the following equations [As shown in quations : (2)G′η0=GpRt and plateau modulus pG are 0.1 s and 150 Pa, respectively. The predicted zero-shear viscosity (based on Equation (4)) and measured zero-shear viscosity for DFNa solution at 75 mM CKC are 15 Pa.s and 32 Pa.s, respectively. The measured zero-shear viscosity for NPNa solution is 2 Pa.s (Rt and plateau modulus pG for NPNa solution are 0.01 s and 200 Pa for NPNa (The corresponding Maxwellian curves from Equations (2) and (3) are plotted as solid lines in s 2 Pa.s b. Similafor NPNa b, which G″ data depart from the Maxwell model at high frequencies. This discrepancy is known to be linked with the presence of wormlike micelles in the solution, because micelles are dynamic entities that break and recombine rapidly [In summary, the dynamic responses of these catanionic aggregates are predicted fairly well by the Maxwell fluid model with a single relaxation time, which is a signature of systems containing wormlike micelles ,47,50,51 rapidly ,47,51.Here, we study two different derivatives of benzalkonium chloride (BKC): CKC and benzyl-di-methyl-octyl-ammonium chloride (C8). CKC is a 16-carbon-unit alkyl benzalkonium chloride derivative, while C8 is an 8-carbon-unit alkyl benzalkonium chloride derivative. BKC is an eye drop preservative used in approximately 70% of commercially available topical ophthalmic formulations . Figure In drug-containing catanionic aggregates, cationic surfactants interact with anionic-charged drugs due to both electrostatic and hydrophobic interactions ,55,56,57P) has been a conventional parameter to study the lipophilic character of drug compounds, and the correlation of lipophilicity to pharmacokinetics and pharmacodynamics [P to evaluate the impact of this parameter in the drug release kinetics from contact lenses.The octanol–water partition coefficient  or not present at all, more drug is available as “free” molecules, which is the percentage of the total drug in the gel that is neither adsorbed into the polymer matrix nor entrapped in the catanionic aggregates . Most ofFor FBNa, as shown in The in vitro release data for NPNa are shown in P). Among the three drugs studied, DFNa has the highest log P, while NPNa has the lowest log P , Contact lenses have important properties, e.g., optical transparency and water content, which should not be compromised with the incorporation of aggregates or any type of nanoparticles. Also, the oxygen permeability ,61 and mThe present work focused on extending the release duration of three NSAIDs drugs, namely DFNa, FBNa, and NPNa, from pHEMA contact lenses. It was shown that the release time can be extended with the formation of drug-participating catanionic aggregates. These aggregates are formed due to the ionic and hydrophobic interactions between a long-alkyl chain cationic surfactant and an anionic drug in aqueous solutions. Steady and dynamic shear rheology showed that highly viscous catanionic aggregates present shear thinning and viscoelastic behavior typical of wormlike micelles, depending on the drug type and CKC concentration. For instance, more hydrophobic drugs such as DFNa showed stronger effects on promoting the formation of viscoelastic catanionic aggregates than less hydrophobic drugs, e.g., NPNa. Furthermore, the Maxwellian fluid model, which represents solutions with wormlike micelles, showed good agreement with the obtained experimental data. We showed that the increase in the release time depends on three factors: (i) the concentration of the cationic surfactant, (ii) the alkyl chain length of the cationic surfactant, and (iii) the octanol–water partition coefficient of each NSAID.P values, e.g., DFNa, was enhanced in duration in comparison to drugs with smaller log P values such as NPNa. Finally, it was shown that the transmittance and water content of the contact lenses were not significantly compromised by the addition of CKC. Future studies should involve the use of small-angle scattering and transmission electron microscopy that can help in the confirmation of the wormlike morphology of the micelles and in the characterization of their size. Additionally, these studies should evaluate the material properties of the contact lenses, such as oxygen permeability, material modulus, and wettability. Biocompatibility studies should also be performed to assess any potential risk of cytotoxicity before proceeding to in vivo studies.It is believed that the mechanism of extended release is due to the entrapment of the drugs in viscoelastic catanionic aggregates, which retards the diffusivity of drugs to the release medium. For all studied drugs, by increasing the CKC concentration, the release duration was extended. Furthermore, C8, which has a shorter alkyl chain length than CKC, was unable to form drug-participating catanionic aggregates. Thus, the release extension for C8-based contact lenses was insignificant. The release time of drugs with higher log


Spondyloarthritis may be increasingly present in older patients as life expectancy increases. We investigated clinical differences between early-onset and late-onset spondyloarthritis in Japan.We retrospectively reviewed 114 patients consecutively diagnosed with spondyloarthritis. The clinical course of each patient was observed for ≥1 year. We defined early-onset and late-onset spondyloarthritis as <57 or ≥57 years at a median age of this study group, respectively. We compared clinical characteristics between these 2 groups.P < .01). Inflammatory back pain (IBP) was significantly more common in the early-onset group (P < .01), whereas dactylitis frequency was significantly higher in the late-onset group. Significantly more patients with early-onset spondyloarthritis were human leukocyte antigen (HLA) B27-positive (P < .01). Articular synovitis, particularly of the wrist, was significantly more common on power Doppler ultrasound (PDUS) in the late-onset group (P < .01). Tenosynovitis or peritendinitis, particularly in the finger and wrist flexors were also more frequent in the late-onset group . Enthesitis of the finger collateral ligament and lateral collateral ligament were significantly more common in the late-onset group (both P < .05). Multiple logistic regression analysis revealed that, comparatively, IBP was significantly and independently much more likely to occur in the early-onset group.Disease duration was significantly shorter before diagnosis in the late-onset group (The patients with late-onset spondyloarthritis had a lower frequency of IBP and HLA B27 and a higher frequency of dactylitis and PDUS findings in peripheral involvement. In the 1970s, Moll and Wright established the concept of a group of interrelated disorders termed seronegative spondyloarthritides. In Japan, the prevalence of HLA B27 is 0.3% in the general population, which is much lower than that in other countries. However, the frequency of HLA B27-positivity in Japanese SpA patients has not been clarified.The frequency of human leukocyte antigen (HLA) B27-positivity, which is associated with SpA, varies substantially among individuals of different countries. but cases are increasingly reported.–14 The number of such patients is expected to increase with longer life expectancy.,13–16 Later- or late-onset SpA (LOSpA),13,15,16 has not been precisely defined, but most studies have used onset at 50 years or older to classify such patients. Previous studies that assessed areas with high HLA B27 prevalence revealed differences between characteristics of early-onset SpA (EOSpA) and LOSpA.,14,15 However, the findings in LOSpA have not yet been reported for Japan, a super-aging society.Onset of SpA in older adults has been considered rare,In the present study, we compared the clinical profiles of patients with EOSpA or LOSpA to describe the clinical characteristics of LOSpA in Japan.22.1 Enthesis tenderness was evaluated on clinical examination according to the Leeds Enthesis Index and the Spondyloarthritis Research Consortium of Canada. The inclusion criteria were any of the various sets of SpA classification criteria that include the following: Amor, ESSG, ASAS criteria for axSpA, ASAS criteria for pSpA, and modified New York criteria. Thus, all patients satisfied 1 or more of the SpA classification criteria. We defined radiographic sacroiliitis as grade ≥2 bilaterally or grade 3-4 unilaterally on pelvic X-ray according to the modified New York criteria. In cases wherein radiographic sacroiliitis was uncertain or there was persistent IBP, MRI of the SIJs was performed. This was defined in the MRI as the presence of active inflammatory lesions such as bone marrow edema or osteitis as proposed by the ASAS MRI working group. An expert radiologist (MU) interpreted the MRIs. We also performed power Doppler ultrasound (PDUS) assessments in patients with peripheral symptoms. These assessments were done by JCR-registered sonographers . Systematic multiplanar gray scale (GS) and power Doppler (PD) examinations of joints, tendons, and entheses were performed with a LOGIQ S8 ultrasound machine  using a multifrequency linear transducer (4–15 MHz). We assessed articular synovia by PDUS at the wrists and the metacarpophalangeal (MCP), proximal interphalangeal (PIP), knee, and ankle joints bilaterally. Articular synovitis on PDUS was defined as GS and PD findings in the synovium. Tendons and tendon sheaths were also bilaterally assessed by PDUS at the extensor and flexor tendon sheaths of the wrist, periextensor tendon and flexor tendon sheaths of the finger, and tibialis posterior tendon sheaths. Tenosynovitis and peritendinitis were defined as GS and PD positivity of the tendon sheath or peritendon. Finally, we assessed the entheseal insertions bilaterally with US at the finger collateral ligaments, common extensor tendons on the lateral epicondyle of elbow, quadriceps tendon on the superior pole of the patella, proximal patellar ligament on the inferior pole of patella, distal patellar ligament on the tibial tuberosity, medial collateral ligament on the medial femoral condyle, lateral collateral ligament on the lateral femoral condyle, and Achilles tendon on the calcaneus. Enthesitis was defined on PDUS as a PD signal with or without structural abnormalities of the enthesis. Patients who did not have medical records including onset of disease, HLA-B antigen, and fulfillment of the SpA criteria were excluded. Further, those who did not have a definitive diagnosis and were diagnosed as palmoplantar pustular arthritis and synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome were excluded.This was a single-center retrospective cohort study approved by the medical ethics committee of Japan Community Healthcare Organization Isahaya General Hospital. The study population consisted of 114 Japanese patients with SpA who were managed between April 2009 and December 2017 at our institution. The present study included both incident and prevalent cases. The clinical course of each patient was observed for ≥1 year. A definitive diagnosis of SpA was made by a Japan College of Rheumatology (JCR)-certified rheumatologist  after excluding other rheumatic diseases by evaluating the following items: medical history, physical examination, laboratory findings, imaging findings, SpA classification criteria, and therapeutic response. Inflammatory back pain (IBP) was defined by the Berlin criteria.The definition of age at onset associated with LOSpA has not been consistent among previous reports. In this study, we defined EOSpA and LOSpA as <57 or ≥57 years at a median age of this study group, respectively. We compared clinical manifestations and laboratory and imaging findings between these 2 groups.2.2The demographic and clinical characteristics were compared with Fisher exact test for discrete variables and with Wilcoxon test for continuous variables. The Kruskal–Wallis test followed by Dunn multiple comparisons test was used to compare the 2 groups.P-values of <.05 on univariate analysis. Statistical analyses were performed with JMP pro 13.0 software . All reported P-values are 2-sided. A P-value of <.05 was considered statistically significant.To independently determine factors associated with LOSpA, we performed multiple logistic regression analysis, using variables with 33.1A total of 114 patients including those with EOSpA (n = 55) and LOSpA (n = 59) were reviewed in this study. Among these patients , 78 (68.4%) were diagnosed as uSpA, 16 (14%) as PsA, 11 (9.7%) as AS, 6 (5.3%) as IBD-related arthritis, 2 (1.8%) as nr-axSpA, and 1 (0.9%) as ReA. The median age at disease onset was 48.0 years for the EOSpA group and 72.0 years for the LOSpA group  and 8 of 11 (72.7%) in all patients with SpA patients and those with AS, respectively. Significantly fewer patients with LOSpA were HLA B27-positive than those with EOSpA. Although all patients with AS in the EOSpA group were HLA B27-positive, only 2 of 5 (40%) patients with AS in the LOSpA group were HLA B27 positive (data not shown).We found no significant differences in terms of the presence of enthesis tenderness, arthritis of the lower limbs, or uveitis. However, IBP was significantly less common in the LOSpA group, whereas dactylitis was significantly more common in the LOSpA group Table . The RF 3.3The Amor criteria were most commonly fulfilled in both groups Table . None of3.4There was no significant difference in the frequency of sacroiliitis on X-ray or MRI between the 2 groups Table . Periphe3.5We selected factors that varied significantly between the 2 groups in the univariate analysis and subjected them to multivariate logistic regression analysis. Only 1 factor was independently associated; IBP was more likely to be associated with the EOSpA group compared with the LOSpA group Table .4Our study clarified the clinical and laboratory differences between LOSpA and EOSpA in a retrospective cohort of Japanese patients. In addition, to the best of our knowledge, this is the first study to describe in detail the differences in PDUS findings in patients with EOSpA and LOSpA. Patients with LOSpA had shorter disease duration before diagnosis, lower prevalence of IBP, and HLA B27-positivity. In contrast, dactylitis was more common in the LOSpA group than in the EOSpA group, as were PDUS findings of articular synovitis (particularly wrist synovitis), tenosynovitis, peritendinitis (particularly finger and wrist flexor tenosynovitis), and enthesitis . Further, a previous study in Japan showed an entire of SpA prevalence of 0.04%, which is low compared with that in other countries. A study in Brazil showed that individuals with EOSpA had a significantly higher frequency of HLA B27-positivity than those with LOSpA, suggesting an association between HLA B27 and early-onset disease. A possible implication of our findings is that the extremely low prevalence of HLA B27 in Japan accounts for the lower prevalence of early-onset disease among Japanese patients with SpA. Moreover, the frequency of HLA B27-positivity is especially high in individuals with AS compared with other forms of SpA. In addition, patients with PsA with axial involvement reportedly had a significantly higher frequency of HLA B27-positivity than those with only peripheral involvement, which suggests an association between the presence of HLA B27 and axial involvement. Given these observations, Japanese patients with SpA probably have a lower frequency of AS and higher frequency of peripheral involvement because of the lower prevalence of HLA B27 in the Japanese population compared with that in other countries. Indeed, while the prevalence of AS is estimated to be between 0.1% and 1.4%, the estimated prevalence of AS is much lower in Japan (0.0065%). In our study, 8 of 11 patients (72.7%) with AS were HLA B27-positive. This frequency was comparable to that reported in the phase 3 trial of infliximab for AS in Japan (72.2%). However, the prevalence of HLA B27 was more than 90% among patients with primary AS in Western European countries. Although HLA B27-positivity in Japanese patients with AS has not been clarified, it may be lower than that in Western European countries.The prevalence of HLA B27 in Japan is much lower than that in other countries. A single-center cohort study in Japan showed that among patients with SpA , 40  were classified as uSpA, 10 (16.9%) as AS, 6 (10.1%) as PsA, 2 (3.4%) as IBD-related arthritis, and 1 (1.7%) as ReA excluding palmoplantar pustular arthritis and SAPHO syndrome. Although average age was higher in our study, the proportion of SpA subgroups was similar. Consistent with the findings of our study, this study suggested a higher prevalence of uSpA in Japan compared with that in other countries. Late-onset uSpA appears to be comparatively more common than late-onset AS.–33 It is important to recognize that even the elderly can develop SpA, particularly uSpA, despite the very low prevalence of HLA B27.Among 106 patients with SpA in Germany, 43.3% were diagnosed with uSpA, 30.2% with AS, and 14.2% with PsA, thus suggesting that uSpA is the most frequent type of SpA. A previous report from Brazil showed that patients with LOSpA had significantly fewer axial symptoms  and, conversely, a higher frequency of peripheral symptoms . Other reports have also suggested that peripheral involvement is more common in LOSpA.,31–33 Late-onset PsA is also more associated with peripheral arthritis, with inflammatory edema of the joints.,35–37 Consistent with a previous study, we found that patients with LOSpA had a shorter disease duration before diagnosis. This is possibly because of a higher frequency of peripheral symptoms in LOSpA with readily visible physical signs that can be more easily evaluated by physicians.Our study showed that wrist synovitis, finger and wrist flexor tenosynovitis, and enthesitis of finger collateral ligament and lateral collateral ligament were more commonly detected on PDUS in the LOSpA group. PD signals of finger flexor tendon and finger collateral ligament are occasionally observed in dactylitis. On the other hand, 50% of women with AS in another study also had fibromyalgia. This suggests that the 2 diagnoses may be confused with each other or that they may accompany one another. Late-onset uSpA might be misdiagnosed as RA, polymyalgia rheumatica (PMR), remitting seronegative symmetrical synovitis with pitting edema (RS3PE) syndrome, crystal-induced arthritis, or osteoarthritis. In a study of 20 patients with late-onset uSpA who were HLA B27-positive, 6 appeared with features mimicking RS3PE  and 3 had a PMR-like syndrome at onset. Therefore, it is important to distinguish other inflammatory rheumatic diseases from SpA. Clinical features, such as tenderness of enthesis, arthritis of lower limbs, and dactylitis are frequently detected in LOSpA. Thus, these findings are useful for distinguishing SpA from other rheumatic diseases. The use of PDUS early on to identify enthesitis and dactylitis may, thus, be advisable to facilitate an appropriate diagnosis in patients with peripheral symptoms.The difficulty of definitively diagnosing SpA may also be a reason for an apparently low prevalence of the disease in Japan. A report from Canada showed that 11 of 35 patients initially considered to have fibromyalgia were eventually diagnosed with SpA. each form of SpA has been reported at the onset of the disease in older patients.,14,41 In Japan, the aging rate is the highest than in other countries, and the population ratio of people over 65 years old was 26.6% in 2015. Our study was performed in an area with a super-aging population, where individuals aged >65 years account for 27.1% to 36.3% of the population. In the future, it is expected that the prevalence of LOSpA will increase as aging progresses. The different characteristics between LOSpA and EOSpA helps in not only making a correct diagnosis but also selecting the optimal treatment, thus avoiding side effects caused by unnecessary medication.Although LOSpA has been considered rare, In our study, each patient was observed for ≥1 year by rheumatologists who performed comprehensive evaluations and excluded other rheumatic diseases. Because the patients were evaluated in routine clinical practice, PDUS findings may have introduced bias in the interpretation of clinical diagnosis. Moreover, we determined age at onset as the age when the patient presented the obvious symptoms associated with SpA. However, if the symptoms are insidious, it is possible that the patient may be grouped into LOSpA. Second, our study was a single-center retrospective study performed at a hospital in an area where the population is progressively aging, which might have caused selection bias. Because the sample size is small, there is a necessity to investigate by increasing the number in a nationwide multicenter research. Finally, there are no established standard values to evaluate disease activity in all forms encompassed by SpA. Therefore, we did not include disease activity as a variable in our study. Prospective multicenter studies are warranted to further compare the clinical profiles of EOSpA and LOSpA, including disease activity.Our study has several limitations. First, there is no gold standard for the diagnosis of SpA.5This is the first study to describe clinical differences between EOSpA and LOSpA in Japan. In particular, we noted differences in the PDUS findings between these 2 groups. We found that patients with LOSpA had a shorter disease duration before diagnosis, a lower prevalence of IBP or HLA B27-positivity, and a higher prevalence of dactylitis, and peripheral PDUS findings included articular synovitis (particularly wrist synovitis), tenosynovitis or peritendinitis (particularly finger and wrist flexor tenosynovitis), and enthesitis . It is important to distinguish LOSpA from other inflammatory diseases with peripheral involvement such as RA, PMR, RS3PE syndrome, and crystal-induced arthritis. Making a correct diagnosis of LOSpA facilitates selection of the optimal treatment and possibly reduces side effects caused by unnecessary medication.The authors wish to thank the patients and medical staff for their contribution to the study.KF and TK had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. YE, KF, and TK helped with the study design. YE, KF, TK, AM, MM, TT, MU, and AK were responsible for acquisition of data. YE, KF, and TK were responsible for the analysis and interpretation of data. YE, KF, and TK prepared the manuscript. YE, KF, and TK performed statistical analysis. All authors read and approved the final manuscript.Supervision: Akinari Mizokami, Masanobu Mine, Toshiaki Tsukada, Masataka Uetani, Atsushi Kawakami.Writing – original draft: Yushiro Endo.Writing – review and editing: Keita Fujikawa, Tomohiro Koga.


For both pump and valve operations low energy requirements (mW range) were estimated for precise control of small flows (iii) Finally, the dynamic response of the pump was investigated by using alternating currents at a range of different frequencies.An electrochemical flow cell with Nafion 212, aqueous LiI/I Transport of solutions and ions in pores and membranes are of interest in many processes in biology ,2,3,4 an2O molecules, and immobilised charges within a charged pore or a membrane [j) is entirely driven by the cation flux and F is the Faraday constant, while for a real membrane a fraction of the current is dissipated transporting anions. Additionally, polar H2O molecules are dragged together with the ions, and it results in a mass flux through the membrane, where electrical power is converted into hydraulic pumping power against a pressure gradient. This processes known as electrokinetic energy conversion (EKEC) [The principles behind EOF are the rather complex electrokinetic interactions between mobile ions, polar Hmembrane . The conectivity . In the n (EKEC) ,15,16,17ficiency .EKEC was originally studied theoretically in the 1960’s for micro channels ,21,22, anterface ,24. Highnterface ,26,27,28 network ,30,31,32 network .In this work we present direct measurements of the power consumption for a voltage-gate functioning with lithium iodide/iodine and a membrane electrode assembly (MEA) based on Nafion 212 and porous carbon paper. Furthermore, we experimentally investigate the dynamic response of the electro-osmotic pumping capabilities, which to the best of our knowledge has not yet been presented in literature.P) versus the volumetric flow , whereas in Ref. [P assumes negative values when power is provided to the system; that is when the system is operated as a pump  ,21,22,34 in Ref.  they werP are opposite, that The efficiencies   P with tquations  and (4) 2 solutions were prepared using Lithium Iodide, LiI , in Milli-Q Water and were saturated with Iodine, I2 . Nafion 212 . On the high-pressure (left-hand) side, a needle valve was installed on the outlet tube, thereby enabling control of the differential pressure difference between the two sides of the cell. As the flow cell was operated in counter-flow conditions, the pressure difference was calculated as the logarithmic mean: The flow-cell  controlled with NI LABview software. Depending on experimental duration, several cycles were run for each frequency. Additionally, four-point Electrochemical Impedance Spectroscopy (EIS)  was performed to analyse the electrical response between 10I) entering the electrochemical flow cell, mass change Δp=−ΔIn Equation  and the ic η∼25% . On the By using the transport coefficients obtained for Nafion 212 the power consumption in different conditions can be calculated. If for instance an active area of 1 cmproof-of-concept we show a video in the As a To investigate in more detail the dynamic response of the voltage-operated gate a series of alternating current experiments were conducted. A similar technique was recently used for studying the fouling on ultrafiltration membranes . The strI, I, The experiments were carried out with the same cell used for the previously pump curves. However, to increase the resolution of the mass transport measurements, the circulation pumps were not running during experiments thus reducing the noises on the measurement from 15 mg down to the resolution of the scale (0.1 mg). In between experiments, the circulation pumps were used to flush the cell to ensure equal concentration in both chambers. During experiments, alternating currents were applied to the cell with a fixed amplitude of 95 mA at different frequencies (+ or I3− in water (With respect to the frequency response of the phase shift (apillary . This no in Ref. ). Hence f, and ultimately limited the data acquisition and analysis for the mass transport to 0.02 Hz. Nonetheless, At frequencies P, versus volumetric flow rate, a, h, and k only dependent on the hydraulic permeability coefficient, An EKEC flow cell equipped with a Nafion/carbon paper electrode assembly was operated as a voltage-operated gate. Input power,


Background: Toxoplasma gondii (T. gondii) is one of the most prevalent infectious parasites in humans worldwide. The diagnosis of toxoplasmosis is based on serological screening through the detection of anti-toxoplasmosis antibodies: IgG, which indicates previous exposure and the presence of active immunity, and IgM, which indicates a recent infection. We aimed to determine the prevalence of anti-toxoplasma antibodies in pregnant women at King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia, and explore the cost-effectiveness of anti-toxoplasma screening.Method: This was an analytic retrospective study of women who underwent serology testing for anti-toxoplasmosis antibodies at KAUH in 2013-2018. Data were collected from hospital documentation and IBM Statistical Package for Social Sciences  version 22 was used for analysis.Result: Of the 9,098 pregnant women seen at KAUH, 2,754 had undergone the test, and 38 had a positive result, i.e., a seroprevalence rate of 1.4%. Most women were Saudis (57.9%), and almost all were multiparous. Of those with a positive result, 36.8% were in the third trimester. Most births were by spontaneous vaginal delivery (65.8%). Twelve (31.6%) of the women with toxoplasmosis experienced obstetric complications. The estimated total cost of screening the pregnancies was US $919,646.00Conclusion: The prevalence of pregnant women with a positive anti-toxoplasmosis test result was low, and we believe there is no net benefit from screening all pregnant women for toxoplasmosis. Primary prevention should be through health education, and we recommend screening only women with high-risk pregnancies. Toxoplasma gondii (T.gondii) is a common intracellular parasite that is considered one of the most prevalent infectious parasites in humans worldwide . TransmiIn pregnancy, the most significant risk occurs during the first trimester; even though the transmission rate of T. gondii is low, the related morbidity is high during this period, and it can result in severe congenital anomalies . This isThe diagnosis of toxoplasmosis is based on serological screening through the detection of anti-toxoplasmosis antibodies; IgG, which indicates previous exposure and the presence of active immunity, and IgM, which indicates a recent infection .In Riyadh, Saudi Arabia, a study conducted in 2015, explored the prevalence of toxoplasmosis among 250 pregnant women from multicenter healthcare and community-based settings. It estimated the seropositivity of anti-toxoplasma IgG and IgM antibodies to be 32% and 6.4%, respectively . A studyIt has previously been observed that prenatal screening resulted in substantial cost savings due to a reduction in congenital toxoplasmosis in Austria, with a total annual saving of US $2.1 million . This stIn contrast, neonatal screening can be added to established Guthrie card-based testing, and is estimated to cost between £1.85 million and £3.5 million for a population of 700,000 live births for each infected child prevented, most of whom would be asymptomatic . HoweverOur study is retrospective and analyses laboratory data on T.gondii screening carried out at KAUH from January 2013 to February 2018. The study group comprised all pregnant women who attended the antenatal care unit and were screened using the anti-toxoplasmosis antibodies serology test. A total of 9,094 women delivered their babies at KAUH during the study period, but pregnant women who had not undergone the test were excluded.After identifying a positive result, our aim was to look for any complications in the mother or their fetus. Samples from all pregnant women who underwent prenatal screening were tested for anti-toxoplasma antibodies using enzyme-linked immunosorbent assay scanning for IgM and IgG. The data were obtained from the electronic medical documentation and registration system . A structured data sheet was used to collect the nationality, maternal age (years), gestational age (GA) at delivery, gravidity, parity, anti-toxoplasmosis level, and GA at which the test was performed. In those with a positive test result the history of complications was examined, which included oligohydramnios, anhydramnios, cardiomegaly, intrauterine growth restriction (IUGR), abortion, fetal hydrops, premature rupture of membranes (PROM) and intrauterine fetal demise (IUFD). The IBM Statistical Package for Social Sciences  version 22, was used to analyze the data. The study protocol was approved by the Institutional Review Board of our university hospital.Of the 9,098 pregnant women attending the antenatal care unit, 2,754 had taken the test, and only 38 were found to be positive - a seroprevalence of 1.4%. Most of these pregnant women were Saudis (57.9%), and almost all were multiparous, with just one woman being primiparous, as shown in Table Most of the births were by spontaneous vaginal delivery (65.8%), as shown in Table Finally, regarding the related costs, from January 2013 to February 2018, a total of 5,053 tests were carried out for 2,754 pregnancies. The total estimated price of these serological tests was US $919,646.00 (IgG US $93 and IgM US $89).KAUH has implemented prenatal screening for toxoplasmosis, but limited studies have been conducted to determine the anti-toxoplasma antibody prevalence in pregnant women attending KAUH. Of the 9,094 pregnant women included in this study, 2,754 were screened for anti-toxoplasma IgM and IgG antibodies. Among the tested women, 38 returned a positive result - a seroprevalence of 1.4%. This is very low compared to results reported in other centers of Saudi: 32.5% IgG seroprevalence in Riyadh,  24.1% inA study conducted in the United Kingdom (UK) showed a lack of evidence and insufficient data on the effectiveness of prenatal and neonatal screening . Also, rA study in Austria  showed tIn this study, 12 (31.6%) of the 38 positive cases were associated with obstetric complications. Two pregnancies were complicated with oligohydramnios and two with IUGR. Other reported complications included two cases of cardiomegaly, which was shown to be associated with T.gondii infection in a case-control study . MoreoveOur study is limited by the fact that the data were obtained from one tertiary hospital and describe the seroprevalence of T.gondii among pregnant women. Hence the results may not be suitable to generalize to the whole community. In addition, seroconversion could not be observed, as only one blood sample from each woman was investigated, with no attempt to confirm such infection using, for example, IgG avidity tests or polymerase chain reaction.Our present data revealed that the prevalence of pregnant women who had positive seroprevalence of anti-toxoplasmosis was low, at 1.4%. Hence there is no net benefit from carrying out this screening test for all pregnant women; screening and treatment will not result in any significant changes in fetal health status, and affected infants will be identified during their early life. Based on the results, we believe that it will be beneficial to perform anti-toxoplasma testing only in high-risk pregnancies, instead of routine prenatal screening. Routine testing reveals so few positive cases, for a relatively high price, that it is not cost-effective and wastes resources. Also, by carrying out this test routinely, patients are exposed to unnecessary work-up and investigations. Instead of carrying out these tests for those with non-risk pregnancies, primary prevention by health education should be used. We recommended utilizing the funds currently spent at KAUH on anti-toxoplasma IgG and IgM tests for low-risk pregnancies for other more essential screening tests.