Aim. Physicochemical mechanical and in vitro biological properties of novel formulations of polymeric calcium phosphate cements (CPCs) were investigated. Methods. Monocalcium phosphate, calcium oxide, and synthetic hydroxyapatite were combined with either modified polyacrylic acid, light activated polyalkenoic acid, or polymethyl vinyl ether maleic acid to obtain Types I, II, and III CPCs. Setting time, compressive and diametral strength of CPCs was compared with zinc polycarboxylate cement (control). Specimens were characterized using X-ray diffraction, scanning electron microscopy, and infrared spectroscopy. In vitro cytotoxicity of CPCs and control was assessed. Results. X-ray diffraction analysis showed hydroxyapatite, monetite, and brushite. Acid-base reaction was confirmed by the appearance of stretching peaks in IR spectra of set cements. SEM revealed rod-like crystals and platy crystals. Setting time of cements was 5–12 min. Type III showed significantly higher strength values compared to control. Type III yielded high biocompatibility. Conclusions. Type III CPCs show promise for dental applications. The setn (–COO)  −1 (−1 disappeared in the IR spectrum of the set cement and two new stretching bands attributable to carboxylate (–COO) formation were observed at 1558 cm−1 and 1401 cm−1 indicative of acid-base reactions involving PMVE-Ma acid and CPC powder (The infrared spectra of the 35% w/w aqueous solution of PMVE-Ma acid and the set cement (Type III) are shown in . The (–C−1 . The banC powder  19]..−1 (−1 d2) which exhibited the characteristic peaks around 2θ = 36.253° and 2θ = 26.611°, respectively, were detected in the XRD patterns of unreacted and reacted zinc polycarboxylate cement (5 (PO4)3(OH), 2θ = 31.773°], monetite , brushite , and calcite . Two phases . Type III cement showed an increase in SDH activity by (>90%) and was statistically equivalent to the negative Teflon control after 1 wk. Type III cement was significantly higher than the Teflon control at week 2 and week 4. Type I cement was severely cytotoxic (>90% suppression) relative to Teflon controls, but significantly (P < 0.05) improved were statistically equivalent to the Teflon control after 4 weeks. Type II cement did not change significantly in cytotoxicity over the entire four week evaluation. Type II cement was significantly (P > 0.05) lower than Teflon control over the entire four week evaluation period. Zinc polycarboxylate cement also significantly suppressed mitochondrial activity throughout the testing but showed some improvement by the fourth week.Cellular mitochondrial suppression induced by the CPCs and zinc polycarboxylate cement (control group) is illustrated in . Aging iThe most difficult challenge in designing and manipulating dental materials is being able to mimic the complex physical and functional characteristics of natural tissues. Development of a replacement material that either mimics natural tissue properties and performance and/or one that is eventually resorbed and replaced by equivalent new tissue is the final goal of restorative dentistry. Thus, hydroxyapatite (HA) materials combined with organic compounds are promising dentin replacing materials. Various calcium phosphate derivatives, for example, hydroxyapatite (HA), tricalcium phosphate (TCP), octacalcium phosphate (OCP), dicalcium phosphate (DCP), and monocalcium phosphate (MCPM) have been studied in the last decade because of their biocompatibility, osteoconductivity, and self-hardening properties which are desirable in a broad range of dental and biomedical application , 29–31. In the present study, experiments have been undertaken in order to develop some nontraditional dental cementing materials. The principal compounds [synthetic hydroxyapatite (SHAp6) and calcium oxide (CaO)] that have been used, proved to have (as far as dentistry is concerned) encouraging properties which will certainly open an avenue for the material scientists to overcome some of the drawbacks encountered with well known dental cements. In the present study, all materials selected for the preparation of polymeric calcium phosphate cements: calcium oxide (CaO) monocalcium phosphate monohydrate (MCPM), and synthetic hydroxyapatite (SHAp6) powders as well as modified polymeric liquids (Polyacrylic acid “PA”), visible light cured polyalkenoic acid (VLC), and polymethyl vinyl ether maleic acid (PMVE-Ma), are all of medical grade, commercially available, and have a well-established compatibility.Calcium oxide (CaO) is known to react rapidly with water and plays and important role in the hydration reaction of the set cement  . As for monocalcium phosphate monohydrate (MCPM) it is often used as the acid calcium phosphate in hydraulic calcium phosphate formulations, but commercial MCPM is not pure, contains a small amount of orthophosphoric acid and moisture, is consequently difficult to mill, and the powder is sticky and presents aggregates. Because granularity influences the mechanical properties of the hardened cement, it was, therefore, necessary to premix MCPM with CaO before grinding it though a rapid decrease in the amount of (MCPM) was observed during mechanical grinding by a solid-solid reaction with CaO . An esse Ginebra et al. (2004) stated that the cement cannot be univocally related to the degree of reaction without considering the microstructural features . The sieTherefore, in the present work, in order to overcome the disadvantages of mixing with water, the aqueous solutions of modified  and polymethylvinyl ether maleic acid) were used. Their setting reaction was found to be biphasic, the first step during the mixing time, (MCPM) reacted with CaO immediately to give dicalcium phosphate dihydrate (DCPD) which in the second step, reacted more slowly with the remaining CaO to give hydroxyapatite.An essential criterion was also considered in relation to the molecular weight and concentration of the aqueous solutions used for mixing the powder, as higher molecular weights tend to result in cements with shorter setting times and a higher compressive, diametral, and biaxial flexural strengths than lower molecular weight counter parts . The polymethyl vinyl ether maleic anhydride (PVME-Ma) is a commercial copolymer offered in several molecular weights and can be dissolved by hydrolysis of the anhydride group in water to form the corresponding maleic acid copolymer . This copolymer has already a number of nondental applications in hair sprays and surgical adhesives, which suggests potential favorable biocompatibility for dental and other biomedical uses . BecauseIn order to optimize the powder liquid ratio of the mixing powder, extensive preliminary testing of various powder mixtures ratios was performed resulting in the optimal ratio of 4 : 1. The setting time of zinc polycarboxylate cement (control group) used in this study agreed with those values previously reported in the literature .The setting time results of the three formulated polymeric cements  indicateAs for Type II polymeric CPC cement mixed with polyalkenoic acid (VLC), the setting time  cannot bIn Type III CPC mixed with (PMVE-Ma acid), a setting time of 9 min was observed. This may be attributed to the multifunctional nature of the PMVE-Ma acid which appears to react forming insoluble products which coat the cement particle. This encapsulation slightly retards their dissolution which may be attributed to residual maleic anhydride units as observed in the IR spectral data  and subs Results of the compressive and diametral tensile strength values of the newly formulated polymeric calcium phosphate cements (CPCs) as shown in Tables  and 4 deIn this study, the reaction between CPC powder and 35% (w/w) (PMVE-Ma) aqueous solution (Type III) resulted in a polymeric CPC with a compressive strength of 71.68 MPa and diametral strength of 11.43 MPa one hour after mixing. These results are in accordance with Matsuya et al. . These eThe cement prepared from CPC powder and visible light cure polyalkenoic acid (VLC) Type II had a compressive strength value of 66.86 MPa and diametral tensile strength of 7.39 MPa, 1 hr after mixing and reached its maximum value at the end of 24 hours. These results coincide with Miyazaki et al. . The resStorage in distilled water at 37°C slightly affected the mechanical properties, therefore suggesting a stable formula that can resist disintegration in the oral environment.Multifunctional acids such as polymethylvinyl ether maleic acid (PMVE-Ma) and polyacrylic acid (PA) are characterized by the presence of carboxylic groups . The calThe use of total reflectance of infrared spectroscopy makes it possible to monitor the setting reaction and the transformation of the COOH group to COO groups. The infrared spectra of the set polymeric CPC Types  24 hours after mixing showed the absence of the stretching peak of carboxylic group (–COOH) and the appearance of two new carboxylate stretching peaks indicating the formation of the polyacrylic salts Figures .2. The presence of tin oxide depends on the commercial cement initially used: it is present in small quantities in some cements and not in others. No zinc polycarboxylate reflections were seen, since this compound is amorphous. Therefore, the cement must be a composite of unreacted oxides ZnO (mainly) and SnO2 and the amorphous zinc polycarboxylate matrix resulting from the setting reaction of these oxides with polyacrylic acid and water. These results are in accordance with the core link structure proposed for such cements [5 (PO4)3(OH)], monocalcium phosphate monobasic hydrate [Ca(H2PO4)2·H2O], calcite [CaCO3], and portlandite [Ca(OH)2].The X-ray diffraction analysis of zinc polycarboxylate unreacted powder identified two crystalline phases ZnO and SnO cements . The X-rDuring the hydration of cements consisting of nominally 60wt % monocalcium phosphate monobasic and calcium oxide and 40 wt% hydroxyapatite, two dibasic phosphate compounds,  were identified in the sample. Monocalcium phosphate monobasic (MCPM) reacted with excess water forming monetite and/or brushite by2 from air forming calcite.Although mechanical and physical properties are of great concern for dentin regenerating pulp capping, lining, or base material, biocompatibility is another critical issue. The current study established that 4 weeks aging of the developed formulations of calcium phosphate cements may significantly change their ability to alter cellular function. However, the effect was not uniform for all formulations. Type I calcium phosphate cement (CPC mixed with polyacrylic acid) showed less mitochondrial suppression with time. It is possible as cytotoxic elements leached from the material, they either complexed with other molecules in the medium or broke down into smaller components, in each case rendering it less cytotoxic.  For Type II calcium phosphate cements mixed with resin modified glass ionomer (Vitremer), suppressed cellular activity was ongoing, suggesting that leaching of components with biological liabilities remained even after 4 weeks of aging. The Vitremer liquid is a light sensitive, aqueous solution of a modified polyalkenoic acid and contains 2 hydroxy ethyl methacrylate (HEMA). In most dental resin modified glass ionomer cements, HEMA is often used as a comonomer to render the resin modified polyacid compatible with water , 45. How We prepared the CPCs by mixing calcium oxide, calcium phosphate monohydrate (MCPM), and synthetic hydroxyapatite. Calcium oxide (CaO) is known to react rapidly with water and plays an important role in the strength and degree of hydration of dental cements. As for monocalcium phosphate monohydrate (MCPM), it is often used as the acid-calcium in hydraulic calcium phosphate formulations. Calcium phosphate biomaterials are thought to generally be biologically well tolerated, because the main inorganic constituents of bone, hydroxyapatite is comprised of calcium and phosphate , 47. Interestingly, the cytotoxicity of the three different formulations is dependent on the composition of the polymeric acid used for mixing. In our experimental design, cytotoxicity was estimated by mitochondrial succinate dehydrogenase activity (SDH) activity in the MTT assay and expressed as a percentage of the Teflon negative control value (100%) being equivalent to Teflon with no evident cytotoxicity. On the other hand, the biocompatibility of zinc polycarboxylate cement (control group) has also been investigated. Our data show that zinc polycarboxylate cement was the most cytotoxic of the tested materials in accordance with previous data , 49.Type III CPC presented reasonable setting time, significantly higher compressive, and diametral tensile strengths when compared to zinc polycarboxylate cement (control group).By virtue of these characteristics coupled with its biocompatibility, Type III CPC cement shows promise for dental applications.


Future increases in global surface temperature threaten those worldwide who depend on rice production for their livelihoods and food security. Past analyses of high-temperature stress on rice production have focused on paddy yield and have failed to account for the detrimental impact of high temperatures on milling quality outcomes, which ultimately determine edible (marketable) rice yield and market value. Using genotype specific rice yield and milling quality data on six common rice varieties from Arkansas, USA, combined with on-site, half-hourly and daily temperature observations, we show a nonlinear effect of high-temperature stress exposure on yield and milling quality. A 1°C increase in average growing season temperature reduces paddy yield by 6.2%, total milled rice yield by 7.1% to 8.0%, head rice yield by 9.0% to 13.8%, and total milling revenue by 8.1% to 11.0%, across genotypes. Our results indicate that failure to account for changes in milling quality leads to understatement of the impacts of high temperatures on rice production outcomes. These dramatic losses result from reduced paddy yield and increased percentages of chalky and broken kernels, which together decrease the quantity and market value of milled rice. Recently published estimates show paddy yield reductions of up to 10% across the major rice-producing regions of South and Southeast Asia due to rising temperatures. The results of our study suggest that the often-cited 10% figure underestimates the economic implications of climate change for rice producers, thus potentially threatening future food security for global rice producers and consumers. Oryza sativa L.) production serves as the primary source of income for nearly one billion people and provides the staple food for more than half the world’s population Rice  models project mean global surface temperature increases between 1.8°C and 4°C over the 21st century at a rate of 0.2°C increase per decade Rice yield and milling quality jointly determine the economic value of rice from the field to the mill and in the market. Production shocks in any given stage affect value creation in later stages. Producer revenue depends on the sale of paddy (unprocessed) rice and miller revenue depends on the sale of milled rice and byproducts to domestic and international markets. High-temperature stress can decrease the quantity of paddy  rice available for milling and change the distribution and quality of milling outcomes. Milling quality aspects affected by temperature include chalkiness, immature kernels, kernel dimensions, fissuring, protein content, amylose content and amylopectin chain length −1) of unprocessed kernels; milled rice yield (MRY) – the mass percentage of milled kernels (milled rice) to an initial mass of paddy rice kernels; head rice yield (HRY) – the mass percentage of milled kernels ≥ three-quarters the length of an unbroken (whole) milled kernel (head rice) to an initial mass of paddy rice; broken rice yield (BKY) – the mass percentage of milled kernels<three-quarters the length of an unbroken (whole) milled kernel to an initial mass of paddy rice; and chalk content (CHK) – defined as the ratio of chalky to non-chalky area of 100 brown rice (kernels obtained immediately after removing the husk) kernels. Definitions of MRY, HRY, BKY, and CHK correspond to the experimental definitions stated in HRY, and MRY given 1°C, 2°C, and 4°C increases in growing season air temperature. The predictions are used to calculate changes in the quantity and value of paddy and milled rice for producers and millers across temperature scenarios.Recent research has shown that modest (1°C) increases in daily maximum and minimum temperatures can decrease paddy yields by as much as 10% HMC) and milled in duplicate. HMC is an in-field measurement of the ratio of liquid to solid kernel mass and is expressed as a percentage. HMC was measured immediately after samples were harvested using a single-kernel moisture meter ; moisture content was expressed on a wet basis HMC and CHK were recorded for each harvest repetition and MRY and HRY were recorded for each milling repetition. Only the MRY and HRY observations associated with the measured chalk content were used in the present analysis.Annual Arkansas Rice Performance Trial (ARPT) experimental data from six locations over four years were used to estimate the yield and milling quality in the models . Three rMRY is calculated as the mass percentage of polished whole and broken kernels remaining after milling a 150-gram sample of paddy rice:HRY is calculated as the mass percentage of polished whole kernels remaining after milling a 150 gram sample of paddy rice and separation of broken kernels using a double-tray sizing device :BKY) is calculated as the difference between the observed MRY and HRY because this difference has not been measured experimentally, but is by definition the difference between the two values. Chalk content represents the percentage chalky area of a 100 kernel sample of brown rice, obtained by Broken rice yield , i on day d at station s in year t, d and k, respectively. Similarly, thermal nights (TN) above 22°C are defined asi on night n at station s in year t, n and, Ambient air temperature and relative humidity recordings were collected at each location at 30-minute intervals using two temperature sensors . The sensors were placed at panicle height, in-between experimental plots, within the 18-plot block of rice cultivar plots grown at each station. Given the randomized block design of cultivar location within each field, this study uses the set of means of each pair of 30-minute temperature observations as the set of temperatures associated with a given year-location combination. We define the thermal time metrics using the half-hourly data points. Thermal days (TD) above 33°C are defined as.www.srrb.noaa.gov/highlights/sunrise/sunrise.html). Vapor pressure deficit (VPD) (kPa) was calculated using these data following iRH is the relative humidity at time i.Daily sunrise and sunset estimates were calculated for each station/year combination using the National Oceanic and Atmospheric Administration’s (NOAA) solar calculator (−2) data for 2007–2010 were obtained from the the NASA Climatology Resource for Agroclimatology Half-hourly weather data were not available until 10 days prior to 50 percent heading  for trial i at station s for cultivar j; a is a vector of weather coefficients; sb is a vector of station intercepts to control for spatially invariant unobserved effects such as soil type; jc is a vector of cultivar intercepts to capture genetic yield differences across cultivars; and isju are the error terms associated with each trial. Heteroskedasticity robust standard errors are used to accommodate non-constant variance across locations and years −2 d−1) during the vegetative stage; kTDN, the sum of thermal degree-days above 33°C 33TD and thermal degree-nights above 22°C 22TN during window k for k = W1, W2, and W3 , and MRY (%), respectively, for trial i at station s; isHMC represents harvest moisture content (HMC) in HMC and 2HMC in sb is a vector of station intercepts; and, 2HMC term allows for a quadratic response of HRY to HMC, a relationship previously documented for these cultivars W2TDN and W3TDN to capture temperature effects during early (W2) and late (W3) grain filling  accounts for the heteroskedasticity of unknown forms and allows calculation and statistical testing of both direct and indirect effects. Given the presence of CHK, HRY and MRY are estimated given 1°C, 2°C, and 4°C increases in mean growing season temperature. Using the means of W2TDN and W3TDN as the baseline, we then attempt to answer the question: How might the observed sample means of chalk content, HRY, and MRY change if the data were resampled under the three warming scenarios? To estimate these changes, we add 1°C, 2°C and 4°C to each observed temperature datum following the procedure used in W2TDN and W3TDN are then recalculated for each hypothetical scenario. Then, the sample means of these hypothetical variables are calculated and used to predict changes in the means of the outcome variables under each scenario. Estimating changes in the sample mean of the temperature variables under these scenarios rather than the prediction of each individual observation across scenarios avoids out-of-sample prediction. Investigation of the changes in the sample means under these scenarios rather than any one observation better fits this examination of the future of Arkansas rice production given a warmer climate.Changes in milling quality given 1°C, 2°C, and 4°C increases in growing season (emergence to harvest) temperatures are estimated using the total effects measure W1TDN is associated with a -0.3 percent change in paddy yield, all else held constant . In paddy yield models with vapor pressure deficit (VPD) as an independent variable, the VPD coefficients were statistically insignificant and/or unstable across alternate specifications  . The effconstant . The TDNications .A 1°C increase in average growing-season temperature implies a 6.2% decline in paddy yield, all else held constant . Given 2W2TDN and W3TDN are positive and statistically significant (P<0.05 to P<0.01) across all cultivars. The magnitudes of the W3TDN coefficients are larger, suggesting that high-temperature stress during late grain filling has a greater impact on chalk formation. In the HRY and MRY equations, W2TDN has a negative, statistically significant coefficient across all cultivars (P<0.10 to P<0.01) and the W3TDN coefficients are relatively small in magnitude and statistical significance   . This esCHK, MRY and HRY largely determine the value of a unit of paddy rice, but the quantity of milled rice output per unit area harvested depends on paddy yield. −1 more non-chalky head rice than XL723 and roughly 1.2 t ha−1 more than LaGrue or Wells.Total milling revenues across cultivars do not follow the same pattern as milled rice quantity because the quantities of broken and chalky kernels are valued less than the quantities of non-chalky kernels. Using the average U.S. FOB price for long- and medium-grain milled head rice and the average price for broken rice over the sample period, the total revenue estimates in TDN metric to capture heat effects on sensitive processes occurring during the reproductive and ripening stage more than do average daily temperatures. The statistical significance of the W1TDN coefficient supports this conclusion. The statistically significant W1TDN coefficient agrees with the experimental literature on the inverse relationship between high temperatures at pollen development and anthesis leading to increased spikelet sterility and lower yields due to loss of pollen viability W2TDN coefficient.The statistically insignificant effect of high temperatures during the vegetative stage differs from TDN metric, which captures the nonlinear changes in high-temperature accumulation (both frequency and magnitude) given shifts in the temperature curve  did not result in an equivalent reduction in HRY and MRY   and MRY , since cUsing a predictive model of chalk content at the prefecture level in Japan, HRY and broken rice tell only part of the story. The total revenue implications associated with changes in this distribution drive the wedge further between high-temperature exposure in the paddy and milled outcome levels with the size of this wedge varying across genotypes. We illustrate this using multiple regression models developed above to estimate the impacts of 1°C, 2°C, and 4°C increases in growing season temperatures on the changes in quantity, quality distribution, and total revenue at the milled level  to maximize edible rice  per unit land area. As mentioned earlier, impact analysis of high temperature under field conditions across the major rice-producing regions of South and Southeast Asia has been restricted to grain yield Climate change necessitates the development of cultivar-specific models capable of simultaneously estimating the relationship between high temperatures and rice yield and milling quality. The omission of rough rice yield or milling quality inevitably leads to an underestimation of the true extent of high-temperature effects on rice production and this could distort policy decisions designed to overcome food insecurity given the changing climate. Biologically, the estimation of both yield and milling quality responses to high temperatures is critical because the detrimental impacts occur across various developmental stages and are genotype-specific. High-temperature stress during the sensitive gametogenesis and early flowering largely determines paddy yield loss while increased chalk formation (white core) during the early grain-filling stage governs head rice loss due to breakage. Genotypic variation illustrated in our milling quality results might provide rice breeders with insight to justify genetic crosses of lower yielding varieties (e.g. Cypress) if the goal is maximization of non-chalky This study provides insight where previous experimental and econometric analyses of rice production outcomes have not: (i) examined, or if they have examined, have not estimated, rough rice yield and milling quality models simultaneously; (ii) extensively addressed multicollinearity issues among explanatory weather variables in econometric models; and (iii) identified the economic implications of these innovations. Continued observation of the interaction between increasingly variable weather conditions and rice production outcomes will allow refinement and enhancement of this modeling approach to, we hope, provide plant breeders, agricultural policy makers, and private enterprises with important direction for sustaining rice production in an increasingly hot future.Figure S1Visual approximation of exposure to high temperatures. The black line represents temperatures at time t, the upper (lower) horizontal line represent the day (night) temperature threshold, and the red (orange) area between the temperature and upper (lower) horizontal line represents the day (night) high-temperature exposure, expressed in this study as 33TD (22TN). The summation of these areas gives the TDN metric used in this paper’s primary analysis. Artificially increasing growing-season temperatures, thus shifting the temperature curve upward, allows examination of non-linear responses to high-temperature exposure.(PDF)Click here for additional data file.File S1Tables S1. Coefficients from primary specification regression. Table S1 contains the coefficients used to generate (DOCX)Click here for additional data file.


In comparison, intranasal (i.n.) inoculation with the same dose of Nipah virus resulted in lethal respiratory disease in all animals. In animals infected with Nipah virus via drinking, virus was detected in respiratory tissues rather than in the intestinal tract. Using fluorescently labeled Nipah virus particles, we showed that during drinking, a substantial amount of virus is deposited in the lungs, explaining the replication of Nipah virus in the respiratory tract of these hamsters. Besides the ability of Nipah virus to infect hamsters via the drinking route, Syrian hamsters infected via that route transmitted the virus through direct contact with naïve hamsters in 2 out of 24 transmission pairs. Although these findings do not directly prove that date palm sap contaminated with Nipah virus by bats is the origin of Nipah virus outbreaks in Bangladesh, they provide the first experimental support for this hypothesis. Understanding the Nipah virus transmission cycle is essential for preventing and mitigating future outbreaks.Since 2001, outbreaks of Nipah virus have occurred almost every year in Bangladesh with high case-fatality rates. Epidemiological data suggest that in Bangladesh, Nipah virus is transmitted from the natural reservoir, fruit bats, to humans via consumption of date palm sap contaminated by bats, with subsequent human-to-human transmission. To experimentally investigate this epidemiological association between drinking of date palm sap and human cases of Nipah virus infection, we determined the viability of Nipah virus (strain Bangladesh/200401066) in artificial palm sap. At 22°C virus titers remained stable for at least 7 days, thus potentially allowing food-borne transmission. Next, we modeled food-borne Nipah virus infection by supplying Syrian hamsters with artificial palm sap containing Nipah virus. Drinking of 5×10 In Bangladesh, outbreaks of Nipah virus occur almost every year, resulting in respiratory and neurological disease with high case-fatality rates. Based on epidemiological data Nipah virus is thought to be transmitted from fruit bats to humans via drinking of date palm sap contaminated by bats that drink from the sap stream or collection vessel during collection. Additionally, human-to-human transmission has been shown to occur. Here, we experimentally modeled the proposed transmission cycle of Nipah virus in Bangladesh in Syrian hamsters. Hamsters that drank artificial palm sap containing high doses of Nipah virus became infected with the virus and developed neurological signs of disease. Virus replication occurred mainly in the respiratory rather than the intestinal tract. Most importantly, hamsters infected with Nipah virus through drinking of contaminated palm sap could transmit the virus to uninfected cage mates. As treatments for Nipah virus are currently unavailable and medical interventions are difficult to implement in rural outbreak areas, our best hope to prevent or intervene in future outbreaks of Nipah virus lies in the potential to block transmission from bats to humans and from human to human. Understanding how Nipah virus is transmitted is essential to achieve this. Nipah virus first emerged in 1998 during a large outbreak of encephalitis and respiratory disease in Malaysia and Singapore, causing 276 cases of encephalitis with 106 fatalities In addition to the initial zoonotic transmission, subsequent human-to-human transmission also plays an important role in the epidemiology of Nipah virus outbreaks in Bangladesh. It was estimated for the outbreaks in Bangladesh between 2001 and 2007 that approximately 50% of cases was the result of human-to-human transmission Thus far, the epidemiological association between drinking date palm sap and Nipah virus infection has not been confirmed experimentally. Therefore, we set out to assess the ability of date palm sap to function as a vehicle for zoonotic transmission of Nipah virus using a well-established small animal model for Nipah virus pathogenesis and transmission, the Syrian hamster 3, 105 and 107 TCID50/ml) of Nipah virus (strain Bangladesh/200401066) in artificial palm sap was determined at 22°C. Of note, between October and March  the average temperature in Bangladesh fluctuates between 20°C and 28°C. No significant difference was detected between the slopes of the three lines . In addition, the slopes of the three groups were also not significantly different from zero . Thus, no significant reduction in Nipah virus infectious titer was observed at any of the dilutions for at least 8 days at 22°C . Eight hamsters were monitored daily for signs of disease for up to 28 days post inoculation (dpi). One hamster succumbed to respiratory disease on 6 dpi; a second hamster was euthanized on 12 dpi , likely indicating an active infection. In comparison, all intranasally inoculated hamsters shed virus from the nose for up to 7 days and from the throat up to 11 dpi .8 TCID50 of Nipah virus (strain Bangladesh/200401066) in artificial palm sap. Nasal, oropharyngeal, urogenital and rectal swabs were collected up to 11 days after supplying the hamsters with artificial palm sap. All eight tested hamsters shed virus for several days, mainly via the oropharynx and, at later time points, the intestinal tract  and singly housed to examine transmission via fomites, direct contact or aerosols as described previously We have recently shown that Nipah virus  is transmitted between Syrian hamsters primarily through direct contact 8 TCID50 of Nipah virus (strain Bangladesh/200401066). After two days, when the hamsters had drunk the artificial palm sap, drinking bottles were replaced with bottles containing water and a naïve hamster was added to each cage. At 28 dpi the naïve hamsters were euthanized and sera were collected. Out of 24 naïve hamsters, 2 hamsters showed presence of antibodies directed against Nipah virus in ELISA, likely indicating that Nipah virus was transmitted to these hamsters.Next, we set out to determine if hamsters infected with Nipah virus through drinking of artificial palm sap containing Nipah virus could transmit the virus to naïve hamsters via direct contact. Of note, a larger number of animals was used in this experiment since the hamsters infected with Nipah virus through drinking of artificial palm sap shed a lower amount of virus than intranasally inoculated hamsters  and tranEpidemiological investigations in Bangladesh suggest that Nipah virus is introduced into the human population via the consumption of raw date palm sap. This study provides the first experimental evidence for the transmission of Nipah virus via the consumption of palm sap containing Nipah virus, resulting in neurological signs of disease in Syrian hamsters. Although these findings do not directly demonstrate that date palm sap contaminated with Nipah virus by bats is the origin of Nipah virus outbreaks in Bangladesh, they provide experimental support for the current hypothesis, based on epidemiological observations, of the zoonotic introduction of Nipah virus via contaminated date palm sap.Nipah virus was very stable in artificial palm sap, likely due to its neutral pH and high sugar content. Nipah virus was preserved much better in artificial palm sap than on the surface of fruit or in fruit juice Based on virus distribution in tissues of infected hamsters and the presence of vRNA mainly in throat swabs rather than urogenital or rectal swabs, the porte d'entrée for the initial Nipah virus infection upon drinking of artificial palm sap containing Nipah virus was the respiratory tract rather than the intestinal tract. This finding was further strengthened using fluorescently labeled Nipah virus to visualize the deposition of virus upon inoculation via the nose, gavage or upon drinking. These experiments clearly showed that during drinking the virus does not only end up in the intestinal tract but some of the volume is also deposited in the lungs, thereby explaining the replication of virus in respiratory tissues. However, the main disease manifestation in hamsters infected through drinking was the development of neurological signs, suggesting that the animals became infected with a relatively low dose, despite the high amount of virus present in the artificial palm sap. Previous studies have shown that inoculation of hamsters with a low dose of Nipah virus results in neurological signs of disease, whereas a high dose results in respiratory disease In one of the hamsters infected with Nipah virus through drinking of palm sap, virus was detected by immunohistochemistry in the olfactory bulb, indicating that virus traveled from olfactory neurons in the nasal turbinates through the cribriform plate into the olfactory bulb and from there further into the central nervous system. Although this route of Nipah virus into the brain has been described before Besides the ability of Nipah virus to infect hamsters via the drinking route, we showed here that Syrian hamsters infected with Nipah virus through drinking of palm sap containing Nipah virus can transmit the virus through direct contact with naïve hamsters. Transmission upon drinking of Nipah virus-containing artificial palm sap was less efficient than upon intranasal inoculation with Nipah (strain Bangladesh/200401066), likely as a result of decreased virus shedding upon infection through drinking. Within the transmission model for Nipah virus in Syrian hamsters no differences were observed in transmission route or efficiency between the Malaysian Prophylactic or therapeutic intervention measures are currently not available to prevent, treat or contain zoonotic transmission of Nipah virus. Moreover, medical interventions might be difficult to implement in rural outbreak areas. Therefore, our best hope to prevent or intervene in future outbreaks of Nipah virus lies in the potential to efficiently block zoonotic and human-to-human transmission and thereby spread of the outbreak. Currently, efforts are underway in Bangladesh to prevent zoonotic transmission of Nipah virus from fruit bats to people by restricting access of bats to date palm collection pots and thereby preventing contamination of the date palm sap with Nipah virus All animal experiments were approved by the Institutional Animal Care and Use Committee of the Rocky Mountain Laboratories, and performed following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) by certified staff in an AAALAC-approved facility.Nipah virus (strain Bangladesh/200401066) was kindly provided by the Special Pathogens Branch of the Centers for Disease Control and Prevention, Atlanta, Georgia, United States. This strain (SPBLOG# 200401066) was isolated from a throat swab collected from patient #3001 on January 22 2004 in Bangladesh. This patient was a 10-year old male who developed altered mental status on January 21 and cough and breathing difficulties later that day. The patient was admitted to Goalando Hospital, Bangladesh, on January 22. None of the patient's contacts developed Nipah virus infection; the patient is presumed to have been infected via direct spillover from the bat reservoir . The virus isolate was propagated in Vero C1008 cells in DMEM (Sigma) supplemented with 10% fetal calf serum, 1 mM L-glutamine (Lonza), 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco). For fluorescent labelling of virus, Nipah virus-containing cell supernatant was cleared by low speed centrifugation and virus was pelleted by spinning 2 hours at 21000 rpm in the ultracentrifuge. The pellet was resuspended in 1 ml PBS and loaded onto a 20%–60% (w/w) sucrose gradient and centrifuged overnight at 39000 rpm. The virus fraction was collected and pelleted once again by centrifuging 2 hours at 21000 rpm; the pellet was resuspended in 1 ml PBS. Purified Nipah virus particles were labeled using an Alexa Fluor 680 Protein Labeling Kit (Molecular Probes). Excess dye was removed by dialyzing against PBS.Artificial palm sap was prepared based on a literature report 7 TCID50/ml, aliquotted into 1 ml aliquots and incubated at 70°C or 100°C for up to one hour.Nipah virus (strain Bangladesh/200401066) was added to artificial palm sap at the desired concentration, aliquotted into 1 ml aliquots and incubated at 22°C or 28°C for up to eight days. The stability data were analyzed using the linear regression model in the GraphPad prism 6 software package. For inactivation of Nipah virus in artificial palm sap, Nipah virus (strain Bangladesh/200401066) was added to artificial palm sap at 10tm:AURA, Harlan Laboratories) were inoculated with Nipah virus via different routes. One group received 107 TCID50 Nipah virus (strain Bangladesh/200401066) via intranasal inoculation in a total volume of 80 µl. One group received 107 TCID50 Nipah virus (strain Bangladesh) via gavage in a total volume of 500 µl. The remaining two groups received 107 and 5×108 TCID50 Nipah virus (strain Bangladesh/200401066), respectively through drinking of artificial palm sap. Animals were housed singly and supplied with 30 ml artificial palm sap containing a total dose of 107 or 5×108 TCID50 Nipah virus instead of drinking water. When animals had drunk all artificial palm sap, in about 2 days, they were again supplied with drinking water. Nasal, oral, urogenital and rectal swabs were collected daily from eight hamsters inoculated via all four different routes. Swabs were collected in vials containing 1 ml DMEM supplemented with 50 U/ml penicillin and 50 µg/ml streptomycin. On days 2, 4, 8, 12 and 28 post inoculation 8 animals from each inoculation group were euthanized and blood, trachea, lungs, heart, liver, spleen, kidney, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon , bladder, brain and nasal turbinates were collected for virological  and histopathological  analysis. Hamsters used for histopathological analysis were anaesthetized using ketamine (80–100 mg/kg) and xylazine (7–10 mg/kg) and perfused with PBS containing 5 mM EDTA, followed by 4% paraformaldehyde. Tissues of interest were then further fixed according to BSL4 standard operating procedures for a minimum of 7 days in 10% neutral buffered formalin.Four groups of 40 6–8 week old female Syrian hamsters (HsdHan7 TCID50 of fluorescently labeled Nipah virus (strain Bangladesh/200401066). To prevent fusion of virus particles with target cells, hamsters were euthanized ten minutes after inoculation and the respiratory tract was excised; ex vivo imaging was subsequently performed on the head and respiratory tract in an IVIS Spectrum imager (PerkinElmer). Images were acquired using an excitation wavelength of 675 nm with an emission scan at 720, 740, 760 and 780 nm at field of view B (6.6 cm) in auto-exposure mode with medium binning, f-stop 3. Following acquisition, images were unmixed in Living Image 4.2 with tissue autofluorescence subtracted. The resulting AF680 image was used for subsequent analysis. Rectangular Regions of Interest were drawn around the entire lung, trachea or nasal tract. The resulting average radiant efficiency was used to determine the quantity of labelled virus that was detected in the respiratory tract (combined trachea and lungs) or in the nasal tract (head).To visualize the deposition of virus in the hamster respiratory tract after inoculation, 3 hamsters per inoculation route were inoculated intranasally, esophageally and via drinking as described above with 107 TCID50 of Nipah virus (strain Bangladesh) in a total volume of 80 µl. On day 4 post inoculation, hamsters were euthanized and a single naïve hamster was placed in each cage. For direct contact transmission experiments, eight 6–8 week old female singly housed Syrian hamsters were inoculated intranasally with 107 TCID50 of Nipah virus (strain Bangladesh/200401066) in a total volume of 80 µl. On day 1 post inoculation, a naïve hamster was added to each cage. For aerosol transmission experiments, eight 6–8 week old female Syrian hamsters were inoculated intranasally with 107 TCID50 of Nipah virus (strain Bangladesh/200401066) in a total volume of 80 µl and singly housed in specially designed aerosol transmission cages. On 1 dpi, a naïve hamster was placed on the opposite side of the inoculated hamster. The hamsters were separated by two stainless steel grids, allowing airflow from the inoculated to the naive hamster but preventing direct contact and fomite transmission. In all transmission experiments, nasal and oropharyngeal swabs were obtained from inoculated and naïve hamsters daily and the bodyweight of naïve hamsters was determined. Upon signs of severe disease, inoculated and naïve hamsters were euthanized; remaining hamsters were euthanized four weeks post exposure.To determine the transmission route for Nipah virus (strain Bangladesh/200401066) we used the recently described Syrian hamster transmission model 8 TCID50 of Nipah virus (strain Bangladesh/200401066) instead of drinking water. When animals had drunk all artificial palm sap they were again supplied with drinking water and a naïve hamster was added to each cage. Nasal and oropharyngeal swabs were obtained from inoculated and naïve hamsters daily and bodyweight of naïve hamsters was determined. On signs of severe disease, inoculated and naïve hamsters were euthanized; remaining hamsters were euthanized four weeks post exposure.To determine whether Nipah virus (strain Bangladesh/200401066) is transmitted via direct contact after infection through palm sap containing Nipah virus, 24 6–8 week old female singly housed Syrian hamsters were supplied with 30 ml artificial palm sap containing 5×1050 was calculated from 5 replicates by the method of Spearman-Karber. Tissue homogenates were prepared by adding 1 ml DMEM to the weighed tissue and homogenizing using a TissueLyzer II (Qiagen). Homogenates were centrifuged to clear the homogenate before inoculating cells.Virus titrations were performed by end-point titration in VeroC1008 cells. VeroC1008 cells were inoculated with tenfold serial dilutions of swab medium or tissue homogenates. One hour after inoculation, the inoculum was removed and replaced with 200 µl DMEM supplemented with 10% fetal calf serum, 1 mM L-glutamine (Lonza), 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco). Five days after inoculation, cytopathic effect (CPE) was scored and the TCIDHistopathology and immunohistochemistry was performed on hamster tissues. Necropsies and tissue sampling were performed according to a standard protocol approved by the Institutional Biosafety Committee. After fixation for 7 days in 10% neutral-buffered formalin and embedding in paraffin, tissue sections were stained with hematoxylin and eosin (H&E) and an immunohistochemical method using a rabbit polyclonal antiserum against the Nipah virus nucleoprotein 50 equivalents in the samples.RNA was extracted from swab samples using the NucleoSpin 96 Virus Core kit (Macherey-Nagel) and a Corbett Robotics model CAS 1820 automatic RNA extractor. RNA was eluted in 100 µl. 5 µl RNA was used in a one-step real-time RT-PCR targeted at the NP gene using the Rotor-Gene probe kit (Qiagen) according to instructions of the manufacturer (primer and probe sequences are available on request). In each run, standard dilutions of a titered virus stock were run in parallel, to calculate TCIDAntibody responses were measured in an enzyme-linked immunosorbent assay (ELISA) using inactivated Nipah virus  as the antigen. Nipah virus-containing cell culture supernatant was concentrated and purified by centrifuging for two hours at 21000 rpm over a 20% sucrose cushion. The pellet was resuspended in PBS and Triton X-100 was added to a final concentration of 1%; the preparation was then inactivated with γ-radiation according to standard operating procedures. This suspension was used to coat immuno 96 microwell maxisorp plates (NUNC) at 4°C overnight. Subsequently, plates were blocked with 5% skim milk in PBS containing 0.05% Tween 20 (PBST) for 1.5 hours at 4°C. After 3 washes with PBST, 50 µL of diluted serum samples were added, and the plates were incubated for 1 hour at 37°C. Bound antibodies were detected after 3 washes using an anti-hamster secondary antibody conjugated with horseradish peroxidase . Following incubation for 1 hour at 37°C, bound HRP was detected using the ABTS Peroxidase Substrate System (KPL). The absorbance at 405 nm was measured using a microplate spectrophotometer. Sera were considered positive when absorbance was higher than three standard deviations above the mean of negative control sera.Figure S17 (black triangles) and 105 (black squares) TCID50/ml in artificial palm sap and left at 28°C for 8 days. Samples were taken at the indicated time points and virus titer in those samples was determined by titration on Vero C1008 cells. The stability data were analyzed using the linear regression model in the GraphPad prism 6 software package. The regression line (solid) is plotted together with the 95% confidence interval (dotted line). (B) Nipah virus (strain Bangladesh/200401066) was diluted to 107 TCID50/ml in artificial palm sap and left at 70°C (green line) or 100°C (blue line) for 1 hour. Samples were taken at the indicated time points and virus titer in those samples was determined by titration on Vero C1008 cells. Geometric mean titers were calculated from three replicates; error bars indicate standard deviation; dotted line indicates the cut off value of the assay.Stability of Nipah virus in artificial palm sap. (A) Nipah virus (strain Bangladesh/200401066) was diluted to 10(TIF)Click here for additional data file.Table S11 Viremia was determined by real-time RT-PCR.Tissue distribution of Nipah virus in hamsters inoculated intranasally, esophageally or via drinking as determined by virus titration. Numbers in the table indicate number of animals in which virus was detected in the indicated tissues at the indicated time points; total number of animals is indicated at the top of the column. (DOCX)Click here for additional data file.Table S21 Tissues were collected when animals were euthanized due to severity of disease signs. 2 nc: not collected.Tissue distribution of Nipah virus in hamsters inoculated intranasally, esophageally or via drinking as determined by immunohistochemistry. Numbers in the table indicate number of animals in which viral antigen was detected in the indicated tissues at the indicated time points; total number of animals is indicated at the top of the column. (DOCX)Click here for additional data file.


Skeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ.We have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification.As gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological conditions. Vertebrate skeletal muscles are complex organs composed by a variety of cell types besides the typical long, multinucleated cells called myofibers: fibroblasts in the connective layers, endothelial and smooth muscle cells in the vessel walls, nerves, and Schwann cells around the axons and blood cells flowing through the vessels. Even considering only the contractile components, still skeletal muscle appears as a complex and versatile tissue since myofibers possess a wide range of molecular, metabolic and physiological properties, as well as diverse size The actual contribution of single myofibers to the muscle transcriptional phenotype may be overshadowed in gene expression studies with whole muscles, just because of the complex anatomy of skeletal muscle and the heterogeneity of myofibers. The problem is exacerbated in pathological states with infiltrating immune cells or replacement of contractile cells by connective tissue, like in muscular dystrophies or during muscle regeneration The goal of our work was to demonstrate the feasibility of scaling down the phenotypic analysis of skeletal muscle by applying transcriptome profiling to the single fiber level (microgenomics) Single fibers belonging to two populations of murine muscle fibers, type 1 and type 2B  were selected for gene expression profiling. Although fibers of the two groups could be easily harvested from the soleus and EDL muscles respectively, it was necessary to identify them among the other fiber types. To do this, electrophoretic separation of MyHC isoforms, the gold-standard method for fiber typing, was applied. Briefly, muscles were incubated with collagenase to dissociate intact, unstrained myofibers that were separated under stereo microscope from hyper-contracted fibers . IsolateOne-class SAM analysis, carried out on the results of the competitive hybridization of single fibers vs. reference preparation, revealed genes with significantly different expression between myofibers and whole muscle. In total, 2,530 up-regulated and 2,488 down-regulated genes were identified  using a A high number of genes up-regulated in myofibers define the identity of muscle cells. GO analysis showed the significant enrichment in genes coding for mitochondrial and cytosolic proteins, as well as for typical muscle structural proteins and muscle specific isoforms of metabolic enzymes  . Novel fAccording to our experimental design, all the arrays are independent and all fibers of the same type form a unique class. By running unpaired two-class SAM analysis we focused on gene expression diversity between the two groups of myofibers. In total 1,505 non redundant DE genes were identified in SO type 1 vs. FG type 2B fibers. In particular, 930 probes were over-expressed in type 1 fibers and 602 in type 2B fibers . Since tTo extend the initial analyses to all DE genes, we performed GO enrichment . It shou2+ is not only essential for muscle contraction, but it is also a primary signaling molecule implicated in the specification of the slow phenotype A complex network of regulatory proteins governs the expression of muscle genes through combinatorial mechanisms acting on specific DNA elements and in several instances the molecular mechanisms involved in the regulation of fiber phenotype remain unclear To focus on metabolic differences between fiber types we queried a dedicated resource available at KEGG. Only by lowering the threshold of the statistical test (FDR 5%), thus extending the analysis to 4,555 genes, we could obtain significant results. Almost all genes in the glycolytic pathway that converts glucose into pyruvate were identified as over-expressed in type 2B fibers while many genes of oxidative phosphorylation and fatty acids oxidation were over-expressed in type 1 fibers . To our 2+ binding protein in the SR of skeletal muscle. Two calsequestrin genes encode different isoforms: Casq2 is expressed in slow skeletal and cardiac muscle, while Casq1 is DE between fast and slow skeletal muscles Prediction Analysis of Microarray (PAM) was implemented in order to find which genes are most useful to discriminate between the two groups of myofibers. The reliability of the PAM test was supported by the presence of well known markers of fiber type. Myostatin, a secreted protein that inhibits muscle differentiation and growth, is strongly associated with MyHC 2b expression in normal muscle To validate the microarray results by an independent method we carried out qPCR experiments on homogeneous pools of 50 fibers. Real-time PCR needs reference genes of invariant expression as internal control. Two canonical references were discarded: Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) had a high expression in FG type 2B fibres and Beta-2-microglobulin (B2m) in the whole muscle control . MitofusqPCR results indeed confirmed significant differences in the expression level for most tested genes : Aox1, CThe structural variability of muscle fibers has been related to differences in relative proportions of membrane structures and to different expression of regulatory and contractile proteins. Although a number of methods have been applied to investigate muscle fiber heterogeneity  and Italian regulations (DL 116/92) concerning the care and use of laboratory animals. Experimental procedures were approved by the local Ethical Committee of the University of Padova.Wild-type CD1 mice (Charles River) were housed in a normal environment provided with food and water. Adult males were killed by rapid cervical dislocation, to minimize suffering, at three months age (weight: 33–35 g).−1; Sigma). The collagenase-treated muscles were sequentially rinsed for 2 min. in 3 ml of DMEM, 3 ml DMEM supplemented with 10% fetal bovine serum (FBS) and 3 ml of DMEM and finally transferred into 50 mm×18 mm well containing 3 ml of DMEM with 10% FBS. All plastic was pre-rinsed with 10% FBS, to prevent sticking. Single myofibers were liberated by gentle physical trituration with a wide-mouth plastic Pasteur pipette (about 4 mm diameter). The triturating process was repeated several times until about 100 intact fibers were obtained. After each physical trituration, the muscles were transferred in a new well, to get rid of collagen wisps and hyper-contracted fibers. Quickly, intact and well isolated fibers were picked under stereo-microscope and washed first in DMEM and then in phosphate buffered saline . About one-third of each fiber was clipped and placed in Laemmli buffer ; the remaining part of the fiber was dissolved in lysis solution for RNA extraction. All samples were collected within 45 min. from the last trituration step.Detailed information is available about fiber composition and length in the mouse soleus and EDL muscles MyHC isoforms were separated in SDS-PAGE as described by Talmadge & Roy Reagents optimized for minute amount of material assured higher RNA yields compared to the classical Trizol reagent. Total RNA was extracted from fiber fragments or pools using the silica membrane technology of RNeasy Micro Kit (Qiagen). Single fibers were disrupted by adding 75 µl Buffer RLT and lysate was homogenized by vortexing for 5 min. The protocol was essentially that suggested by the manufacturer, with the following modification: RNA elution was performed with 14 µl RNase-free water pre-heated at 37°C and repeated a second time to avoid loss of RNA in the column. Due to the dead volume of the column, we recovered about 20–24 µl. We estimated that the amount of total RNA purified from a single fiber is in the range of one to few nanograms.Purified RNA samples were lyophilized and amplified twice using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion), in accordance with the manufacturer's instructions. First strand synthesis with an engineered reverse transcriptase should produce virtually full-length cDNA, which is the best way to ensure reproducible microarray results. The use of a modified oligo(dT) primer bearing a T7 promoter Both RNA extracted from single fibers  and aRNA (200 ng) were analyzed using the RNA 6000 Pico or Nano LabChip on a 2100 Bioanalyzer (Aligent). The sample (1 µl) was separated electrophoretically as described by the manufacturer and data were displayed as a gel-like image and/or an electropherogram. All poor quality RNA samples were discarded.2+ ATPase  or Glyceraldehyde-3-phosphate dehydrogenase  consisted of 13,443 70mer oligonucleotide probes and it was purchased from the Gene Expression Service available at CRIBI. Each oligo was spotted in two replicates on MICROMAX SuperChip I glass slides (Perkin-Elmer) using Biorobotics Microgrid II (Apogent Discoveries). We produced an updated and careful annotation of all sequences by querying three databases: ENSEMBL (version 56), RefSeq (version 38) and UniGene (version 183). About 1,500 probes did not find significant hits. The updated platform (version 2.0) has been submitted to the GEO Database, with Accession Number GPL10688.All microarray data is MIAME compliant and the raw data is available in the GEO database (accession number GSE23244).Labeled targets from single fibers and muscle control were mixed and ethanol precipitated. After dissolving the pellet in 120 µl of hybridization buffer , samples were denatured at 90°C for 2 min and added to the microarrays. Prehybridization was for 20 hours at 46°C in the presence of 5X SSC, 5X Denhardt, 0.1% SDS, 100 ng/µl ss-DNA. Competitive hybridizations were carried on for 44 hours at 46°C in an ArrayBooster microarray incubator , followed by a series of post-hybridization washings.Microarray slides were inserted into a VersArray ChipReader dual confocal laser scanner (Bio-Rad) for fluorescence detection at 5 µm resolution.Raw scanner images were processed with ScanArray Express Software (Perkin-Elmer) for fluorescent quantification.Global mean normalization was performed across element signal intensity and expression values were transformed into Log2 ratio of normalized intensities. Positive values correspond to genes over-expressed in myofibers, whereas negative values refer to genes over-expressed in whole muscles, and therefore under-expressed in myofibers. All statistical analyses were performed with MIDAW Hierarchical cluster analysis was performed by MultiExperiment Viewer , a part of TM4 Microarray Software Suite Significance Analysis of Microarrays (SAM) is a non-parametric statistical test based on a permutation approach specifically implemented for microarray data Gene Ontology enrichment was performed with the Gene Ontology Tree Machine tool (GOTM) using a P-value of 0.1 Supervised class-prediction analyses were performed by applying Prediction Analysis of Microarrays (PAM). This program uses the method of the nearest shrunken centroids to identify a subgroup of genes that best characterizes a predefined class The details of real-time quantitative PCR (qPCR) were described in a previous study Figure S1Flow chart of the experimental strategy.(PDF)Click here for additional data file.Text S1Protocol for microarray sample processing.(PDF)Click here for additional data file.Dataset S1Expression data presented in the main paper.(XLS)Click here for additional data file.Dataset S2Additional information for data presented in (XLS)Click here for additional data file.Dataset S3Additional information for data presented in (XLS)Click here for additional data file.Dataset S4Additional information for data presented in (XLS)Click here for additional data file.


Although three-dimensional (3D) β-tricalcium phosphate (β-TCP) scaffolds serve as promising bone graft substitutes for the segmental bone defect treatment, no consensus has been achieved regarding their optimal 3D architecture.In this study, we has systematically compared four types of β-TCP bone graft substitutes with different 3D architectures, including two types of porous scaffolds, one type of tubular scaffolds and one type of solid scaffolds, for their efficacy in treating segmental bone defect in a rabbit model.Our study has demonstrated that when compared to the traditional porous and solid scaffolds, tubular scaffolds promoted significantly higher amount of new bone formation in the defect regions as shown by X-ray, micro CT examinations and histological analysis, restored much greater mechanical properties of the damaged bone evidenced by the biomechanical testing, and eventually achieved the complete union of segmental defect. Moreover, the implantation of tubular scaffolds enhanced the neo-vascularization at the defect region with higher bone metabolic activities than others, as indicated by the bone scintigraphy assay.This study has further the current knowledge regarding the profound influence of overall 3D architecture of β-TCP scaffolds on their in vivo defect healing performance and illuminated the promising potential use of tubular scaffolds as effective bone graft substitute in treating large segmental bone defects. Treatment of large segmental bone defects caused by severe trauma, non-union fractures or tumor resection remains as a major clinical challenge. Nowadays, the usual strategy for large bone defect treatment involves the use of autologous or allogeneic bone grafts, with more than 500,000 bone grafting procedures conducted annually in the United States alone, making bone tissue the second largest transplanted tissue in the world In order to fabricate a favorable 3D scaffold as bone graft substitute to promote bone defect healing, various types of materials with different chemical properties were carefully investigated, including metal, polymeric material, ceramic material and so on. Bioactive ceramic materials such as β-tricalcium phosphate (β-TCP) have been demonstrated as attractive material candidates for 3D scaffold fabrication, because of their chemical similarity to the inorganic phase of natural bone, favorable biocompatibility, osteoconductivity and bioresorbable properties Commercial β-TCP powder was obtained from Edward Keller . The β-TCP scaffolds were manufactured as described previously All animal experiments were performed in strict accordance with protocols approved by the Institutional Animal Care Committee of Xijing Hospital. Forty New Zealand rabbits  were randomly divided into six experimental groups: (Group A-D) β-TCP scaffold group (n = 8 in each group), in which defects were implanted with four types of β-TCP scaffolds respectively according to a randomized complete block design; (Group E) autologous bone control group (n = 4), in which defects were implanted with autologous radius; and (Group F) control untreated group (n = 4), in which defects were left untreated to validate that the defects created here are critical sized defect. In order to analyze the biological performance of β-TCP scaffolds for large bone defect treatment, X-ray, gross view, fluorochrome marker, micro-computer tomography (micro-CT), biomechanical testing, histology and emission computed tomography (ECT) analysis were carried out in the present study .General anesthesia was performed on rabbits before the surgery using an intramuscular injection of a mixture of Ketamine and Xylazine. Limbs were shaved, skin was disinfected with iodine solution and a longitudinal incision was made to expose the radius, and a 16-mm segmental bone defect was created with a miniature oscillating saw At 12 weeks post-surgery, under the anesthesia, the defect region of rabbits was subject to the radiographic imaging  to assess the healing of bone defect. The status of bone repair and growth of callus were studied in samples taken out through the original incision after the animals were sacrificed.Sequential fluorochrome markers were administered 2 weeks and 3 days before the animals were sacrificed. Tetracycline  was administered using intramuscular injection. After the animals were sacrificed at 12 weeks post-surgery, the implants were retrieved for fluorescence analysis.2 pixels were acquired for each tomogram. The X-ray source voltage was set at 80 kV and beam current at 200 mA using filtered Bremsstrahlung radiation. The scanning angular rotation was 180°, and the angular increment was 0.40°. To minimize beam hardening artifacts a 1 mm aluminum X-ray beam filter was used to attenuate soft X-rays at the source. The projections were reconstructed using a modified parallel Feldkamp algorithm, and segmented into binary images (8-bit BMP images). For determination of the 3D micro-architectural properties within the bone regeneration area, specimens were evaluated using 3D analysis software . The percentage of bone volume out of total volume (BV/TV) and degradation rate were calculated using the threshold of 2000 for bone tissue and 3000 for scaffold.Specimens containing the scaffold construct and some surrounding tissue were harvested (n = 8 in Group A-D) and fixed in 10% neutral buffered formalin, placed in the sample holder and scanned under the micro CT . About 1600 projections of 1024Immediately after the animals were sacrificed, the mechanical properties of the implants (n = 4 in Group A–D) were evaluated with the use of a destructive compression test. Cubes of 8mm×4mm×4mm from each specimen were sawed and tested under wet conditions at room temperature. The compressive load was applied at cross-head speed of 1 mm/min using the material testing machine  until fracture occurred. From the load-displacement curve, the compressive strength was calculated from compressive load and geometric area of the specimens. The compression test of the β-TCP scaffold was also done before implanted.Specimens from bone defect sites (n = 4 in Group A–D) were fixed in 10% formalin solution for 7 days. All implants were dehydrated in a graded ethanol series (70–100%) and transferred into a methylmethacrylate (MMA) solution that polymerized at 37°C within 1 week. Three slices of histology section (about 50 µm in thickness) were conducted longitudinally alone the long axis of tibia at the central region using the modified interlocked diamond saw . Histological sections remained unstained first and were subjected to the fluorescence analysis using epifluorescence microscopy. Then they were stained with 1.2% trinitrophenol and 1% acid fuchsin (Van Gieson staining) for light microscopy analysis. The qualitative analysis of bone formation and fluorochrome markers were performed using a light/fluorescence microscope . Prior to histomorphometry analysis, bone and material were pseudocoloured respectively using Adobe Photoshop 6.0 and then measured using an image analysis system . Bone formation was quantified from the pixels that represented bone tissue, while the total area was defined as the implanted bone site (4 mm×16 mm). The rate of new bone formation was presented as the percentage of bone area in total implant area [×100%]. In addition, the bone mineralization apposition rate  was analyzed from the images of fluorochrome labeling.99mTc-methylene- diphosphonate (MDP) was injected through the ear vein at a dose of 5 MBq/kg. ECT images were acquired (140 Kev maximum) 4 h after injection, and delayed images were obtained (512×512 acquisition matrix). During the dynamic imaging, manually drawn rectangular regions of interest (ROIs)  were established on the implanted bone sites. The uptake ratio of 99mTc-MDP (T/NT) between the implant site and the contralateral radius was determined for quantitative analysis.ECT was performed at 2, 4, 8 and 12 weeks after surgery. p<0.05 were considered to be statistically significant.The results were expressed as the means ± SD. Statistical analysis was performed using one way ANOVA followed by a SNK-q test for multiple comparisons. Values of Four types of cylindrical β-TCP scaffolds with different architectures were manufactured and characterized by micro-CT respectively . Two typ[TIGHTER All rabbits exhibited normal diets and movements after surgery, without any surgical complications and sign of infection. X-ray images were taken to evaluate the position of the scaffolds and the development of bone regeneration within the defects  and autologous bone graft (Group E) showed well integration of implants with the surrounding tissue (p>0.05), indicating a similar growth rate of bone tissue in these four kinds of scaffolds.Fluorescent labeling was detected in the regenerated bone in all specimens. Bone remodeling was validated by interrupted bone labeling and new bone deposition. The bone labeling was detected in the pores of both porous scaffolds (Group A–B) and tubular scaffolds (Group C), while only limited amount of new bone formed around the solid scaffolds (Group D) . It was p<0.05), indicating better osteogenesis of tubular scaffolds than porous or solid scaffolds , while Group D showed the least amount of degradation (15.9±1.8%)  . The resp<0.05) or Group B , indicating better mechanical property and improved osteointegration capability of the tubular bone grafts.In order to evaluate the mechanical properties of the scaffolds and the integration of the new bone tissue within scaffolds, we carried out compression tests on the native and implanted scaffolds 12 weeks post transplantation. The solid shaped scaffolds (Group D) showed the highest compression strength of native scaffolds but achieved the lowest strength of harvested scaffolds after 12 weeks implantation, indicating little bone formation and osteointegration with scaffolds . Tubularp<0.05), indicating better bone ingrowth in tubular scaffolds than other ones  in the defect region tended to increase with time after the surgery throughout the whole study in each group, while the rate of increase became gradually steady after 4 weeks post-surgery. The solid scaffolds (Group D) had the lowest uptake ratio which changed slightly after implantation, indicating limited infiltration of capillary vessels. Group A, B and C displayed comparable 99mTc-MDP uptake ratio (T/NT) at 2 weeks, while tubular scaffolds (Group C) had a higher degree of blood flow perfusion from 4 weeks post-surgery than that of porous scaffolds (Group A and B) (p<0.05).Bone scintigraphy with radioactive tracers has been widely used in the imaging of vascularization and metabolic activity of bone tissue. In this study, ECT was conducted at 2, 4, 8 and 12 weeks after surgery to analyze the vascularization of the TCP scaffolds. Four hours after injection of 99mTc-MDP, about half of the tracer was deposited in the bone and delayed images were obtained . The uptA and B) . At 12 wPorous scaffolds as bone graft substitutes have been widely investigated for segmental bone defect treatment Scaffolds that mimic natural bone structures could be used as a favorable bone graft substitutes for cell and bone ingrowth when implanted, with reported efficacy in promoting in vivo osteogenesis and integration with surrounding bone tissue When compared to porous scaffolds, tubular scaffolds demonstrated to be better bone graft substitute design in treating the segmental defect, achieving considerably better quantity and quality of new bone formation. Generally, it is more challenging to repair the segmental bone defect than a cavity bone defect, due to poor bone formation environment of segmental defect, which is surrounded predominantly by soft tissue. To prevent the rapid invasion of surrounding soft tissue and protect the defect space for subsequent bone tissue ingrowth has become the key for successful defect healing 99mTc-MDP, the most commonly used tracers in clinical bone research, will be accumulated mainly into the bone tissue after injection, especially in the highly vascularized region and the accumulation dosage is closely correlated to the degree of vascularization. The capacity and accuracy of ECT to evaluate the vascularization has been validated by a number of other methods such as magnetic resonance imaging (MRI) Moreover, tubular scaffolds facilitated higher volume of vascularization within the grafts than porous ones, as indicated by the radionuclide bone imaging. Radionuclide bone imaging is a well-established clinical examination technique to assess the abnormalities of bone tissue Despite of its osteoconductive properties, the in vivo application of β-TCP is generally fraught with the problem of rapid degradation after the implantation In this study, we have systematically compared the efficacy of tubular scaffolds with porous scaffolds for segmental bone defect treatment, and demonstrated that compared with the traditional porous structure, scaffolds with tubular structure can promote much better defect healing with significantly higher amount of bone tissue formation and neo-vascularization, greater in vivo biomechanical strength and eventually achieving the complete union of segmental defect. These findings have provided a further knowledge about the influence of scaffold architecture on their in vivo defect healing performance and illuminated the great potential to use tubular scaffolds as effective bone graft substitutes in the segmental defect treatment.


Chronic kidney disease is a long-term complication in acute intermittent porphyria (AIP). The pathophysiological significance of hepatic overproduction of the porphyrin precursors aminolevulinate acid (ALA) and porphobilinogen (PBG) in chronic kidney disease is unclear. We have investigated the effect of repetitive acute attacks on renal function and the effect of total or five-sixth nephrectomy causing renal insufficiency on hepatic heme synthesis in the porphobilinogen deaminase (PBGD)-deficient (AIP) mouse. Phenobarbital challenge in the AIP-mice increased urinary porphyrin precursor excretion. Successive attacks throughout 14 weeks led to minor renal lesions with no impact on renal function. In the liver of wild type and AIP mice, 5/6 nephrectomy enhanced transcription of the first and rate-limiting ALA synthase. As a consequence, urinary PBG excretion increased in AIP mice. The PBG/ALA ratio increased from 1 in sham operated AIP animals to over 5  and over 13  in the 5/6 nephrectomized mice. Total nephrectomy caused a rapid decrease in PBGD activity without changes in enzyme protein level in the AIP mice but not in the wild type animals. In conclusion, high concentration of porphyrin precursors had little impact on renal function. However, progressive renal insufficiency aggravates porphyria attacks and increases the PBG/ALA ratio, which should be considered a warning sign for potentially life-threatening impairment in AIP patients with signs of renal failure. Acute intermittent porphyria  is an inherited metabolic disease characterized by partial deficiency of hepatic porphobilinogen deaminase (PBGD). The disease is inherited in an autosomal dominant manner and is the most common of the acute porphyrias Chronic kidney disease may occur as a long-term complication of symptomatic disease in acute porphyrias, leading to vascular complications, progression of peripheral neuropathy and eventually need for dialysis. Chronic arterial hypertension and renal impairment become more common after middle age in AIP, especially in patients with frequent porphyric attacks There are few studies in which biopsies have been performed in patients with AIP. In one large population study in vivo study to address the liver-kidney interaction during acute porphyric attacks. Although the mouse is a predictive model for AIP, extrapolation to human disease is considered and discussed in the paper.In the present work, using an AIP mouse model th phenobarbital challenge was significantly higher than that after the 3rd challenge. Urinary porphyrin precursors and porphyrins were unchanged within the normal range in wild type animals . These studies demonstrate that end stage renal insufficiency may aggravate the acute porphyria state, hypothetically by substrate inhibition. Physiologically, in a post-translational step, a dipyrromethane cofactor is assembled to apo-PBGD to become the holoenzyme PBGD This hypothesis may be supported by this The reduced hepatic ALAD activity associated with renal impairment did not modify ALA accumulation as suggested by unchanged urinary ALA excretions in the AIP mice. In fact, the inhibition of ALAD is only significant for ALA accumulation when important ALAD deficiency is observed. This is the case of the homozygous patients with ALAD deficiency, lead poisoning and hereditary tyrosinemia deficiency where the hepatic ALAD activity is less than 1% of the reported normal activity In conclusion, consecutive phenobarbital challenge in mice caused slight degrees of renal lesions unrelated to porphyria and indicated that massive porphyrin or porphyrin precursor excretion, or their passage through the kidney, did not modify renal function in AIP mice. However, conclusions obtained in the AIP mouse model cannot be extrapolated to chronic AIP disease and we cannot disregard the potential deleterious effect of high porphyrin and porphyrin precursor accumulation maintained during years. Indeed, once end stage renal disease was established we cannot rule out the deleterious effect of porphyrin accumulation since dialysis membranes display a limit efficiency to filter these molecules Our results indicate that progressive renal insufficiency in AIP mice may aggravate the acute porphyria state. These data may illustrate the pathophysiology in AIP patients afflicted by recurrent acute attacks and renal failure. This is an extremely vulnerable clinical condition in which the development of severe neuropathic complications is very likely. Even though dialysis membranes are able to clear porphyrin precursors The increase in PBG/ALA ratio should be considered a warning sign for potentially life-threatening aggravation of the porphyric condition. These patients should be candidates for combined kidney and liver transplantation, in order to correct the primary enzyme deficiency as well as restore renal function. These data and other recent cases have clarified previous concerns and could help to formulate the indications for and the timing of transplantation in AIP.tm1(neo)Uam) and T2 strain (C57BL/6-pbgdtm2(neo)Uam) mutations. To biochemically imitate a human porphyria attack, AIP mice were injected intraperitoneally with increasing doses of phenobarbital  for four consecutive days . Urines (24-hour) were collected in metabolic cages.Animal studies. Experimental protocols were performed according to European Council Guidelines. Acceptable standards of humane animal care and treatment employed in these mice (ref. no. CEEA022-06) and the experimental design of this study (ref. no. CEEA029-09) were approved by the Ethics Committee for Animal Testing of the University of Navarra. AIP mice Partial or total nephrectomies were performed in 4- to 6-month old mice of both sexes. Acute renal failure was induced surgically. Mice were anaesthetized and kidneys were exposed by dorsal flank incision. In the 5/6 nephrectomy model, the renal artery was briefly clamped and two thirds of the left kidney (upper and lower poles) were excised, leaving the upper pole renal capsule and adrenal gland intact. One week later, the right kidney was removed after ligation of the renal artery, vein and ureter. A phenobarbital challenge was administered one month after extirpation of the right kidney. One day after the last phenobarbital injection, 24-hour urine and serum samples were collected, animals were sacrificed, and the three first enzymatic steps in hepatic heme biosynthesis were investigated.In the bilateral nephrectomy groups, both kidneys were removed in the same operation, after ligation of the renal artery, vein and ureters. The nephrectomy was performed after the last dose of phenobarbital challenge and animals were killed 10 hours later. Serum and liver samples were collected at each time.4/CH3OH  and measured in a Spectrofluorometer . Urinary porphyrin concentration was measured according to Westerlund et al. Biochemical analysis. Renal impairment was estimated by serum blood urea nitrogen levels  or serum cystatin C concentration  5′-CAAAGAAACCCCTCCAGCCAA -3′, reverse primer: 5′-GCTGTGTGCCGTCTGGAGTCTGTG -3′, product length:101 bp). The amount of ALAS1 transcript was calculated as the n-fold difference relative to the control gene actin as an internal control . Results were expressed according to the formula 2ΔCt(Actin)−ΔCt(gene), where ΔCt represents the difference in threshold cycle between the target and control genes.RNA extraction and hepatic liver enzyme expression analysis. Total RNA was extracted from liver tissues using TRIzol Reagent (Invitrogen life technologies). Total RNA was used to make cDNA using the Stratascript first strand cDNA synthesis kit (Stratagene). The steady state mRNA level of the ALAS1 was analyzed by quantitative RT-PCR using iQ SYBR green supermix in an iQ5 real-time PCR detection system . PCR amplification was performed under the following conditions: one cycle of 3 min. at 95°C; followed by 35 cycles of 15 s at 95°C, 30 s at 60°C, 25 s at 72°C, and 10 s at 70°C, 10 s at 75°C and 30 s at 80°C (detection temperature of 82°C); followed by a single final extension cycle of 72°C for 4 min. Relative transcript level was determined using primers annealing specific cDNA sequences of ALAS1  of right and left kidneys were processed for hematoxylin and eosin and sirius red stainings. Lesions and prognostic score of renal biopsies were evaluated by a trained pathologist (ES) using a semiquantitative score adapted from the Banff's consensus criteria for the evaluation of renal allograft biopsies (doi: 10.1111/j.1600-6143.2006.01688.x). Briefly, glomerular, tubulointerstitial and vascular lesions were scored . The main features scored were: chronic glomerulopathy, tubulitis (t), tubular atrophy (a), interstitial inflammation (i), interstitial fibrosis (sirius red positive) (f), vasculitis (v), subendothelial thickening (th), and arteriolar hyalinosis (ah). In addition, presence of crystals or deposits was ascertained, and occasional findings were reported under ‘observations’.Figure S1Expression levels of ALAD and PBGD in the liver of AIP mice suffering from different degrees of renal insufficiency. The cDNA samples were obtained as described in 5′-ACGTCTGCTTGTGCCCCTAC -3′, reverse primer: 5′-ACAGCGTCGGTCTCCAAAAG -3′, product length: 311 bp) or PBGD . The amount of each transcript was calculated as the n-fold difference relative to the control gene actin as an internal control . The amount of each transcript was expressed according to the formula 2ΔCt(Actin)−ΔCt(gene), where ΔCt represents the difference in threshold cycle between the target and control genes. The non-parametric Mann–Whitney U-test was used for comparison of two groups of mice. Nx5/6, 2/3 nephrectomy of the left kidney and extirpation of the right kidney; Nx 6/6, total nephrectomy. ALAD, aminolevulinate acid dehydratase; PBGD, porphobilinogen deaminase; AIP, Acute Intermittent Porphyria.(TIF)Click here for additional data file.Figure S2Lack of histological abnormalities in the remnant kidney pole from porphyric mice one month after 2/3 nephrectomy of the left kidney and extirpation of the right kidney. A) This image indicates well organized histoarchitecture of the renal cortex from a porphyric mice. Glomerulus (arrow) was surrounded by glomerular capsule including proximal (*) and distal tubules (#), B) Cross sections of tubules in the medulla. Kidney samples were taken three days after the last phenobarbital dose. No heme precursor deposits or vascular atrophy were observed .(TIF)Click here for additional data file.Figure S3Unchanged hepatic PBGD protein level in wild type and AIP mice ten hours after total nephrectomy. Male mice data are presented in left panel and female animals in the right panel. Immunoblot assay was performed as described (TIF)Click here for additional data file.