Consumption of added sugars (AS) has been associated with increased risk for liver disease and risk factors for cardiovascular disease. The objective of this study was to further understand the relationship of AS intake with liver enzymes and risk factors for cardiovascular disease in adults  participating in the National Health and Nutrition Examination Survey (NHANES) 2001–2012.Individual usual intake (IUI) of AS was estimated using the Markov Chain Monte Carlo ratio method using two days of 24-hour dietary recalls gathered using standardized protocols. Subjects were separated into six consumption groups: 0 to <5%, 5 to <10%, 10 to <15%, 15 to <20%, 20 to <25% and ≥25% of energy as added sugars. Linear and group trends were determined using regression analyses for liver enzymes, cardiovascular risk factors, weight parameters, glucose, metabolic syndrome, and C-reactive protein. Logistic regression calculated odds ratios for these biomarkers above established risk levels (p < 0.01).2 across AS intake groups); % overweight/obese or % obese also showed inverse group trend . Liver enzymes did not show a significant association with AS IUI. Mean plasma glucose levels (mg/dL) showed an inverse linear (beta = −0.13 mg/dL per AS intake); and group association with energy from AS IUI (beta = −0.71mg/d L across AS intake groups). There was no association of AS intake with the risk of elevated uric acid levels; however, the odds of reduced uric acid levels was 17% lower with increasing intake of energy from AS IUI in group trend analysis only .There was an inverse group trend association with AS IUI for lower body mass index (beta = −0.12 kg/mAlthough it is sensible to consume AS in moderation, results suggested that higher intakes of AS were not consistently associated with physiologic parameters. Since the data were cross-sectional, they cannot be used to assess cause and effect. Thus, additional studies are warranted to confirm these findings with more rigorous study designs. The Institute of Medicine's  2002 recommendation was that no more than 25% of energy should come from added sugars. This recommendation was based primarily on nutrient dilution As mentioned above, AS intakes have been decreasing in recent years and assessing AS relationship with physiological variables in the context of lower intakes remains relevant. The objective of this study was to further understand the relationship of increasing levels of intakes of AS as a percent of energy with physiologic parameters using a large nationally representative data set: the National Health and Nutrition Examination Survey (NHANES) 2001–2012.2.2.1.An overview of the NHANES, including the purpose 2.2.Intake data were obtained from two 24-hour dietary recalls using an Automated Multiple-Pass Method (AMPM) Individual usual intake (IUI) of AS as a percent of energy was estimated using the Markov Chain Monte Carlo (MCMC) ratio method, as implemented in v1.0 of the SAS programs published by the National Cancer Institute (NCI) 2.3.Most demographic information was collected via interview using cycle appropriate questionnaires >30 kg/m2The National Heart Lung and Blood Institute Guidelines The National Heart Lung and Blood Institute Adult Treatment Panel III criteria was used to define metabolic syndrome as having three or more of the following risk factors: abdominal obesity, WC >102 cm  or >88 cm ; hypertension, systolic blood pressure ≥130 mmHg or diastolic blood pressure ≥85 mmHg or taking anti-hypertensive medications; HDL <40 mg/dL  or <50 mg/dL ; high triacylglycerols ≥150 mg/dL or taking anti-hyperlipidemic medications; high fasting glucose ≥110 mg/dL or taking insulin or other hypoglycemic agents 2.4.Model II). For Model II, there was only one variable that had a significant interaction of AS intake and NHANES cycle: HDL. Even in this case the range of difference across NHANES cycle and AS intake was quite small 52 to 53.5 mg/dL in HDL. Therefore, only results from Model I are presented. Given the differences in physical activity across added sugars intake groups, we also assessed whether there was a significant interaction of added sugars intake with physical activity. Significance was set at a p value <0.01 for all analyses and all analyses were adjusted for the complex sample design of NHANES and used appropriate subject sample weights Means/percentages and standard errors of demographic data by AS intake groups were determined. Regression analyses were used to assess if there was a significant difference in demographic variables across AS intake and pairwise comparisons of groups using t-tests were obtained using the SUDAAN (v 11.0). For the liver enzyme and cardiovascular risk factor levels, beta coefficients were generated from linear models regressing levels against added sugars intake as a percentage of total energy intake from AS; additionally a group trend analyses across AS intake groups was determined. Logistic regression was used to determine if the levels of added sugars intake had a lower odds ratio of having adverse physiologic outcomes. In model I, covariates used in the regression models included race/ethnicity, gender, physical activity level, alcohol intake, age, and poverty index ratio. Since there have been recent secular changes in consumption of foods and beverages with AS, a second model, included the NHANES cycle and an interaction terms of AS intake X NHANES cycle along with covariates in Model 1.  and a lower prevalence of being overweight or obese  or being obese  than those consuming less energy from AS. Finally, the group trend beta for energy intake was 19 kcals (p = 0.0070) with 110 kcal difference from the lowest and highest groups.When assessing group trends , there w3.2.th percentile CI]; p = 0.0083). No difference was seen for the linear trend analysis for reduced uric acid levels; no other differences were found . Logistic regression showed that the odds of reduced uric acid levels were 17% lower with increasing intake of AS for group trend analysis only. All other results suggested no association of usual AS intake with risk factors.There was a small, but significant difference, in mean BMI in the group consuming more than 25% of energy from AS when compared with all groups consuming less than 25% of energy from AS. There were also small, but significant differences in the percentage of the population of overweight or obese or obese individuals at the higher levels of intake (≥25% of energy) from AS. This is counterintuitive to what one might expect, based on the literature AS consumption, particularly in the form of fructose alone or as a component of sugar sweetened beverages, has been associated with a number of metabolic derangements, including inflammation of the liver and the development or exacerbation of NAFLD There has been a paucity of studies that have looked at liver enzymes and AS intake; Shimomy et al. That none of the cardiovascular risk factors, with the exception of the inverse association of AS intake with fasting plasma glucose levels, were significantly different across the groups of increasing percent energy of AS intake was surprising. Welch et al. In some This study had a number of strengths, notably the large sample size of a nationally representative dataset. The NHANES has a large representative sample and uses validated methods to collect two 24-hour dietary recalls, which can then be used to determine IUI This study also had a number of limitations. Data used in this study did not include the more recent NHANES 2013–2016 cycles because they were not available at the time the study was conducted. However, given the large sample size  from the 2001–2012 cycles was clearly large enough to complete the study. As with all cross-sectional studies cause and effect relationships could not be determined. Twenty-four hour dietary recalls are memory-dependent and there is an underreporting bias in energy intake that is higher in overweight and obese individuals, and women 5.Higher intakes of AS were not consistently associated with liver enzymes, cardiovascular disease biomarkers, including blood pressure, lipid levels, and glucose levels; or elevated uric acid levels. Data suggest that current recommendations for AS intake may be overly restrictive, at least for some people, and restrictive AS intake may lead to some Americans not meeting recommended micronutrient intakes Click here for additional data file.


Ciona robusta is a model organism characterized by a transparent body, exhibiting peculiar physiologic and evolutionary characters. In vitro fertilization and breeding of sea squirts is possible, in order to preserve consistent genetic pools. However, some aspects of its biology, as the feeding efficiency according to diet quantity and quality, are still scarcely known. Here we test the effects of three experimental diets on survival and growth, to detect physiological and molecular responses to various types of alimentary suspended particles and the effects of feed concentrations. We also aimed at determining rearing conditions able to limit handling operations, save artificial seawater and control water pollution. Molecular analyses of growth-related genes were performed to detect stressful effects due to feed quality and quantity. A strong effect of doses was highlighted, but water pollution may represent a major concern. A compound diet containing both live algae and non-live particles of a correct size is indispensable to assure development, low stress and high survival rates. Overall, our findings suggest protocols for an easier rearing of Ciona robusta in the laboratory, increasing the potentialities of these organisms as models for research.The sea squirt  Ciona robusta, exhibiting an almost transparent body, is characterized by a wide geographical distribution1. It is a euryhaline and eurytherm species3 living in areas of high turbidity and in coastal lagoons6. Several descriptions of C. robusta are available from various locations around the world, along with studies on its taxonomy and the main synonymies7. This organism may be a dominant member of benthic communities along European coasts8, reaching high densities , especially in colder seasons6. Its wide environmental adaptability permits to live in polluted harbours, as well as in dense seagrass (Posidonia oceanica) meadows11. It is sensitive to various types of pollution, especially in warmer seasons12, when its population naturally decreases13 but information about the factors influencing winter blooms is still incomplete14. However, the alternation of demographic explosions and consistent numerical reductions is typical of other species of solitary ascidians16, representing an aspect of their life cycle, still poorly known17, that is considered age and density-independent20.The solitary ascidian 21. It is a hermaphroditic broadcast spawner, but cannot self-fertilize22. C. robusta is mostly self-sterile and it has been used for studies on the mechanism of self-incompatibility23. Its genome has been fully sequenced and annotated24 and various mechanisms, including the biology of endostyle25 (precursor of a human thyroid), the fertilization process26 and the ability to react to various allelochemicals27, have been investigated. It is an easy-to-breed organism that can be reared in relatively small tanks26. For this reason, it is considered a model organism for scientific research28 especially for genetic29, genomic, regeneration30, and various medical studies31. In fact, genetic screens, germline transgenesis, electroporation of plasmid DNA and microinjection of morpholinos are all routinely employed on this species and complemented by targeted mutagenesis, homologous recombination, and RNAi30. In addition, many lines of transgenic animals expressing fluorescent markers in a wide array of patterns, under the control of endogenous or exogenous promoters, have been generated and are available for medical studies32.Its semi-transparent body not only permits the observation of the internal anatomy, but also to follow the embryo development . The body colour at the distal end of siphons is the main external character distinguishing sister species within its species complexC. robusta is becoming an endangered resource in some areas33, due to global and local environmental changes impacting its survival. Despite its abundance and the plethora of scientific investigations on its ecology and molecular biology, our knowledge of its feeding physiology is still incomplete35, and this limits our possibilities for culturing genetically consistent populations for scientific purposes. In fact, improvements of culture methods for marine invertebrates in closed system are prerequisites for a generalized use of transgenic lines26.However, Ciona robusta is a suspension filter-feeder that removes particles from the water column36. Food particles are further conveyed into the intestine, which bends upwards, from the base of the stomach, to join the rectum and opens into the wall of the atrial cavity, expelling faecal pellets through the atrial siphon37. It produces a mucous lattice38 that captures the collected food particles forming a string, further transported to the oesophagus and the stomach, which is located beneath the gill basket, moving from the languets of the dorsal lamina39. Various experiments demonstrated a lower retention limit of 1–2 μm for captured particles, and that such small particles as bacteria can also be captured40. Most laboratory studies on the filtration rate and size of ingested particles were performed using uniform-size unicellular algal cultures, not reflecting the actual size variability of particles recorded in nature34. However, several reports on inefficient retention rates of larger particles were based on comparisons of pumping and clearance rates34, rather than direct measurement of retention efficiency. In our case, we tested the efficiency on growth rates and mortality rates of such different particles with average size ranging from yeast cells (3–40 µm) to larger algae contained in the Shellfish diet and Algamac (300–600 µm).C. robusta but feeding rates considerably intensify with increasing concentration of algal cells and drop at the highest concentrations 41. Feeding studies on marine invertebrates demonstrated the importance of dietetic regimes (both their composition and their particle size distribution) to promote the growth of post-larvae and adults44.Overall, filtration rates depend on the size of Ciona robusta has been maintained in scientific laboratories since the last century, kept on a diet of diatoms, so demonstrating the feasibility of long-term culture for such model invertebrates. In addition, some laboratories set ascidian culture facilities close to the sea, facilitating large-scale productions. In this case, seawater naturally containing plankton was pumped into the tanks26. However, several points are of critical importance in closed systems for establishing healthy populations in continuous culture, while reducing costs of production. Food is a key element for the culture26 of Ciona and it must be cheap and easy to obtain and to prepare; in addition, it must provide indispensable compounds for its growth and reproduction, assuring low levels of water pollution. Previous authors considered a live natural diet as the most appropriate, especially for juveniles, due to its small particle size and low levels of pollution. This may represent a major advantage over artificial foods, particularly when seawater availability is limited. However, this method imposes the cultivation of live algae, considered as an expensive practice in aquaculture42. Moreover, periodical contamination requires efforts to re-establish mother cultures. To avoid these problems, some authors applied simple artificial diets based on commercially available mixtures of dry or suspended microalgae. The results in terms of growth and health of mature individuals, however, have never been directly compared with those made possible by natural diets36. In addition, filter-feeders have been successfully cultured simply by improving bacterial proliferation, using simple substrates and yeasts, but this strategy was never attempted for culturing C. robusta26. Here we propose a direct comparison of results made possible by the adoption of three culture strategies: (a) easy administration of a conserved algal complex commercially available; (b) adoption of a complex dietetic regime containing both live algae and organic suspended particles; (c) administration of a simple feed made of dry milk and dried algae, to take advantage of both plant and animal feeding principles in their simplest formulation. Experiments were performed by employing an automatic culture device, at two levels of particle concentration, in order to provide for the first time a comprehensive and direct comparison of growth rates promoted by different strategies and identify the best feeding efficiencies according to production costs. The efficiency of diets was evaluated by comparing growth and survival rates, but we also used qPCR to detect dose-dependent stressful conditions and test the hypothesis that dietetic regimes might affect the expression levels of genes involved in developmental processes of sea squirts.C. robusta during our experimental trials  out of 1,000 fertilized embryos settled on each plate at the stage of postlarva, morphologically identical to an adult. Thus, a reproductive success of 0.63 was recorded. In addition, a prompt decrease of densities due to early mortality was exhibited by all treatments after 24 h from the transfer of plates in the experimental tanks. In particular, at day 1, individuals fed on Shellfish diet at low concentration exhibited a survival rate of 60.9%; individuals under Compound feed treatment exhibited a survival rate of 54.5%; individuals under M&S treatment exhibited a survival rate of 44.8% . In parallel, average widths of individuals  were lower at lower doses of feeds. Length was a good descriptor of growth, since it exhibited the largest intervals of variation  while high concentration treatments yielded K values of 0.067 (Shellfish), 0.074 (Compound) and 0.070 (M&S). Thus, M&S produced slight faster growth at low concentrations, while Compound diet promoted the best growth results at higher concentration (Table t test).Size increases of sed Fig. . Length sed Fig. . Clear dsed Fig. C,D, follsed Fig. E,F and Ssed Fig. A,B. In ased Fig. . Thus, hion Fig. ; howeverion Fig.  in the aion Fig. , low conCi-HSPA2/8 (2.7), Ci-HSPA9B (3.1), Ci-HSPA4/4L/HSPH1 (3.9) and Ci-HYOU1 (2.3)  and Ci-HSPA5b (− 4.0). Otherwise, the compound diet only affected the expression levels of a single gene, down-regulating Ci-HSPA12 (− 3.2). At high feeding doses, only the gene Ci-HSPA12 was down-regulated by both Compound diet and M&S. Moreover, we followed the expression levels of ten genes involved in the endostyle formation and in the developmental processes  and Ci-Ends4 (3.9), with respect to Shellfish diet. No variations in the expression of genes involved in juvenile development were detected. Otherwise, M&S induced gene expression variation only in the gene Ci-Ends4 (3.9).The gene expression of juveniles fed on the Compound diet and M&S were compared with those fed on Shellfish, considered as the control diet. The analysis of gene expression of eight heat shock proteins at low feeding doses shows an increase of the expression levels of four heat-shock proteins after feeding on M&S with respect to the Shellfish diet: .3) Fig. . This diSPA2/8 2., Ci-HSPAses Fig.  to detec26. The experimental design herein described, set as a modification of a patented automatic rearing system for the culture of model organisms43, was sufficient to reduce the need for operator times, since it automatically provided water changes and feed administrations, and only required the periodical restocking of feeding reservoirs. The automatic system assured continuous control of the chemical quality of water at low concentration of feeds and when Compound feed was tested. However, it was almost unsatisfactory in the treatment M&S at higher feed concentrations, as demonstrated by the continuous increase of nitrate and phosphate during the experimental period. This might explain the episode of mortality influencing the treatment M&S after the third week of experiment. Similarly, the diet Shellfish produced sufficient growth and survival rates, especially at higher concentrations, but also led to an increase in nitrates and phosphates that the automatic system hardly controlled during the second half of the experiment. The automatic system performed at its best when feed doses were lower and produced acceptable results, at higher concentrations, in the treatment Compound, evidently promoting lower pollution of water, although most chemical and physical parameters were kept in the range preferred by this species35. Particle concentrations tested in this study represent sub-threshold levels for most tunicates, because negative effects of particle additions would be expected at concentration greater than 46 mg l−1 according to previous investigations44.Culture methods and experimental designs may fit various locations, available space, funding and manpowerCiona at minimum levels. This conclusion is also supported by records of organic particle concentrations at the end of the 3-h feeding periods45. In fact, suspended particles were quickly depleted at low concentration of feeds, and cultured individuals were partially starved. Due to this evident deficiency, the final size of cultured individuals differed even within low-concentration treatments. Growth constants were slightly different, showing an increasing efficiency of Shellfish vs. Compound vs. M&S. In these conditions (low doses of feeds), M&S maximized its efficiency, possibly due to microbial proliferation as an alternative food source, while minimizing the pollution of water, due to scarcity of food. In fact, previous authors46 observed that C. robusta exhibited a positive relationship between ingestion rate and cell concentration of Isochrysis galbana and that the largest filter-feeding efficiency was recorded at 5 × 103 cells/mL. Similarly, investigations44 indicated that ingestion rates increased linearly with food concentration in another tunicate . However, other studies35 did not detect a clear maximum in the feeding rates of tunicates, according to the concentrations of particles. In our case, when cultures were managed at higher doses (ad libitum) residual suspended particles were still present before water changes, demonstrating that individuals never starved. The gut of Ciona reaches an intake threshold at high food concentrations, above which the clearance rate is reduced as a form of protection against gut saturation47. At high loads of suspended particles a reduction in ingestion rates was associated with satiation46 and an active rejection48  was observed, while a reduction in lateral cilia beat frequency47 influenced the ingestion rates. Consequently, in conditions of high particle concentrations48 the best growth performances (expressed by K) were exhibited by the treatment Compound, followed by M&S and Shellfish, but in this case the differences among treatments were quite slight. In contrast, differences among treatments were significant when feeds were dosed at low concentrations, indicating that the first factor influencing growth is the concentration of feeds and that the quality of feeds50 becomes important at lower concentrations of suspended particles. In addition, M&S produced high mortality events at higher concentrations, probably due to a proliferation of microorganisms  that negatively influenced the clearance rates and the physiology of Ciona51. Following these evidences we may conclude that, although a diet able to maximize bacterial production may be beneficial for Ciona at lower concentrations of particles, its deleterious effects in terms of pollution of water and triggering of opportunistic bacteria blooms may drastically reduce survival rates and growth patterns at higher concentrations, as previously observed for various ascidians52. Also Joly26 observed that artificial feeds might be scarcely suitable for juveniles, as Ciona failed to grow and died, due to water pollution.Survival rates and growth curves indicate animal functional responses to food concentrations, and show the performances under various dietetic regimes. Functional responses under low concentration of feeds did not differ, although the quality of water was kept in ideal conditions in these treatments. Evidently, the stress produced by insufficient feeding kept both the size and the survival of C. robusta juveniles , and only one of them was also targeted by M&S, even if at low rates. Also in this case, molecular data supported morphological observations, since we detected an increase in the expression levels of two endostyle-related genes in juveniles fed on the Compound diet, which produced the best growth rates.Morphological data are explained by molecular investigations. In the present work, for the first time, we demonstrated that diets provided at low rates induced stress in 44 and we demonstrated that C. robusta follows such a general mechanism. Ideal feeds for culturing Ciona must fulfil some important criteria: they must be easy to obtain and to prepare, provide sufficient nutrients for growth and reproduction, and pollute as little as possible54. Although previous reports on the effects of feed concentration are slightly contradictory and comparisons are sometimes difficult to make, the basic principles of filter-feeding are well-known and they have been confirmed here. Differences in species, temperature, conditions , the units used to describe the particle concentration and the use of different feeding measurements produce wide scattering among results by various authors40. Carver33 and Petersen47, both pointed to the variability in filtration estimations for C. robusta, even at similar temperature regimes. Overall, our preliminary hypotheses were confirmed, since the Shellfish feed produced a low level of survival at low doses, but it produced less than 20% of mortality after 1 month of experiment at higher doses and assured a moderate growth and it was actually the easiest and cheapest feed tested; the Compound feed containing live prey was the most effective, both for survival and growth, but it was more complex to be produced and administered. The fermented feed was simple and effective, but it demonstrated to be almost dangerous at higher concentrations, producing bacterial diseases and mortality, as revealed by microscopy observations. In fact, bacterial films covering most individuals were detected under the optical microscopy and blooms of ciliates invaded the bodies of cultured specimens. In addition, our hypotheses about doses were confirmed, taking into account previous papers indicating the feeding activity of tunicates48 and morphological data along with molecular investigations demonstrated that the growth constants of various feeds are comparably low at low concentration of suspended particles, because growth is fundamentally limited by starvation. However, at low concentration of feeding particles the association of a fermentable substratum (milk) with a protein-rich algal item (Spirulina) provides the best results in terms of growth (K = 0.031) because it maximizes the presence of food items suspended in the water. In contrast, when C. robusta was fed ad libitum, only slight differences among feeds emerged and a compound feed, containing both live particles and organic detritus, maximized both growth and survival. Such a nutritive feed assured a growth constant as high as 0.065, indicating that mature individuals suitable for experimental purposes (about 20–25 cm length), may be obtained in less than 2 months in an automatic device. In contrast, working at lower concentration of feed particles, the best results in terms of growth were obtained adopting the diet M&S (doubling time 12.81 days). In this case, to obtain mature individuals of only 6 cm length, a growth period of 3 months should be forecasted but, as demonstrated by our experimental trials, this diet must be kept at lower density of particles to avoid larger mortality due to bacterial blooms, although bacterial diseases could be further controlled using Gentamycin, according to Joly26. Additional dietetic adjustments could be attempted in future55, as a key prerequisite for the isolation, maintenance, and experimental use of stable transgenic lines26, taking into account the variable growth patterns prompted by different diets and the advantages provided by an automatic rearing system , as shown by this investigation.Substantially, the feeding processes of ascidians are well establishedCiona robusta reared in aquarium cultures. To this end, we produced C. robusta larvae and post-larvae by in vitro fertilization of its gametes, and tested the effects of three diets, at two concentrations (Table This investigation aimed at defining (a) the effect of particle concentration and (b) the effect of diet composition on the growth and survival of ns Table , on theiAdult sea squirts were collected by scuba divers on mussel culture ropes at depths between 6 and 20 m in the Ionian Sea off Taranto  in October 2018. Individuals showing no signs of damage were manually selected on site, stored in refrigerated containers and transferred to Napoli  at the Stazione Zoologica facility for the culture of model organisms. They were reared in 400 L open cycle aquariums up to the collection of gametes.Mature specimens were identified according to the size of gonoducts, as observed through the transparent body: eggs and full oviducts are reddish or brownish; sperm and sperm ducts are whitish. Continuous illumination was applied until the collection of gametes, to prevent spontaneous spawning. Gametes were collected by dissecting the body wall of three individuals with a scalpel, to reach ventrally located oviducts and sperm-ducts. Eggs were collected by a Pasteur pipette and transferred into a 200 mL glass vessel for complete mixing of gamete pools. Sperm was obtained from three sperm-ducts after dissection of body walls, pooled, and diluted with 2 mL of filtered (Millipore 0.22 µm) seawater. The concentration of gamete suspensions was determined by means of a cell counting chamber. Sperm was added to pools of 1,000 eggs poured in plastic Petri dishes filled with 150 mL of filtered (Millipore 0.22 µm) seawater, in order to fertilize at a ratio of 5,000:1 of sperm to eggs, to prevent polyspermy. Subsequently, Petri dishes containing fertilized eggs were incubated for three days in a thermostatic chamber  to allow embryo development. A partial change of the culture medium was provided using filtered seawater after the complete settlement of larvae, and 150 µL of Shellfish food were added in each dish to avoid larvae starvation prior to start the feeding experiments. The density of settled larvae in each Petri was assessed at this stage, under stereomicroscopy, prior to moving culture dishes to experimental tanks .Three diets were set and compared:Ready-to-use commercial diet  surrounded by a dense liquid, sufficient to assure its long storage at low (4–5 °C) temperatures. It provides nutrition for bivalve species, from first feeding larvae to adult broodstock, increasing both growth rate and survival, thanks to the presence of diatoms with a high fatty acid profile58. Shellfish diet is also suggested as an excellent feed for ascidians, sea cucumber larvae, soft corals, Artemia, and some copepods, because it assures absence of toxic metabolites60, with respect to live cultures of microorganisms. The dry weight of Shellfish diet corresponds to 8% of its fresh weight. Cell counts in the suspension vary due to the natural origin of the product, but it roughly corresponds to 2 × 109 cells mL−1.(a) Compound diet  with dry feeds  with the addition of a fat-rich algal derivative (Algamac), increasing also the levels of such unsaturated fatty acids as DHA and EPA. The actual composition of live feeds may influence the physiology of consumers at various levels64; thus, any mixture of live feeds must be produced in controlled conditions and accurately tested on target consumers.(b) Simplified compound diet  we tested this diet made of milk powder  and Spirulina (a protein-rich cyanobacterium).(c) The three formulations were administered at two different doses  in order to assess the effect of particle concentrations. These doses contained, on average, about 300 (low dose) or 8,000 (high dose) food particles per mL of culture medium.43 modified for the culture of Ciona  along with concentrations of nutrients, . In parallel, average length and width of post-larvae, and their density in each plate were measured under the stereomicroscopy, using a micrometric 10 × ocular. The experiment lasted 30 days, up to complete development.This experiment was conducted using a patented (E.U. Patent 102015000012043) automatic rearing systema Figure . It cons65 and 1 μg of total RNA for each sample was retro-transcribed with an iScript cDNA Synthesis kit . The complete coding sequences of ten genes were retrieved from NCBI (available at https://www.ncbi.nlm.nih.gov/) and Joint Genome Institute (JGI) Genome Portal (https://mycocosm.jgi.doe.gov/Cioin2/Cioin2.home.html). Specific primers were designed on the nucleotide sequences, and selected fragments (Table −1/slope) were used to calculate the efficiency of primer pairs, determining Ct values vs. the logarithm of each dilution factor, by testing five serial dilutions . PCR efficiencies were calculated for control and target genes. Diluted cDNA was used as a template in a reaction containing a final concentration of 0.3 mM for each primer and 1 × Fast Start SYBR Green master mix  . PCR amplifications were performed in a ViiATM7 Real Time PCR System  thermal cycler using the following thermal profile: 95 °C for 10 min, one cycle for cDNA denaturation; 95 °C for 15 s and 60 °C for 1 min, 40 cycles for amplification; 72 °C for 5 min, one cycle for final elongation; one cycle for melting curve analysis (from 60 to 95 °C) to verify the presence of a single product. Each assay included control for each primer pair with no-template. Primers for the eight genes for HSP70 superfamily proteins were those reported in Fujikawa66. All qPCR reactions were carried out in triplicate to evaluate intra-assay variability. Fluorescence was measured using ViiATM7 software Version 1.0 . The expression levels of each gene were internally normalized against the housekeeping geneCi-CA6, which encodes cytoplasmic actin66, using REST software  based on the Pfaffl method68. Relative expression ratios above 2.0 cycles were considered significant when comparing M&S and Compound diets versus Shellfish diet. Experiments were repeated at least twice, in order to obtain an experimental replicate of each qPCR plate. All used software was not freely available , according to RuoccoC. robusta in each treatment were obtained according to the Malthusian model69:0 is the size at time 0 and K is the Malthusian growth rate of the population70. Growth curves were compared by means of the Friedman test and the Dunn’s multiple comparison post hoc tests in order to evaluate the differences among treatments and doses. Chemical measures and survival data were tested for their normality using a Shapiro–Wilk normality test and, when normality was confirmed, the significance of differences among treatments was assessed by means of one-way ANOVA followed by Tukey’s post hoc tests. When normality was not assured, Mann–Whitney test was applied. Paired t tests were applied to evaluate the significance of differences in growth descriptors, between couples of dietetic treatments. All statistical analyses and graphs were performed using GraphPad Prism 8.0.0 for Mac, GraphPad Software, San Diego, California USA (http://www.graphpad.com), not freely available.Growth curves of Supplementary information


Land use and hunting are 2 major pressures on biodiversity in the tropics. Yet, their combined impacts have not been systematically quantified at a large scale. We estimated the effects of both pressures on the distributions of 1884 tropical mammal species by integrating species’ range maps, detailed land‐use maps (1992 and 2015), species‐specific habitat preference data, and a hunting pressure model. We further identified areas where the combined impacts were greatest (hotspots) and least (coolspots) to determine priority areas for mitigation or prevention of the pressures. Land use was the main driver of reduced distribution of all mammal species considered. Yet, hunting pressure caused additional reductions in large‐bodied species’ distributions. Together, land use and hunting reduced distributions of species by 41% (SD 30) on average (year 2015). Overlap between impacts was only 2% on average. Land use contributed more to the loss of distribution (39% on average) than hunting (4% on average). However, hunting reduced the distribution of large mammals by 29% on average; hence, large mammals lost a disproportional amount of area due to the combination of both pressures. Gran Chaco, the Atlantic Forest, and Thailand had high levels of impact across the species (hotspots of area loss). In contrast, the Amazon and Congo Basins, the Guianas, and Borneo had relatively low levels of impact (coolspots of area loss). Overall, hunting pressure and human land use increased from 1992 to 2015 and corresponding losses in distribution increased from 38% to 41% on average across the species. To effectively protect tropical mammals, conservation policies should address both pressures simultaneously because their effects are highly complementary. Our spatially detailed and species‐specific results may support future national and global conservation agendas, including the design of post‐2020 protected area targets and strategies. Article impact statement: Tropical mammals have lost on average 40% of their original distribution due to the combined effect of hunting pressure and human land use. Overexploitation and habitat loss due to agricultural activities are major pressures on biodiversity in the tropics  and extirpation . We retrieved the distance to access points from the hunting database , and species to account for between‐country variation in hunting laws and policies, culture, taboos, and traditions , human population density, and the species’ body mass. We fitted the model based on a data base with 3281 mammal abundance estimates of 296 species (from 51 families and 14 orders) from 163 studies and 114 papers that systematically compared abundance between hunted and unhunted sites within the tropics  resolution. Our predictions were based on the taxonomic identity of the species (captured by the random effect intercept species) and its body mass (species’ vulnerability to hunting pressure) combined with the distribution of context‐dependent drivers of hunting pressure in the species’ initial distribution . We retrieved data on human population density specific to 1992 (average between 1990 and 1995) and 2015 from CIESIN  of species i, Aloss,i,LU is the area loss (square kilometers) due to land use only, Aloss,i,H is the loss (square kilometers) due to hunting only, Aloss,i,LU∩H is the overlap in loss between the 2 pressures (square kilometers), and iA is the initial distribution (square kilometers).To quantify the total reduction in the species’ distributions, we overlaid the maps of both pressures and identified the area lost due to hunting only, land use only, and the overlap of the 2 pressures Fig. . We thenn = 979 species), small , medium , and large . We further calculated the average area loss across mammals from different continents . Finally, we compared the area losses from 1992 to 2015 to identify possible changes in the magnitude and relative importance of the 2 pressures over time.We grouped our area‐loss results by species group based on body size: very small  in cell y , Aloss,i,y is the area loss of species i in cell y (square kilometers), and i,yA is the initial area of species i in cell y (square kilometers). All the calculations were done using a Mollweide equal‐area projection in R 3.5.3 (R Development Core Team https://doi.org/10.17026/dans-zah-vs6x.We defined hotspots and coolspots of area loss as areas with great (>90%) or small (<10%) distribution loss due to the combined pressures across the species per 0.25° (∼25 km) grid cell . In each 0.25° cell, we divided the cumulative area lost by the cumulative initial area across all the species present in that cell:On average across the species, distributions of the species declined 41% (SD 30) due to the combined impacts of hunting and land use (2% overlap between the 2 pressures [Supporting Information]). Land use resulted in an average loss of 39% of the initial distribution (SD 30) and hunting in a loss of 4% (SD 11). The smallest mammal species (<0.1 kg) were mostly affected by land use (loss of 42% [SD 31])  across the species were identified in the Gran Chaco, Atlantic Forest, El Cerrado, northwestern part of South America, East Africa, Madagascar, Thailand, and Java. Areas with small loss (coolspots) were in the Amazon Basin, the Guianas, the Congo Basin, central Borneo, and Papua New Guinea Fig. .Estimated area losses were the greatest in Africa (average loss 46% [SD 30]), followed by the Americas (40% [SD 31]), and Asia (37% [SD 28]) Fig. . These l2 in 1992 to 288,000 km2 in 2015 (FAO Losses in distribution increased from a mean of 38% (SD 31) in 1992 to 41% (SD 30) in 2015 (Supporting Information). Some species increased their distribution , for example, in Ethiopia where the area of pasture decreased from 448,000 km2015 FAO . Yet, moTo our knowledge, we are the first to quantify the combined impact of land use and hunting pressure on the distributions of mammals across the entire tropical region. Our results suggest that tropical mammals lost on average 40% of their distribution due to these 2 pressures combined Figs. , wherebyCercopithecus lowei), a generalist species, was primarily affected by hunting (but see Linder & Oates [Ateles fusciceps), a forest specialist, was affected most by deforestation and land‐use change. We found a relatively small overlap between the impacts of the 2 pressures , whereaes Figs. , 2019. Hes Figs. . Additioes Figs. , 2019. TOur results further showed clear spatial variation in the effects of the 2 pressures. At the level of continents, we found that pasture may remove 24% of natural habitat in the Americas, whereas only 7% may be removed in Asia Fig. . FurtherIn contrast, the Amazon and Congo Basins, the Guianas, Borneo, and Papua New Guinea had relatively small loss of distribution across the species (coolspots), which is in line with results of previous efforts to map human impacts on biodiversity  and of crosswalk between the GLOBIO land‐use map and the IUCN habitat classification (Appendix S2), comparison between hunting database and selected tropical mammal species (Appendix S3), area loss by land use and hunting with 2 thresholds (Appendix S4), area loss due to different pressures (Appendix S5), patterns of tropical mammal species richness (Appendix S6), species affected by land use or hunting pressure as main driver of distribution reduction (Appendix S7), model selection results for the binomial hunting model (Appendix S8), mean area loss due to different pressures (Appendix S9), and cross‐walk from IUCN habitat classes to ESA CII and GLOBIO classes 11 (Appendix S10) are available online. 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Periodontal diseases are some of the most widespread oral afflictions, and they are labeled as chronic infections caused by the accumulation of bacteria in dental plaque that produces localized inflammation of the periodontium. The use of local drug delivery systems to treat periodontal diseases has received greater attention, because the active substance is targeted directly to the affected area, which minimizes its systemic side effects. Therefore, the purpose of the investigation was to develop and characterize different types of gel formulations—bigel, hydrogel and oleogel—as local delivery systems containing metronidazole (MET), which can be applied to the oral mucosa. The influence of the formulation type on the mechanical, rheological and mucoadhesive properties were examined. Moreover, in vitro release of metronidazole, its ex vivo permeation through buccal porcine mucosa and antimicrobial activity measured by the plate diffusion method were estimated. It was found that the gel formulations obtained were non-Newtonian systems, showing a shear-thinning behavior and thixotropic properties with good textural features such as firmness, compressibility and adhesiveness. Moreover, the preparations designed possessed beneficial mucoadhesive properties. The formulated hydrogels and bigels containing micronized MET were considered as better formulations in terms of drug release and antimicrobial activity compared to commercially available metronidazole ointment. An ex vivo permeation study with the use of porcine buccal mucosa demonstrated that the bigel formulation was characterized by higher initial permeability rate providing a fast therapeutic effect with simultaneous moderate retention in mucosal tissue to decrease the risk of local cytotoxicity. Periodontal diseases are very common oral afflictions that may affect as much as 90% of the worldwide population . They arMany local drug delivery systems such as implants, films, fibers, ointments or gels prepared using biodegradable or nondegradable polymers have been tested for periodontal therapy. Among these carriers, mucoadhesive gel formulations have received considerable attention in treating periodontitis ,8,9,10. Antibiotics and antiseptics have been commonly used to treat moderate to severe periodontal diseases ,6. AmongThe purpose of this study was to enhance local delivery of MET using three types of gel formulations, namely hydrogel, oleogel and bigel, to cure periodontal diseases. The physicochemical, rheological, textural and bioadhesive properties of the designed gels were studied. Moreover, the antimicrobial activity of prepared gel carriers was estimated. Furthermore, in vitro MET release and its ex vivo permeation were examined to select the optimal gel formulation that may be used as the most effective local drug delivery system applied to the oral mucosa.® 200  was a gift from Evonik Industries AG, Hanau, Germany. Tocopheryl acetate (vitamin E), linseed oil , methyl paraben and propyl paraben , Tween 80 , low viscosity sodium alginate , 61% mannuronic acid and 39% guluronic acid , sodium azide , sodium chloride, sodium hydroxide potassium, potassium phosphate monobasic, sodium phosphate dibasic, methanol , ethanol 99.9% , acetonitrile HPLC grade and ethyl alcohol 96%  were used as received. All chemicals and solvents used for the study were of analytical grade. Simulated saliva solution  was prepared by dissolving 8 g sodium chloride, 0.19 g potassium phosphate monobasic and 2.38 g of sodium phosphate dibasic in 1 L of water. Mueller–Hinton agar was provided by Biomaxima . Cuprophan® was received from Medicell . Metronidazol 100 mg/g ointment with a composition of PEG 400 Da, PEG 4000 Da and cetostearyl alcohol used as a control was a product of CHEMA-ELEKTROMET . The control strains Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were from TCS Biosciences Ltd., Buckingham, United Kingdom. Cellulose acetate membrane filters (CA 0.45 μm) were received from Millipore . Buccal porcine mucosa was derived from a local veterinary service. This procedure did not require the approval of the local ethical committee on animal testing. Samples of the mucosa were stored at −20 °C and before the experiment were defrosted and cut into 5 mm diameter pieces.Metronidazole (MET) and glycerol 85% were purchased from PPH Galfarm Sp. z.o.o . Aerosil® GmbH, Haan, Germany) testing different conditions; time from 5 to 60 min and at rotational speed of 20 Hz 1 g of powder with three ZrO2 balls having diameter of 7 mm were charged in a cylindrical jar of the mill to fill one third of the volume of the jar. For the best process conditions of metronidazole micronization (MMET), 30 min and 20 Hz were chosen.Milling of MET for particles size reduction was performed in an oscillating ball mill MM 400 . Samples of MET and MMET (in the amount of 5 mg) were placed in aluminum pans and heated in the range 25–300 °C under a 20 mL/min nitrogen flow .The solubility of MET and MMET was determined by using the shake-flask method. Briefly, an excess amount of MET/MMET was added to each vial containing 5 mL of the selected solvents . Mixtures were mechanically shaken for 24 h at 25 ± 0.5 °C and allowed to stand for 24 h to attain equilibrium. Then mixtures were centrifuged at 3000 rpm for 15 min, followed by filtration through a CA membrane filter (0.45 μm), diluted appropriately with methanol and analyzed by HPLC method at 319 nm against a standard ,23.Different types of gel systems, namely hydrogel, oleogel and bigel, were prepared using an RZR 2020 mechanical stirrer . The compositions of the designed formulations are listed in w/w) in an aqueous-based solution containing glycerol 85% (as a humectant) and parabens, previously dissolved in 96% ethanol, with the help of mechanical stirrer at a moderate speed. Mixing was continued until transparent hydrogels were received.Alginate hydrogel (H) was prepared by gradually dispersing gelling agent , Tween 80 was added to the heated oil (70 °C) with constant mechanical stirring at 300 rpm, and next the gelling agent AerosilBigel (B) was developed by separately forming oleogel and hydrogel and then mixing them using a mechanical stirrer (800 rpm) at room temperature. Bigel formulations were made at different hydrogel to oleogel weight ratios, namely 30:70, 50:50 and 70:30. Stable and homogeneous bigel was obtained only using the hydrogel to oleogel weight ratio of 70:30.w/w concentration was uniformly dispersed in gel vehicles. The concentration of active ingredient was set based on commercially available product. Control gel formulations without MET (placebo) were also initially prepared.MET or MMET (micronized metronidazole) at 10.0% v/v); flow rate 1.0 mL/min; detection at 319 nm; retention time 3.0 min [2 = 0.999).MET content was determined after extraction of gel formulation samples in ethanol (99.9%) and analyzed by HPLC method using an Agilent Technologies 1200 HPLC system equipped with a G1312A binary pump, a G1316A thermostat, a G1379B degasser and a G1315B diode array detector  in the following conditions: Zorbax Eclipse XDB-C18, 4.6 × 150 mm, 5 µm column ; mobile phase: acetonitrile—0.01M phosphate buffer pH 4.7 , which was calibrated before each use with standard buffer solutions. Each measurement was carried out three times and average pH was calculated.Gel formulation samples (in quantity corresponding to 10 μg of MET/MMET) were observed under 100× magnification, and particles size was analyzed by using a Motic BA 400 optical microscope equipped with a camera  .−1 were recorded, and the rheograms were evaluated by plotting the obtained values of shear stress versus shear rate (2.00–20.00 s−1) [The viscosity was determined using a Brookfield viscometer  equipped with a cone/plate type CPA52Z  measuring system, at a temperature of 25 ± 1 °C. The viscosity values of gel formulations at a shear rate of 10.00 s−1 for a distance of 5 mm into the sample (30 g) and redrawn. Data collection and data analysis were performed using the Texture Exponent software package. Parameters such as firmness, compressibility and adhesiveness were determined from the force–time plots [Texture properties of prepared hydrogel, oleogel and bigel formulations were examined using a TA.XT Plus texture analyzer  for backwards extrusion measurements. A disc (35 mm diameter) was pushed at a speed of 2 mm·sme plots ,26,27.−1, test speed 0.1 mm∙s−1, contact time 120 s, post-test 0.1 mm∙s−1, applied force 0.5 N. The adhesive properties were determined as the maximum detachment force (Fmax), and the work of adhesion (Wad) was calculated from the area under the force versus distance curve, expressed in µJ. The work of adhesion (Wad) was calculated using the following formula:−1) and the multiplication by 1000 is in order to express the result in units of work (µJ) [Evaluation of bioadhesiveness was performed using a TA.XT Plus texture analyzer  on the porcine buccal mucosa model. Samples of the mucosa were frozen at −20 °C and stored no longer than 4 weeks. On the day of the experiment, mucosa was defrosted and cut into 5 mm diameter pieces. Next, samples were thawed in physiological saline solution (0.9% NaCl) at 25 ± 0.5 °C for 30 min; mucosa was attached to the lower end of a cylindrical probe using a cyanoacrylate glue, and gel formulations in the amount of 0.5 g were placed below the probe. The experiment was carried out at 37 ± 0.5 °C (water bath) to mimic in vivo conditions. Experimental parameters of the process were chosen during preliminary tests and set as follows: pre-test speed 0.5 mm∙sork (µJ) ,28.®, Medicell, London, UK) using an enhancer cell with a surface area of 3.80 cm2. The enhancer cell consisted of a Teflon load ring, a cap, a membrane and a drug reservoir. This study was performed using the USP dissolution apparatus 2  with mini vessels (250 mL) and mini paddles. Samples, each of about 3 g, were placed in the enhancer cell, which was then immersed in the dissolution vessel containing 100 mL of the release medium–simulated saliva solution (SSS), pH 6.8 (the sink conditions were maintained), previously warmed to 37 ± 0.5 °C. Agitation was provided by mini paddles at 75 rpm, and aliquots each of 2 mL were withdrawn at different time intervals . Withdrawn samples were replaced by equal volumes of fresh release medium [The release of MET/MMET was measured through a cellulose membrane  bacteria Escherichia coli ATCC 25922. Broth cultures (100 µL), adjusted to yield approximately 107 CFU/mL , were inoculated on plates containing Mueller–Hinton medium. After 15 min of drying at room temperature, wells with a diameter of 5 mm were cut out in agar plates, into which prepared gel formulations (100 mg) were placed. The plates were incubated at 37 °C for 24 h. The results were recorded by measuring the zones of growth inhibition surrounding the wells using a caliper  with an accuracy of 0.1 mm [The antibacterial activity of designed gel formulations was evaluated by the plate diffusion method on Muller–Hinton agar. Tested microorganisms included the Gram(+) bacteria f 0.1 mm . Hydroge2. After the equilibration period, a proper amount of tested formulations with 50 mg of MMET was carefully applied over the tissue surface and protected with parafilm. Commercially available ointment with MET was used as a control. The acceptor medium (phosphate buffer with addition of 0.005% of sodium azide) was then recirculated beneath the buccal mucosa at a constant rate of 30 mL/h. The sink conditions were confirmed during preliminary studies. At predetermined time intervals, samples of acceptor medium were collected, filtered through CA 0.45 µm filters and analyzed for drug content using the HPLC method. The samples were replaced by the same volume of acceptor solution to assure sink conditions.Gels containing MMET were selected for the ex vivo permeation study due to their better release profile and antibacterial activity. Permeation studies were accompanied in a flow through cell system equipped with thermostatic Teflon Bronaugh diffusion chambers and a peristaltic pump . Porcine buccal mucosa from large white pigs weighting ~200 kg were from the veterinary service of a local slaughterhouse . Freshly excised tissue was preserved in the isotonic saline solution and kept at −20 °C for no longer than 60 days. Prior to experiments, tissue was thawed at room temperature and cut into pieces (weight 80–100 mg). In brief, porcine buccal mucosa was sandwiched between the two compartments with the mucosal side facing the donor compartment and equilibrated with phosphate buffer solution (pH 6.8) for 1 h. The diffusion area of the tissue was 0.81 cmAt the end of the studies, the suspension from the donor compartment was carefully aspirated to a flat-bottom flask and washed with simulated saliva solution  and methanol until complete MMET removal from the top of the tissue. The drug retained in the buccal mucosa was extracted by methanol at 30 °C for 4 h. After having been filtered through 0.45 µm nylon filters, the extract was analyzed for the amount of drug retained in the human vaginal epithelium by the HPLC method. Passive mucosal permeability and retention behavior were expressed as the amount of drug per unit area of the tissue ,33.P < 0.05 was considered as significant.Results are presented as the mean ± standard deviation (SD) based on six independent experiments. Statistical analysis was done by one-way analysis of variance (ANOVA) using Statistica 10.0 software . A probability level of Micronization by the milling technique is a conventional method for particle size reduction and is frequently used for improving the solubility of drugs. This process does not enhance the equilibrium solubility of the drug itself, but it raises the dissolution rate by decreasing their size and increasing the surface area of the active compound ,35. DiffAs the most appropriate milling process conditions, 30 min at 20 Hz were considered. The ball milling technique increased the solubility of MET, especially in the case of solubility in linseed oil. In addition, homogeneous particle sizes of approximately 10 µm were obtained. Many compounds may undergo polymorphic changes during different technological processes, including milling. For this reason, the effect of milling on the crystal transformation of MET was investigated. DSC  is a thermoanalytical technique that can provide information regarding polymorphic changes in the milled material. It was found that the milling process under the chosen conditions (30 min at 20 Hz) did not influence MET crystallinity. The substance retained its properties, as confirmed by the DSC thermograph . As is v®, fumed silica) has been successfully used to increase viscosity and impart thixotropic character to lipophilic ointments and non-aqueous suspensions and create oleogels for pharmaceutical use. These effects are due to the formation of a three-dimensional network between the silica particles via hydrogen bonding, thus imparting a paste-like consistency, especially with oils, depending on the polarity of the oil [During the design of semi-solid formulations for administration to the oral mucosa, the choice of appropriate vehicle and the selection of adequate excipients play essential roles. They influence the quality of the dosage form, its stability, drug release and efficacy. The choice of an optimal vehicle depends on many factors, such as the characteristic of the active substance, required therapeutic activity and stability of the final product. The appropriate vehicle should be smooth, inert, odorless, tasteless, physically and chemically stable and compatible with the active ingredient. It should neither irritate nor sensitize the oral mucosa. Moreover, it should be of such a consistency that spreads easily when stress force is applied and should also remain in the application site for a long time . In gel  the oil ,39. Algi the oil .The prepared gel formulations with MET and MMET were inspected visually for their color, homogeneity, consistency and phase separation. Hydrogels and oleogels were white or yellow (as a result of linseed oil used in the formulation). Formulations showed good homogeneity, smooth consistency with the absence of syneresis and they were easily spreadable. MET or MMET were evenly suspended in the bases. Stable and homogeneous bigels  were obtained only using the hydrogel to oleogel weight ratio of 70:30. Prepared bigels were smooth, homogenous, slightly yellow in color, creamy in appearance and they were not greasy in touch. In bigels formulated with a 50:50 weight ratio, syneresis (phase separation) 24 h after preparation was observed. In the case when a 30:70 proportion of hydrogel to oleogel was used, the consistency of the bigel was too firm and it was characterized by poor spreading.The average content of active substance was in the acceptable USP pharmacopoeial limit (90%–110% of the labeled amount) for semisolid formulations containing metronidazole . The ranThe pH of a product may influence the solubility and stability of an active ingredient in the formulation. The pH values of prepared formulations with MET and MMET were in the range from 6.0 to 8.9, and the lowest values were observed in the case of oleogels . MoreoveSignificant differences in the size of suspended particles were observed . In prepThe dosage form designed for local administration to the oral mucosa should be characterized by proper viscosity to provide semi-solid consistency, which will guarantee good spreadability and enable sufficiently long contact time with the mucosa. It may also influence the textural properties  and the release of the drug. Prepared gel formulations were found to have significantly different viscosity values  defines the work required to deform the product during compression of the probe, and it is correlated with the spreadability on the oral mucosa. Firmness and compressibility quantify sample deformation under compression and shear. They are related to sample consistency. Low values of firmness and compressibility ensure that minimum work is required to remove the formulation from the container and to administer it at the site of application. Adhesiveness is the negative area covered by the force–time curve, and its value represents the work required to remove the probe from the sample, which is connected with the breaking of cohesive bonds. In the case of semi-solid formulations for use on the oral mucosa, higher values are more favorable to ensure prolonged adhesion ,27,28.Placebo formulations possessed slightly lower firmness, compressibility and adhesiveness compared with gel formulations containing MET . The addBioadhesion refers to the adhesive interaction between the polymeric drug delivery system and the mucus covered oral cavity. If the adhesion is on a mucosal surface, then the phenomenon is called mucoadhesion. Strong adhesion can occur if two surfaces are able to form either covalent or ionic bonds. Additionally, weaker forces, such as polar (dipole–dipole), hydrogen bonding or van der Waals interactions are involved in bonding the two surfaces . DifficuIt was observed that all formulations were characterized by bioadhesive properties, which might be explained by the formation of hydrogen bonds between the proteins present in the mucosa and the polymer chains used as gelators. The results of the ex vivo study carried out using the porcine buccal mucosa as a model adhesive layer are presented in max and Wad were noticed in the case of bigels, characterized by high viscosity and high values of mechanical parameters. This is in accordance with other authors who showed a correlation between rheological and textural results, that is, an increase in the interaction between a highly viscous sample and mucus [max values in bigels and hydrogels, while in the case of oleogels, significant growth of Fmax was observed. Moreover, bigels containing drug were characterized by modestly higher Wad compared to placebo, while for hydrogels and oleogels, a decrease in the examined value was noted. Higher values of bioadhesion parameters in case of some placebo gels compared with MET loaded formulations might indicate that active substance hinder polymer contact with the mucous membrane.The greatest values of Fnd mucus ,45,46. Fnd mucus . Furthernd mucus ,49. On tnd mucus . AdditioFrom the in vitro dissolution studies, it is possible to determine how the type and composition of the formulation affects drug release. Drug release from semi-solid preparations depends on several parameters such as the nature of the vehicle, its viscosity, solubility of the active compound in the vehicle and acceptor medium, as well as drug partition coefficient between the vehicle and water. The type of the semi-solid vehicle determines the ability of the acceptor fluid to penetrate into the formulation; it is considered that the release rate of hydrophilic and moderately hydrophilic active substances usually increases with hydrophilicity of the vehicle in the following order: hydrophobic base  < emulsion  < hydrophilic base (hydrogels) .The release of MET from different types of gel formulations is demonstrated in 2 and 22 mg/cm2. Moreover, these results are due to the increase of hydrophilicity of formulations that facilitated the penetration of the release medium into the vehicle and promoted diffusion of the active substance. Oleogels demonstrated the slightest drug release . The release rate from hydrophobic oleogels was slower than from hydrogels or bigels, owing to the partitioning of the MET between aqueous (release medium) and oil phase (oleogel). It should be also noted that micronization process improved release of MET for all prepared gel formulations, which was particularly evident in the case of bigel.It was shown that the amount of released MET from oleogels was significantly the lowest and it can be ranked in the following descending order: C  > HMMET > HMET ≈ BMMET > BMET > OMMET > OMET. The largest amount of released MET was observed for the control formulation (ointment base on PEG) and hydrogel containing micronized substance (HMMET); after 5 h, the cumulative amount of released metronidazole was, respectively, 25 mg/cm2) and the values of the release exponent (n) were used to determine the best fit model. Zero-order kinetic describes the drug release rate from dosage forms independent of the drug concentration. First-order kinetic illustrates the dependency on the drug concentration i.e., when the drug concentration is high, the release rate is faster. In the case of Higuchi and Korsmeyer–Peppas models, release rate is variable in time, and these models are founded on the assumption of the accordance of the release with Fick’s law [Mathematical models play a crucial role in the interpretation of the mechanism of drug release from a dosage form and aid understanding of the drug release kinetics. In order to better characterize the drug release behavior, the mechanism of drug release from designed gel formulations was analyzed according to various mathematical models, namely zero order kinetic, first order kinetic, Higuchi model and Korsmeyer–Peppas. The correlation coefficient . The most fitted model to explain the drug release from all designed preparations was the Higuchi equation  were in the range of 0.992–0.999. Higuchi’s model expresses diffusion of the drug in accordance with Fick’s law, where the rate of diffusion through the membrane is affected by the difference in drug concentrations across the membrane.The Higuchi kinetic plots were found to be fairly linear, as indicated by their highest regression values, and for all prepared formulations correlation coefficients values .It was found that the highest antibacterial activity against both The drug permeation profile from tested semi-solid formulations and commercially available product across an excised porcine buccal mucosa is presented in 2, which may be confirmed by the high mucosal permeability of MMET, as reported in the literature [2/h) than from all other tested formulations, which most probably resulted from the presence of PEG acting as penetration enhancer and solubilizer. Interestingly, the bigel formulation with 30% lipid component (considered as a factor limiting drug solubility and in consequence its absorption), displayed high initial drug penetration rate. Moreover, despite higher viscosity and more firmness structure (2/h) was comparable to the value obtained for hydrogel (280 µg/cm2/h), suggesting enhanced diffusion of drug through the bigel matrix. Significant differences in MMET permeation from tested formulations were observed after 5 h. Hydrogel exhibited a higher level of cumulative permeation (approximately 1200 µg/cm2 after 5 h of incubation), whereas significantly lower permeation of MMET through mucosal tissue was observed for the oleogel preparation (about 330 µg/cm2). This observation may be attributed to profound differences in drug solubility in aqueous media and linseed oil  showed lower drug release rate than the hydrophilic (hydrogel) and the mixture of hydrophilic and hydrophobic bases (bigel). It was found that higher antibacterial efficiency against tested strains was observed in the case of hydrogels and bigels. An ex vivo permeation study using porcine buccal mucosa as a model membrane demonstrated that bigel formulation was characterized by high initial permeability rate to assure fast therapeutic effect with simultaneous moderate retention in mucosal tissue to decrease the risk of local cytotoxicity. Results of the study showed that the tested bigel containing micronized drug seemed to be a promising and effective form for local delivery of metronidazole.


Cell growth is driven by the synthesis of proteins, genes, and other cellular components. Defining processes that limit biosynthesis rates is fundamental for understanding the determinants of cell physiology. Here, we analyze the consequences of engineering cells to express extremely high levels of mCherry proteins, as a tool to define limiting processes that fail to adapt upon increasing biosynthetic demands. Protein-burdened cells were transcriptionally and phenotypically similar to mutants of the Mediator, a transcription coactivator complex. However, our binding data suggest that the Mediator was not depleted from endogenous promoters. Burdened cells showed an overall increase in the abundance of the majority of endogenous transcripts, except for highly expressed genes. Our results, supported by mathematical modeling, suggest that wild-type cells transcribe highly expressed genes at the maximal possible rate, as defined by the transcription machinery’s physical properties. We discuss the possible cellular benefit of maximal transcription rates to allow a coordinated optimization of cell size and cell growth. Cell physiology is characterized by global parameters such as cell growth rate and cell size. These global parameters depend on the integrated function of biochemical, molecular processes that function inside cells to synthesize its various components. Cells coordinate synthesis rates by controlling the fraction of proteins allocated to each process. This allocation is defined by the proteome composition that best optimizes cellular fitness. In microorganisms, maximal growth rates are particularly important for fitness, which is optimized in combination with additional demands such as a rapid response to changing environments. Given these constraints, a fundamental, yet poorly understood question is what restricts the typical values of cell growth rate and size: why do rapidly growing bacteria or yeast divide at time scales of tens of minutes, rather than seconds or days? What sets the typical size of these microorganisms to tens of microns, rather than millimeters or centimeters?One possibility is that the typical quantitative parameters characterizing cell physiology are set by mechanistic constraints that limit the biosynthesis rates within the cell. Ribosome elongation rate, for example, defines the absolute minimal division time: during balanced growth, doubling time must be long enough to allow one translation complex to reproduce itself. This theoretical upper boundary cannot be realized in cells since the translation of additional endogenous proteins other than ribosomes is required for performing the various cellular activities. Still, the measured growth rate in rapidly growing yeast and bacteria do fall in the range (30–50% deviation) predicted by this theoretical maximum of ribosome-only production , SAGA complex, and the SWI/SNF complex. By contrast, the transcription signature of burdened cells had no apparent resemblance to that of cells deleted of translation factors, including ribosomal components. We confirmed the phenotypic relevance of the correlation between burdened cells and mediator mutants by demonstrating epistatic interactions between the burden and mediator mutants. Of note, only ∼5% of the DNA-bound Mediator localized to the engineered mCherry locus. The relative mediator binding to the rest of the genome was mostly unchanged, refuting the possibility that the burden phenotype resulted from competition for limiting Mediator. Examining the overall absolute transcript abundance, we find that burdened cells increase the amounts of endogenous transcripts, perhaps as a consequence of their larger size. The increase in endogenous transcript abundances was mostly uniform between genes, but the proportional increase failed at highly expressed genes, and genes associated with bursty transcription. This lower relative abundance of rapidly transcribed genes explained the similar transcriptional signature between burdened cells and mediator mutants.Our results, together with data from the literature and mathematical modeling, suggest that transcription is limiting in wild-type cells growing in standard conditions. We demonstrate that wild-type cells transcribe some genes at rates that are close to the maximal possible rate, as defined by the elongation velocity of the RNA polymerase and its footprint on the DNA. Consequently, transcription rates at rapidly transcribed genes cannot increase further, together with the general transcriptional increase we observed in the burdened cells. To rationalize this finding, we asked what the benefit of maximizing mRNA production could be. Modeling these effects, we suggest that transcribing close to the biochemical limit allows cells to maximize cell size while maintaining the evolutionarily-optimized proteome composition that defines the allocation of proteins between the different cellular functions.S. cerevisiae used in this study were constructed on the genetic backgrounds of: BY4741 (MATa his3-∆1 leu2-∆0 met15-∆0 ura3-∆0), BY4742 , or Y8205  in corresponding amounts. Nitrogen limiting medium was prepared from YNB without amino acids and ammonium sulfate (BD 3101130) by supplementing 50µM of ammonium sulfate and the essential amino acids. The various media’s pH values were: SC = 5.0 .All strains of 3pr-LEU2) using st3pr-LEU2) or SC megeneΔ::KANMX) using UPTAG and DNTAG primers as described in the Yeast Deletion Project (http://www-sequence.stanford.edu/group/yeast_deletion_project/usites.html). The deletion was then validated with primers A, B, and kanB.Deletion and double deletion strains created for validation experiments were derived from BY4741 using the LiAc/SS DNA/PEG method described . In eachp34_TDH3 and p69_TDH3 were crated as described in . mCherry flow cytometry was conducted with excitation at 488nm and emission at 525 ± 25nm for GFP samples. For mCherry markers, excitation was conducted at 594nm and emission at 610 ± 10nm. The average number of cells analyzed was 30,000.C. The initial OD was set to ∼0.05, and the WT initial frequency was ∼50% of the total population. Following growth in the specific condition, the number of generations was calculated from the dilution factor. Frequencies of GFP vs. mCherry cells were measured by flow cytometry. The cells were diluted once a day and may have reached a stationary phase. A linear fit of the log2 for the WT frequency dynamics was used to calculate the slope for each competition assay. The relative fitness advantage is derived from the slope divided by log2. The ‘% of WT division rate (µ)’ is 1 + fitness advantage. Each strain percentage of µ-WT was presented against its mCherry levels from the second day of the experiment or against its copy number calculated from the mCherry levels. Experiments were performed in 96 well plates.Cells were grown overnight to stationary phase. A wild-type reference GFP positive strain was then co-incubated with each of the mCherry burden strains at 30°S), Figure S4C) according to the equation:Epistatic interactions were performed as described previously , 100 mM EDTA 0.5 M, and 100 U/ml lyticase]. The lysis mixture was transferred to a 96-well plate that was incubated at 30° for 30 min. The plate was then centrifuged for 10 min at 3000 rpm, and the supernatant was transferred to a 96-well plate provided by the Nucleospin 96 RNA kit, followed by extraction as described in the kit protocol. Labeled cDNA was created from RNA extracts, and cDNA was barcoded and then sequenced in the Illumina HiSequation 2500 system, using a Truseq SR Cluster Kit v3 -cBot-HS cluster kit and a Truseq SBS Kit v3-HS run kit (50 cycles).As described in Processing and analysis of sequenced RNA were as described in med12Δ as follows: several double deletion strains were created as described above in Strains in addition to burden library generated as described above in Protein burden libraries creation. Strains were grown overnight at 30° with shaking until saturation. Next, at time point 0, the tubes were strongly vortexed for 30sec following OD600 measurement every few seconds, as indicated in Flocculation assays were performed on the background of oC, with shaking to ≈OD0.6-0.8. Next, cells were washed in ice-cold PBS without Ca++ and Mg++ followed by resuspension in 2mM DSG  and agitation for 30min at room temperature. 1% formaldehyde was added, and cells were crosslinked for an additional 5 min. The crosslinking was stopped by adding glycine to a final concentration of 125mM and incubating at room temperature for 5 min. Cells were washed twice with ice-cold DDW  and flash frozen. ChIP was performed as in oC and subsequently incubated with antibody-coupled beads overnight. Later, lysates were washed on magnet with five rounds on lysis buffer, twice with cold buffer W1 , twice with cold buffer W2  and twice with cold TE . Then lysates were eluted with direct elution buffer  at 65oc, O.N with maximal shaking. Finally, DNA was purified by the addition of 2µl RNaseA (10mg/ml), 37oc, 1hr, followed by the addition of 1µl glycogen and 2.5µl Proteinase K (20mg/ml) to each sample, 37oc, 2 hr. Proteinase K Inactivation by incubation at 80°c for 20min.Cells were grown overnight at 30DNA from the previous step subjected to SPRI cleanup with SPRI beads 2.3x and eluted with 10mM Tris-HCL pH 8. DNA libraries for Illumina NextSeq 2500 sequencing were prepared as in S. cerevisiae  and pBS69 plasmid. Genomic tracks were created from the sequence reads, representing the enrichment on each position of the joined genome. Physical fragment length was estimated by the shift best aligning the mapped sequenced reads from both ± strands, and single-end sequence reads were then lengthened accordingly (in the range of ≈100-130bp). The signal regions are defined as -500bp before the TSS to the TTS . A fixed amount of ODs of S. paradoxus cells was added to twice as many ODs of each S. cerevisiae sample, such that the OD ratio between them is constant throughout the samples. The mixed samples were then flash frozen.S. cerevisiae and S. paradoxus sum-of-reads and then to the number of cells as measured in the experiment, as described earlier. Twelve repeats in SC and six repeats in Low Pi/N. Shown is the mean value with +- SE.RNA extraction and library preparation were performed as described above, and the fastq files were then processed by a pipeline for RNAseq data that was created by Gil Hornung , as described in  were constructed on a synthetic genetic array ready strain and were integrated into yeast libraries using the synthetic genetic array method . A RoToRC, and a single quarter was replicated separately into 80µl SC 386 well plate. The 386 well plates were left shaking at 30oC overnight. The following day, the plates were diluted 1:80 and mixed 1:3 (low:high burden) using TECAN Freedom EVO© robot. The mixed plate was left shaking at 30oC for ∼4-4.5 hr for a final OD of ∼0.5-1.5. Then the plate was read in FACS in a “high-throughput” mode for GFP and mCherry with an average of ∼50,000 cells.For the screening, two 1536 well plates (plates corresponding to the same genes in the control library and the protein burden library) were taken out from the 4°Two biological repeats of the whole GFP-burden libraries were produced  and S5F.In Silico according to their mCherry level by manual gating, and for each subpopulation, the median GFP was calculated. The ratio of the two libraries was plotted against the control protein abundance, and a trend line was calculated using MATLAB MALOWESS function for the proteins whose fluorescence was above the autofluorescence (∼200[A.U]).For each protein in the two biological repeats, cells were divided https://doi.org/10.6084/m9.figshare.12014937 and fileS2-4 at https://doi.org/10.6084/m9.figshare.12093624.Strains and plasmids are available upon request. The paper’s raw data are available in fileS1 at: Cells modify their gene expression when subjected to genetic or environmental perturbations. Often, the expression signature of such cells provides a sensitive probe of the perturbation. Accordingly, the expression signature of cells forced to express excessive amounts of inert mCherry protein could reveal the internal pathways and limitations inflicted by this burden. We previously constructed a library of budding yeast cells, with each strain containing a different copy number (between 1-20 copies) of genomically integrated pTDH3-mCherry constructs. These strains produce mCherry proteins at increasing levels , peakinggE, Previous studies described genes whose expression correlates with the growth rate over a wide range of genetic or environmental perturbations . Mutants that affect the same process often exhibit epistatic interactions . To examNext asked whether burdened cells show phenotypes that are similar to those exhibited by mutants of the Mediator’s head or tail sub-complexes. Cells deleted of the Mediator Kinase inhibitory sub-complex are pseudo-hyphal and flocculate when growing in liquid media . We, theTaken together, the pattern of epistatic interactions between burden and mediator mutants is consistent with the similarities in their gene expression profiles and flocculation phenotypes.The similarities in gene expression and phenotypes between burdened cells and mediator mutants may be explained if mCherry production depletes the Mediator from endogenous promoters. To examine this, we measured the genome-wide binding profiles of three Mediator head and tail subunits using ChIP-Seq. Binding patterns at endogenous genes were insensitive to the burden . The fact that burdened cells increase in size and in protein content .S. paradoxus spike-in as a normalization standard. As we hypothesized, total absolute mRNA content in the burdened cells was significantly  higher than in wild-type . We found that protein-burdened cells increase the amount of endogenous mRNA, probably as a consequence of their larger size, caused by perturbed size-regulation. This increase, however, is limited in highly expressed and bursty genes. The transcription signature of burdened cells can, therefore, be explained by their inability to induce further the expression of genes that are already transcribed close to their limit. However, we do not think that the majority of growth defects we describe come from this change in expression. Multiple factors contribute to growth reduction, including the (passive) dilution of ribosome concentration, as discussed in our previous study . Available data suggest that this limit is relevant for Why would cells transcribe genes close to this upper bound of maximal transcription? Could there be a functional benefit in maximizing mRNA production?We suggested that this optimization allows cells to grow in steady-state conditions to maximize their overall protein content (and cell size) while maintaining the internal distribution of proteomic groups that are compatible with optimal growth. Indeed, as part of this optimal growth, the proteome fraction dedicated to translating ribosomes is defined. The ribosomes will be able to translate efficiently only as long as sufficient mRNAs are available as a substrate (Note that the relationship between cell size (protein content) and cell growth relates to evolutionarily optimized conditions. It does not, however, capture changes in cell growth or cell size caused by genetic perturbations. Indeed, the later does not comply with optimal conditions, and accordantly, shows varying relationships depending on the precise perturbation.Whether cells work close to this limit of maximizing the ribosome number is not clear: Budding yeast expresses an estimated 200,000 ribosomes, compared to 35,000 transcripts  are also caused by cell cycle arrest , for which cells can still maintain optimal growth. The maximal possible initiation rate, which limits this production, may, therefore, serve as a fundamental physical constraint, limiting cell size. This is analogous to the time of ribosome translation, which is the fundamental unit defining the cell growth rate. These two physical constraints on transcription and translation, set by the basic biochemical parameters inherent to these processes, may define the characteristic values of the division time and size of rapidly proliferating cells.


To investigate conditions that cause temporal lens opacity, we tested chemical and physical factors, such as anaesthesia dose, ocular surface dryness, and infrared (IR) light exposure in anaesthetised C57BL/6 N mice. Mice were anaesthetised with a low  or high  dose of anaesthetic and examined every 5 min from 10 to 30 min after anaesthesia was induced. Lens opacity levels were assessed and graded (1–6) using the standard classification system. Regardless of the anaesthetic dose, lens opacity grade was 1–2 in moisturised eyes with application of 0.5% carboxymethylcellulose, and 5–6 in dry ocular surface conditions. Lens opacity in mice with high-dose anaesthetic in the dry ocular surface condition was not different from that of mice with low-dose anaesthetic. Lens opacity grade 1–2 was noted in eyes in the wet ocular surface condition, regardless of IR light exposure. During IR light exposure in eyes in the dry ocular surface condition, lens opacity (grade 6) in mice with high-dose anaesthetic was not different from that (grade 6) in mice with low-dose anaesthetic. We demonstrated that ocular surface dryness might be a relevant factor for the formation and progression of lens opacity in anesthetized C57BL/6 N mice. Anaesthesia dose and IR light exposure did not strongly influence lens opacity formation. Furthermore, eyes with corneal dryness-induced lens opacity recovered to normal status without additional intervention. During observation of the posterior segment of the eye, transparency of the ocular media is essential, because opaque medium, especially lens opacity, significantly affects accuracy and results of measurement. Lens opacity is induced by genetic, developmental, and environmental cues. It is sometimes difficult to discriminate short-term lens opacity from cataracts that permanently cloud the lens and impair vision. Interestingly, 90 genes were annotated as associating with abnormal lens morphology in the International Mouse Phenotyping Consortium database, whereas 51 genes were similarly annotated by the German Mouse Clinic.Because many genes have been annotated as associating with lens abnormalities, there is a need to determine the various factors causing them. It has been reported that various chemical and physical factors, including drugs, anaesthetics, oxygen supply, calcium, pH stress, and dehydration, affect the formation of lens opacity in mice and rat –4. FurthTherefore, in the present study, we aimed to investigate the effect of anaesthetic dose, ocular surface dryness, and the presence or absence of infrared (IR) light on the formation of lens opacity in C57BL/6 N mice. We documented the time course of lens opacity formation by using the Micron Image-Guided Spectral-domain optical coherence tomography (OCT) system  and slit-lamp biomicroscopy incorporated into the OCT device (Phoenix Research Labs) Fig. . We alsoMale C57BL/6 N mice, aged 14–16 weeks, were supplied by Jackson Laboratories . Mice were maintained in an experimental animal facility under specific pathogen-free conditions at Yonsei University College of Medicine . Mice were housed under a 12-h light/dark schedule  with ad libitum access to autoclaved food and water; they were treated humanely and with regard for the minimisation of suffering.Mice were anaesthetised with a low  or high  dose of anaesthetic and examined every 5 min from 10 to 30 min after anaesthesia was induced. Pupils were dilated with 0.5% tropicamide/0.5% phenylephrine mixed eye drop  immediately after anaesthesia. Then, eyes with clear ocular media, as defined by OCT and slit-lamp biomicroscopy, were assessed. Lens opacity were imaged by the Micron Image Guided Spectral-domain OCT system after applying special instrument for examining the anterior segment and slit-lamp biomicroscopy which is incorporated with OCT system.All experimental procedures were completed in the Department of Laboratory Animal Resources of Yonsei Biomedical Research Institute and Yonsei University College of Medicine. First, we modified the previous simple classification system described by Bermudez et al. to develop our own lens opacity severity classification system . Fig. 2 Eight groups of mice (at least three mice per group) were used to investigate the effect of three factors on the formation of lens opacity, including anaesthetic dose, ocular surface dryness, and IR light exposure Fig. . AnaesthBriefly, to test whether ocular surface dryness affects lens opacity in the absence of IR light exposure (only exposed during OCT examinations), we did not apply preservative-free artificial tears for a total of 30 min. Conversely, the corneas were kept moist with application of preservative-free 0.5% carboxymethylcellulose every 1 min after anaesthesia was induced with a low or high dose of anaesthetic. The same experiments were performed in the presence of IR light exposure (continuous exposure from 10 to 30 min after anaesthesia). Next, to examine whether anaesthetic dose affects lens opacity, mice were anaesthetised with a high or low dose of anaesthetic and examined without IR light exposure, every 5 min from 10 to 30 min after anaesthesia was induced in each ocular surface condition. The same experiments were performed in the presence of IR light exposure.n = 5). Then, lens transparency was assessed at 0, 1, 2, 4, and 6 h after induction of lens opacity of grade 5–6. Additionally, moderate lens opacity was induced by IR light exposure for 40 min in mice without anaesthesia (n = 5). In the same manner, lens transparency was assessed at 0, 1, 2, 4, and 6 h after induction of lens opacity of grade 3–4.Finally, we explored the reversibility of lens opacity. Lens opacity of grade 5–6 was induced by ocular surface dryness after anaesthesia was induced with a low dose of anaesthetic . Differences were considered to be statistically significant at This study was conducted in strict accordance with and adherence to the relevant national and international guidelines regarding animal handling as mandated by the Institutional Animal Care and Use Committee (IACUC) of the Yonsei University Health System . The committee has reviewed and approved the animal study protocol (#2011–0137). All experimental protocols were conducted in accordance with the tenets of the Declaration of Helsinki and the Association for Research in Vision and Ophthalmology (ARVO) Statement on the Use of Animals in Ophthalmic and Vision Research.To evaluate degrees of lens opacities resulting from various factors, we collected sample pictures from all image data and established classification standards consisting of six grades of lens opacity. Lens opacity initially formed in the centre of the anterior lens and progressively expanded towards the periphery Fig. . A modifNext, we investigated whether anaesthetic dose affected the formation of lens opacity. Mice were separated into two groups and administered a low (80%) or high (120%) dose of anaesthetic, respectively. First, without IR light (exposure solely during OCT examinations), mice were examined every 5 min from 10 to 30 min after anaesthesia was induced with application of preservative-free 0.5% carboxymethylcellulose  every 1 min. The grade of lens opacity in eyes with a high dose of anaesthetic was similar (grade 1–2) to that of eyes with a low dose of anaesthetic   was observed in the dry ocular surface condition at 20 min after anaesthesia, despite the application of a low dose of anaesthetic .Next, mice were anaesthetised with a high dose of anaesthetic and examined bilaterally every 5 min from 10 to 30 min after anaesthesia. The right eye was kept moist with regular application of preservative-free artificial tears, and the left eye was kept dry throughout the experiment. Compared with mice that received continued application of moisture to the eye  began to be observed at 20 min after anaesthesia; lens opacity of grade 6 (three of three mice) appeared at 30 min after anaesthesia  of eyes with lens opacity of grade 5–6 induced by ocular surface dryness began to regain transparency at 1 h; all lens opacity was completely reversed to grade 1 by 6 h Fig. a. These In the present study, we investigated conditions which cause formation of lens opacity in anaesthetised C57BL/6 N mice using an OCT system and slit-lamp biomicroscopy incorporated within the OCT system. The results demonstrated that ocular surface dryness might contribute to the formation and progression of lens opacity in anaesthetised mice. Furthermore, the formation of lens opacity induced by ocular surface dryness was a reversible phenomenon.Among many factors, anaesthetic drug, dehydration of the ocular surface, and temperature are involved in the formation of lens opacity , 2, 4. IMany anaesthetic drugs are known to inhibit the natural blink reflex and to retract the eyelids, consequently disrupting tear films –4, 13. TWe also investigated the effect of IR light exposure on the formation of lens opacity. In both wet and dry ocular surface conditions, IR light exposure and non-exposure resulted in similar levels of lens opacity. Taken together, our results indicate that the avoidance of tear film dehydration is crucial for effective reduction of the incidence and severity of the formation of lens opacity during mouse eye experiments , 5.The phenomenon of reversible lens opacification in mice has been published by many researchers –4, 14. IA previous study reported that the formation of lens opacity significantly increased with lower body temperature, concluding that exposure to ≤23 °C caused cataracts in wild-type SV129 male mice . Bec. Bec1]. The methodological limitation of this study was its small sample size. To validate our results, a larger sample size for each group is warranted. Considering the importance of ocular surface hydration and ocular fluid homeostasis in the formation of lens opacity, further investigations of the relationships between ocular surface dryness and other confounding factors should be performed; moreover, analyses of changes in ocular fluid content and ion concentrations are needed. Because ketamine/xylazine anaesthesia in rats can produce acute hyperglycaemia, associations between lens opacity and a variety of metabolic changes after anaesthetisation with ketamine/xylazine also need to be explored .We demonstrated that ocular surface dryness might be a primary factor in the formation and progression of lens opacity in anaesthetised mice. This opacity was restored to normal status spontaneously after several hours. We believe that our results may enable researchers to minimise the formation of lens opacity during ocular phenotype studies in mutant mice. In addition, our data provide an important element of the protocol to maintain optical transparency during mouse eye experiments.