In the last years, difficulties occurring in corn cultivation  have been forcing dairy farmers to consider alternative silages. Some experiments conducted on lactating cows have proven that the total replacement of corn silage with sorghum silage did not reduce milk yield. However, this kind of substitution involves supplementing sorghum-based diets with grains, to compensate for the lower starch content of sorghum silage compared to corn silage. Change of silage type and inclusion of starch sources in the diet would influence rumen fermentations, with possible effects on milk composition  and coagulation properties. A worsening of milk coagulation properties would have a negative economic impact in Italy, where most of the milk produced is processed into cheese.This study was designed to compare milk composition and quality, with emphasis on fatty acid profile and coagulation properties, in dairy cows fed two diets based on corn or sorghum silage.P = 0.043) but not 4% fat corrected milk (P = 0.85). Feeding sorghum silage did not influence milk contents of protein (P = 0.07) and lactose (P = 0.65), and increased fat content (P = 0.024). No differences emerged for milk concentrations of saturated (P = 0.61) and monounsaturated fatty acids (P = 0.50), whereas polyunsaturated fatty acids were lower (P < 0.001) for the sorghum diet. Concentrations of n-6 (P < 0.001) and n-3 fatty acids (P = 0.017) were lower in milk of cows fed the sorghum diet. Milk coagulation properties did not differ between the two diets, except the “a30” , that was lower (P = 0.042) for the sorghum diet.The sorghum diet reduced milk yield (Feeding a forage sorghum silage, properly supplemented with corn meal, as total replacement of corn silage maintained milk composition and did not influence negatively milk coagulation properties, which have a great economic relevance for the Italian dairy industry. Thus, silages obtained from forage sorghums could have a potential as substitute of corn silages in dairy cow diets. Corn silage is the main ingredient of diets fed to lactating cows in the farms of the Po Valley , with exception of those producing milk processed into some Protected Denomination of Origin cheeses . However, because of some difficulties occurring in corn cultivation , in the last few years dairy farmers have been considering the use of alternative forages for ensiling. Experiments conducted on lactating cows have proven that the total replacement of corn silage with sorghum silage did not affect milk yield –4. HowevIn this framework, this study was designed to compare milk yield, composition, and quality, with particular emphasis on milk FA profile and MCP, in dairy cows fed two experimental diets containing silages obtained from corn or forage sorghum.Medicago sativa). The soil belonged to a farmhouse located in San Martino di Lupari, Province of Padova, latitude 45.3°N, longitude 11.5°E; elevation: 40 m above sea level. The corn was fertilized with 200 units of Nitrogen, 140 units of Phosphorous, and 180 units of Potassium/hectare, and weed control was applied with 1.5 L/hectare of herbicide Adengo . Corn was harvested at two-thirds milk line stage of maturity on 30 September, 2014. Plants of forage sorghum  were sown on an adjacent field in the same farmhouse as above on 17 June, 2014 on a soil that previously hosted barley. The sorghum was fertilized with 160 units of Nitrogen, 80 units of Phosphorous, and 100 units of Potassium/hectare, and weed control was applied with 0.3 kg/hectare of herbicide Casper . Sorghum was harvested at the beginning of the early bloom stage, on 30 September, 2014. No irrigation was needed for either forages due to the favorable climate situation. For both corn and sorghum a forage harvester with 8 line-head for chopping  was used. Corn plants were harvested with knives adjusted to a 13-mm theoretical length of cut and processed with a 3-mm roller clearance. Sorghum plants were harvested with knives adjusted to a 20-mm theoretical length of cut, without any processing. After harvest, corn and forage sorghum were ensiled into bunker silos without inoculation and covered with nylon film for 60 d. Sample of silages (n = 4 per corn and sorghum) were obtained collecting about 3 kg of silage from 4 different points in the front of each bunker silos. The chemical composition of the two silages is given in Table Corn plants  were sown on 29 May, 2014 on a soil that previously hosted alfalfa . Eighteen Holstein-Friesian dairy cows , housed in a tie-stall commercial dairy farm belonging to the farmhouse described above , were used. Animals were randomly assigned to 2 groups subjected to 2 treatment sequences in a cross-over experimental design of 28-d periods. Each experimental period was preceded by a 10-d preliminary period, thus the whole experiment lasted 76 d. Each group received a total mixed ration (TMR) based on corn silage (CS diet) or forage sorghum silage (SS diet). Two silages were included in different proportions , according to their chemical composition. The SS diet contained an amount of corn meal that was almost double compared to the CS diet (254 vs. 142 g/kg DM), in order to compensate for the lower starch content of forage sorghum silage compared to corn silage. The two TMR were prepared using a total mixer wagon. During preparation of TMR, the two experimental diets were only mixed, without a further chopping of silages. The diets were formulated to be isoenergetic and isonitrogenous using the Plurimix system® , and according to NRC system [The amount of diet distributed in the manger of each experimental group was measured daily and recorded by the weighing station of the mixer wagon. The orts were daily collected and analyzed for DM in order to obtain the daily DM intake (DMI), on a group basis, as difference between consumed and residual DM. Individual milk yield was recorded and individual milk samples (50 mL/each) from the morning milking only were collected from each cow to be analyzed for composition, FA profile, and MCP . Representative samples (about 3 kg as fed) of the two silages and of the two diets were collected to be analyzed for chemical composition and particle size distribution. Feed and diet samples were collected at the beginning and at the end of each period (n = 4 samples for each silage and diet).220 Fiber Analyzer . The ADF, inclusive of residual ash, and ADL contents were sequentially determined according to [Samples of silages and diets were analyzed in duplicate for DM  according to the standard FIL-IDF148a [2 (somatic cell count × 10−5). Milk lipids were extracted according to the procedures described by [2 as carrier gas (flow of 0.22 mL/min). The second was a 3.8 m × 250 μm  × 0.25 μm film thickness column  and used H2 as carrier gas (flow of 22 mL/min). A Computerized Renneting Meter  was used to determine MCP within 5 h after collection of samples. Milk samples (10 mL) were preheated at 35°C, and 200 μL of rennet  were diluted to 1.2% (v/v) in distilled water and then added to the milk. This analysis provided measurements of rennet coagulation time , k20 (the time to observe a curd firmness of 20 mm after the rennet was added to the sample), and a30 (curd firmness 30 min after rennet addition). Measures of pH  were conducted before measuring MCP.Fat, protein, and lactose contents of the milk were analyzed using the FIL-IDF procedure  by Milkoribed by . Fatty aribed by . The firThe particle size distribution of silages and diets was determined by dry sieving using a Penn State Particle Separator equipped with three screens . Approximately 250 g of feed sample was placed on the upper screen of the separator and sieved according to the procedure described by .Mean particle size of silages and diets was calculated according to . The phyAll individual data were preliminarily analyzed using the TTSET procedure of  to invesCompared to the corn silage, the sorghum silage showed, as expected, a lower DM content  for the SS diet, whereas yields of 4% fat corrected milk (P = 0.85), fat (P = 0.78), protein (P = 0.23), and lactose (P = 0.07) did not differ between the two diets. The yield of SCM was comparable for the two diets (P = 0.59) and resulted 31.6 and 31.0 kg/d, for the CS and the SS diet, respectively. In terms of milk composition, the SS diet resulted in a greater fat content  and a nominally greater protein content  compared to the CS diet. Lactose, urea and SCS in milk were not influenced by the diet. Except for the value of a30, that was lower  in the SS diet, milk coagulation properties did not differ between diets (Table P = 0.008).The DMI did not differ between the two diets (P = 0.97).Feed efficiency, expressed as the ratio between the SCM and the DMI, was similar for the two groups of cows  for the SS diet in comparison to the CS diet (Table P < 0.001) for the SS diet compared to the CS diet. Among saturated FA, concentrations of butyric , caprylic , palmitic , and stearic acid  were greater in the milk of cows fed the SS diet; opposite trends were observed for capric (C10:0), and lauric acid (C12:0). Among polyunsaturated FA, concentrations of n-6 and n-3 were lower in milk of cows fed the SS diet . The ratio n-6/n-3 also resulted lower (P < 0.001) for the SS diet compared to the CS diet. Conjugated linoleic acid isomers (CLA) content, expressed as the sum of the single isomers, was greater (P = 0.004) in the milk of cows fed the SS diet.Total concentrations of saturated FA and monounsaturated FA in milk did not differ between the two diets  when cows received the CS diet . However, differences disappeared when milk production was corrected for milk fat content, as the SS diet favored a + 7% increase of fat percentage compared to the CS diet. Positive effects of sorghum silage on milk fat percentage have been observed by others [In this experiment milk yield was greater (+1.8 kg/d) when cows received the diet based on corn silage. Such a result is in accordance with findings of  and it cy others . In the y others  for cowsThe absence of effects on milk protein, in terms of percentage and production (kg/d), is confirmed by . In the Results of this study highlighted that milk FA profile was clearly influenced by the diet, with notable changes in proportion of the individual fatty acids. Milk was richer in PUFA when the cows were fed the CS diet, whereas the total contents of SFA and MUFA were not influenced by the dietary treatment.The current literature encompasses several studies that evaluated effects on milk FA composition due to the replacement of corn silage with grass silages . HoweverEffects of corn silage on milk FA profile are well documented, but their interpretation is often complicated by the inference of chemical characteristics of corn silage and other feed ingredients included in the ration .In this study, the two experimental diets differed for NDF and starch concentrations. Such a result was mainly related to the use of a forage sorghum that, as expected, had contents of NDF and starch, respectively, much greater (711 vs. 349 g NDF/kg DM) and much smaller (26 vs. 352 g starch/kg DM) compared to corn silage. In this context, inclusion of a greater amount of corn meal in the SS diet was not sufficient to compensate chemical differences between the two diets.The addition of starch sources  to the ration is often associated with a decrease in milk FA with 6 to 16 carbons, and to an increase in unsaturated FA with 18 carbons . AccordiIn addition to starch and NSC, also the dietary fat content and composition exert an influence on milk FA profile . In thisPossible effects on milk FA profile due to the lactation stage, parity, or farming conditions can be excluded, because the two groups of animals were balanced for the first two parameters and farming conditions were the same for all the cows.Despite the different effects on milk FA profile, the SS diet produced some improvements on the nutritional value of milk in comparison to CS diet. Particularly, the SS diet increased concentration of some FA with anticarcinogenic properties, such as butyric (C4:0), which is mostly protective against colon cancer, and CLA . On the The role of feeding on influencing MCP is still uncertain, even if there is evidence that these properties are more influenced by genotype of animals than by phenotypic factors such as feeding . HoweverFeeding a forage sorghum silage, properly supplemented with corn meal, as total replacement of corn silage, maintained DMI, milk yield, and feed efficiency intended as the ability of cows to convert feed to milk. The substitution of corn silage with sorghum silage did not change the concentration of saturated and monounsaturated fatty acids, but reduced the concentration of polyunsaturated fatty acids in milk. In addition, n-6 and n-3 fatty acids resulted lower in milk of cows fed the sorghum diet as compare to cows fed the corn diet. Milk coagulation properties, which have a great economic relevance for the Italian dairy industry, were not altered by the substitution of the corn silage with the sorghum silage in dairy cows. These preliminary results suggest that forage sorghum silages could have a potential as substitute of corn silages in dairy cow diets.


Results show that bioaerosols induce Reactive Oxygen Species (ROS) production, varying along the microorganism type, species, and concentration. Fungal spores show up to 10 times more ROS generation than bacterial cells. At the highest concentrations, fungal spores present as much oxidative reactivity as the most redox-active airborne chemicals . Moreover, bioaerosols substantially influence OP of ambient PM and that of its chemical constituents: in presence of A. fumigatus spores, the OP of Cu/NQ is increased by a factor of 2 to 5, whereas, 104 and 105 S. epidermidis bacterial cells.mL−1 halves the OP of Cu/NQ. Finally, viable and gamma-rays-killed model bioaerosols present similar oxidative reactivity, suggesting a metabolism-independent cellular mechanism. These results reveal the importance of bioaerosols for PM reactivity. PM toxicity can be modified due to bioaerosols contribution or by their ability to modulate the OP of toxic chemicals present in PM.Bioaerosols represent up to 15–25% of PM by mass, but there is currently no assessment of their impact on Oxidative Potential (OP), or capacity of particulate matter (PM) to produce damaging oxidative reactions in the human lungs. Here, the OP of selected bioaerosols (bacteria cells  Exposure to PM has been linked with a wide range of deleterious health effects in both humans and animals. These include among others, cardiopulmonary disease, lung cancer, or asthma5. Atmospheric concentration of PM is regulated , but more than mass, detrimental effects are strongly associated with PM composition, surface area and size8. The atmospheric research community is currently working on alternatives to the mass concentration of PM to find relevant health and exposure metric, involving more PM properties10. One underlying mechanism explaining many health effects of PM is its capacity to carry out or catalyze the formation of reactive oxygen species (ROS) within lung cells, responsible for further oxidative stress and airway inflammation12. This intrinsic property of PM called oxidative potential (OP) is easily revealed in biochemical and acellular assays by monitoring anti-oxidants depletion when in contact with PM.Atmospheric particulate matter (PM) is defined as a complex and dynamic mixture of particles from both chemical and biological origins15. They comprise living and dead microorganisms such as bacteria, fungi, viruses, bacterial and fungal spores, and microbial fragments, endotoxins, mycotoxins, pollens etc.16. Microorganisms in bioaerosols can survive as single cells or attached to dust particles or hydrated aerosol particles. Most bioaerosols range in size from micrometer to submicrometer19. Due to their light weight and size range, bioaerosols can be transported over long distances19, for example, transferred by wind, making them complex, highly variable and ubiquitous in both indoor and outdoor environments. Cultivable bioaerosols  have been detected at high concentration in indoor and outdoor air23. Recent findings indicate that in most areas, bioaerosols can account for up to 15–25% of total PM mass24 and higher concentrations have been reported in Amazonian areas (from 74 to 80% of PM mass)24. Their concentration and composition fluctuate primarily depending on the sources impacting the site under consideration , the aerosolization mechanisms, and the physical and environmental conditions  prevailing at each site25. Usually, airborne fungi exist as spores while bacteria are cells26. Bioaerosol concentration in indoor/outdoor air ranges from 101 to 105 microorganisms.m−327. Higher concentrations (up to 105–107 microorganisms.m−3) have been measured in rainforest environments or polluted areas 28.While the physical and chemical properties of PM have been extensively investigated, relatively little is known about primary biological airborne particles (PBAP) also known as bioaerosols10 bioaerosol fraction since evidence is growing about their implication in negative effects of PM both on climate 16 and on human health29. Inhalation is the primary route of exposure to bioaerosols30. Inhalation of high concentrations of fungal spores, bacterial cells, and their derivatives in indoor/outdoor environments is associated with serious inflammation-related health risks such as asthma, chronic obstructive pulmonary disease, aspergillosis, and immunological reactions26. Bioaerosols can also be responsible for infectious diseases such as tuberculosis, meningitis, and legionellosis25. In addition, dose-response relationships exist between inhalation of airborne microorganisms and the development or exacerbation of asthma symptoms in humans32. As for the chemical components of PM, it is worth noting that the pathogenicity of bioaerosols also depends on the number of inhaled microorganisms, their chemical composition, and their size33.More recently, attention has been given to the PM34, while the impact of bioaerosols are unaccounted for. For example, Charrier and Anastasio (2015) attributed about 30% of overall OP of PM to unidentified components of submicron fine particles collected in Fresno during summer12. So, a reasonable hypothesis could be that bioaerosols may account for part of this unexplained fraction. In support of this, Vaitlingtom et al., (2013) among others have shown biodegradation of hydrogen peroxide (H2O2) by cloud-borne microorganisms36, highlighting that cloud-borne microbial communities could also interact with H2O2 oxidants (or other kinds of ROS) involved in the oxidative stress.Studies addressing the OP of PM are currently focusing only on the chemical fraction34 was firstly adapted to viable bioaerosols and reliability of the results was evaluated with ascorbic acid (AA) and the 2′,7′-dichlorofluorescin diacetate (DCFH) cell-free methods. Then, OP of isolated model bioaerosols (cells and spores) and that of model bioaerosols associated with real ambient PM and highly toxic model airborne chemicals (copper and 1.4-naphtoquinone) were assessed based on the acellular DTT assay, using realistic environmental concentrations of both bioaerosols and ambient or model PM. Linearity as a function of concentration of PM  was also investigated. The final purpose of this work was to begin to explore some of the main processes by which bioaerosols may contribute to the overall OP of PM.Therefore, our study was conducted in order to initiate the assessment the contribution of bioaerosols to the oxidative potential (OP) of ambient PM. Within this framework, measurement of OP with the acellular DTT assay38. Bacterial cells induced a very low DTT oxidation (~10–20 pmolDTT.min−1) within the 30 min. experiments, but this rate tended to increase with increasing concentrations. Figure All of the tested model bioaerosols induced DTT depletion, which varied among the tested microorganisms and concentrations Fig. . LikewisA. fumigatus presented a significant oxidative reactivity for all tested concentrations, consistently with the results of several in vivo and in vitro studies assessing the inflammatory potential of non-pathogenic fungal spores involved in airway inflammation40. Shahan et al., (1998) demonstrated that the secretion of pro-inflammatory proteins  by rat alveolar macrophages is differentially induced by fungal spores39. For instance, in this last study, among the tested Aspergillus species, the spores of A. fumigatus were able to stimulate the production of pro-inflammatory cytokines  while those of A. terreus were not. Other studies assessing the toxicological impacts of bioaerosols collected in various occupational backgrounds indicated that all sampled bioaerosols  are able to increase the production of ROS of the “granulocyte-like HL60” cell lines42. The intensity of ROS production was dependent upon the nature and amount of bioaerosols42. Indeed, Timm et al., (2009) demonstrated through a multiple regression modeling that the inflammatory potential induced by fungal spores better explains the total inflammatory potential of bioaerosols41, thus supporting the significant differences in antioxidant depletion observed between the bacterial cells and fungal spore tested in our study.Among the tested fungal spores, only those of 25. To the best of our knowledge, there is currently no regulated exposure limit for bioaerosols although several recommended maximum limits have been proposed44. For instance, based on inflammatory respiratory effects in animal models, Eduard et al.,40 have proposed a “lowest observed effect level” (LOEL) of 105 spores.m−3 for non-pathogenic and non-mycotoxin producing fungal species. When converted to liquid concentrations  and 1 µM naphtoquinone (NQ) in absence or presence of 48. Once deprotonated, these groups become anionic and therefore available for complexation of cationic chemicals (such as Cu2+) in aqueous solution. The processes involved in the limitation of quinones’ oxidative potential could be different, as quinones can diffuse freely into the lipid bilayers of the cells walls, where they can be trapped51, thus becoming less available for reacting with DTT. Moreover, some studies have evidenced the formation of hydrogen bonds between the reactive functional groups present on bacterial cells walls and quinones or derivatives49. These two types of interactions between S. epidermidis cells walls and NQ or Cu may explain the lower reactivity of NQ (or Cu) in presence of important concentrations of S. epidermidis cells.This protective effect may be explained by the interaction of these chemicals (adsorption or uptake) with bacterial cells, thus reducing their availability for reacting with DTT. Numerous studies have shown that bacterial cells walls present huge amounts of reactive sites  capable of chemical complexationP. fluorescens and Micrococcus sp. but only at a concentration of 105 cells.mL−1 . However this was not the case for NQ, for which intrinsic oxidative reactivity decreased in the presence of the bacterial cells. For example, in the presence of Micrococcus sp., the oxidative reactivity declined by almost 40% with the mixture as compared to that calculated as an individual sum from NQ and Micrococcus sp. The discrepancy in effects of the 3 tested bacterial species on oxidative reactivity of Cu or NQ could be due to differences in cell wall composition . The cell wall of Gram positive bacteria  is known to contain important amounts of peptidoglycan  as well as of teichoic and teichuronic acids (containing carboxyl groups)46. On the contrary, Gram negative bacterial cells (e.g. P. fluorescens) present external membranes with many proteins , much less peptidoglycan and lack teichoic and teichuronic acids47, thereby being potentially less reactive towards Cu or NQ.The same experiment was performed with A. brasiliensis, A. fumigatus, S. chartarum and Penicillium sp. The results observed for A. brasiliensis are shown in Fig. 4 and 105 spores.mL−1 of A. brasiliensis, respectively. Similar increases in oxidative reactivity, but of variable intensity, were observed with the other fungal spores  was assessed in the presence of increasing concentrations of airborne spores of see Fig. . These oA. brasiliensis spores and Cu, NQ, or both chemicals, with the sum of DTT depletions measured with individual biological and chemical species used at the same concentrations. The results show good agreement between the DTT depletion measurements obtained with both methods  remained equal to the sum of individual reactivity. Similar patterns were also obtained with ambient PM collected in a very different environment which is a subway platform in Toulouse, France, , based on the in vivo study of immune competent mice, which demonstrated that A. fumigatus spores induce comparable inflammatory cell influx before and after being heat-killed52. This last study also showed that active metabolism is not critical for the underlying process, which is probably mediated by β-glucans exposed at the outer spore-walls52. Other findings suggested more complicated processes implying an “unknown” protein54. For instance, by comparing control groups with mice inoculated with viable and non-viable P. chrysogenum spores, Cooley et al., (2000) indicated that mice exposed to viable spores produce significant total IgE serum whereas that exposed to non-viable spores did not53. This suggests that the process is not related to the spore/cell wall constituents, because the same constituents are found onto both active and non-viable microorganism wall. Noteworthy is that the precise underlying mechanism is still not clearly understood55.Several vs fungal spores), the species, and the cells/spores concentrations. At the highest tested concentrations of 104 and 105 spores.mL−1 , fungal spores present oxidative reactivity similar to those of the most redox-active airborne chemicals (e.g. Cu and NQ) and up to 10 times more reactivity than bacterial cells. At concentrations equal or lower than 103 microorganisms.mL−1 (which could translate into the most current indoor/outdoor bioaerosol levels), all of the tested model bioaerosol presented insignificant OP, except A. fumigatus which is an opportunistic pathogens. (2) The OP of mixtures of fungal spores with natural PM or model airborne chemicals (Cu or NQ) are similar to the sum of individually measured OP values, thus confirming a cumulative effect of the OP of mixed reactants. (3) Bacterial cells (S. epidermidis) strongly reduced the OP of Cu and NQ, in relation with a protective action of these bacteria against oxidation, probably related to cell wall chelation of these chemicals, which thereby decreases their availability for reacting with DDT. This mechanism has been widely considered for modeling interactions between bacterial cells and heavy metals, referring to a surface complexation process. (4) Finally, experiments with gamma-rays inactivated airborne microorganisms have shown that oxidative mechanisms occurring at the surface of fungal spores and bacterial cells are probably non-metabolic (passive) as viable and gamma-rays inactivated cells or spores presented similar OP values. Although further studies are needed to better understand the underlying mechanisms, these results suggest that ROS production by bioaerosols is probably independent of cell′s metabolism.This study aimed at evaluating the intrinsic oxidative reactivity of model bioaerosols  through the use of an acellular assay (DTT depletion), and their role in the overall Oxidative Potential of atmospheric PM. Four main results were obtained. (1) The tested model bioaerosols presented intrinsic oxidative reactivity, which varies with the type of microorganisms  and their byproducts , pollen, plants debris etc. which can positively or negatively modulate the OP of PM. It would be important to fully assess the impact of bioaerosols on health for the initiation of health alerts by authorities, since both the type of airborne microorganisms and their concentration were shown in the present study to control the oxidative reactivity of natural PM.57. For bacteria, two Gram positive cocci (Staphylococcus epidermidis and Micrococcus sp.) and one Gram negative rod (Pseudomonas fluorescens) were selected. Concerning fungi, spores of Stachybotrys chartarum and Penicillium sp. and of Aspergillus brasiliensis and Aspergillus fumigatus were used. These fungal species are commonly used in exposure models because of their frequent occurrence in indoor air contamination55.Some common atmospheric bacterial and fungal species have been selectedA. fumigatus is involved in various health issues such as severe invasive aspergillosis in immune-compromised people, or in allergic bronchopulmonary aspergillosis. S. chartarum is of particular interest in indoor environments due to its ability to colonize damps and to produce mycotoxins (trichotecenes) responsible for respiratory and neurological effects58. It should be emphasized that all selected microorganisms are commonly found in outdoor or indoor environments.P. fluorescens, S. epidermidis and A. brasiliensis. The other species were isolated from urban and natural environments: indoor for Micrococcus sp., S. chartarum and Penicillium sp., and outdoor for A. fumigatus. Air samples were collected using a one stage Andersen impactor at a flow rate of 28.3 L.min−1 for 4 minutes59. These microorganisms were directly impacted onto solid culture media: Malt Extract Agar culture for fungi and Trypticase Soy Agar for bacteria. Plates were incubated at 27 °C for 5 days. Colonies were identified by macroscopic and microscopic examinations, using lactophenol blue stain for fungi and Gram staining for bacteria.Commercial reference strains  were used for For each previously isolated and identified microorganism species, a single colony was cultured on biological culture media: Luria-Bertani  for bacterial cells and Malt agar  for fungal spores. The inoculated culture plates were incubated at 30 °C, for 2 and 5 days, for bacterial cells and fungal spores, respectively. This step was intended to collect the cells and/or spores in their exponential growth phase See Fig. .4] = 1 M; pH = 7.4 ± 0.1). This cells-cleaning process was repeated three times.For bacterial cells, a single colony of each isolated strain was inoculated in 10 mL Luria Bertani liquid medium  and grown at 30 °C for 2 days, with agitation at 150 rpm. Preliminary experiments indicated that the liquid culture media were not suitable for the cell-free OP assays, due to very fast and important depletion of antioxidant in the negative controls. These media contain reactive chemical compounds, such as transition metals (e.g. Fe), which are known to cause high DTT depletion. To avoid such interferences during the DTT assay, bacterial cells suspensions were centrifuged for 10 min at 5000 g (at ambient temperature). The bacterial pellet was suspended aseptically in 10 mL of sterile chelex treated phosphate buffer , which were not investigated in the present study. Hence, after fungi culture onto solid culture media for 5 days, fungal spores were collected in sterile ultrapure deionized water (UP Water), by gently scraping the surface of culture medium with sterile swabs. The swabs were soaked in 10 mL UP water and stripped by vortexing, in order to recover the maximum of spores. This process was performed several times to get enough spores without incorporating culture medium in spores’ suspensions.60, in order to obtain enumerations with estimated errors lower than 15%.To ensure that there was no contamination during the cells/spores culture steps, culture purity was visually controlled by optic microscopy : 10 µL of microbial suspensions and serial dilutions (1:10 to 1:10000) were loaded between glass slides and coverslips. The presence of single shape cells was verified systematically and the number of cells/ spores was determined in order to determine liquid concentrations: at least 100 bacterial cells or fungal spores were counted in several microscope fieldsTo evaluate if the oxidative potential of airborne microorganisms is linked with active or passive metabolic mechanisms, some model bioaerosols were inactivated with gamma-rays. Briefly, microbial suspensions prepared in glass tubes were irradiated with gamma-rays at 10 kGy for 16 h . After irradiation, cell wall integrity was checked by optical microscopy and the absence of cell viability was also tested by culturing aliquots of the gamma-irradiated samples, which revealed always negative.10 samples were collected on quartz filters with a high-volume air sampler (DA-80) at a flow rate of 30 m3/h over 24 h. Sampling was achieved in different environments in France: Passy 61 and Toulouse (subway station).PMTo evaluate the effect of bioaerosols on the OP of ambient PM, 5 mm diameter punches of filters were extracted in UP water by shaking for 2 hours at 37 °C in the dark. Filter extracts were then centrifuged (at 1500 rpm for 5 min) at 4 °C, to separate the filter residue and the supernatant, which was used for the OP measurement.34.For model chemical compounds, appropriate amount of copper (Cu) and 1,4-naphtoquinone (NQ) were also dissolved in UP water by shaking the for 40 min at 37 °C in the dark condition. These chemical species were selected since they are known to be highly redox-active and widely used as positive controls in OP studies63. Briefly, DTT consumption is continuously monitored when in contact with PM, and the depletion of DTT (in excess) is proportional to the concentration of redox-active species present in PM. The protocol established by Charrier and Anastasio, (2012) was adapted to fit our constraints, and significant changes were brought in the sample pretreatment step34.The dithiothreitol (DTT) assay is a cell-free method commonly used to assess the oxidative potential of airborne redox-active chemical compoundsvs time, as shown in Fig. DTT indicates the amount of DTT at time t (nmol), N0: is the amount of DTT (expressed in nmol) at time t = 0, Abs0: is the absorbance corresponding to the initial amount and Abst: is the absorbance measured at time t.The loss of DTT was monitored with a semi-automated method conducted in 48 wells plates: intrinsic light absorbance of the mixture was measured at 412 nm on a plate-reader (TECAN spectrophotometer Infinite® M200 pro). The procedure was as follows: 50 µL of model bioaerosols suspensions and/or 50 µL of PM extracts were incubated with an appropriate quantity of 1 M phosphate buffer  in a final volume of 550 µL. Triplicates of each sample were used for kinetic studies. The reaction was initiated by adding 50 µL of DTT (0.5 mM DTT solution in phosphate buffer) to each well (including the negative controls). The reaction was stopped at 3 specific times  by adding 100 µL of 1 µM 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB). The reaction between the remaining DTT and DTNB gives rise to a yellowish color  which can be monitored at 412 nm (maximum absorbance). Measurement repeatability was assessed by calculating the coefficient of variability (CV) between triplicates. The calculated CV were always lower than 5%. The rate of DTT loss, which indicates the ability of model bioaerosols to produce ROS, was determined from the slope of the linear regression of DTT remaining amount The intrinsic rate of DTT loss for all samples was calculated by subtracting blank values (or negative controls) from each sample value within the same experiment.2 to 105 microorganisms/ml. Such concentrations could correspond to the exposure levels currently observed in both indoor/or outdoor or/and occupational backgrounds (e.g. composting facilities). Table −1 and corresponding values in microorganisms per cubic meter of inhaled air. This conversion was performed with the following equation:3: is the concentration of viable microorganisms in air (microorganisms.m−3); Nb/mL: is the concentration of microorganisms in liquids (microorganisms.ml−1); 20 ml is the estimated volume of lung fluid and 11m3 is the volume of inhaled air within a 24 h, by an adult human30. To simplify the calculation, we assumed that inhaled microorganisms remain into the lung fluid for 24 h, are viable and fully transferred to the deep lungs.The rate of DTT depletion is assessed in presence of increasing realistic environmental concentrations of bioaerosols. Tested concentrations ranged from 10Differences between the rate of DTT loss from single mixture of PM components  and those calculated as the sum of individual rates were analyzed with the non-parametric Wilcoxon rank-sum test. This non-parametric method was chosen because of the small sample size (n = 3). Statistical differences are indicated with asterisks (p ≤ 0.1).Supporting information


Three Mg alloys, Mg–1.34% Ca–3% Zn (MCZ), Mg–1.34% Ca–3% Zn–0.2% Sr (MCZS), and Mg–2% Sr (MS), were examined to understand their bio-corrosion behavior. Electrochemical impedance spectroscopy and polarization scans were performed after 6 days of immersion in cell culture medium, and ion release and changes in media pH were tracked over a 28 day time period. Scanning electron microscopy (SEM) of alloy microstructure was performed to help interpret the results of the electrochemical testing. Results indicate that corrosion resistance of the alloys is as follows: MCZ > MCZS > MS. Previously published work has provided a detailed review of the suggested Mg corrosion mechanisms [Metallic biomaterials commonly employed in orthopaedic applications include titanium and its alloys, stainless steels, and a class of cobalt–chromium–molybdenum based alloys. Although these devices offer superior mechanical strength, they are bioinert, and permanently remain as a foreign material in the body. It is not uncommon that a second surgery for removal of the device, especially in the case of fracture fixation, is necessitated . In ordechanisms .2+ ions has been shown to increase cell adhesion and osteoblastic activity in vitro [Magnesium possesses mechanical properties similar to those of bone tissue . As the in vitro ,7,8,9, ain vitro ,11,12. Rin vitro ,14,15,162 as a partially protective byproduct, leaving Mg materials susceptible to continued corrosion. Uncontrolled Mg corrosion can have detrimental effects in vivo, where it is possible for released hydrogen gas to accumulate in the surrounding tissue, as well as OH− ions increasing the local pH [Although these advantages make Mg and its alloys promising candidates for use as orthopaedic materials, their widespread clinical use has been hindered, based on low corrosion resistance in the physiological environment. Mg corrosion is accelerated in chloride containing solutions , and a hlocal pH . Effortslocal pH . In termlocal pH  and AZ31local pH . More recently, a range of Mg alloys have been created and tested specifically for their use as biomaterials in both orthopaedic and cardiovascular applications . Some prIn order to avoid concerns associated with potential toxicity, Mg alloying elements should be restricted to those that have already demonstrated long-term biocompatibility. For this reason, three Mg alloys will be focused on herein: Mg–1.34% calcium–3% zinc (MCZ), Mg–2% strontium (MS), and Mg–1.34% calcium–3% zinc–0.2% strontium (MCZS). Zinc (Zn) may lead to increased bone formation through enhancing alkaline phosphatase and collagen production , and cal2Sr performed the most favorably when testing a range of Sr additions [The goal of the study was to characterize the bio-corrosion of three Mg alloys containing combinations of Ca, Zn, and Sr in vitro, to contribute to the development of Mg materials for biomedical applications. Previous studies have shown MCZ to display acceptable in vitro corrosion rates , and thidditions , whereasdditions . MS is sEDS point analysis corresponding to the regions indicated in Although the immersion media contained a sodium bicarbonate buffer system, Mg alloy corrosion was still able to produce an alkaline shift in the solution . Media wCumulative ion release as measured by inductively coupled plasma mass spectroscopy (ICP-MS) corresponds to the trends observed for the pH measurements. The results  reflect The open circuit potential (OCP) of the alloys shows a shift towards increasingly electropositive potentials over time . The inicorr and Icorr derived from the polarization scans are presented in corr and more electropositive Ecorr indicate enhanced corrosion resistance of a material. Both MCZ and MCZS have a significantly smaller Icorr and are more electropositive Ecorr when compared to MS. These results point to the MS alloy being the most susceptible to corrosion.Representative polarization scans of each alloy after 6 days of immersion in cell culture medium are presented in Nyquist plots corresponding to the EIS  spectra collected for the Mg alloys after 6 days of immersion in cell culture medium are displayed in S corresponds to the electrolyte resistance, CPE1 models the capacitance of the double layer, and R1 is related to the initial metallic corrosion. The remaining circuit elements can be assigned a physical meaning based on Mg corrosion taking place through a mechanism involving intermediate surface coverage dependent on potential [2 is related to an adsorption pseudo-capacitance whose value is determined by the charge stored in the intermediate film [2 models the resistance to discharge of the intermediate [3, represents the presence of a second adsorbed intermediate able to enhance material corrosion [P) using Equation (1) [PR is inversely related to the corrosion current, and is an important parameter in understanding the electrochemical behavior of a material. Within the circuit model, Rotential ,35. CPE2ate film ,36,37, worrosion . Resultstion (1) :(1)RP=111) than MS, and greater resistance to discharging of the intermediate (R2) than both MS and MCZS. Q2 is significantly greater for both MCZ and MCZS in comparison to MS, perhaps indicating the adsorbed intermediate has an increased capacity for charge storage on these materials. Finally, the calculations for PR suggest that out of the investigated alloys, MCZ displays significantly greater resistance to corrosion processes than MS, with MCZS at intermediate levels.The EIS results show that MCZ has greater resistance to the initial charge transfer step  light for 30 min, for sterilization.SEM was used to examine the microstructure of the alloys. Samples were further prepared by incrementally polishing with glycol diamond suspensions to 1 μm, followed by final polishing with 0.04 μm colloidal silica suspension. Samples were sputter coated with carbon and examined in backscattered electron (BSE) mode with a SEM  equipped with energy dispersive X-ray spectroscopy (EDS) using mapping technique and point analysis. 3) buffer system was present in the αMEM, which should maintain the pH of the solution at physiologically relevant levels, near 7.4. The test electrolyte was replenished every 48 h over the duration of all immersions in an attempt to maintain relevant near neutral pH levels. In order to simulate the conditions of the human body, a cell culture medium (CCM) containing proteins and a bicarbonate buffer was used as the test electrolyte for corrosion studies. The CCM was composed of alpha minimum essential medium  supplemented with 10% fetal bovine serum , Atlanta Biologicals, Flowery Branch, GA, USA), and 1% penicillin/streptomycin (Gibco) . A sodiu2, served as the working electrode, and were positioned between a nylon base and glass tubing with an O-ring providing a watertight seal. 8 mL of the test electrolyte was added to the glass tubing before covering with either a rubber stopper or breathable membrane. The rubber stopper, which included holes for gas exchange, suspension of a graphite counter electrode, and suspension of a silver/silver chloride (Ag/AgCl) reference electrode was used for samples dedicated to electrochemical testing. All potentials reported are with respect to the Ag/AgCl reference electrode. The breathable membrane was used to seal samples for solution measurements. The final surface area to volume ratio for all samples was close to 30 mL/cm2, and the corrosion cells were maintained in a humidified cell culture incubator at 37 °C with 5% CO2. The test electrolyte was exchanged every 48 h during testing.A custom three-electrode corrosion cell was used to assess the electrochemical behavior of the alloys . Resin ecorr, Icorr, and corrosion rate (mm/y). EIS was carried out by applying a 10 mV sinusoidal oscillation about the OCP, over a frequency range of 100 kHz to 5 mHz. The resulting impedance data was fit to an appropriate equivalent circuit model using a complex nonlinear least-squares method . After modeling the EIS data, PR was calculated as described in the results section [PR and Tafel slopes derived from the polarization scans [Appropriate connections were made to a potentiostat  and the open circuit potential (OCP) of the alloys was monitored continuously, over the initial 24 h of exposure, to test the electrolyte. Following 6 days of incubation, electrodes were again connected to a potentiostat. At this point, the OCP was monitored for 20 min, before performing either a polarization scan or electrochemical impedance spectroscopy (EIS). Potentiodynamic polarization scans were executed at a scan rate of 1 mV/s from −0.15 V vs. OCP to 0.5 V vs. OCP. The resulting curves were analyzed to estimate the E section . The Steon scans . The coron scans . Both polarization scans and EIS were conducted on a minimum of 4 samples of each alloy. The electrochemical tests were considered terminal and incubation of the samples was discontinued following test execution.Three samples of each Mg alloy were maintained in the test electrolyte for 28 days, for analysis of solution pH and ion release. At each exchange of the CCM test electrolyte (performed every 48 h), 1 mL was set aside for pH measurement using a micro-pH meter, and 2 mL aliquots underwent an acid digestion in preparation for analysis by inductively coupled plasma mass spectroscopy (ICP-MS) as previously described . Samplesm is the released mass in grams, n = 2 is the charge transfer number, F is the Faraday constant, t is the total time in seconds, and M is the molecular weight. The calculated current was subsequently converted to a corrosion rate (mm/y) to allow for comparison to results obtained from electrochemical testing.Measured ion release of the materials after 6 days of immersion (days 0–6) was used to calculate the corrosion current by applying Faraday’s Law (Equation (2)).Following the 28 day immersion period, alloy samples used to obtain solution measurements were removed from their corrosion chambers, rinsed with distilled water, and allowed to air dry. The resulting surface morphology of metal samples was then examined with a stereo microscope . corr and Icorr as determined by the polarization scan. A p-value of less than 0.05 was considered significant. Statistical software (SPSS) was used to perform all statistical calculations.A Welch’s ANOVA with Games Howell post hoc tests, where appropriate, was used to identify differences in equivalent circuit model elements as determined by EIS, as well as E17Sr2.2% Sr addition to Mg in a binary alloy, MS, is able to cause rapid dissolution of the material, likely through microgalvanic corrosion initiated by increased volume of the second phase MgMCZ showed the lowest corrosion rate of the investigated materials.MCZS alloy displayed slightly increased corrosion in comparison to MCZ, but significantly lower corrosion in comparison to MS.All materials investigated showed evidence of fast corrosion in the physiological environment, likely due to microgalvanic corrosion between second phases and intermetallics formed by the high alloy loading.In order to identify an optimal Mg material for biomedical applications, alloying additions should be reduced during future Mg alloy development.


Rat somatosensory cortex contains a large sexually monomorphic genital representation. Genital cortex undergoes an unusual 2-fold expansion during puberty. Here, we investigate genital cortex development and female rat sexual maturation. Ovariectomies and estradiol injections suggested sex hormones cause the pubertal genital cortex expansion but not its maintenance at adult size. Genital cortex expanded by thalamic afferents invading surrounding dysgranular cortex. Genital touch was a dominant factor driving female sexual maturation. Raising female rats in contact with adult males promoted genital cortex expansion, whereas contact to adult females or nontactile  male cues did not. Genital touch imposed by human experimenters powerfully advanced female genital cortex development and sexual maturation. Long-term blocking of genital cortex by tetrodotoxin in pubescent females housed with males prevented genital cortex expansion and decelerated vaginal opening. Sex hormones, sexual experience, and neural activity shape genital cortex, which contributes to the puberty promoting effects of sexual touch. We recently identified the somatosensory representation of rat genitals; remarkably, this cortical region—genital cortex—is sexually monomorphic, despite the marked sexual dimorphism of external genitals in rats. Most intriguing was the observation that genital cortex doubles in size during puberty. In order to shed light on this unusual expansion, we studied genital cortex development and sexual maturation in the female rat. We first showed that sex hormones are likely to cause the pubertal expansion of genital cortex. Next, we examined whether sexual experience affects the development of female genital cortex. Raising females together with adult males advanced genital cortex expansion, but cohousing with adult females or exposure to nontactile male cues was not sufficient to drive genital cortex growth. Surprisingly, artificial genital touch led to an early onset of female puberty and growth of genital cortex. In line with this finding, we find that if genital cortex activity is blocked, the advancing effects of adult males on puberty and genital cortex growth are inhibited. Together, our results point to an important role of genital cortex in the puberty-promoting effects of sexual touch. Early analysis of the development of visual cortex by Hubel and Wiesel focused on binocular interactions and showed that both anatomy and physiology of ocular dominance is plastic and shaped in a visually-driven, activity-dependent process . In a siWe were also interested in the relationship between somatosensory genital cortex development and female sexual maturation. In rodents, puberty has been intensely studied and much is known about changes in subcortical structures, such as the hypothalamus, that are associated with puberty ; there iIn primates, the evidence for pheromonal effects on puberty is rather mixed . Sexual Here we ask the following questions: (1) Are sex hormones required for the pubertal expansion of female genital cortex? (2) What are the structural events underlying genital map changes? (3) Does sexual experience affect the development of female genital cortex? (4) Does activity in female genital cortex affect female puberty and does it mediate the female puberty advancing effects of male sexual touch?P < 0.01, Student t test). In contrast, the size of the entire S1 did not differ significantly between young and old females .As a first step in our analysis we performed additional experiments to confirm our recently reported data on the growth of genital cortex during puberty . To thisP < 0.01, Student t test) smaller than the genital cortex size in animals, which were ovariectomized after puberty  and after puberty (at P42). Twenty days after the ovariectomy animals were killed, brains were removed and tangential sections through the S1 were prepared and stained for cytochrome oxidase activity . We obtaWe conclude that sex hormones are required for the pubertal expansion of female genital cortex but not for the maintenance of an adult size female genital cortex.n = 7 animals; 0.05μg estradiol, dissolved in 0.4 ml of sesame oil, per 100 g body weight) or with sesame oil alone . During the fifth day of treatment animals were killed and cortical hemispheres were flattened, tangentially sectioned and processed for cytochrome oxidase activity  and the mean percentage of genital cortex of S1  was significantly greater in estradiol injected animals compared with control animals (oil treated). The size of S1 was not different .The lower panel in P < 0.001, Mann–Whitney U test). We also removed and weighed the uteri. On average, the oil-treated animals had lighter uteri . Taken together these data suggest that systemic estradiol accelerates genital cortex growth and advances the onset of puberty.We also assessed parameters indicative of sexual maturation and the onset of puberty. Thus, we evaluated vaginal opening  and theWhich cellular changes underlie the unusual genital cortex expansion during puberty? To answer this question, we first stained alternating tangential sections of flattened cortices for cytochrome oxidase activity  and with2 ± 0.06 and in old animals 0.56mm2 ± 0.1). Interestingly, the ratio of the genital cortex to the interlimb cortex significantly increases during puberty . The simultaneous increase of genital cortex and decrease of interlimb cortex suggest that the pubertal genital cortex growth reflects an invasion of dysgranular interlimb cortex by putative genital thalamic afferents.Next, we asked, how the genital cortex is able to increase its thalamically innervated area so drastically. Does the genital cortex expand like a balloon, pushing the neighboring cortex aside? Alternatively, do genital afferents invade neighboring territories? To address this issue, we analyzed S1 maps from young and old animals in more detail . SpecifiAlthough sex hormones have been identified as important players during the onset of puberty, there is also evidence that somatosensory stimuli (olfactory or tactile) can influence sexual development. Thus, we assessed if sexual experience affects the development of female genital cortex. To this end, we chose the following experimental paradigm, which is inspired by the seminal work of Bronson and Maruniak . PrepubeP = 0.01). The overall size of S1 . Note that the relative size of genital cortex of the young females, which were cohoused with sexually experienced males  and the relative size . The difference between hemispheres from control  and TTX treated animals  was marked . These results immediately suggest 2 conclusions. First, the fact that there was a difference between control and TTX treated animals indicates that our TTX treatment was effective. Second, the data indicate that the pubertal expansion of genital cortex requires cortical activity.In order to better understand the role of female genital cortex during puberty, we blocked genital cortex activity over a certain time during prepuberty P23–P30). To do so, P21 animals received Elvax implants over the area of genital cortex. Elvax sheets were developed for the slow, gradual release of drugs [–P30. To ive size  was smaln = 11), whose genital cortices were blocked with TTX, had an open vagina. Next, we wondered if an intact genital cortex is required for female sexual maturation. Therefore, we determined the status of puberty in control and TTX treated animals. Accordingly, pictures of vagina and clitoris were taken before implantation and after the behavioral experiment. Five out of 13 animals, which received a ringer soaked Elvax sheet above genital cortex, showed an open vagina at the end of the experiment. The vagina before (left panel) and after the experiment (right panel) for one of those females is shown in In order to obtain insight in the relationship between female sexual maturation and genital cortex development, we plotted genital cortex size against uterine weight in all experiments, in which we collected such data . As showOur data confirm a major pubertal expansion of female genital cortex, which—unlike the maintenance of large genital cortex in adults—requires intact ovaries and sex hormones. The larger area of genital cortex in adults reflects an invasion of adjacent territories by thalamic afferents. Such changes in female genital cortex are advanced by sexual touch and require cortical activity. Most interestingly, blockade of genital cortex delays female sexual maturation, suggesting that genital cortex is the neural structure that mediates the puberty advancing effects of male sexual touch.The expansion of layer 4 of genital cortex in puberty  is an unThe data discussed below indicate that at least some stage of cortical activity seems to be required for genital cortex expansion. The visualization of thalamic afferents by VGluT2 antibodies showed that cytochrome oxidase staining reveals maps completely congruent with thalamic innervation. Thus, the size increase of genital cortex in adults indicates that a larger cortical area is innervated by thalamic afferents. The detailed analysis of body maps in young and adult animals is strongly suggestive that the size increase of genital cortex results from an invasion scenario, in which putative genital afferents innervate neighboring dysgranular cortex.Female genital cortex development is strongly affected by tactile sexual experience. Housing pubescent females with a sexually experienced adult male strongly advances the growth of genital cortex. Even though we killed such females at P30 , they had genital cortices larger than average adult size .pm. Rats had ad libitum access to food and water.All experiments were conducted on Wistar rats purchased from Janvier Labs. All animals were kept on a 12 hour to 12 hour, normal light/dark cycle with lights off at 10:00 Ovariectomies were performed in prepubescent (P21) and adult Wistar rats (P42). For the surgery, animals were anesthetized by injection of an initial dose of 100 mg/kg ketamine and 7.5 mg/kg xylazine. Respiration, blink, and pinch reflex were observed throughout the surgery and, if needed, animals were injected with an extra shot (25%) of ketamine/xylazine mixture  or a 25% dose of ketamine (Sigma-Aldrich) alone. Monitoring of temperature was done using a rectal probe and could be maintained with a heating pad  to 34°C–36°C.To remove ovaries a small incision was made bilaterally on the animal’s bag. The white fat pad, to which the ovaries are attached to, was pulled out through the body wall. Ovaries were grabbed with forceps and removed from the fallopian tube using small scissors. After successful removal, the white fat pad with fallopian tube was put back through the body wall. The incision was sutured using a self-resorbing thread. The wound was disinfected and looked after in the days following surgery.Immature Wistar rats (P26) were divided randomly into groups of 3–4 animals. All animals were weighed daily and the vagina opening was assessed by visual inspection and defined as a complete separation of the membranous sheath covering the vaginal orifice . Photos Animals in the testing group were injected subcutaneously every morning with 0.05 μg 17β-estradiol (Sigma-Aldrich), dissolved in 0.4 ml of sesame oil (Sigma-Aldrich), per 100 g body weight. Rats in the control group received the same amount of sesame oil. On day 5 of the treatment, animals were deeply anaesthetized, perfused, and their brains processed, as described below. In addition, uteri of every animal in experiment was taken out while perfusion and weighed.Wistar rats aged P21 were divided into 3 groups. Each animal of group 1 was housed for 9 days together with a sexually experienced adult female rat (approximately P60). Every rat of group 2 was put for 9 days together with a sexually experienced adult male rat (approximately P60). Group 3 rats were also housed together with a sexually experienced adult male rat (approximately P60), but the cages were separated by a wire-mesh. Whereas the animals of group 1 and 2 were able to interact fully with their housing partner, the rats in group 3 could not touch the male they were housed together with. However, they were able to smell, see, and hear the male through the wire-mesh.Vaginal opening was documented as described above, and pictures of clitoris and vagina were taken before and after the experiment. After 9 days, at the age of P30, animals of all groups were anaesthetized, perfused, and their brains histologically processed as described below. Uteri of all animals were taken out while perfusion and weighed.Prepubescent female Wistar rats (P23) were divided into 2 groups. Control animals were placed 3 times for 10 minutes in a small U-shaped arena over 7 days without further treatment and were then returned to their home cage. Animals in the artificial genital touch group were placed 3 times for 10 minutes in a small arena over 7 days, and their clitoris and vulva were contacted with a lubricated brush by a human  experimenter . After Animals (P21) were divided into 2 groups. The control group received a chronic Ringer-impregnated Elvax sheet above genital cortex and the rats in the testing group were implanted with a TTX-impregnated Elvax sheet above genital cortex.Elvax implants were prepared as described before , 37, 38.For surgical implantation P21 Wistar rats were anesthetized by injection of an initial dose of 100 mg/kg ketamine and 7.5 mg/kg xylazine. Respiration, blink, and pinch reflex were observed throughout the surgery and, if needed, animals were injected with an extra shot (25%) of ketamine/xylazine mixture or a 25% dose of ketamine alone. Monitoring of temperature was done using a rectal probe and could be maintained with a heating pad to 34°C–36°C. Lidocaine was locally injected in the scalp, which was then removed. A craniotomy was performed above genital cortex, and the dura was removed using a bend syringe. The TTX or Ringer impregnated sheets were placed on the brain surface and covered with silicone . The exposed skull was finally covered with dental cement. Animals were allowed to recover from surgery for 2 days. Photos of clitoris and vagina were taken before the behavioral experiment. At the age of P23, implanted animals were put together with a sexually experienced male. After 7 days (at the age of P30), TTX and control animals were anaesthetized and their vaginal opening was documented as described above. Along with the perfusion, the uteri of the animals were cut out and weighed. Brains were histologically processed as described below.At the end of the above-described experiments, animals were anaesthetized using a 20% urethane solution and perfused with phosphate buffer followed by a 2% paraformaldehyde solution (PFA). Brains were removed, hemispheres were separated, and cortices were flattened between 2 glass slides separated by clay spacers . Glass sFor the alternating staining procedure with cytochrome c and VGluT2, immunohistochemical labeling was performed using standard procedures. Briefly, brain sections, which should be labeled for VGluT2, were preincubated for an hour at room temperature in a blocking solution . Afterwards, primary antibodies were diluted in a solution containing 0.5% Triton X-100 and 1% bovine serum albumin. The primary antibody against VGluT2 was incubated with the free-floating sections for at least 24 hours under mild shaking at 4°C. Incubations with the primary antibody was followed by detection with a secondary antibody coupled to the fluorophore Alexa 488. The secondary antibody was diluted (1:500) in 0.5% Triton X-100 and the reaction was allowed to proceed for 2 hours in the dark at room temperature. After the staining procedure, sections were mounted on gelatin coated glass slides with Mowiol mounting medium.The area of various somatosensory regions was measured by outlining the anatomical region of interest and calculating its area using Neurolucida area calculating tool. The area of the following cortical representations was measured: hindpaw, forepaw, trunk, interlimb cortex, and clitoris. The fraction of genital cortex of the whole S1 area was calculated by dividing the clitoris area by the value of the S1 area. The same was done for the fraction of genital cortex of the interlimb cortex. All statistic tests were conducted in Matlab.In order to quantify the state of vaginal opening after the above described experiments, we assigned different states with vaginal opening scores. A score of 0 points to a closed vagina, whereas a score of 0.5 describes a vagina, which was about to open. Finally, a score of 1 represents an open vagina.S1 FigA, Upper panel: Tangential section through S1 of a P25 old animal stained for cytochrome oxidase activity. Lower panel: The corresponding map drawn from the section shown above, genital cortex shown in black.B, Same as A, but the section and map stems from an animal aged P42. The genital cortex is larger than in the P25 animal. C, Same as A for an animal aged P42, which was ovariectomized before puberty at P20. Note that the size of the genital cortex is comparable to the genital cortex of the youngfemale shown in A. D, Same as A for an animal aged P60, which was ovariectomized after puberty at P42. The clitoris representation is greater than in the young ovariectomized animal (C) but comparable in size to the P42 aged animal (B).Figure S1 related to (TIF)Click here for additional data file.S2 FigA, Fraction of genital cortex of the entire S1 in hemispheres of P25, P42 females and females which were ovariectomized at either P20 or P42. B, Same as A but fraction of genital cortex of the PMBSF is shown. Note that the same effects are seen as in A, namely a substantial growth of the genital cortex between P25 and P42 animals. Female rats ovariectomized during prepuberty had smaller genital cortices than animals ovariectomized after puberty. Graphically distribution looks similar in A and B and no big difference in the variability can be make out. See also C, Absolute area of PMBSF in hemispheres of P25, P42, in prepuberty (P20) ovariectomized and postpuberty (P42) ovariectomized female rats. See also Figure S2 related to (TIF)Click here for additional data file.S3 FigA, Upper panel: Tangential section through S1 of a hemisphere obtained from an animal which received subcutaneous sesame oil injections over 5 days. The section was stained for cytochrome c and shows the reconstructed clitoris area best. Lower panel: Corresponding reconstructed map from the section plotted above. B, Same as A, but the section and drawn map are obtained from an animal which received daily estradiol injections. Note that the clitoris area is greater compared to the example section and map shown in A.Figure S3 related to (TIF)Click here for additional data file.S4 FigA, Upper, tangential section through S1 of an animal aged P14. The section was stained for cytochrome oxidase activity. Lower, map of the somatosensory areas drawn on the section shown above. B, Upper, adjacent tangential section through S1 obtained from the same hemisphere, which is shown in A. The section was stained with antibodies (green fluorescence) against VGluT2 (vesicular glutamate transporter 2), which is expressed in thalamocortical afferents. Lower, map of the somatosensory areas drawn on the section shown above. Note that the reconstructed areas correspond in size when comparing A and B.Figure S4 related to (TIF)Click here for additional data file.S5 FigA, Genital cortex size is plotted against uterine weight for all experiments. No tight relationship can be observed. Note that a large uterine weight (≥ 0.15 g) is rarely associated with a small (≤ 1%) relative genital cortex size.B, Same as A, but for specific experimental conditions See also Figure S5 related to Figs (TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S1 DataIndividual data for Figs (XLSX)Click here for additional data file.


To evaluate ocular fundus biological changes after scleral collagen cross-linking (CXL) with riboflavin/ ultraviolet A (UVA) on rhesus monkeys in vivo by analyzing retina and choroid biological parameters. Six 3-year-old male rhesus monkeys (12 eyes) were observed in this study, with scleral CXL procedures applied on superior temporal equatorial sclera on random eyes of all rhesus. Optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA) examination were performed before and 1 week, 1 month, 3 months and 6 months after CXL. The thickness of retina and choroid and the flow density of retinal superficial vascular networks were analyzed respectively in different regions after CXL. As for retina thickness and flow density of retinal superficial vascular networks, no statistical difference was noted between CXL eyes and control eyes at 1 day, 1week, 1 month, 3 months and 6 months (P>0.05). Among choroid parameters, the choroidal thickness in 1500μm temporal to the fovea center of CXL eyes revealed a significant reduction in 1 week postoperatively (P<0.05), but it subsequently increased from 1 month postoperatively, and no statistical difference was found between two groups in the following periods (P>0.05). The choroidal thickness nearby crosslinked region may change temporarily following scleral CXL, and it might recover gradually after 1 month postoperatively. The vascular flow density and thickness of retina were not affected by scleral CXL. Further study should be performed to evaluate the potential adverse effects at the direct vicinity of the application site and the long-term effect of scleral CXL in clinical application. Myopia is one of the most common ocular disorder and has become the leading cause of visual impairment and blindness in Asian countries –2. The p2 for 30 min could lead to serious side effects in retinal tissue [2 for 30 min, there were no structural damage in retina of cross-linked rabbit eyes [2 for 200s) effectively prevents occlusion-induced axial elongation in a rabbit model [Since corneal cross-linking (CXL) using riboflavin and ultraviolet A (UVA) became an increasingly common therapy for stabilizing the cornea and preventing keratoconus progression –9, this l tissue . When thbit eyes . Recentlit model . The expit model .However, previous researches mainly focused on changes of scleral biomechanical properties and histopathological evaluation after scleral CXL to estimate the efficacy and safety of this treatment. Some literature also investigated changes of intraocular pressure (IOP) on scleral reinforced eyes, since it was known that IOP is closely associated with myopia –17. But The experimental study included 6 male Rhesus macaque monkeys (Macaca mulatta) with a mean age of 3.0±0.4 years (range 2.8–3.5 years) and a mean weight of 4.9±0.5 kg (range 3.6–6.2 kg). All rearing and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Capital Medical University (AEEI-2014-127) and were in compliance with the Association for Research in Vision and Ophthalmology Statement for the use of animals in ophthalmic and vision research. All the animals used in this study were housed in groups of between 10 and 25, with windows allowing the animals to view the outside. Animals were in a 4.3 square foot cage measuring 24 (L) x 27 (D) x 32 (H). Cages were made of stainless steel. The temperature of the housing room was maintained at 25–27°C and the relative humidity was 45~55%. Regular changes of equipment and toys were provided. Wood chip substrate was on the floor to provide opportunity for foraging. Foods were provided three times a day and water was available ad libitum. Facilities were in the rom to allow swinging and jumping and engaging in activities from floor to ceiling. Monkeys were health checked daily throughout the experiment. Before recruiting into the experiment, all rhesus monkeys were given comprehensive ocular examination, including anterior segment and fundus examination, to exclude any ocular disease. Rhesus monkeys were not sacrificed in order to be followed up for a long term observation.Both eyes of the monkey were examined clinically before, and 1 week, 1 month, 3 months and 6 months after the scleral CXL operation. For all examinations and surgeries, the animals were anesthetized with an intramuscular injection of ketamine hydrochloride  and xylazine hydrochloride (0.2 mg/kg body weight), with repeated injections of ketamine (10 mg/kg) as needed during the examination. While the measurements were being taken, the eyelids were gently held apart by eye speculum and the tear film was maintained by the frequent application of artificial tears . Cycloplegia was achieved by topically instilling 1–2 drops of 5% tropicamide (Tropicamide phenylephrine eye drops) at 5 min interval 30 min before performing retinoscope, OCT and OCTA.According to our previous scleral CXL laboratory technique , 20, eac2 for 30 min  at a distance of 5 cm from the sclera. The control eyes were exposed and dropped by photosensitizer solution without UVA irradiation. After surgery, the sutures around muscles were removed, and the conjunctiva was closed using a vicryl 7–0 thread . The animals were monitored till awakening and kept in cages. 0.3% Gatifloxacin eye gel was applied after surgery and four times a day in 1 week postoperatively to avoid infection.Photosensitizer solution containing 0.1% riboflavin  was dropped minutely onto the irradiation zone 20 min before and during the 30 min irradiation period. UVA irradiation (365 nm) was applied perpendicular to the sclera of CXL eyes by a UVA device  with a surface irradiance of 3.0 mW/cmThe intraocular pressure (IOP) was measured using the TonoVet™ rebound tonometer  at about 9 AM. According to the manufacturer’s recommended procedures, the equipment was programmed to average the IOP values of six consecutive, acceptable measurements and produce a reading of the mean IOP. Five readings  were obtained per eye, and the mean IOP value was calculated and reported. Each animal was positioned in a seated posture in the process of measuring IOP.The refractive status of each eye, including spherical and cylindrical components, was measured by an experienced investigator using streak retinoscope  and recorded. Refractive errors were reported as the spherical equivalent (SE) in diopters (D).The axial length of each eye was measured by A-scan ultrasonography implemented with a 10-MHz transducer . Rhesus monkeys were in the supine position with the head immobilized by a head holder. The device was programmed to average the values of eight acceptable measurements and produce a reading of the mean. Three readings were recorded and averaged.Both eyes of each rhesus monkeys were scanned using the Spectralis SD-OCT device . The scan was performed on ART 30 frames including 768 A scans. The automatic eye tracking technology maintains fixation on the retina. Only well-centered images with a signal strength of >20db were used for analysis.Macular and retinal layers thickness were reported in an Early Treatment of Diabetic Retinopathy Study macular map (ETDRS), and the 1, 3 and 6 mm rings were considered for the analysis. The 1 mm ring was defined as central thickness. The intermediate and outer rings were divided into four zones designated as superior, nasal, inferior, and temporal. The numerical values recorded for each of the nine zones were used in the analysis of retina thickness.With enhanced depth imaging (EDI) and manual caliper provided by the software, choroidal thickness (CT) values were measured from the outer border of the hyper-reflective RPE to the inner aspect of the sclera at seven locations: beneath the foveal center and at 500 -μm intervals up to 1500μm temporal and nasal to the fovea center.All images were taken by the same trained examiner and all measurements were repeated 3 times at each point.Both eyes of each rhesus monkeys were scanned using the RTVue XR with AngioVue , with a light source centered at 840 nm, a bandwidth of 50 nm, and an A-scan rate of 70,000 scans per second. Scans of macular areas were acquired and the scan size was 6.0 mm × 6.0 mm.Flow density of retinal superficial vascular networks was calculated automatically using the above software and was defined as the average de-correlation value as previously described –23. FiveAll images were taken by the same trained examiner and all measurements were repeated 3 times at each point.Statistical analysis was performed using SPSS for Windows, version 19.0 . The measurements were presented as mean ± standard deviation (SD). Paired t-tests were used to evaluate ocular biological parameters differences between cross-linked and control eyes in different pre-/post-operative periods. In all tests, statistical significance was defined at a level of P < 0.05.A total of 12 eyes of 6 male rhesus monkeys were enrolled into this study, 6 eyes in scleral CXL group and 6 eyes in control group. At the initial measurement, there were no statistically significant differences in the thickness of retina and choroid and the flow density of retinal superficial vascular networks between the two groups (each P>0.05).No signs of inflammation were observed after scleral CXL and throughout the follow-up period. After surgery, all rhesus monkeys had normal corneas, anterior chambers and clear lenses. No vitreous or retinal lesion was observed clinically.The optical coherence tomographic images in CXL eye and control eye were shown in Figs 2, 30 min) was applied in the present research for it has been generally studied and applied both in vivo experiments of scleral CXL [Controversy exists regarding the safety of scleral collagen cross-linking (CXL) by riboflavin/ultraviolet A (UVA), such as the ultraviolet-induced retinal damage. Compared with accelerated CXLs using shorter treatment times with higher UVA irradiations –25, the eral CXL , 13, 20 eral CXL –27. Accoeral CXL –13, the eral CXL . In thisIn the analysis of retinal thickness, SD-OCT was performed pre- and postoperatively. There was no statistical difference between CXL eyes and control eyes at different pre-/post-operative periods in retinal thickness of the nine ETDRS subfields (each P>0.05). These outcomes indicated that the retinal thickness in measured area was not affected by scleral CXL in rhesus monkeys.Flow density is the index reflecting quantitative measurement of vascular density . In the The choroidal thickness in 1500μm temporal to the fovea center of CXL eyes revealed a significant reduction in 1 week postoperatively (P<0.05), but it subsequently increased from 1 month postoperatively, and no statistical difference was found between two groups in the following periods (each P>0.05). In other zones, there was no statistical difference between two groups postoperatively (each P>0.05). In previous studies –32, manyIn this study, no statistical difference was noted in IOP, spherical equivalent and axial length between two groups in different pre-/post-operative periods. Thus, considering the sclera is a kind of tissue with sparse vasculature and penetrated by blood vessels supplying the choroid, we speculate that local capillaries within scleral tissue might be affected temporarily by CXL surgery, leading to reduced blood flow in irradiation zone. Therefore, the local choroidal circulation could be transiently impaired. It is possible that the decrease choroidal circulation caused thinner choroidal thickness in acute postoperative phase, and it may return to the normal level concomitant with the autonomic regulation , 35 subsPotential limitations of our study should be mentioned. First, retinal and choroidal parameters measured in vivo in this study is limited to posterior pole of eyeball, where is not underlying the treatment. However, there has not been any OCT equipment providing the software for measurement in equatorial region so far. Potential adverse effects at the direct vicinity of the application site should be investigated in further histological examination. Second, whereas manual choroidal thickness measurement is one of drawbacks of this study, until now, no automated measurement software is supplied by existing equipment. Finally, these results might be valid for rhesus monkeys, and it has not been clarified whether the results are applicable in other species.In conclusion, the present study found that the choroidal thickness near crosslinked region may change temporarily following scleral CXL. This may be the result of reversible transient microcirculatory dysfunction of the choroid, and it might recover gradually after 1 month postoperatively. The vascular flow density and thickness of retina were not affected by scleral CXL. The potential role of scleral CXL in the effect of this change remain to be investigated. Further study should be performed to evaluate the pathological result and long-term effect of scleral CXL in clinical application.S1 Table(XLSX)Click here for additional data file.


Anesthesia management for patients with severe ankylosing spondylitis scheduled for total hip arthroplasty is challenging due to a potential difficult airway and difficult neuraxial block. We report 4 cases with ankylosing spondylitis successfully managed with a combination of lumbar plexus, sacral plexus and T12 paravertebral block.Four patients were scheduled for total hip arthroplasty. All of them were diagnosed as severe ankylosing spondylitis with rigidity and immobilization of cervical and lumbar spine and hip joints. A combination of T12 paravertebral block, lumbar plexus and sacral plexus block was successfully used for the surgery without any additional intravenous anesthetic or local anesthetics infiltration to the incision, and none of the patients complained of discomfort during the operations.The combination of T12 paravertebral block, lumbar plexus and sacral plexus block, which may block all nerves innervating the articular capsule, surrounding muscles and the skin involved in total hip arthroplasty, might be a promising alternative for total hip arthroplasty in ankylosing spondylitis. Ankylosing spondylitis (AS) is a chronic and progressive autoimmune disease . It mainCase 1: A 38-year-old male  was scheduled for right total hip arthroplasty (THA) due to severe bilateral rigid and sore hip joints. He had a history of ankylosing spondylitis for more than 18 years. Despite a long term usage of sulfasalazine and “painkiller”, the patient developed a rigid kyphotic and scoliotic deformity. An overall physical examination revealed rheumatoid arthritis involved most articles in the body resulting in limited movement and swelling of interphalangeal joints, metacarpophalangeal joints, elbow joints, shoulder joints, and ankle joints, with rigid bilateral hip and knee joints preventing any walk. The airway assessment revealed a Mallampati class III, interincisor distance of 3 cm, and rigid neck incapable of any movement. X-ray of his cervical, thoracic and lumbar vertebrae and pelvis revealed a loss of physiological curvature of the vertebral column, kyphosis, hyperplasia at the edge of the vertebrae, “bamboo” like bony bridge, blurred facet joints, and calcification of ligament, a fusion of both sacroiliac joints, rough articular face and narrowed articular cavity and hyperplasia of both hip joints  suffered from AS for 20 years, and was scheduled for left total hip arthroplasty (THA). He had a history of coronary heart disease and received coronary stent implantation 7 years ago. A physical examination revealed rigidity of cervical and lumbar spine, severe press pain and immobilization of left hip joints. An X-ray of the neck, chest and lower back revealed bamboo-like changes of the spine. CT of the hip joints reported necrosis of the left femoral head, and ankylosis of left hip joint. He had taken aspirin for anticoagulation therapy for a long term, and was replaced by low molecular weight heparin.Case 3: A 26 years old female  was scheduled for left total hip arthroplasty. She was diagnosed with ankylosing spondylitis 14 years ago, with pain on almost all major joints of her body. She could not stand or walk because of bilateral femoral head necrosis. She had a family history of ankylosing spondylitis, with her father also suffered from this disease. Physical examination revealed rigidity of neck and left hip joint. X-ray of the thoracic and lumbar vertebrae reported rigidity of the spine, calcification of interspinous ligament. X-ray of bilateral sacra-iliac joints, hip joints, knees and ankles reported narrow joint space, osteoproliferation, bone defect and joint deformity. The chest X-ray reported texture enhancement of bilateral lungs and infection of right lower lobe.Case 4: A 65 years old male  was scheduled for left THA. He was diagnosed with AS for 10 years, and developed necrosis of both femoral heads, and could not walk because of pain in the left hip joint. He had a history of hypertension for 10 years, and diabetes mellitus for 7 years, and smoking history for 50 years with nearly 50 cigarettes per day. A chest X-ray revealed enhanced texture of bilateral lungs and right pleural effusion. X-ray of his pelvic revealed bilateral femoral head necrosis, narrowed joint space, osteoproliferation and bone defect under articular surface of bilateral sacra-iliac joints and hip joints, and bamboo-like changes of lower lumbar spine.In the operation room, the patients were settled in a lateral decubitus position with surgical side upmost, and were given oxygen through a face mask. 500 ml of Ringer's solution was infused while pulse oximeter, NIBP and ECG monitors were connected. Difficult intubation carts, including LMA, bougie, fibre optic bronchoscopy, cricothyrotomy needle and tracheostomy set, were standby.We performed all of the peripheral nerve blocks guided by the ultrasound  combined with nerve stimulator , with the patient in the same lateral decubitus position (Fig. th rib, and then rotated at the level that showed the 12th rib connecting to its corresponding transverse process and creating an acoustic shadow (Fig. We performed T12 paravertebral block via a transversal in-plane technique at the level of inferior articular process, as previously described by Krediet, A.C., et al.  A low-frdow Fig. . Scannindow Fig. . The conFor lumbar plexus block, the low-frequency ultrasound probe was placed along the Tuffier’s line, lateral to spinous process, to achieve an axial scan of the psoas major muscle Fig. . At the For sacral plexus block, we used a modified technique described by Taha . The lowTwenty minutes later, sensory loss to pinprick had been achieved between T10 and S3 unilaterally Table .Table 1AIn the PACU, all the patients did not complain of discomfort in the surgical side, and the visual analog scale scores were 0–1. The patients were given a patient-controlled intravenous analgesia for 24 h postoperatively, and were discharged 7–10 days later uneventfully.Ankylosing spondylitis is a common inflammatory rheumatic disease that affects the axial skeleton, causing characteristic inflammatory back pain, which can lead to structural and functional impairments and a decrease in quality of life . The disAnesthesia management for ankylosing spondylitis is a challenge due to management of difficult airway, respiratory and cardiovascular complications, as well as the medications for disease and pain control . Both aiAt present, general anesthesia is administered for most of the patients with ankylosing spondylitis undergoing hip surgeries. Fixed neck may make regular endotracheal intubation and tracheostomy difficult . In patiAn update to evidence-based guidelines for the management of hip fractures in older persons suggests that regional anesthesia is recommended for most patients with a grade A of evidence . HoweverDisadvantages of spinal anesthesia may include urinary retention, hypotension, and issues involving perioperative anticoagulation , the risOur patients were all severely affected by ankylosing spondylitis for decades. As for case 1, the patient had no movement in his spine and had difficulty walking or sitting up from the supine position, and he was always kept on a rigid sitting position with bilateral hip and knee joints fixed at about 90° flexed. With an anticipated difficult airway and impairment of pulmonary function, general anesthesia with tracheal intubation is a big challenge. An ultrasound examination of lower back showed extreme narrowing of his lumbar vertebral spaces. Because calcification of the ligamentum flavum impeded the penetration of ultrasound, epidural space and ‘posterior complex’ could not be identified through lumbar interlaminar space with a paramedian oblique sagital scan described by Karmakar . So a ceAccording to Miller’s Anesthesia, the upper dermatome level of sensory block to T10 is recommended for hip surgery with spinal anesthesia . Recent The potential advantages could be anticipated for our combined peripheral nerves block for THA include avoided stressful awake intubation, eliminated multiple-attempted lumbar puncture, improved hemodynamic stability , reducedThe combination of T12 paravertebral block, lumbar plexus block and sacral plexus block may provide reliable surgical analgesia, adequate muscle relaxation, and satisfied postoperative pain control for total hip replacement in patients with ankylosing spondylitis.