Solanum lycopersicum) consumption has been one of the most common causes of produce-associated salmonellosis in the United States. Contamination may originate from animal waste, insects, soil or water. Current guidelines for fresh tomato production recommend the use of potable water for applications coming in direct contact with the fruit, but due to high demand, water from other sources is frequently used. We sought to describe the overall bacterial diversity on the surface of tomato fruit and the effect of two different water sources (ground and surface water) when used for direct crop applications by generating a 454-pyrosequencing 16S rRNA dataset of these different environments. This study represents the first in depth characterization of bacterial communities in the tomato fruit surface and the water sources commonly used in commercial vegetable production.Tomato (Pantoea and Enterobacter were the most abundant.The two water sources tested had a significantly different bacterial composition. Proteobacteria was predominant in groundwater samples, whereas in the significantly more diverse surface water, abundant phyla also included Firmicutes, Actinobacteria and Verrucomicrobia. The fruit surface bacterial communities on tomatoes sprayed with both water sources could not be differentiated using various statistical methods. Both fruit surface environments had a high representation of Gammaproteobacteria, and within this class the genera Despite the major differences observed in the bacterial composition of ground and surface water, the season long use of these very different water sources did not have a significant impact on the bacterial composition of the tomato fruit surface. This study has provided the first next-generation sequencing database describing the bacterial communities living in the fruit surface of a tomato crop under two different spray water regimes, and therefore represents an important step forward towards the development of science-based metrics for Good Agricultural Practices. Salmonella and E. coli strains implicated most frequently. Lettuce and tomatoes were the commodities associated with the most outbreaks, followed by cantaloupe and berries [An increasing number of epidemic outbreaks caused by contamination of produce by human pathogens have been observed in the United States . Between berries . In rece berries .The phyllosphere has found itself at an intersection of food safety concerns and research that examines the microbial ecology of agricultural environments -6. HumanSalmonella [E. coli O157:H7 [Salmonella contamination. Groundwater sources, on the other hand, are less likely to support enteric pathogens because of the natural filtering mechanisms of soils, although poorly managed wells are susceptible to contamination [In the United States, the tomato industry's Good Agricultural Practices guidelines, which are focused on improving the food safety of the product, recommend the use of potable water for applications that come in direct contact with the crop . Given tlmonella  and whit O157:H7 . This remination .The type of irrigation system can influence the risk of crop contamination: overhead irrigation, for instance, is more likely to produce virus contamination than are furrow and drip irrigation . StudiesDenaturing gradient gel electrophoresis studies have indicated that variables such as plant species and stage of development can affect the composition of phyllosphere microbial communities. In addition, it was found that these communities are far more complex than culture-based methods used in the past had indicated ,15,16. RMore recently next-generation sequencing technologies, including 454-pyrosequencing, have provided more comprehensive descriptions of bacterial communities in different environments due to the increased number of sequence reads obtained -26. A stps), or groundwater (pg), three samples of surface water (ws), and three samples of groundwater (wg). Using these data, we sought to 1) compare the bacterial profile of ground and surface water that was used for pesticide applications and 2) assess the impact of water quality on the fruit surface bacterial profile of a tomato crop. A smaller preliminary dataset of 2008 fruit surface samples generated through Sanger sequencing is also included for comparison. Despite the significant differences between bacterial communities in surface and groundwater, the surface communities on the tomato fruits treated with these water sources could not be differentiated by a variety of statistical methods.We utilized 454-pyrosequencing to generate 34,016 16S rRNA gene sequences from 16 field samples: 10 tomato fruit samples that had been sprayed with either surface water (After screening our data for poor sequences and contaminants (see Methods), we recovered 27,757 high-quality 16S rRNA gene sequences with an average of 1,734 ± 471 (SD) sequences per sample (results refer to the 2009 data unless otherwise stated).ps4) was dominated by Firmicutes sequences, most likely as a result of contamination with internal fruit material. While the wg samples displayed similar 16S rRNA profiles dominated by Proteobacteria, the ws samples had a more even representation among four dominant phyla. In addition, ws samples contained a large number of sequences that could not be classified even at the phylum level  using the RDP Bayesian classifier with a confidence threshold of 80%. Examining the phylum level distributions across samples, we found that nearly all fruit surface samples appeared to have very similar 16S rRNA profiles. In these, Proteobacteria dominated the observed sequences, with smaller representations of Firmicutes and Actinobacteria. One surface water treated sample , most notably Betaproteobacteria, which makes up approximately 86% of sequences on average in the 5, most npg and wg were compared (10 vs. 86%), among nine differentially abundant bacterial classes (FDR ~0.07). Fourteen bacterial classes were differentially abundant between ws and ps (FDR ~0.06), most notably Clostridia, which was enriched for in ws. Both fruit surface environments were enriched for Gammaproteobacteria. Despite the differences observed between water sources, no significant differences were found between the two fruit surface environments (this includes an attempt in which the ps4 outlier was removed).Betaproteobacteria was also the most differentially abundant class when Pantoea, Enterobacter, Sphingomonas, Leuconostoc, Pseudomonas and Burkholderia . Table ps samples, and Gammaproteobacteria is enriched in pg  indicated that in both the 454 and Sanger data, Bacilli is enriched in the g Figure . At the s Figure . Enterobpg), 399 (ps), 228 (wg) and 1342 (ws). After computing rarefaction curves for each sample  using Mothur  and a sie Figure , we immel Figure .Good's Coverage Estimator on the OTUs from each sample using Mothur. Results indicated that we captured between 93 and 98% of the species in all of the samples except for ws samples, where we only identified between 70 and 73% of the species.To assess the diversity captured with the samples, we calculated the We then examined shared OTUs between individual replicates and treatments. Fruit surface environments shared approximately half their OTUs, and these represented more than 90% of the sequences in both samples. In contrast, water environments shared only 31 OTUs, which represented 2% of the OTUs present in surface water and 14% of those in groundwater. These shared OTUs corresponded to 62% of the sequences in groundwater, but only 6% of the sequences in surface water. These results again point to the greater differences between water-based microbial communities as compared to those in the treated tomato fruit surfaces.A hierarchical clustering of all samples was performed using the Jaccard index based on shared OTU composition Figure . This trhttp://clovr.org). CloVR is a virtual machine designed to run large-scale genomic analyses in a cloud-based environment such as Amazon EC2. The CloVR-16S track runs Mothur [To test the sensitivity of the above results to any particular methodology, we re-ran our analysis using the new automated 16S rRNA pipelines provided by the CloVR software package  included organic managed plots (2008) and use of an additional pond as a source of surface water (2009). Standard agricultural practices for the production of shipping tomatoes in the region were used.Field plots were established at the University of Maryland Wye Research and Education Center in Maryland's Eastern Shore . The soil was a Nassawango silt loam. Tomato transplants were planted in the field on June 9 2008 and June 10 2009. 'Sweet olive' (2008) and 'Juliet' (2009) grape tomato plants were planted on black plastic mulch and trained using stakes and a four-tier string system. The experimental design was a randomized complete block design with five blocks and three treatments. Seedlings were planted in paired rows (only one of them used for this study), 1.8 m apart. Each paired row was 9.0 m apart from the next set of paired rows. Within each row, each experimental unit was 9.0 m from the next. An experimental plot was composed of 3 grape tomato plants alternated with 2 'Brandywine' shipping tomato plants, which were not used for sampling (2008) or 5 grape tomato plants (2009) at an in-row spacing of 60 cm. In 2008, pesticides mixed in either ground or surface water were sprayed on: June 21, June 29, July 7, July 15, July 23, July 30, August 10 and August 30. In 2009, pesticides were sprayed on July 2, July 14, July 28, August 9, August 20 and August 30. Spray treatments were applied with a CO® bags (2008) or Whirl-Pak® bags (2009) by using new gloves for each replicate and ethanol disinfection of pruning shears between samples. Samples were then transported back to the laboratory at 4°C. One hundred milliliters of sterile water were added to the bags, and samples were agitated for 1 minute by hand and then sonicated for 2 minutes. The microfloral wash was then transferred to polypropylene tubes and centrifuged at 30,000 × g overnight at 4°C. The pellet was then transferred to a microcentrifuge tube and stored at -80°C until DNA extraction was performed. Three liters of groundwater and 50 ml of surface water collected on August 31 2009 were filtered through 0.45 μm Fisherbrand® filters . Filters were aseptically divided into four microcentrifuge tubes and stored at 80°C. DNA extraction from filters and pellets was performed using the Promega Wizard DNA extraction kit  in 2008, and the Zymo Research fungal/bacterial DNA extraction kit  in 2009.Samples consisting of 6 tomato fruits were aseptically collected on September 1 2008 and August 31 2009. Fruits were systematically harvested from different locations within the experimental unit and placed in Ziploc®-T Easy Vector (Promega) according to manufacturer's instructions. Bacterial colonies were frozen in 100 μl aliquots of Luria broth (Miller) solution with 10% glycerol in 96-well plates and shipped on dry ice to Agencourt Genomic Services, Beverly, MA, for Sanger sequencing.PCR amplification of the 16S rRNA bacterial gene was performed using forward primer GM5F 5'-CCTACGGGAGGCAGCAG-3'  and reve2, 0.60 units AmpliTaq Gold LD, 10 × buffer provided by manufacturer). PCRs used a denaturation step at 95°C for 5 min, followed by 30 cycles of 95°C 1 min, 55°C 1 min, 72°C 1 min, with a final extension step at 72°C for 5 min. Four independent PCR amplifications were performed for each sample. After a gel based confirmation of PCR amplification, PCR products were purified using the AMPure kit  following manufacturer's recommendations, and quantified using a Qubit flurometer (Invitrogen). PCR products were pooled and the average fragment size was assessed on a 2100 Bioanalyzer  using a DNA 7500 chip. Emulsion-based clonal amplification and sequencing on the 454 Genome Sequencer FLX-Titanium system were performed at the W. M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign according to the manufacturer's instructions . The PCR products were sequenced on two regions of a 16-region 70 × 75 picotiter plate. Signal processing and base calling were performed using the bundled 454 Data Analysis Software version 2.0.00.PCR amplification of the 16S rRNA bacterial gene was performed using forward primer Bact-8F (AGAGTTTGATCCTGGCTCAG)  and reveRecent validation studies have demonstrated several biases in analyses of 16S rRNA sequence datasets produced using 454-pyrosequencing technology . We haveSequences were first selected for length and quality according to the following criteria:(i) ≥100 nucleotides in length (not including sample-specific barcodes)(ii) a perfect match to a sample-specific barcode(iii) reads were trimmed at the beginning of a poor quality region - defined as a 10 bp window containing 8 bp with a Phred-score ≤ 20.http://microbiomeutil.sourceforge.net/- Broad Institute) and contaminants such as chloroplast and eukaryotic DNA using BLAST [Reads meeting the above criteria underwent rigorous screening for chimeric reads  depends on the total number of sequences in the sample (Ni) and the number of singleton OTUs within that sample, n1i.where Good's coverage of the Statistical comparisons between environments were made using Metastats  . The trimmed MSA was imported into Arb to create a neighbor-joining phylogenetic tree, using Staphylococcus aureus as an outgroup.We then searched all sequences from our samples against this database using BLASTN with default parameters and isolated any reads matching one of the reference genes with ≥ 98% identity along ≥ 95% of its length. NAST was then used to create a multiple sequence alignment of all matching reads and a reference set of 68 Enterobacteriaceae species that spanned http://clovr.org - Institute for Genome Sciences - University of Maryland Baltimore). These methodologies run similar analyses using Mothur [To investigate the sensitivity of our major results to our particular methodology, we ran two alternate analyses employed by the CloVR virtual machine software package , standard error of the corresponding average (SE) and p-value for the comparison between environments.Click here for fileTable S2: Bacterial genera abundance in tomato fruit surface and water samples. Average relative abundance of sequences assigned to that genus (mean), standard error of the corresponding average (SE) and p-value for the comparison between environments.Click here for file


Polyvinyl Alcohol (PVA) hydrogel plugs were implanted in artificial osteochondral defects on the trochlear groove of rat knees. After 0, 3, 6, 12, and 24 weeks of followup, samples containing the implants were mechanically evaluated by creep indentation test, chemically, and histologically by optical microscopy. The mechanical test pointed towards an increase of the implant creep modulus and the chemical analysis exhibited an increasing concentration of calcium and phosphorus within the implants over time. Optical microscopy showed no foreign body reaction and revealed formation, differentiation, and maintenance of new tissue at the defect/implant interface. The absence of implant wear indicated that the natural articular lubrication process was not disturbed by the implant. The performance of the irradiated and acetalized PVA was considered satisfactory for the proposed application. The articular cartilage consists of a high-specialized, low friction tissue that covers the epiphyses with the function of enabling bones do glide over each other and to absorb impacts within the joint without causing wear; this cartilage has a poor capacity of renewal –3. Its vThe articular cartilage can suffer degeneration, beginning with a degradation of the cartilage itself  that may reach and expose the subchondral bone  . The damNowadays, treatment options are based on abrasion, microfractures, mosaicplasty, first- and second-generation autologous chondrocyte implantation, and allogeneic osteochondral grafting –16. ThesIn view of this, the development of osteochondral implants that mimic the physical, chemical, and mechanical properties of the articular cartilage has been proposed , 17, 18.α-hydroxy ester), poly(ethylene glycol) (PEG), and poly(caprolactone) (PCL) [Among the materials used for osteochondral defect repair, bioceramics, such as bioglass, hydroxyapatite, and calcium phosphate, occupy a prominent place . Notewore) (PCL) . These pe) (PCL) . Amongste) (PCL) , 21–26. e) (PCL) –30.in vitro and in vivo tests of PVA hydrogel have exhibited promising results [in vivo performance of PVA cross-linked by electron beam irradiation implanted in Wistar rats' knees by means of mechanical, chemical, and histological tests.Preliminary  results , 32, quaAqueous solution of PVA was obtained by 10% Sigma-Aldrich mW 89,000–98,000 g/mol, 99% hydrolyzed and was prepared and homogenized at 70°C for 1 h in magnetic stirrer. The solution was transferred to a Petri dish and kept at room temperature for 7 days resulting in 1 mm thick membranes . The memThereafter, the samples were hydrated and swelled in 0.9% sodium chloride (NaCl) solution for 48 h and osteochondral implants (plugs 2 mm in diameter and 1 mm in height) were obtained by punched cut outs . The pluForty-four male rats (~380 g) were anesthetized by intravenous injection of 25 mg/kg pentobarbital according to the ethical protocol approved by the Ethics Committee in Animal Experimentation of the University of Campinas, São Paulo, Brazil (protocol n° 1047-1/2008). The knee joint was accessed by a medial parapatellar incision and a twist drill fitted with a depth stop was used to produce cylindrical osteochondral defects (2 mm in diameter and 1 mm deep) in the intercondylar region.The animals were divided into 6 groups: 5 experimental groups with 8 animals each, for implantation of the PVA samples  and a “cartilage” control group (CG) with 08 animals, whose articular cartilage was kept intact.ad libitum until the animals were sacrificed.The hydrogel plugs were inserted under pressure into the defects with the aid of a trephine and sit flush with the surface of the adjacent articular cartilage . The join = 36) were randomly selected and stored in 0.9% NaCl solution until the test, in order to avoid tissue autolysis. The test was carried out on a universal testing machine EMIC DL300 . A load of 4.905 N (0.5 kgf) was applied to the implant surface by a hemispherically ended indenter (1.4 mm diameter) and kept for 180 seconds [in situ at 180 seconds after load application was calculated according to the equation below  and three nonimplanted plugs EG00 (n = 3) were subjected to XRF analysis, in order to assess the incorporation of calcium (Ca), phosphorus (P), and sulfur (S) by the PVA. After removal, the plugs were dried at room temperature, put into XRF polyethylene sample cups (Chemplex), and analyzed on an energy dispersive X-ray fluorescence spectrometer (Shimadzu EDX 700) with rhodium X-ray tube and semiconductor Si (Li) detector (energy resolution of 165 eV (Mn Ka line), under the following conditions: 3 mm diameter incident beam collimator, X-ray tube voltage of 15 kV, exposure time of 100 s, and detector dead time of 25% [Three hydrogel plugs from each EG  were fixed in buffered 10% formaldehyde solution (pH 7.5) for 48 h, decalcified in a solution of ethylenediaminetetraacetic acid disodium (EDTA) 7% (w/w), hydrochloric acid 100% 140 mL (w/w), sodium tartarate 0.15% (w/w), sodium, and kaulium tartarate 0.9% (w/w) in distilled water for 15 days, washed in distilled water for removal of residual decalcifying solution, immersed in ethanol 70%, and finally embedded in paraffin. The samples were prepared for OM according to the previously described protocol [After the mechanical test, fourteen samples from the EG group (protocol .μm thick) were cut on a rotatory microtome Leica 2155 and stained with hematoxylin eosin (HE). Images were obtained with a Leica DMLB 100 S microscope (magnification 200 μm). The images were analyzed for the absence, moderate, or intensive presence of the following structures: granulation tissue (GT), collagen fibers (CF), mineralized bone matrix (MB), neoformed bone tissue (NB), and collapse of neoformed tissue (CN).Semithin serial sections , moderate (++), and (+++) intensive as described in According to Mow et al.  implantsFigures in vitro and in vivo compression cannot be compared with each other. Contrary to in vitro, in vivo tissue recovers its original dimensions after load removal thanks to a number of factors that define the elasticity and water absorption capacity of its complex structure, such as pressure, viscosity, temperature and macromolecular content of the interstitial fluid, and the concentration and type of dissolved ions [Several studies have shown that the mechanical behaviors of the articular cartilage under ved ions , 3, 4.Comparing the creep modulus obtained in this study for rat cartilage  and thatAnother contribution to the increase of the creep modulus over time could be assigned to the absorption of calcium and phosphorus by the implant, evidenced by the results of the EDXRF analysis .The EDRFX analysis  revealedEDRFX results indicated an increase in Ca and P levels over follow-up time. Further studies are being carried out to determine if the analyzed elements were detected in or on the surface the PVA.According to the macroscopic results of the EGs , the intAs observed in the macroscopic images Figures , the PVAFrom the OM , the quaThe implant surface appears to have maintained its integrity due to the presence of synovial fluid. That was possible because the electron beam crosslinking of the PVA promoted the formation of a polymeric structure that was strong, yet still complacent and capable of absorbing and retaining synovial fluid, which kept low the friction between the articular surfaces and counter surfaces in all EGs.The absence of signs of implant rejection in almost all samples attests the good compatibility between the hydrogel and the tissues surrounding the artificial osteochondral defect Figures –13.The PVA hydrogel implant bore the load without wear or rupture, offered a stable and suitable environment for growing new bone at the interface with the host tissue, and did not disturb the natural articular lubrication. The results of these mechanical, chemical, and histological analyses indicate PVA hydrogel irradiated and acetalised as a promising biomaterial for repairing osteochondral defects.


Interbody fusion is an established procedure to preserve disk height and anterior fusion, but fusion with autografts, allografts, and metallic cages has its endogenous shortcomings. The objective of this study is to investigate whether a biphasic scaffold model, the native demineralized bone matrix cylinder in conjunction with degradable biomaterial poly, can be employed as a biological graft for interbody fusion. The poly was synthesized by polycondensing malic acid and polycaprolactone and then the concentric sheet of poly was fabricated into the demineralized bone matrix cylinder derived from rabbit femurs. Rabbit chondrocytes were loaded onto the three-dimensional constructs with 1-day in vitro culture and implanted into the subcutaneous dorsal pocket of nude mice. The chondrocytes/scaffold constructs are approximately two folds bigger than the scaffold-alone constructs after 12 weeks of implantation. X-ray and micro-computed tomography imaging showed endochondral bone formation in the chondrocytes/scaffold constructs as early as 4 weeks and showed that the bone intensity increased over time. Histological staining confirmed the above observation. By week 8, lamellar bone tissues were formed inside the demineralized bone matrix cylinder. In addition, the compression biomechanical test showed that the chondrocytes/scaffold constructs produced a significant higher compressive strength compared to the scaffold group. These results demonstrated that the inner-phase poly degraded over time and was replaced by new bone in an in vivo environment. Tricortical iliac crest autograft (bone from the same patient) serves as a gold standard for interbody fusion, since the graft has strong osteoconductive and osteoinductive potentials for bone remodeling2; however, the harvesting from the iliac crest can be associated with short- and long-term morbidity in up to 22% of the cases.3 This donor site morbidity has fueled the search for various forms of allograft materials as alternatives for cervical interbody fusion. Yet, allograft incorporates more slowly and has risk of disease transmission and immunogenicity.4 Nowadays, metallic cage devices have gained increasing popularity; however, titanium, one of the major components, has an elasticity modulus six times larger than the cortical bone.5 The altered biomechanics of the cervical spine from insertion of a metal scaffold may lead to unfavorable effects. In addition, some shortcomings of metallic cages, such as cage migration, subsidence, adjacent level degeneration, stenotic myelopathy, and nonunion, have already been reported.68 Long-term assessment of these cage devices, especially in multilevel applications, has been less than satisfactory with the incidence of revision procedures increasing over the last several years. Tissue engineering is an attractive technique for bone replacement,911 which has several advantages, including no donor site morbidity, decreased operating time, and conformation to the bone shape.Since Cloward began anterior interbody fusion of the cervical spine with autogenous iliac bone grafts, anterior cervical discectomy and fusion has been established as one of the operative procedures to distract the disk distance, enlarge the neural foramen, and obtain anterior interbody fusion.12 In our previous study, we have demonstrated that poly (PPCLM) (obtained by polycondensation of polycaprolactone (PCL) and malic acid) had good biocompatibility in a foreign body response in vivo assay.13 Incorporation of demineralized bone matrix (DBM) into the scaffold enhanced the compressive strength compared with PPCLM alone. In addition, the tensile stress of the DBM/PPCLM scaffold was 50-fold greater than that of PPCLM alone.Most synthesized bone scaffolds have good osteoconductive but not osteoinductive property. Some natural materials such as collagen, hyaluronic acid, and chitosan have the regeneration qualification but with inadequate mechanical properties.In the present study, we proposed a biphasic scaffold model for the interbody fusion by combining decalcified allograft and biodegradable materials. The inner phase of the scaffold is composed of a biomaterial based on PPCLM, oriented in a concentric sheet and seeded with chondrocytes. The outer phase of the scaffold is composed of DBM derived from rabbit femurs, which enclose a high content of type I collagen. The PPCLM and DBM were fabricated into a three-dimensional (3D) scaffold. Thus, the DBM/PPCLM constructs not only stimulate the intervertebral disk structure to fuse the vertebrae but also have both osteoconductive and osteoinductive properties. To explore the usefulness of this scaffold in interbody fusion, we characterized the cell proliferation, bone formation, and mechanical properties of the construct by subcutaneous implantation of the constructs in nude mice.13 Briefly, the malic acid (0.1 mol/L) and PCL (0.05 mol/L) chemicals were mixed and melted at 140°C with a constant stirring of nitrogen gas. After melting, the prepolymer was formed by lowering the temperature to 120°C for 1 h under nitrogen gas stirring. Then the PPCLM prepolymer was dissolved in tetrahydrofuran to form a 25 wt% solution, followed by addition of sieved salt. The resulting slurry was cast into poly(tetrafluoroethylene) dishes. After evaporating for 24 h, the molds were then transferred into an oven for postpolymerization. The salt in the composites was washed out by successive incubations in deionized water  every 12 h for 96 h. The porous sponge-like foams were freeze-dried and stored in desiccators for future use.PPCLM was synthesized as previously described by our group.14 The femurs were harvested from New Zealand white rabbits, and dissected free from connective tissues and washed with sterilized water. The femurs were then extracted with a mixture of chloroform and methanol (1:1 ratio) for 1.5 h followed by demineralization with hydrochloric acid for 15–18 h at 2°C and were sequentially washed with deionized water, 2 M CaCl2 at 2°C for 1 h, 0.5 M ethylenediaminetetraacetic acid (EDTA) for 1 h, 8 M LiCl for 1 h, and, finally, deionized water at 55°C. The DBM tubes (about 8 mm in diameter and 5 mm in height) were then equilibrated with Dulbecco’s modified Eagle’s medium (DMEM) containing 100 units/mL of penicillin and 100 mg/mL of streptomycin for 1 h at 37°C, and were stored at −70°C until use.DBM cylinder tubes were prepared following our published method.13 Briefly, the long stripes (2 mm × 5 mm × 40 mm) of PPCLMs were rolled over and inserted into the DBM tubes to form the DBM/PPCLM scaffold.The DBM/PPCLM scaffolds were fabricated by introducing the PPCLM scaffold into the DBM tubes.14 The rib cartilages were dissected from the male New Zealand rabbits. The cartilages were cut into pieces and digested for 3–6 h with 0.1% collagenase type II  at 37°C and then were resuspended in DMEM with 4.5 g/L glucose, 10% fetal bovine serum, 50 mg/L sodium ascorbate, 10 mm (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 units/mL penicillin, and 100 mg/mL streptomycin. The primary chondrocytes were cultured in a humidified incubator with 5% CO2 for 5–7 days. The cells were marinated in subconfluent, and the medium was changed every other day. The second passage of cells was used for experiments.The chondrocytes were isolated with enzyme digestion following previous published method.15 The strips of PPCLM were presoaked with 75% ethanol for 2 h and washed with an excess amount of phosphate-buffered saline (PBS) in a sterile condition. Approximately 5 × 106 cells/mL of chondrocytes were seeded onto the strips and were then incubated for 4 h prior to the addition of the culture medium. The complexes were cultured for another 24 h, and then, the rolled PPCLM sheets were inserted into the DBM scaffolds for subcutaneous implantation.Rabbit chondrocytes were cultured onto scaffolds as previously described.Animal protocols were approved by the University of Virginia Animal Care and Use Committee. The athymic NCr-nu/nu mice were purchased from Jackson’s laboratory. The scaffolds and chondrocytes/scaffolds were subcutaneously implanted in the dorsal space of the nude mice. Briefly, a 15-mm incision was cut in the dorsal skin of the mice between the scapulars using a size 15 scalpel blade, and a scaffold was embedded into the pocket. The samples were harvested at 1, 2, 4, 6, 8, and 12 weeks after implantation and fixed with 4% paraformaldehyde. Each group has four implants at each time point.16 The mice were anesthetized by isoflurane and placed in an in vivo µCT system . Following reconstruction of the 2D slices, an appropriate threshold was chosen. From 2D slices, 3D images were created using a feldkamp back projection algorithm . The 3D image was sectioned with Image J software , with one sagittal image per pixel. The volume of the bone tissue was selected and defined as the region of interest (ROI). Quantitative data of bone mass and volume were normalized to the data from the DBM at the same specimen.At 4, 6, 8, and 12 weeks after implantation, 2D radiographs of the specimen (four samples of each time point) were taken to qualitatively assess bone formation. For the quantitative evaluation of bone formation, in vivo micro-computed tomography (µCT) was performed at the same time points.17 The images were captured with a Nikon Eclipse E600 microscope .The implants were embedded into paraffin and cut into 7-µm-thick sections. The sections were subjected to standard hematoxylin and eosin (H&E), and Safranin-O staining as previously described.13 To test this tensile property of the scaffold, the implants were cut into a rectangular shape and the longitudinal tensile stress was measured on an Instron 5544 mechanical tester equipped with a 500-N load cell . The samples were compressed at a rate of 10 mm/min and stopped at 60% strain. All samples were tested in triplicate, and data are presented as mean ± standard deviation (SD).The compression properties were tested as our published method.The data are presented as mean ± SD at a significance level of p < 0.05. The statistical differences between groups were calculated using a Student’s t-test.13 We hypothesized that this cell-integrated scaffold can form bone-like tissue in an in vivo environment. To address this question, we implanted the constructs subcutaneously into the dorsal space of the nude mice. There were no significant size difference between the chondrocytes/scaffold and scaffold-alone construct at the first 2 weeks after subcutaneous implantation. However, the sizes of chondrocytes bearing constructs started to distinguish from the scaffold-alone constructs from the fourth week postoperatively, and size differences became gradually obvious until the samples were harvested at 12 weeks. The sizes of the chondrocytes/scaffold constructs were about two folds bigger than the scaffold-alone constructs at postoperative 12 weeks as showed by the morphology  and Safranin-O (right panel) is shown in The biomechanical property is critical for bone function. We, therefore, tested the biomechanical features of these implants. The compression test showed that chondrocyte-integrated scaffolds produce a significantly higher compressive strength (fourfolds) compared to the scaffold-alone ones  at week 1822 Herein, we have developed a biphasic scaffold using a native DBM as an outer layer and the PPCLM as an inner layer loaded with chondrocytes in the shape of intervertebral disk for interbody fusion. We demonstrated that the chondrocytes proliferated on the biphasic engineered constructs and endochondral new bone formation developed 4 weeks postoperatively in scaffold with chondrocytes in subcutaneous environment.As the metallic cages have many shortcomings, various biodegradable cages have been developed with tissue engineering approaches.24: (a) the DBM cylinder is a natural biomaterial with superior biocompatibility properties to most of the synthetic scaffolds developed thus far. Unlike most commercial DBM, the DBM cylinder provides structural strength; (b) DBM contains proteins and growth factors that necessitate for chondrogenesis and osteoinductivity. As we know, external growth factor(s) appliance has been well reported in spine fusion. Anterior cervical fusion with recombinant human bone morphogenic protein-2 (rhBMP-2) has an estimated 40% greater risk of adverse events with rhBMP-2 in the early postoperative period, including life-threatening events. After anterior interbody lumbar fusion, rates of implant displacement, subsidence, infection, urogenital events, and retrograde ejaculation were higher after using rhBMP-2 than controls.25 The DBM has different combination of growth factors/noncollagenous proteins in physiological levels, while no aforementioned side effects associated with high concentration of rhBMP-2 were reported; (c) the DBM cylinder has the potential to be partially decalcified to provide the sufficient mechanical competence according to different biomechanical requirement during bone formation; and (d) It does not evoke any appreciable local foreign body immunogenic reaction as antigenic surface structure of bone is destroyed during demineralization.The biphasic scaffold in the current study contains an outer DBM cylinder to simulate the native bone tissue, which has several advantages26 and that cells, either chondrocytes or stem cells, are critical for solid bone formation. Cells may serve as the targets of noncollagenous protein and growth factors released from the DBM. One of the shortcomings of this study is the lack of a chondrocytes/PPCLM/nonactive DBM control and a fibroblasts/PPCLM/active DBM control to define the role of active DBM and chondrocytes. For the future interbody fusion animal study, we will have both control groups to characterize the roles of chondrocytes and/or DBM in bone formation. We believe that chondrocytes/PPCLM and active DBM have synergic effects on the bone formation, which deserves further investigation.Our results showed that chondrocytes/scaffold implants have much more bone formation  to 5 and27 Malic acid–derived polymers are also used in tissue engineering as well as drug delivery.28 In addition, the intermediate product of malic acid is mammalian tricarboxylic acid, which is completely degraded into carbon dioxide and water. Thus, we had hypothesized that PPCLM produced by combination of PCL and malic acid was suitable for chondrocytes proliferating. Indeed, our previous in vitro data showed that chondrocytes could adhere and proliferate on both the surface and inside of the scaffolds.13 Consistently, in the current study, we demonstrated that chondrocytes proliferated in the scaffold as the size of the chondrocytes/scaffold constructs is much bigger than the scaffold-alone constructs after 12 weeks of implantation (30 In the histological sections, we did not observe any detectable necrosis area and inflammatory responses in both scaffold-alone and chondrocytes/scaffold constructs. Safranin-O staining showed that PPCLM degradation was observed since 1 week postoperatively, and chondrocytes speeded up the process, which was evidenced by loss of layered PPCLM structure in the chondrocytes/scaffold constructs versus the blurred PPCLM layers in the scaffold-alone constructs 1 week postoperatively. All these results suggest that the DBM/PPCLM is suitable for chondrocytes proliferation and osteogenesis, which is the base for bone tissue engineering.One of the fundamental features of scaffolds is to support adherence and proliferation of cells. In this biphasic scaffold, the inner phase was built by polycondensing the PCL and malic acid. The PCL polymer has been widely used in bone, cartilage, cardiac, vascular, and skin tissue engineering. The degraded products of PCL are neutral in pH and thus do not alter the local environment.antation . More imantation  and 5. Tantation  and µCT antation . The bonantation . Althoug31 Fifth, the tissue engineering design still uses a two-step approach, in vitro culture and then in vivo implantation. In the future interbody fusion in vivo experiment, we will use autograft bone marrow aspiration from either vertebral body or iliac crest loaded on PPCLM/DBM construct directly. Despite these limitations, the use of a DBM/PPCLM biphasic scaffold appears promising and worthy of further investigation.Certain limitations existed with the current technique and require further investigation before this approach can be applied to interbody fusion in vivo. First, the interface between the DBM and PPCLM remains a potential weak area. µCT showed clear spaces between DBM and PPCLM/chondrocytes . FurtherAltogether, this study demonstrated that the biphasic scaffold with the native DBM as outer layer and the biodegradable PPCLM as inner phase, which is in the shape of an intervertebral disk, is a promising candidate for interbody fusion as evidenced by abundant endochondral bone formation and increased biomechanical properties in a subcutaneous implantation model.


Soil microbial communities are in constant change at many different temporal and spatial scales. However, the importance of these changes to the turnover of the soil microbial communities has been rarely studied simultaneously in space and time.Archaea, Bacteria and Fungi along with key soil chemical parameters. We questioned how these abiotic variables influence the turnover of bacterial, archaeal and fungal communities and how they impact the long-term patterns of changes of the aforementioned soil communities. Interestingly, we found that the bacterial and fungal β-diversities are quite stable over time, whereas archaeal diversity showed significantly higher fluctuations. These fluctuations were reflected in temporal turnover caused by soil management through addition of N-fertilizers.In this study, we explored the temporal and spatial responses of soil bacterial, archaeal and fungal β-diversities to abiotic parameters. Taking into account data from a 3-year sampling period, we analyzed the abundances and community structures of Our study showed that management practices applied to agricultural soils might not significantly affect the bacterial and fungal communities, but cause slow and long-term changes in the abundance and structure of the archaeal community. Moreover, the results suggest that, to different extents, abiotic and biotic factors determine the community assembly of archaeal, bacterial and fungal communities. Understanding temporal and spatial patterns in the abundance and distribution of communities has been a fundamental quest in ecology. Such an understanding is crucial to allow an anticipation of responses of ecosystems such as soil to global changes Temporal variation of conditions is a very common feature of ecosystems. Ecologists have long been interested in how such variation structures natural communities The number of studies employing the concept of β-diversity to understand how microbial communities respond to biotic and abiotic parameters has increased substantially in soil ecology. Martiny and co-workers 3 to up to 106 per g soil 4 fungal propagules per g of dry soil were observed in Antarctic soils 7 per g of soil in soil crusts Of the soil microbial groups, bacteria have been mostly studied, as they exhibit an estimated species diversity of about 10In this study, we explored the temporal and spatial fluctuations of soil microbial communities and their relation to local environmental conditions. In order to do so, we investigated the spatiotemporal dynamics of the soil microbiota by analyzing the patterns of α- and β-diversity of archaea, bacteria and fungi in eight agricultural soils across the Netherlands. We sampled the soils eleven times, from 2009 to 2011. Furthermore, to complement the analyses, we applied TLA  The eight soil sites sampled are located in the Netherlands. Their characteristics and geographical coordinates are found in 3− in mg/kg of soil), ammonium (N-NH4+ in mg/kg of soil), organic matter (OM in %) and clay content (in %). The pH was measured in CaCl2 suspension 1∶4.5 (g/v) . Organic matter (OM) content is calculated after 4 hours at 550°C. Nitrate (N-NO3−) and ammonium (N-NH4+) were determined with a colorimetric method using the commercial kits Nanocolor Nitrat50  and Ammonium3  according to manufacturer's protocol.The environmental variables measured included pH, concentrations of nitrate , according to the manufacturer's instructions, after the addition of glass beads  to the soil slurries. The cells were disrupted by bead beating  three times for 60 s. Following extraction, the DNA preparations were electrophoresed over agarose gels in order to assess DNA purity, quality (average size) and quantity. The quantity of extracted DNA was estimated on gel by comparison to a 1-kb DNA ladder  and quality was determined based on the degree of DNA shearing (average molecular size) as well as the amounts of coextracted compounds.Burkholderia terrae BS001 or ITS region of Rhizoctonia solani AG3. Dilutions ranged from 107 to 102 gene copy numbers/µl. The archaeal standard curve was obtained by serial dilution of PCR product generated from Cenarchaeum symbiosum with the aforementioned archaeal specific primers Absolute quantification was carried out in four replicates on the ABI Prism 7300 Cycler . The 16S rRNA gene was amplified by qPCR using diluted extracted DNA as template. Specific primers for archaea (group 1 crenarchaeota) 771F/957R Archaea-specific forward primer at position 344 with a 40-bp GC clamp For DGGE analysis, bacterial 16S rRNA genes were PCR amplified using the forward primer F968 The diversity of each of the soil bacterial, archaeal and fungal communities was determined on the basis of the PCR-DGGE profiles. Total diversity (α) of the dominant community members was estimated from these data using the Shannon index, as recommended by Hill et al. To test how α-diversity, β-diversity and microbial abundance varied in relation to environmental variables, parametric Pearson correlation coefficients were calculated between α and β diversities, soil pH, organic matter, nitrate, ammonium, clay content and soil moisture, as well as between total abundances and TLA slopes using SPSS v18.0.3 . All variables except pH were transformed (Log(x+1)) prior to all analyses. Moreover, we applied variance partitioning to evaluate the relative contribution of the drivers of the microbial assemblages. Forward selection was used on CCA  to select a combination of environmental variables that explained most of the variation observed in the species matrices. For that, a series of constrained CCA permutations was performed in Canoco  to determine which variables best explained the assemblage variation, using automatic forward selection and Monte Carlo permutation tests (permutations = 999). The length of the corresponding arrows indicated the relative importance of the chemical factor explaining variation in the microbial communities.Soil pH, nitrate, ammonium and organic matter levels were determined in triplicate across all soil samples. Soil pH was significantly higher (P<0.05) in soils K, G and L  than in soils B, V, D, W and S  during the whole experimental period and no significant variation over time was observed. In all soils, significant changes were observed in the levels of nitrate, with lower values at the end of the growing season for most of the soils  and higher at the beginning . Levels of ammonium also varied over the whole period, with higher values being observed at the beginning of the season  and lower values at the end  .Considering each soil individually, they had characteristically different values, with higher levels of nitrate and ammonium found in soils B, V, D and S than in soils W, K, G and L . In 2009We studied the variations in the abundances of archaeal, bacterial and fungal communities over time and across all samples in three years. The total bacterial abundance showed significant temporal variation during the whole period, ranging between 8.12±0.23 (mean ± standard error) (September 2011) and 10.93±0.06 (June 2010) log copy numbers per g dry soil and showing comparable copy numbers in sandy (9.65±0.13) and clayey soils (9.64±0.16). The archaeal abundance (crenarchaeota) ranged between 6.96±0.14 (April 2009) and 8.78±0.07 (April 2011) log copy numbers per g dry soil, and showed significant differences between sandy and clayey soils across almost all sampling times, with lower numbers in the sandy soils (7.77±0.13) than in the clayey soils (8.22±0.13). Fungal abundance varied between 8.76±0.16 (February 2011) and 10.00±0.09 (April 2011), and significantly higher abundance was observed in the sandy soils depending on the sampling time . OverallWe used Pearson's correlation to examine how soil parameters influenced the abundances of bacterial, archaeal and fungal communities. Whereas the archaeal abundances were positively correlated with soil pH , they were negatively influenced by nitrate . A positive relationship was observed between fungal abundance and soil organic matter , and a negative relationship was observed between fungal abundance and archaeal abundance . Relationships between the abundance of bacteria and fungi, as well as between bacteria and archaea, were not significant. Interestingly, none of the soil parameters measured influenced bacterial abundance significantly.Understanding how species are distributed in space and time may yield a first avenue towards their assembly rules Concerning correlations with edaphic factors, a positive effect of OM content was observed on the archaeal α-diversity  . When usThe patterns of temporal β-diversity of the archaeal, bacterial and fungal communities  showed small but significant variations across soils . BacteriAlthough chemical parameters might show variability over time, significant correlations could still be observed. The patterns in the archaeal temporal β-diversities observed were mainly due to positive correlations with nitrate  . None ofSignificant relationships were observed between variation in β-diversity and H′ for bacterial  and fungal communities  but not for archaeal communities.The temporal changes of the microbial guilds were quantified and statistically tested via TLA. TLA analyses were performed separately per year and also considering all three years. For both analyses, the results and conclusions were similar. Therefore, we decided to include only the whole three year dataset. A statistically significant regression line (P<0.05) was observed for the archaeal community, with an overall slope of 1.835 . MoreoveIn our study, population sizes of archaea, bacteria and fungi, estimated using quantitative PCR, were found to be within the range observed in other soil systems A high abundance of crenarchaeota in soils has been previously observed The variations in microbial abundances could be explained by several parameters, depending on the target group. Soil pH and nitrate explained more than 75% of the variation in archaeal abundance. Previous studies have reported negative effects of pH on group 1.1c Crenarchaeota Acidobacteria and ActinobacteriaInterestingly, the bacterial abundances didn't respond to soil pH or any other measured abiotic parameter, although several studies have reported pH as the main determinant of bacterial community composition None of the soil parameters measured in this study were able to explain the patterns of α-diversity observed for bacteria and fungi. It might be that the taxonomic scale was too broad and a deeper analysis would allow a better understanding of the observed patterns, as observed by Rasche et al. Crenarchaeum symbiosum and Nitrosopumilus maritimus suggested that these organisms are capable of mixotrophy Archaeal α-diversity, on the other hand, was shown to respond to nitrate and OM levels. Nitrate had opposite effects on archaeal richness and diversity, depicting a community that responds to increasing nitrate with an increase of richness but a great decrease of evenness. This most likely indicates the outgrowth of previously undetectable OTUs. Thus, in addition to the strong negative effect on archaeal abundance observed by qPCR, nitrate availability seems to be a crucial factor determining archaeal community structure. The positive correlation between archaeal diversity and soil OM content indicate that the OM provides a substantial fraction of carbon to the local archaeal communities. Recently, genomic analyses of To assess how dynamic each soil microbial group was over time, we calculated the temporal β-diversity (average Bray Curtis dissimilarity) for each soil over time and for each microbial group. We observed a higher temporal β-diversity for archaea than for bacteria and fungi across all soils. This indicates that the archaeal communities are much more dynamic than the bacterial or fungal ones along a time gradient. These differences are probably due to the differential physiologies and sensitivities to environmental perturbations of these microorganisms. It has been shown that changes in temperature, moisture One main finding of this study is that, although the β-diversity patterns of the three microbial domains investigated are related with the same set of abiotic factors, the total percentage of variation able to explain those patterns was much higher for archaeal (45.0%) than for bacterial (6.6%) or fungal (6.9%) communities. This suggests that the archaeal communities might be much more sensitive to environmental changes than the bacterial or fungal ones. Based on these results, we hypothesize that the archaeal communities of agricultural soils with a long history of N-fertilization are more sensitive to disturbances than the corresponding bacterial or fungal communities.To be able to quantify community dynamics, allowing comparisons and providing a general overview of long-term trends in the complex soil system, we used the slopes obtained from TLA. TLA has been intensively used to identify directional changes and to quantify temporal dynamics of macroorganisms Changes in environmental variables within soil sites determine how time affects turnover (β-diversity), as different microbial interactions are favored if prevailing conditions change In this study we demonstrate that changes in the community composition of bacteria and fungi could be linked to both environmental and biotic factors (e.g. species-species interactions), as their (α-) diversity co-varied significantly with their β-diversity over the time period of the study. Conversely, archaea showed no significant correlation between α- and β-diversity, and the community shifts were mainly driven by the surrounding environment, mostly by the effects of soil pH and nitrate concentrations. This might indicate that changes in archaeal community are mostly driven by environmental factors, as previously observed by Zinger et al. No specific permits were required for the described field studies. The locations are not protected. The field studies did not involve endangered or protected species.Figure S1Biplots of canonical correspondence analysis (CCA) of Archaeal, Bacterial and Fungal similarity matrices and vector fitting of the environmental variables. Similarity matrices from DGGE data were obtained from eight soils over three years . Physico-chemical data, soil moisture (Humidity), soil nitrate (NO3), soil ammonium (NH4), organic matter (OM), clay content (clay + silt) and soil pH (pH) are presented with black arrows.(DOCX)Click here for additional data file.Table S1Soil chemical parameters measured in this study.(DOCX)Click here for additional data file.Table S2PCR mixtures for real time quantification of Archaeal 16S rDNA, Bacterial 16S rDNA and Fungal ITS region.(DOCX)Click here for additional data file.Table S3PCR mixtures for DGGE analysis of Archaeal 16S rDNA, Bacterial 16S rDNA and Fungal ITS region.(DOCX)Click here for additional data file.


Wounds and wound healing have always been one of the most important subjects that experimental researches were dedicated to. Simvastatin has been used for long as a common lipid lowering agent which was reported to have some pleiotropic effects such as antioxidation, anti-inflammation and immunomodulation. In this study we aimed to determine the effect of simvastatin on wound healing process in laboratory rats by means of stereological and histopathological analyses.2 circular full-thickness wound on their back were divided into three groups: SS group that received a gel with 2% concentration of simvastatin; UW group that received no treatment but daily irrigation with normal saline; Base group that was treated with the gel base. Duration of the study was 12 days and at the end, wound closure rate, grade of inflammation, granulation-tissue formation, ulceration, epithelization, fibroblast proliferation, collagen-bundles synthesis, and vascularization were determined.36 male Sprague-Dawley rats (220 ± 20 g) with a 1 cmOutcome of this study revealed that Simvastatin improves the wound healing by its anti-inflammatory and epithelization induction effect as well as statistically significant induction of fibroblast proliferation and collagen bundle synthesis which were reported by our stereological and histopathological investigations. Results of the present study demonstrated that topical Simvastatin enhances the wound healing process through affecting different aspects of tissue regeneration; however, further researches are needed to find the exact mechanism, advantages and disadvantages of this chemical agent. In addition, some complications such as disruption of blood vessels, infection and advanced inflammation might be occurred during the repair process , a HMG-CoA reductase inhibitor, is one of the statin derivatives commonly used as lipid lowering agent. Moreover, different studies demonstrated the various properties of SS such as immunomodulation, anti-inflammation, endothelial function improvement, endothelial nitric oxide synthase inhibition, and anti sepsis effect. Different experimental studies suggested that pleotropic effects of SS are independent of its lipid lowering property. Recent studies also reported possible improving influence of SS in the wound healing process; however, the safety profile of SS was declared excellent especially in local treatments and major side effects were rarely observed and were mostly reversible -7.Since SS presented positive effects on mechanisms that are contributed to wound healing promotion, in this study we aimed to determine the healing effect of this agent on full thickness skin wounds in laboratory rats. There was no report of using unbiased stereological methods  beside histopathological analyses for determining the effects of SS on full thickness skin wound.Preparation of SS gelSS was obtained from the Pharmacology Department of Shiraz University of Medical Sciences, Shiraz, Iran. In order to facilitate its application, we prepared 2% SS gel by dissolving 2 g SS in 2 cc ethanol (70%), and then transferring the solution into 2% carboxymethylcellulose (CMC) (2 g CMC dissolved in 98 cc distilled water). The base of gel was also supplied by the same method but without SS component.Animals and Excision of wound model2 circular full-thickness wound was created on the posterior surface of animal’s neck. The rats were randomly divided into three groups. One group was treated with vehicle gel (Base group), a group treated with SS gel (SS group), and the untreated wounded group (UW group) that received no-treatment except cleaning of the wound surface with normal saline every day. After 12 days, the animals were euthanized with ether overdose and full thickness skin biopsies (1 cm×1 cm) were taken from the wound site and were fixed in buffered formaldehyde (pH = 7.2) for histopathological and stereological evaluations. In an experimental study, 36 male Sprague-Dawley rats (220 ± 20 g) of 2-3 months of age were chosen. General anesthesia was induced by intramuscular injection of mixture of Ketamine  and Xylazine . A 1 cmThe study protocol was approved by the Animal Ethics Committee of Shiraz University of Medical Sciences and the animal care was in accordance with their guidelines.Histopathological studyAll the specimens were fixed in formalin, paraffin blocks were provided from the specimens, and 15 µm slides were created from the skins and stained with Hematoxylin-Eosin. All the slides were inspected by a pathologist who was unaware of the groups. Scoring was done for acute and chronic inflammation, ulceration, granulation tissue formation and re-epithelization as presented in Wound closure analysis2)  . ThereafWound closure rate (%) = ((area at visit 1 – area at each visit) / area at visit 1) ×100Tissue preparing and processing2. The pieces were embedded in a cylindrical paraffin block. The cylindrical blocks were sectioned using orientator methods for generating isotropic uniform random sections , nine pieces of the skin samples were cut and prepared for stereological analysis, each about 1 mmsections . The secStereological analysisThe volume densities of the collagen bundles  were estimated at final magnification of «×450» by using point counting method and the following formula , 11: Vv (collagen/dermis) = P (collagen)/P (dermis) (collagen) was the number of points hitting the profiles of the collagen and P(dermis) was the number of points hitting the dermis (The Pe dermis .The length density of the vessels (Lv) and their mean diameter were estimated at final magnification of «×450» and by using the following formula , 11: v = 2×ΣQ/ ((a/f) ×Σf) LQ" was the total number of vessel profiles counted per rat sample, (a/f) was the area of the counting frame, and "∑f" is the total number of frames counted per animal. Mean diameters of the vessels were calculated by measuring the short axis of a sampled vessel as its diameter  of the fibroblasts was estimated by employing 15 μm sections at magnification of ×2000 , the “opNv = ΣQ/ΣA× hWhere "ΣQ" was the number of nuclei coming into focus in the dissector height, "ΣA" was the total area of the unbiased counting frame in all microscopic fields and "h" was the height of disector (5 μm here). Statistical analysis of the dataData were collected, analyzed and reported as mean and standard deviation (mean ± SD). Statistical comparisons between stereological outcomes of groups were carried out by using SPSS software . One-way analysis of variance (ANOVA) followed by Tukey’s post test were used to analyze the data. P ≤ 0.05 was considered as statistically significant.Pathological study resultsAccording to pathological comments, ulceration, acute inflammation, granulation tissue formation, and slight epithelization (score 1) were the dominant presentations in the UW group. In base group, acute inflammation was observed in the rats as the governing type of inflammation; ulceration, granulation tissue formation and slight epithelization (score 1) were also dominant in base group. Group SS had no sign of inflammation at the day 12 except one of the animals with acute inflammation; full epithelization (score 3) was seen in the rats except one of them with score 2 epithelization.Wound closure rate2 with insignificant difference among the experimental groups. The rate of wound closure in SS group was significantly higher in comparison with the UW and base groups (P < 0.05)  . The basFibroblast populationNumerical density of the fibroblasts (Nv) in the dermis of SS group was reported to be 103% higher than UW (P > 0.001) and 95% higher than the base group (P > 0.001) .Volume density of the collagen bundlesThe volume densities of the collagen bundles were significantly higher in SS group comparing to those of the UW and base groups (P < 0.001) . Volume density, Length density and diameter of the vesselsThe mean of volume densities of vessels in SS group were insignificantly higher than UW and base groups by 26% (P = 0.79) and 52% (P = 0.36) respectively. Statistical analysis of length densities of the vessels revealed lower mean values in SS group (P = 0.79) and base group (P = 0.83) comparing to the UW group .Comparing the means of vessel diameters demonstrated that this parameter was higher than the base group by 57% (P = 0.01) in UW group and by 182% (P = 0.005) in SS group .Cutaneous wound healing is a complex process involving inflammation, fibroblast proliferation, angiogenesis, collagen bundle formation, and finally tissue remodeling . The reset al. it was shown that topical administration of SS on open skin wounds not only had anti-inflammatory effect, but also reduced bacterial infection of the wound site  had shown that oral administration of SS improved intestinal wound healing in rats; they also mentioned the anti-oxidative and immunomodulatory impacts of this agent in their investigation (Statins, particularly SS, are commonly used clinically for treatment of hypercholesterolemia. Moreover, various studies indicated different pleiotropic properties of statins such as anti-inflammatory, endothelium protective, anti-thrombotic and anti coagulant, immunomodulatory, and antioxidant activity -, , 14. Preund site . Resultsund site , which wtigation .In the present study, we showed that topical administration of 2% SS gel improved the rate of wound closure in laboratory rats. Pathological analyses also confirmed the anti-inflammatory effect of the agent as well as positive influence on granulation tissue formation and re-epithelization which hastened the healing process. Based on stereological outcomes, SS augmented the proliferation of fibroblasts, collagen bundles and also vessel formation which all accelerate the healing course; however, stereological parameters suggesting the vascularization induction influence were not statistically noticeable in contrast to other experimental groups.To come into conclusion, SS revealed positive effect on the wound healing process considering pathological and stereological outcomes that were used in this experimental study; however, further investigations, particularly clinical studies, are needed to approve this agent as a treatment in skin injuries and wound healing and to determine the exact mechanisms involved.


The clinicopathological significance of cyclin D1 overexpression and prognosis of oral squamous cell carcinoma has not been fully quantified. We performed a comprehensive meta-analysis for evaluation of cyclin D1 overexpression in oral squamous cell carcinoma to determine the strength of this association.Using both medical subheadings and free terms, we searched PubMed, Embase and the Institute for Scientific Information Web of Science for all eligible studies published before Nov. 2013. We retrieved 1674 citations, determining that 15 met the selection criteria. We used the odds ratio (OR) and hazard ratio (HR) as the common measures of association to quantitatively determine the correlation between cyclin D1 overexpression and outcomes of oral cancer. We performed a meta-analysis and heterogeneity, sensitivity, and subgroup analyses to clarify and validate the pooled results.The pooled results provided compelling evidence that cyclin D1 overexpression was significantly correlated with increased tumor size , lymphoid node metastasis , tumor differentiation , and advancement of clinical stages , and adversely influenced overall survival of OSCC patients . The strength of association varied in different oral cavity subsites.Our findings indicated that cyclin D1 expression correlates with detrimental clinicopathological outcome and poor prognosis in oral squamous cell carcinoma. Our results may be useful in the management of oral cancer. Oral squamous cell carcinoma (OSCC) is a common malignancy in the head and neck region, with significant incidence and mortality. In 2008, there were an estimated 263,900 newly diagnosed oral cancer patients and 128,000 deaths from oral cancer worldwide In clinical practice, the conventional prognostic tool for cancers is the tumor-node-metastasis (TNM) system, in which lymph node metastasis is the most relevant More recently, attention has focused particularly on a panel of molecular markers, which includes cell cycle regulators, as possible predictors of biological behaviors in oral cancer Therefore, we performed a systematic review and meta-analysis of all eligible studies published published to date to gain deeper insight into the clinicopathological and prognostic significance of cyclin D1 in OSCC. We found a significant correlation between cyclin D1 overexpression and clinicopathological outcome and prognosis.We searched PubMed, Embase and the Institute of Scientific Information Web of Science for eligible studies published before November 2013. Searches were carried out using both medical subheadings and free terms. We used a combination of the following search string:  AND (“cancer” OR “carcinoma” OR “neoplasm” OR “tumor”) AND (“cyclin D1” OR “CCND1”) AND . In addition, we manually screened the reference lists of included studies for further relevant studies. If the identified studies reported on overlapping populations, we selected the study that was published more recently or that contained more information.Two reviewers (Zhao Y. and Yu D.) screened the study selection process independently and in duplicate. Inter-reviewer agreement of the eligibility of the studies between reviewers was good, the kappa value was 0.9. Any disagreement was resolved by arbitration until consensus was achieved. Studies were eligible for inclusion if: (1) they were original articles published in either English or Chinese; (2) they focused on the association of cyclin D1 overexpression with high-risk clinicopathological factors and OSCC prognosis (3) they used immunohistochemistry (IHC) as the main method to examine the cyclin D1 expression in OSCC specimens. We restricted the included studies to those on Asian populations as previous studies have reported on these populations extensively. We excluded studies with no clinicopathological data. Studies on cutanous, verrucous and lip carcinoma as well as other types of carcinoma were also excluded.All data for selected full-text articles were extracted by two independent reviewers (Zhao Y. and Yu D.) using standardized Excel 2007 worksheets . Discrepancies were resolved by discussions and referring to the contents of the articles. We extracted the basic study information , participant characteristics , IHC methodology  and clinicopathological parameters  from each study, and recorded the survival results of each study. From studies that reported hazard ratios (HR) in both univariate and multivariate models, we extracted the latter because these results were more convincing, as there had been adjustment for potential confounders.http://digitizer.sourceforge.net/).We used the odds ratio (OR) as a common measure of association to determine the correlation between cyclin D1 expression and clinicopathological outcomes of oral cancer. The HR was used for quantitative evaluation of the impact of cyclin D1 expression on survival rate. Some studies did not include the point estimates and HR variance, therefore we used the data available in such studies and applied the method reported by Tierney et al. to determine the HR and its 95% confidence interval (CI) 22 represented the proportion of inter-study variability attributed to heterogeneity rather than systematic error, which ranges from 0% to 100% 2 values of 25%, 50% and 75% indicated low, moderate and high bias, respectively For meta-analysis, the statistical significance of pooled estimates was determined using the Z-test. Heterogeneity across studies was checked by a chi-square based Q test and the Higgins IAll statistical tests in this meta-analysis were performed using Stata 11.1 software  with two-sided p values. A p-value <0.05 was considered statistically significant.Eleven studies investigated the association between cyclin D1 expression and primary tumor size, which involves 1063 participants. Cyclin D1 overexpression was more prevalent in larger tumors  than in smaller tumors , and with OR = 1.617 (95% CI = 1.046–2.498) . Some suWe also summarized information associating cyclin D1 overexpression with other clinicopathological parameters as the topic of interest reported in the included studies, including nodal metastasis, histological grade and clinical stage . The ovehet >0.1), while there was some heterogeneity among studies following analysis of other clinicopathological features, with I2 ranging from 44.1 to 66.8%  .To investigate whether cyclin D1 overexpression was a prognosis factor in oral cancer patients, we meta-analyzed the HR data extracted from individual studies or derived using the calculations described in the. Most of the studies indicated a stronger link between cyclin D1 overexpression and poor survival. For OS, mortality was higher in cyclin D1-positive groups than in cyclin D1–negative groups , with no potential heterogeneity across studies (p = 0.0%) . SignifiThe overall pooled estimates of the relation of cyclin D1 expression to clinicopathological and prognostic outcomes were not substantially altered following the exclusion of any individual study, indicating the reliability of our results.Analysis of clinicopathological features and survival data determined no obvious asymmetry in the funnel plots for publication bias (p>0.05) . The morBased on 1251 Asian OSCC patients from 15 studies, we explored the relation of cyclin D1 expression to clinicopathological features and survival in OSCC. Our pooled results are compelling evidence of a significant correlation between cyclin D1 overexpression and increased tumor size, tumor differentiation, lymphoid nodes metastasis, and advancement of clinical stage, which may adversely influence survival in OSCC.Our findings are in agreement with a recent meta-analysis of esophageal carcinoma that combined the clinicopathological data of 2041 patients In the subgroup meta-analysis, the strength of association of cyclin D1 expression in tongue SCC was stronger than in mixed tumor sites of the oral cavity. There may be two reasons for this difference: First, compared to other subsites in the oral cavity, tongue SCC is characterized by a more aggressive biological phenotype, with a high-degree of cervical lymph node spread that might be reflected at molecular level, such as with cyclin D1 expression. Second, cyclin D1 may be differentially expressed in various anatomical sites in the oral cavity Certain heterogeneities were observed in our pooled analysis of the clinicopathological data. Heterogeneity across studies can likely be attributed to different IHC methodology, including the primary antibody used, antibody dilutions, and the scoring system applied. Asymmetric labeling of cyclin D1 expression in different parts of a specimen may also contribute to heterogeneity Although our systematic review was robust in identifying a correlation between cyclin D1 expression and poor clinical outcome in OSCC, this study should be interpreted in view of its limitations. First, this review was restricted to studies published in English and Chinese, which may have caused a potential bias. However, studies in other languages were not always available to us. Second, heterogeneity was not eliminated entirely although we conduct subgroup and sensitivity, which may have distorted the pooled results. Third, due to the lack of original data in the included studies, it was not possible to evaluate the combined effect of and interaction between cyclin D1 and other molecular markers on clinical outcomes. As the carcinogenesis of oral cancer involves a multi-hit process Despite these limitations, our findings may have important clinical implications. Clinicians would find our conclusions helpful, as there is currently no molecular tool for evaluating the development and progression in oral cancer. As a vital cell cycle regulator, Cyclin D1 could be considered a valuable prognostic tool in OSCC treatment, when combined with conventional TNM staging. Much caution is advised when managing aberrant cyclin D1 expression in OSCC, as it indicates a high risk of regional metastasis and recurrence following surgical treatment. Such patients, however, may undergo a more thorough treatment modality and be followed more closely in clinical practice. Moreover, as preoperative evaluation of cervical metastasis lacks accuracy, we concur with the opinion that IHC analysis of cyclin D1 expression in diagnostic biopsy samples could be deemed a routine to select patients who can be probably treated with elective neck dissection The current study also highlights the need for future research in this area. Studies associating cyclin D1 expression and oral cancer prognosis are warranted to better ensure ample sample size with varying in the study population, better control for potential confounders  and to take into account the interaction between cyclin D1 expression and environmental factors or personal habits . Such studies may have many advantages over the current study which mined small- samples individual studies. In addition, as the biological behavior of SCC in different oral cavity subsites may vary, more attention should be focused on investigating cyclin D1 expression and prognosis in individual oral cavity subsites. The prognostic value of cyclin D1, either alone or combined with alternative molecular markers, would be validated in clinical practice.Our meta-analysis indicates that cyclin D1 overexpression correlates with poor clinicopathlogical outcome and prognosis in OSCC. The results obtained may aid the management of oral cancer patients.Checklist S1PRISMA Checklist.(DOCX)Click here for additional data file.


Drosophila melanogaster for glucose-6-phosphate dehydrogenase (G6pd), cytosolic isocitrate dehydrogenase (Idh), and cytosolic malic enzyme (Men) along with seven different genetic backgrounds to lend biological relevance to the data. The responses of the NADP-reducing enzymes and two downstream phenotypes (lipid and glycogen concentration) were compared between the control and stress conditions. In general, responses in NADP-reducing enzymes were greater under conditions of oxidative stress, likely due to an increased demand for NADPH. Interactions between the enzymes were altered by environmental stress in directions and magnitudes that are consistent with differential contributions of the different enzymes to the NADPH pool: the contributions of G6PD and IDH seem to be accentuated by oxidative stress, and MEN by starvation. Overall, we find that biological network interactions are strongly influenced by environmental conditions, underscoring the importance of examining networks as dynamic entities.Interactions across biological networks are often quantified under a single set of conditions; however, cellular behaviors are dynamic and interactions can be expected to change in response to molecular context and environment. To determine the consistency of network interactions, we examined the enzyme network responsible for the reduction of nicotinamide adenine dinucleotide phosphate (NADP) to NADPH across three different conditions: oxidative stress, starvation, and desiccation. Synthetic, activity-variant alleles were used in  Despite their independent functions, studies have shown that the activities of these enzymes are co-regulated, likely due to their shared function in maintaining cellular pools of NADPH for use in a number of downstream processes, such as lipogenesis, antioxidation, and immune response  enzyme network in response .G6pd, Idh, and Men on the activities of one another and on lipid concentration in adult D. melanogaster. These studies found significant interactions among IDH, MEN, and G6PD [measured to represent both NADP-reducing PPP enzymes as per e.g., reduction in one enzyme was accompanied by increases in another) and support the theory of the enzymes interacting to maintain a cellular pool of NADPH. However, in some situations, the responses were counterintuitive and would appear to exacerbate the loss of NADPH. Lowered IDH activity, for example, was found to be associated with lower MEN activity , NADPH provides the reducing power for a number of enzymatic and small-molecule antioxidants, which work to detoxify ROS [reviewed in D. melanogaster. Enzyme activity-variant flies, mimicking natural variation seen in wild flies, were used for G6pd, Idh, and Men under each of the conditions in order to quantify the response (or interaction) in other enzymes as well as the two downstream phenotypes. We find that, in general, stress conditions amplify the interactions between NADPH network members and can also change the directionality of the response. Overall, these results highlight the dynamic nature of genetic interactions and emphasize the importance of examining interactions across multiple conditions.Here we report our examination of the consistency of metabolic interactions between the NADP-reducing enzymes and two downstream phenotypes (lipids and glycogen content) under oxidative stress, starvation, and desiccation in D. melanogaster lines were created from isofemale lines collected from the wild in the eastern United States . The isothird chromosomes used were CT21, HFL53, JFL12, JFL29, MD76, MD80, and VT26. Idh and Men enzyme activity-variant allele lines were from sets previously created using P-element excisions . Multiple third chromosomes were used to strengthen biological significance of our results by accounting for possible variation in responses across genetic backgrounds, as previous studies have found that genetic background may affect the way in which network members interact  were established for each cross genotype, and flies were allowed to lay eggs for four days. To test the consistency of network interactions across varying environments, the experiment was repeated under different environmental conditions. Emerging male flies were collected, aged for five days, and exposed to one of four conditions: paraquat-induced oxidative stress, starvation stress, desiccation stress, or benign laboratory (control) conditions. Male flies were used exclusively based on findings from previous work that found similar trends in both males and females, although the trends tended to be more prominent in males , flies with 100% and 50% activity for each of the NADP-reducing enzymes were generated. Specifically, a series of 16 paired crosses were made using the interact . We chosFlies were fed 20 mM paraquat  incorporated into a cornmeal-yeast-agar-corn syrup diet in order to simulate oxidative stress as previously described in Flies were starved for 24 hr in vials containing 2 mL of 0.5% agar at 25° following established protocols  to standardize enzyme activities, triglyceride, and carbohydrate concentrations for differences in fly size.+) at a “concentration” of one fly per 100 µl of buffer and spun at 13,000 RPM for 5 min at 4° to pellet all solids. In general, assays were conducted using samples of four flies. In a few cases, insufficient flies of a specific genotype were available, and fewer flies were assayed; the homogenization buffer volume was adjusted accordingly. The supernatant was collected and vortexed, and 250 μl was added to one well of a 96-well plate. Aliquots were taken from this master plate for each of the analyses.Flies were homogenized in ice-cold grinding buffer . Absorbance for all assays was measured at 340 nm, every 9 sec, over 3 min at 25°. Samples were assayed twice, and the means were used in further analysis. The assay buffers were previously optimized to give maximum activities  and wereG6PD: 100 mM Tris-HCl, 0.32 mM NADP, 3.5mM D-glucose-6-phosphate (pH 7.4)IDH: 100 mM Tris-HCl, 0.10 mM NADP, 0.84 mM MgSO4, 1.37 mM DL- isocitrate (pH 8.6)MEN: 100 mM Tris-HCl, 0.34 mM NADP, 50 mM MnCl2, 50 mM malate (pH 7.4)+ reduced per minute per microgram soluble protein times 10,000.Enzyme activity is expressed as micromoles NADP500 and total soluble triglyceride concentrations were determined by comparison with a commercially available standard . Each sample was assayed twice, and the mean used in analysis. Results are reported as micrograms of triglyceride per sample.Soluble triglyceride was measured using a commercially available kit  following the manufacturer’s protocol. Triglycerides are a strong correlate of lipids  and werei.e., glycogen) was measured as previously described . Results are reported as milligrams per liter.Total carbohydrate content . Each sample was assayed twice, and the mean was used in analysis. Results are reported as milligrams per liter. Enzyme activities were standardized by soluble protein content to account for differences in size between individual flies and possible differences in the degree of homogenization between samples.Soluble protein was measured by the bicinchoninic acid (BCA) assay using a commercially available kit  following the manufacturer’s protocol. Briefly, the assays contained 10 µl homogenate and 100 µl reagent and were incubated at 37° for 30 min. Sample absorbance was measured at ODln E2vs. ln E1 for each of the seven different third-chromosome backgrounds, where E1 is the enzyme activity of the gene that was genetically modified, and E2 is one of the other NADPH enzymes or triglyceride or carbohydrate concentration. In calculating elasticity coefficients, enzyme activity was expressed as nanomole NADP reduced per minute per microgram soluble protein to allow comparison with previous studies. The above analyses test whether stress conditions modify the effect of reducing NADPH enzyme activity on the NADPH network , but they do not test whether the impacts of the stress conditions are significantly different . To test whether the different stressors significantly differed in their impacts on the NADPH network, enzyme activity results were pooled for each genotype  under each stressor, and an ANOVA on the ratio of i.e., reducing statistical power), this statistical test is a conservative estimate of the presence/absence of a difference in enzyme response across conditions.All crosses were replicated in two independent bottles, with four samples taken from each bottle for each environmental condition tested. Analysis of covariance (ANCOVA) and Tukey’s honestly significant difference (HSD) multiple-comparison tests were performed using JMP 7.0 software (SAS Institute) to determine whether there were significant differences in enzyme activity or triglyceride or carbohydrate concentration between environmental conditions using protein concentration and weight as covariates. ANCOVAs were also performed to determine whether there were significant differences in NADPH enzyme activities or triglyceride or carbohydrate concentrations between the wild-type and reduced-activity genotypes for each of the four environmental conditions tested. In cases where a significant difference was found between the two genotypes, elasticity coefficients (ε) were calculated, as previously described . ElasticG6pdEx40/i, IdhEx4/i, MenEx3/i). Although the primary focus of this research was to examine the consistency of interactions in response to reduction in activities of the NADP-reducing enzymes, our use of environmental stressors also allowed us to quantify the response of the NADPH network to the environmental stressors themselves. The NADPH enzymes have previously been included in studies of stressors in a variety of organisms  with increased activity in desiccated flies : IDH activity was significantly lower under oxidative stress and higher under desiccation , having lower activity under paraquat-induced oxidative stress but significantly higher activity under desiccation (Tukey’s HSD). The reduction in NADP-reducing enzyme activities under oxidative stress is surprising given the role of NADPH in combating oxidative stress, but it is consistent with recent work that also found reductions in these enzymes under oxidative stress generated by knocking out superoxide dismutase enzyme activity , with lower triglyceride concentrations under both oxidative stress and starvation compared with control conditions , and all treatments had significantly lower carbohydrate stores compared with flies maintained under benign laboratory conditions (Tukey’s HSD). The decrease in carbohydrate, but not lipid, stores under desiccation is consistent with trends observed in previous studies  averaged approximately 41.8 ± 3.04% of the G6PD activity of wild-type flies  across all three stress treatments and controls. Elasticity coefficients and statistics are summarized in Table S1.Reduced G6PD-activity flies , consistent with previous studies that suggested that the NADP-reducing enzymes work concurrently to maintain a cellular pool of NADPH ; the response in MEN activity under oxidative stress was significantly larger than that under any other condition (Tukey’s HSD). This significantly larger response  suggests that this condition generates the greatest need for NADPH, consistent with the substantial role for G6PD in the reduction of NADP+ under oxidative stress proposed by others  across genetic backgrounds. IDH activity was significantly higher in the low G6PD activity flies under conditions of starvation and desiccation, although the differences were small: +5.3% and +3.3%, respectively (Table S1). Directly measuring enzyme activity enables us to accurately quantify such small differences, but it is unclear what the biological impact of such small changes in enzyme activity is. There was no statistically significant response in IDH activity to the reduction in G6PD activity under control or oxidative stress conditions (Table S1), although some of the genetic backgrounds did show small compensatory increases in IDH activity, and others showed decreases in activity. This heterogeneity in response stresses the importance of examining genetic interactions across multiple genetic backgrounds, it and also suggests that, although no statistical change was observed overall, the change in G6PD activity has an effect on IDH activity even if the effect is small and not consistent across genetic backgrounds. Statistical tests examining the responses in IDH activity across environmental conditions did not find any significant differences, suggesting that overall IDH response to reduction in G6PD remains fairly consistent across conditions.Like MEN activity, IDH activity varied in response to the reduction in G6PD under the tested environmental conditions, but responses varied  were found to vary with reduced G6PD activity across all conditions (Table S1). In particular, carbohydrate stores were significantly lower in the low G6PD activity flies under control, starvation, and desiccation conditions (Table S1). These reductions in carbohydrate concentration were fairly consistent, ranging from decreases of 11.3–17.6%. Surprisingly, under paraquat-induced oxidative stress, carbohydrate stores were found to be 13.2% higher in the low G6PD activity flies (Table S1). The responses in carbohydrate concentration were found to differ between conditions ; the carbohydrate concentration under oxidative stress differed from all conditions except starvation (Tukey’s HSD).Carbohydrate stores (G6pd genotypes (Table S1). For example, under starvation, four of the seven genetic backgrounds show fairly large decreases in triglyceride concentration, whereas the remaining three show large increases in concentration (Table S1). This type of variability between genetic backgrounds is consistent with triglyceride being a complex downstream phenotype of the NADPH enzyme network with a number of factors outside of the network influencing the concentration, and it is consistent with previous studies  averaged 45.9 ± 0.47% of that of the full-activity flies (IdhEx4/i heterozygotes) across all treatments. Responses to lowered IDH activity were analyzed identically to that of reduced G6PD activity, described above. Elasticity coefficients are summarized in Table S2.IDH activity in the reduced-activity flies (Table S2). There was no significant change in G6PD activity in response to the reduction in IDH under any of the other conditions (Table S2), and when we compared the responses across conditions, the small compensatory increase in G6PD activity under oxidative stress did not significantly differ from the other conditions in which no response was observed. The lack of a response in G6PD activity under benign conditions is consistent with the findings of previous studies . This compensatory increase is consistent with IDH playing an important role in providing NADPH under conditions of oxidative stress as documented in previous studies (Table S2). This decrease counters an expectation of NADPH compensation, but it is consistent with previous studies .In response to the reduction in IDH activity, the magnitude and directionality of MEN activity changed depending on the environmental condition. As expected, under conditions of oxidative stress, there was a significant increase in MEN activity in the low IDH activity flies (Table S2) across the environmental conditions, and were all significantly different from concentrations in wild-type flies; however, none of the increases is significantly different across conditions . This uniform increase in carbohydrate concentration was not observed in response to a decrease in G6PD or MEN (described below) in which reductions in carbohydrates were observed. In contrast to carbohydrates, no significant differences were observed in triglyceride concentration between the two IDH activity groups under any of the environmental conditions (Table S2), suggesting the changes in carbohydrate concentrations are not a general feature of metabolism for energy storage. Instead, this carbohydrate increase may indicate that the decrease in IDH activity may cause metabolic dysregulation.Interestingly, carbohydrate concentration was increased in the low IDH activity flies across all conditions: increases ranged from 7.9 to 36.0% (MenEx55/i heterozygotes) averaged 68.8 ± 2.12% the MEN activity of the full activity flies (MenEx3/i heterozygotes). Responses to lowered IDH activity were analyzed identically to the responses to reduced G6PD and IDH activity described above. Elasticity coefficients are summarized in Table S3.The reduced MEN activity flies (Table S3). The increase in IDH activity varied from 9.5% to 18.8% across conditions, but the amount of IDH increase was not significantly different across these conditions .IDH activity was significantly higher in the lower MEN activity flies under all tested environmental conditions, in apparent compensation for the decrease in MEN activity (Table S3). There was no change in G6PD activity in response to the variation in MEN activity under benign or oxidative stress conditions. This apparent difference in response suggests that MEN may be a larger contributor of NADPH under conditions of starvation, but the different responses in G6PD activity were not significantly different across environmental conditions.G6PD activity was only significantly different in the low MEN flies under conditions of starvation and desiccation: under starvation, G6PD activity was 12.1% higher in the low MEN flies, and under desiccation, G6PD was 5.1% higher in the low MEN flies (Table S3) and were significantly different across conditions . Specifically, the reduction in carbohydrate concentration between Men genotypes under starvation was statistically greater than what was observed under control conditions (Tukey’s HSD test). Although the lack of a change in carbohydrates under oxidative stress was not found to differ from control conditions (in which the reduction in carbohydrates was lowest), the change in carbohydrates under oxidative stress did differ from starvation and desiccation (in which the reduction in carbohydrates was greater). The trend of the response in carbohydrate stores in response to a reduction in MEN is similar to observations made in response to the reduction in G6PD; carbohydrates decreased under every condition except oxidative stress.Carbohydrate concentrations were lower in the low MEN activity flies under all conditions except oxidative stress in which no change was observed. The reductions in carbohydrate stores in response to the reduction in MEN activity ranged from 15.8% to 34.9% (Table S3). Statistical tests indicated that the response in triglyceride concentrations varied across conditions , and the change in triglyceride under starvation differed from that observed under control and oxidative stress conditions (Tukey’s HSD).In contrast to the change in carbohydrates and consistent with earlier work , there wWe examined a simple metabolic network to directly quantify the consistency of genetic interactions across varying environmental conditions and found that interactions between network members change both in directionality and magnitude under different environmental conditions. Overall, this study emphasizes the importance of moving away from mapping biological networks as static snapshots; using multiple conditions and genetic backgrounds can uncover new, and previously undetected, interactions.A combination of reduced G6PD activity and oxidative stress was associated with a larger increase in MEN than that observed under any other conditions. Previous work under benign conditions had found little impact of variation in G6PD, suggesting only a minor role for G6PD in NADPH generation . It appei.e., reduction in IDH activity was accompanied by a parallel reduction in MEN activity). The parallel reduction suggests some form of co-regulation of MEN and IDH, but not in a compensatory fashion. The parallel decrease was only statistically significant under desiccation; however, the pattern was visible under other conditions  and is consistent with previous findings . First, this finding indicates that although G6PD is a major contributor of NADPH under oxidative stress (above), IDH also plays an important role in generating NADPH under these conditions, consistent with findings of previous studies  and lipid content (via triglycerides), two complex phenotypes linked to metabolic enzyme activity and NADPH concentrations  in Drosophila melanogaster, which, surprisingly, also showed a decrease in glycogen content (D. melanogaster.Interactions within the NADPH enzyme network are not restricted to the enzymes; we also found that changes in network member interactions affected downstream phenotypes. In particular, we examined carbohydrate (trations . We founMen genotype starvation. Apparently, even with the compensation seen in both G6PD and IDH activity in response to the reduction in MEN under starvation, NADPH availability for any basal lipogenesis is decreased under these conditions. This reduction is consistent with MEN being the major contributor of NADPH under conditions of starvation.In contrast to the extensive changes in glycogen content, changes in total lipid were observed only under a single condition: lipids decreased in the reduced e.g., see directly act on the network. Although we did not identify the specific differences between the genetic backgrounds, the variation in the third chromosome is likely a reasonable estimate of the type of genetic variation present in wild populations. Often studies take a simplified approach by focusing on a specific mutation in a specific genetic background and do not consider that phenotypic variability can be found when a mutation is studied in different backgrounds. The pronounced variations in responses across backgrounds that we demonstrate suggest that such studies in a single background should be interpreted with caution.In almost all cases, we observed differences in the responses of the enzymes and metabolite concentrations when these were quantified in different genetic backgrounds (This study provides evidence that interactions between members of the NADPH enzyme network can change, both in directionality and magnitude, in response to differing environmental conditions. This conclusion is consistent with studies of other, larger-scale genetic networks (