We report in this paper the case of a 45-year-old woman with neurological deficit due to ischemic brain infarct, multiple sclerosis, antiphospholipid syndrome, and  The choice of anesthetic technique in patients with preexisting neurological disease may be a specific concern for anesthetists . Neurolo A 45-year-old, ASA physical status IV woman  with ovarian cancer was scheduled for abdominal hysterectomy and bilateral salpingoophorectomy with epidural anesthesia, after her refusal for general anesthesia. The patient had been successfully subjected to cesarean section under epidural anesthesia four years ago.β-thalassemia. On her admission, normotensive sinus tachycardia (115 bpm), fever and intense lower abdominal pain along with ascites and nausea were noted. The patient presented with 26.5% hematocrit, a platelet count of 465.000/L, and normal bleeding tests. Neurological examination revealed right hemiparesis, dropping of the left corner of the mouth, impaired oropharyngeal function with dysphagia to both solids and liquids, and altered walking gait. The patient had been receiving cinnarizine 10 mg daily, bromazepam 3 mg three times per day, and paracetamol occasionally.  Her clinical history revealed an ischemic infarct of the left frontal lobe since the age of 19, multiple sclerosis since the age of 20, antiphospholipid syndrome (heterozygote for the methylenetetrahydrofolate reductase (MHTFR) gene), and heterozygous 1-L2 intervertebral space; the epidural space was found using the loss-of-resistance technique. A polyamide catheter was easily introduced and placed at the 12 cm catheter mark. A test dose of 3 mL of 2% lidocaine was injected. After catheter placement the patient turned supine and was given 15 mL of ropivacaine 0.75% and 100 μg fentanyl. A sensory block to T3 was established 15 minutes later. During the 2-hour surgery, another 5 mL of ropivacaine 0.75% and 10 mL of lidocaine 2% were given as the patient complained of discomfort during peritoneal traction. Entonox inhalation was used to alleviate patients' discomfort during peritoneal traction and abdominal wall closure. At the recovery room, 1/2 an hour after the last epidural dose, the patient experienced severe pain and was administered 8 mL of ropivacaine 0.2% and 2 mg of preservative free morphine followed by 6 mL of ropivacaine 0.375% fifteen minutes later. A sensory block to T12 was present 11/2 hours later with no motor block at recovery.  In the operating room, with the patient positioned in the left lateral decubitus position, a 18-gauge Tuohy needle was inserted at Lμg fentanyl hourly. The patient also received 3 g of paracetamol daily and triple antiemetic drug combination . On the second postoperative day, pain was effectively managed with the continuous pump infusion but nausea persisted. Neurological examination revealed no signs of deterioration of the preexisting disease. The epidural catheter was removed on the fifth postoperative day. Nausea was not alleviated despite rigorous treatment. The patient developed paralytic ileus on the sixth postoperative day that resolved spontaneously two days later. Postoperative analgesia with 8 mL ropivacaine 0.2% four times daily, 2 mg morphine twice a day, and rescue dose of 6 mL ropivacaine 0.375% failed and the regimen was replaced by a continuous epidural pump, set to infuse 2 mL of ropivacaine 0.2% and 8  Neurological deficit, chronic neuropathic cancer pain, antiphospholipid syndrome, and hematological disorders along with the patients' refusal for general anesthesia form a noteworthy clinical profile, challenging for every anesthetist. In the present case, we report the anesthetic management and the medical questions raised during the clinical course of a patient with the combination of the abovementioned uncommon diseases. μM of lidocaine and may well contribute to the fast changes of symptoms. Worsening of neurological symptoms in patients with preexisting neural compromise has always been a fear for the neuraxial anesthesia and analgesia performing anesthetist –3. Disor Epidural anesthesia is commonly thought to be less harmful than spinal anesthesia; however secondary parameters such as local anesthetic neurotoxicity and needle or catheter induced trauma may cause relapse of the dormant underlying neurologic pathology. Furthermore, many perioperative factors such as fever, infection, stress, fatigue, and the operation itself may result in deterioration of the preexisting neural compromise. To this direction, Hebl et al.  in their The patient has also been diagnosed with antiphospholipid syndrome; an autoimmune disorder characterized by vascular thrombosis. The need for anticoagulant therapy for the patient with antiphospholipid syndrome may prove higher postoperatively and this unanticipated anticoagulation regimen explains the reluctancy in performing neuraxial blockade . Given the intense postoperative pain experienced by the patient in the recovery room and the ward along with the patients' characteristics, the roles of opioid-induced hyperalgesia and preemptive analgesia come into question. The cancer patient with neurological deficit gathers several potential preoperative risk determinants of postsurgical pain; anxiety, depression, and catastrophising personality along with impaired pain modulation have been postulated as primary causal factors leading to postsurgical pain . The int2 agonists or ketamine [ Preemptive analgesia could probably play a role in the clinical setting of this patient –11; howeketamine  could ha Another skepticism that emerges from this case report is whether spinal anesthesia with profound sensory and motor block intraoperatively could have resulted in reduced postoperative pain. Recent studies on spinal anesthesia in patients with preexisting central nervous system disorders report complication-free procedures with patient satisfaction .  Although epidural anesthesia to this patient may have been a common final procedure, balancing the risks and benefits of the anesthetic technique and critically questioning the problems raised by her clinical course was a copious work in the hope that definitive conclusions will be made after new prospective studies.


Anser anser, n = 191) and purple gallinule  were collected from three different impacted sites  during the winters of 2004 to 2008. Lead and aluminium (an indicator of sediment ingestion) and Pb isotope signatures (to discriminate between sources of Pb exposure) were analyzed in freeze-dried, acid digested samples. The concentrations of fecal porphyrins and biliverdin were determined as noninvasive biomarkers to study Pb exposure effects. Results showed a decrease in Pb exposure over time in wintering greylag geese. In contrast, for purple gallinule resident in the Entremuros a clear trend was not evident. For both species, sediment ingestion appeared to be the main source of exposure to Pb. In the Entremuros, some samples from purple gallinule were detected with higher Pb levels than expected for simple soil ingestion, and these had Pb isotopic profiles compatible with mining sludge or Pb shot. Whilst fecal Pb isotopic profiles were effective in differentiating between samples from sites with different levels and sources of pollution, the combined use of element ratios (such as Pb/Al) and other non-traditional stable isotope signatures may also prove worthwhile. Overall, the fecal Pb levels detected were below those described in feces for waterfowl from other uncontaminated areas(<10 µg/g d.w.). Despite this, for both species fecal Pb levels were positively correlated with porphyrin excretion, and for purple gallinule, with the coproporphyrin III/I ratio, suggesting some subtle effects on heme synthesis in birds. Ten years after the mine spill, Pb contamination in birds by this pollution source was still detectable and subtlethal effects may persist.Lead exposure in waterfowl was studied using noninvasive fecal sampling in the Guadalquivir Marshes in Spain, an area affected by the 1998 Aznalcóllar mine disaster. Feces of greylag geese ( The Guadalquivir marshes are an extensive mosaic of wetlands of deltaic origin, located in south-western Spain. Due to its geographical location and environmental characteristics, these marshes are one of the most important wintering sites for migratory waterbirds in the Western Palearctic In April 1998, the Guadalquivir marshes were severely affected by the Aznalcóllar mine accident Anser anser) during the 2001/2002 wintering season, analyzing the concentrations of various metals/metalloids  in feces from 5 different sites within the Guadalquivir marshes After the spill, sick and dead birds with high levels of heavy metals in tissues were found in Doñana National Park and surroundings Porphyrio porphyrio) in Doñana, using feces as noninvasive samples. These two bird species were amongst those most affected by the Aznalcóllar spill Here, we aim to study trends over time in Pb exposure in wintering greylag geese and resident purple gallinule  in successive years from 2004 to 2008 . Feces oAll necessary permits were obtained for the field studies. Permits required to enter Doñana National Park were granted by the Consejería de Medio Ambiente, Junta de Andalucía.Feces (n = 282) and sediment (n = 35) samples (0.2–0.3 g) were freeze dried, acid digested and analyzed using graphite furnace (Pb) and nitrous oxide-acetylene flame (Al) atomic absorption spectroscopy , following methods described previously 206Pb, 207Pb, and 208Pb were also analyzed by inductively coupled plasma mass spectrometry 206Pb, of 22.0833±0.0027% for 207Pb, and of 52.3470±0.0086% for 208Pb. All isotope ratios determined for SRM 981 during analysis were within 1% of the certified value .In a sub-selection of the acid digested feces (n = 72), the stable isotopes Finally, another sub-selection of feces (n = 71) was used for porphyrin and biliverdin analysis. This was conducted using liquid chromatography single quadrupole mass spectrometry (LC/MS) Where necessary, data were log-transformed prior to statistical analysis to meet parametric assumptions. Data were analyzed using General Lineal Models (GLMs) considering Pb or Al concentrations, Pb isotopes signatures, and porphyrin and biliverdin concentrations as dependent variables; sampling site, season and/or species as factors; and Al or Pb concentration as covariates. Lead and Al concentrations in feces of greylag geese collected in the Entremuros during the 2001/2002 sampling season . Mean Pb levels showed a positive relationship with Al . Higher mean Pb concentrations were detected in fecal samples from Cerro de los Ánsares than from Entemuros and Caracoles .Purple gallinule feces differed in terms of Pb concentrations between winters .For both species, fecal Pb levels were positively and significantly correlated with Al in all sites , and between species . For both ratios, lower mean values were detected in fecal samples from geese than in purple gallinule , and a negative relationship was found with Pb concentrations . For geese a positive relationship was detected between fecal Pb and coproporphyrin I/biliverdin and coproporphyrin III/biliverdin ratios . Although no differences in porphyrin levels were detected between sites, correlations showed that the positive relationship with fecal Pb was clearer in samples collected in the Entremuros . For purple gallinule, a positive relationship was detected between fecal Pb and coproporphyrin III/biliverdin ratio , the coproporphyrin III/I ratio .Finally, the statistical analysis regarding biliverdin concentrations showed significant differences between species, with higher values in geese than in purple gallinule , this study shows that there had been a progressive decrease in exposure to Pb in greylag geese in various parts of the Doñana National and Natural Parks. In the Entremuros, the site most affected by the spill, the mean fecal Pb concentration in geese had decreased to 3 µg/g d.w. in 2005/2006, well below the 15 µg/g d.w. previously reported in the 2001/2002 wintering season 206Pb/207Pb and 208Pb/207Pb isotopic ratios were confined between those described previously for the Aznalcóllar sludge and for sediment affected by the spill Scirpus sp. and Plantago sp. in this area Typha sp. and seeds of Cyperaceae species , but also non-plant material, and mineral matter can represent up to 63% of their gizzard contents As expected, differing Pb isotope profiles were detected in the feces of geese from each site sampled. In Entremuros, the 206Pb/207Pb and 208Pb/207Pb isotopic ratios obtained showed values closely related to those previously described for sediment affected and unaffected by the spill. Furthermore, a strong relationship between fecal Pb and Al was also detected for this species.For greylag geese, the mean Pb concentration in feces from the Entremuros was within the range expected when considering the average percentage of estimated ingested sediment (%EIS) described for this species in this area 8%; . If we a206Pb/207Pb isotopic ratio of these outlier samples lay between the ratio described for Pb shot in Spain and the Aznalcóllar spill contaminated sediment, this relationship was less clear for the 208Pb/207Pb ratio. Here, these outliers plotted more within the range for Entremuros sediment affected and unaffected by the spill (Scirpus sp. and Typha sp. Scirpus sp.) and purple gallinule  in Doñana S. maritimus roots from polluted areas within the Entremuros Despite the importance of sediment ingestion as a Pb source, some samples analyzed in the Entremuros showed Pb levels far higher than expected via this exposure route alone. These samples fell well above/outside the expected Pb/Al regression line representing the Pb level expected due to sediment ingestion, and Pb concentrations were >34 µg/g d.w. This level suggests ingestion of point sources of Pb, such as Pb shot he spill . In the Given that the Pb isotopic profile in iron plaque associated with macrophyte roots is likely to be similar to that found in the surrounding sediment, this may be acting as a point source of Pb for the birds studied here which is nevertheless difficult to distinguish isotopically from the bulk sediment. As suggested by previous studies Through the analysis of fecal porphyrins and biliverdin, we have also detected potential changes in heme synthesis related to Pb exposure in geese and purple gallinule in our study area. The positive relationships we found between Pb and coproporphyrin I/biliverdin and coproporphyrin III/biliverdin ratios in feces from geese, especially in those collected in the Entremuros, and with the ratios coproporphyrin III/biliverdin, coproporphyrin III/I and mesoporphyrin/biliverdin in feces from purple gallinule collected at the same site, all suggest that heme synthesis is being affected by Pb. Previous studies have reported a general increase in the levels of bile porphyrins in poisoned mallards, with the increase in coproporphyrin III being higher than in coproporphyrin I Ciconia ciconia) due to the widespread low-level contamination left as a consequence of the Aznalcóllar spill. Furthermore, Baos et al. The inhibition of the enzyme ALAD is considered one of the most sensitive effects related to Pb exposure Further work is now needed to understand the nature of the apparent point source of Pb pollution that is being recorded in the feces of some of these birds, and to pinpoint its exact source . In this context, although the study of Pb isotopic profiles in feces appears to be an effective tool to help to differentiate between pollution sources the combined use of element ratios (such as Pb/Al or Fe/As) and perhaps other non-traditional stable isotope signatures may yet prove worthwhile


Cooked bean powders are a promising novel protein and fiber source for dogs, which have demonstrated potential to alter microbial composition and function for chronic disease control and prevention. This study aimed to determine the impact of cooked navy bean powder fed as a staple food ingredient on the fecal microbiome of healthy adult pet dogs.Fecal samples from healthy dogs prior to dietary control and after 4 wk of dietary treatment with macro- and micronutrient matched diets containing either 0 or 25% cooked navy beans  were analyzed by 454-pyrosequencing of the 16S rRNA gene. There were few differences between dogs fed the control and navy bean diets after 4 wk of treatment. These data indicate that there were no major effects of navy bean inclusion on microbial populations. However, significant differences due to dietary intervention onto both research diets were observed . After 4 wk of dietary intervention on either control or navy bean diet, the Phylum Firmicutes was increased and the Phyla Actinobacteria and Fusobacteria were decreased compared to baseline.No negative alterations of microbial populations occurred following cooked navy bean intake in dogs, indicating that bean powders may be a viable protein and fiber source for commercial pet foods. The highly variable microbial populations observed in these healthy adult pet dogs at baseline is one potential reason for the difficulty to detect alterations in microbial populations following dietary changes. Given the potential physiological benefits of bean intake in humans and dogs, further evaluation of the impacts of cooked navy bean intake on fecal microbial populations with higher power or more sensitive methods are warranted. A more in depth understanding of the companion canine microbiome, including the phylogenetic structure, functional capacity, and how they are altered by diet, is needed. Evidence supports a role of the gut microbiota in digestion and energy harvest, defense against pathogens, nutritional support for enterocytes, and immune stimulation. Molecular approaches, including 454-pyrosequencing, have shown utility for improving our understanding of the complex and diverse microbial communities that exist in the gut. Results from studies characterizing the canine fecal microbiota were recently reviewed Few studies have examined the impact of diet on canine fecal microbial populations utilizing 454-pyrosequencing and metagenomic techniques Given that the composition, dynamics, and functionality of the canine gut microbiota have been largely evaluated in colonies of research dogs living in controlled environments, we sought to examine the influence of a bean-based diet on the gut microbiome of healthy free-living client-owned dogs. Control and navy bean-based diets were formulated and matched for total macronutrient and micronutrient content. This approach allowed us to test whether the source of these nutrients and the phytochemicals present in navy beans were responsible for changes in the fecal microbial communities.The Colorado State University (CSU) Institutional Animal Care and Use Committee approved all trial operations, animal care procedures, and collection of biological samples for safety and digestibility of experimental research diets before beginning study (Protocol ID # 10-1932A).Twenty-one clinically healthy, adult, normal weight, male and female client-owned dogs were recruited to participate in a randomized, double-blinded, and placebo-controlled canine dietary intervention study at the CSU Veterinary Teaching Hospital’s Animal Cancer Center as described by In depth diet description is available in Dogs were fed to maintain a stable body weight (less than 2% change per wk). Owners were instructed to feed only the research diet in the prescribed daily amount. Food offered and refused was measured and recorded by owners daily. Any intake besides that instructed was included in the owner records. Water was provided ad libitum.CGTATCGCCTCCCTCGCGCCATCAG), a reverse ‘LinkerB’ sequence (CTATGCGCCTTGCCAGCCCGCTCAG), a multiplex identifier (MID) unique to each sample, and a Eubacterial-specific sequence [530F (5′-GTGCCAGCMGCNGCGG) or 1100R (5′-GGGTTNGNTCGTTG)] for the V4−V6 region of the 16S rRNA gene. PCR reactions of 25 µl were performed for each sample using a barcoded forward primer (10 µM), barcoded reverse primer (10 µM), dNTP mix (10 mM), FastStart 10X buffer with MgCl2, fastStart HIFi Polymerase (5U/µl) and 5 µl of genomic DNA. The PCR conditions were: 94°C for 3 min; 32 cycles of 94°C for 15s, 62°C for 45s, and 72°C for 1 min extension; followed by 72°C for 8 min. PCR amplicons of all samples were further purified using AMPure XP beads  to remove smaller fragments. Finally, the amplicons were combined in equimolar ratios to create a DNA pool that was used for pyrosequencing. Pyrosequencing of the PCR amplicons was performed at the W. M. Keck Center for Biotechnology at the University of Illinois using a 454 Genome Sequencer and FLX titanium reagents . After sequencing was completed, all reads were scored for quality and any poor quality reads and primer dimers were removed.Fecal samples were collected at baseline and after 4 wk of treatment. Fecal samples were collected within 5 h of being voided and were stored at −20°C until shipment for analysis.Genomic DNA was extracted using the repeated bead beater method described by Yu and Morrision The sequences were selected to estimate total bacterial diversity of DNA samples in a comparable manner and were trimmed to remove barcodes, primers, chimeras, plastid, mitochondrial, any non-16S bacterial reads, and sequences < 350bp. Sequences were then denoised and chimeras removed using Black Box Chimera Check (B2C2) www.krakenblast.com) against a 06-12-11 version database curated from NCBI to include >350,000 high quality 16S bacteria and archaeal sequences as well as quality control screening sequences for mitochondria, plastid, and chloroplast screening sequences. Blast output based upon top hit designations were compiled to generate percentage files at each taxonomic level as described previously Bacterial ID community structure was evaluated using Phred25 quality reads, including both 530F and 1100R oriented , trimmed to remove tags and primer sequence collections, then depleted of chimera, plastid, mitochondrial, sequences with ambiguous base calls, sequences with >6bp homopolymer runs, and any non-16S reads  and sequences < 250bp. The final sequence data were evaluated using Kraken . For 4-wk microbial data, the fixed effect of treatment was tested. Means were separated using LSD with a Tukey adjustment to control for multiple comparisons. Post hoc contrasts were utilized to compare 0-wk vs. 4-wk microbial data. A probability of P≤0.05 was accepted as being statistically significant and P≤0.10 was accepted as a trend.http://www.ncbi.nlm.nih.gov/Traces/sra/) under accession numbers SAMN02117000 to SAMN02117041. The PCA revealed no separation due to treatment at any taxonomic level (data not shown). The OTU, ACE, and Chao estimates of diversity were not different (P>0.05) among groups (data not shown). Similarly, diet did not have a significant impact on the Shannon Index, which indicates that the consumption of navy bean did not alter the overall fecal bacterial diversity at 4 wk.Pyrosequencing of 16S rRNA barcoded amplicons resulted in a total of 560,308 high-quality sequences, with an average of 13,340 sequences per sample. Sequences are available at the NCBI sequence read archive . The remaining phyla  were present in only a few (1 to 3) dogs and were present as < 0.1% of all bacterial sequences. Firmicutes was the most abundant bacterial phylum . Fusobacteria and Actinobacteria were the next most abundant phyla . Proteobacteria and Bacteroidetes contributed to about 0 to 13% and 0.01 to 5.8% of all bacterial sequences, respectively.Firmicutes was largely comprised of the classes Clostridia , Bacilli , and Erysipelotrichi .Lactobacillus , Streptococcus  and Enterococcus . Within the class Clostridia, the major genera were: Clostridium , Blautia , Ruminococcus , and Faecalibacterium . Within the class Erysipelotrichi the major genera were Catenbacterium  and Allobaculum . Many species were present at an average of greater than 2% of all bacterial sequences and with a maximum greater than 10%: Blautia glucerasea, B. hansenii, B. product, Clostridium hiranonis, Catenibacterium mitsuokai, Eubacterium biforme, Ruminococcus gnavus, and Megamonas funiformis. However, none were present in all dogs at high proportions .The Bacilli class was predominantly comprised of the genera Fusobacterium . The predominant genus from the Actinobacteria phylum was Collinsella , and was primarily comprised of C. intestinalis . The predominant genus in the Proteobacteria phylum was Shigella . The predominant species was Shigella sonnei .The predominant genus in the Fusobacteria phylum was Clostridium saccharogumia was lower (P = 0.03) and Clostridium ramosum tended to be higher (P = 0.09) in dogs fed NB compared to those CON. Fecal Coprobacillus catenformis were lower (P = 0.03), while Blautia wexlerae was higher (P = 0.03) in dogs fed NB compared to those fed CON.There were no diet-induced differences detected at the phylum or family level . The abuBlautia coccoides were higher (P≤0.01), the proportion of B. producta tended to be higher (P = 0.10) at 4 wk compared to baseline. Additionally, within the family Clostridiaceae, the proportion of Clostridium leptum was lower (P = 0.02), and the proportion of C. indolis tended to be lower (P = 0.06) at 4 wk compared to baseline. Within the Ruminococcaceae family, Ruminococcus gnavus was lower (P<0.01), while the proportion of R. obeum tended to be higher (P = 0.06) at 4 wk compared to baseline. Within the phylum Fusobacteria, the proportion of Fusobacteriaceae was lower (P≤0.04), and proportions of Fusobacterium and Fusobacterium perfoetens tended to be lower (P≤0.10) at 4 wk compared to baseline. Within the phylum Actinobacteria, the proportion of Coriobacteriaceae was lower (P = 0.05) and the proportions of Collinsella and Collinsella intestinalis tended to be lower (P = 0.06) at 4 wk compared to baseline.There were significant changes in the fecal microbiota between baseline and wk 4 of dietary intervention regardless of diet fed . At the Dry beans have not been included in any commercial pet foods to date, thus if appropriate, it could be a novel protein, carbohydrate and fiber source. Forster et al. Blautia wexlerae, a gram-positive bacteria, was increased by inclusion of cooked NB powder. Martinez B. wexlerae lacks enzymes with glucanase activity, it was hypothesized that whole grain barley inclusion impacted utilization of nutrients by bacteria at higher trophic levels resulting in increased substrate availability for B. wexlerae. Given these results, it may be of interest to examine the functional changes of the gastrointestinal microbiome. The authors are not aware of any research that links this bacterial species to negative or positive health consequences.Clostridium saccharogumia and Coprobacillus cateniformis were decreased in feces of dogs fed NB diets. The ability to metabolize lignans  have been reported for C. saccharogumia and Coprobacillus species with > 90% sequence similarity to C. cateniformisC. saccharogumia, this includes the capacity to metabolize the dietary phytoestrogen secoisolariciresinol diglucoside, which is high in wheat The proportions of The high variability at baseline and low number of animals may have masked small shifts in microbial populations due to navy bean inclusion. The micro- and macronutrient matching of the diets did allow us to determine that cooked navy bean inclusion elicited few alterations in fecal microbial populations. These data provide additional support for the safety of including dry beans as a viable staple pet food ingredient for canines. There are a number of studies supporting the chronic disease-fighting properties of dietary bean intake in humans and using rodent models Bifidobacterium, Lactobacillus, Clostridium, and Fecalibacterium genus indicate that n = 51, 51, 55 and 82 dogs per treatment, respectively, would be needed Micro- and macronutrient matching of the CON and NB diets allowed us to determine that navy bean as a source of carbohydrate, protein, fiber and phytochemicals did not alter the fecal microbial community. It is possible, however, that the diets may have been too similar to elicit large changes in microbial populations, and/or the small shifts which occurred were undetectable with this sample size, study design and technique utilized. For example, post-hoc estimation of the sample size necessary to detect differences due to diet for Clostridium hiranonis was 0.2 to 41% of sequences. Adding an initial adaptation period to studies in which all dogs are fed a similar diet may minimize variability in baseline measurements. Sample collection technique utilized herein  could have also introduced variability. Shorter windows of time from voiding to collection and subsequent freezing would increase consistency of sample handling and environmental exposure post voiding and potentially decrease variability. Another way to account for the variation in the study design in the future would be to provide diets in a cross-over design, so that each dog would act as its own control. Additionally, this would increase the number of dogs receiving each treatment. Many factors may potentially impact fecal bacterial populations  Consistent with results from other studies, variability in the proportions of fecal microbial populations among dogs was high The alterations in fecal microbial populations due to dietary control may have obscured the ability to define changes due to dietary treatment. At baseline, owners reported feeding one or two of 14 different commercial diets, including 13 dry diets  and one wet diet . Data are not available on the actual nutrient composition of these diets. Dietary protein, nitrogen free extract and fiber concentrations and sources, and the ratio of protein to carbohydrate  have all been shown to alter microbial populations of cats and dogs This study evaluated the fecal microbiome of healthy pet dogs before and after 4 wk of feeding macro- and micronutrient-controlled diets containing either 0 or 25% dried navy bean powder. Few significant differences were observed in microbiome composition between 0 and 25% cooked navy bean treatments after 4 wk. Continued research on the microbial metabolic impacts of dietary dry bean intake in dogs undergoing weight loss and with chronic diseases that are similar to human conditions is warranted.Alterations in fecal microbial populations from baseline due to 4 wk dietary control macro- and micronutrient-controlled diets were noted. These data highlight the potential to alter microbial populations of pet dogs with diverse backgrounds and microbial populations in the same direction. The proportions of gastrointestinal microbes are altered in dogs with some diseases compared to healthy animals . Understanding the impacts of dietary changes on microbial populations in healthy dogs may help to treat or prevent these diseases in the future.The proportions of microbes are highly variable in pet dogs, which may impact the ability to detect differences when treatments are similar. Studies performed in pet dogs utilizing 454-pyrosequencing should attempt to decrease variation, or pair 454-pyrosequencing with targeted assays . Variation would likely have been decreased in this study by utilizing a cross-over design, adapting animals to the control diet prior to baseline measurements, or selecting animals with more similar life histories .


Arabidopsis thaliana and Marcgravia caudata were immersed in a selection of contrasting agents used to treat samples for transmission electron microscopy. Grayscale values for floral tissues and background were measured as a function of time. Contrast was quantified via a contrast index. The thick buds of Marcgravia were scanned to determine which contrasting agents best penetrate thick tissues. The highest contrast increase with cytoplasm-rich tissues was obtained with phosphotungstate, whereas osmium tetroxide and bismuth tatrate displayed the highest contrast increase with vacuolated tissues. Phosphotungstate also displayed the best sample penetration. Furthermore, infiltration with phosphotungstate allowed imaging of all plants parts at a high resolution of 3 µm, which approaches the maximum resolution of our equipment: 1.5 µm. The high affinity of phosphotungstate for vasculature, cytoplasm-rich tissue, and pollen causes these tissues to absorb more X-rays than the surrounding tissues, which, in turn, makes these tissues appear brighter on the scan data. Tissues with different brightness can then be virtually dissected from each other by selecting the bracket of grayscale to be visualized. Promising directions for the future include in silico phenotyping and developmental studies of plant inner parts  via virtual dissection as well as correlations of quantitative phenotypes with omics datasets. Therefore, this work represents a crucial improvement of previous methods, allowing new directions of research to be undertaken in areas ranging from morphology to systems biology.Computed tomography remains strongly underused in plant sciences despite its high potential in delivering detailed 3D phenotypical information because of the low X-ray absorption of most plant tissues. Existing protocols to study soft tissues display poor performance, especially when compared to those used on animals. More efficient protocols to study plant material are therefore needed. Flowers of  The most complete way to describe the morphological phenotype of a given object is to build a three dimensional (3D) model. The immense amount of data contained in an accurate 3D model can then be mined in order to explore correlations with other types of data, e.g. metabolite content The phenotype, via two-dimensional sections or via whole mount studies. Reconstructing an object via sections is highly facilitated by existing semi-automatic methods to align the sections ca. 15–10 µm Reconstruction of the 3D structure of a sample can either be done Although originally developed for medical diagnosis in the early 1970s SM, with the topic “computed tomography”, was refined per “Subject Area”, over the last decade ; therefore, very low-energy X-Rays have to be used for proper imaging. Under such conditions, typical X-ray sources have low yields, therefore requiring very long scanning times. Long scanning times not only decrease throughput but also decrease scan quality by increasing the probability of motion artefacts. These problems are especially acute in the case of very young and meristematic tissues without well-differentiated cell walls.ca. 1000, each carried out at a different rotation angle. These pictures are then processed by algorithms that reconstruct a volume model of the sample An HRXCT scan consists of a large number of pictures of X-ray shadows of an object, typically The above-mentioned problems can be solved by infiltrating the samples with heavy metals, since X-ray absorption increases with atomic number. Protocols were already described to increase the X-ray absorption of animal For each infiltration protocol, the following properties were assessed: (1) contrast improvement, (2) repeatability and uniformity of the contrasting agent, and (3) selectivity of contrasting agent. Penetration of the tissues was qualitatively assessed.Arabidospsis thaliana  were used as a study object because they are small, easy to obtain, very well-described, and comprise both complex organs and a wide range of tissues. Furthermore, A. thaliana is by far the most favoured plant model. Late buds of Marcgravia caudata  were scanned in order to qualitatively assess the penetration power of the contrasting agent through thick and dense tissue barriers (see below). In order to demonstrate the versatility of our methods, larger and more complex flowers were also scanned from two other species: Calycanthus floridus , and Haplophyllum lissonotum (Rutaceae).Anthetic flowers of Arabidopsis thaliana samples  and buds of Marcgravia caudata were fixed in FAA  and washed with 70% ethanol (EtOH) prior to infiltration. All other plants samples were fixed and preserved in 70% EtOH.Flowers were infiltrated for ½, 1, 2, and 8 days. For each infiltration protocol, five flowers were scanned for each infiltration time . The investigated contrasting agents are summarized in Arabidopsis thaliana flowers (medium sized samples with diameter 1–10 mm) were washed three times with 1 ml 70% EtOH, and were mounted individually in 250 µl pipette tips . Inside the pipette tips, the samples were submersed in 70% EtOH. On the lower, narrow end of the tip, parafine wax  was used to stabilize the samples by embedding the flower stem in the wax, and to seal the lower end of the pipette tip. The upper, broader end of the tip was cut off at ca. 7 mm above the sample. In order to keep the samples immersed in 70% EtOH during storage and scanning, an additional 5 mm of 70% EtOH were added in the tip as a reserve. PARAFILM (©Pechiney) was used to seal the upper end of the pipette tip  were mounted in 50–200 ml cylindrical containers , stabilized with acryl pillow foam (DACRON®COMFOREL® INVISTA Ltd.). After mounting, two millilitre 70% EtOH were poured along the vial wall, in order to keep the samples in an ethanol atmosphere. The containers were sealed with PARAFILM . The samSmall samples (>1 mm) were critical point dried  and sputtered with gold for three minutes . They were mounted in a drop of epoxy glue (UHU PLUS®), directly on aluminium stubs . The convia scintillator crystals (XRadia Inc. in-house) that convert X-rays into visible light. The light was then focused  and detected via a CCD camera. The X-ray source was a Hammamatsu L9421-02 90 kV Microfocus X-Ray source. Scanning conditions are detailed in A. thaliana samples was scanned in 11h30, therefore allowing two batches to be scanned daily.The scans were performed on an XRadia MicroXCT-200 system. X-Ray detection was performed ca. one thousand per sample.XMReconstructor 8.1.6599 (XRadia Inc.) was used to perform the 3D reconstruction from the scanning data. The AMIRA-based XM3DViewer 1.1.6 (XRadia Inc.) was used to visualize the scans. The reconstructed 3D data were then exported with XMController 8.1.6599 to series of pictures (tiff format) of reconstructed sections through the sample, typically 2 for the tissues and 200 µm2 for the solvent.The abovementioned series of pictures were imported into the MIMICS Innovation Suite 14.1 , in order to measure the grayscale values of the different organs. Grayscale values were measured for the following floral parts: sepals, petals, filaments, pollen grains, ovary wall, ovules (integuments), and stigmatic papillae. Each measurement was repeated three times on each tissue, on each sample. Measurements of the grayscale value of the same tissue within the same sample were performed on different reconstructed sections. Grayscale values were measured on circular areas of 50 µmThe measured grayscale values of a tissue “Gsc(tissue)” were transformed into a Weber contrast index (CI) CI values were then used to quantify contrast improvement.It was assumed that the concentration of the contrasting agent in the tissues followed a simple exponential saturation curve.∞ = concentration after infinitely long infiltration time; k = constant; t = time. In order to assess and compare the penetration powers of the different contrasting agents, the time that contrasting agents required to reach half-saturation (t1/2) was calculated. It was assumed that 90% saturation was attained after 8d infiltration (C8d = 0.9 C∞). The half-saturation time is therefore:With C(t) = concentration at time t; C1/2) from these relations. The distribution of the 1000 half-saturation times was then plotted, for each contrasting agent, for each tissue. Median and interquartile range of the half-saturation time distributions were used as proxies for infiltration speed and reliability respectively.In order to obtain a measure of infiltration speed and the reliability of this speed, Arabidospsis thaliana flowers are open  and very small, with organs only a few cell layers thick. Therefore, contrasting agents do not need to diffuse across large distances to reach all the organs of the flower. M. caudata buds possess a thick and hermetic protection layer made of fused petals. Thus, in order to test diffusion of the contrasting agents into tightly closed samples with many cell layers, M. caudata buds were infiltrated with a subset of the contrasting agents in Under the employed scanning conditions, samples could not be detected, unless infiltrated with potassium permanganate, Lugós solution, phosphotungstate, lead citrate, bismuth tatrate, or uranium acetate. Infiltration with iron diamine, aqueous iodine, and alcoholic iodine did not allow detection of the samples.Contrast improvement varied significantly among contrasting agents and among tissues. e.g. filaments , and Lewis bases (e.g. DNA bases).Differences in contrast increase between tissues is best explained by the nature of the contrasting agents: relatively small, charged molecules that bind relatively unspecifically to charged surfaces (membranes), charged solubilized molecules (The contrast index (CI) values after two days infiltration which were higher than after 8 days could be due to increased leaching of the contrasting agent from the sample into the solvent. An increase of the grayscale of the solvent would lead to a decrease of the CI values see ). LeachiArabidopsis, the contrasting agents with the fastest  and most constant speed  are the following: osmium tetroxide, bismuth tartrate, osmium tetroxide with ferrocyanate, and permanganate. All are comparatively reactive contrasting agents: osmium tetroxide and permanganate are relatively strong oxidants and bismuth tartrate is in an extremely basic 2N sodium hydroxide solution. The long median infiltration times observed for phosphotungstate could be due to three factors: (1) leaching from the sample into the mounting solvent for long infiltration times, (2) leaching from the sample during sample washing, and (3) the fact that saturation concentrations of phosphotungstate can be very high . For such samples, it is recommended to infiltrate 1–8d in phosphotungstate, to dehydrate by drying , to sputter coat with gold, and to mount in a drop of glue directly on a metal stub. Such a protocol allows the visualization with unprecedented resolution of small, soft plant samples with a commercial HRXCT system , which are usually scanned at high resolution, movements of the sample during the scan are an acute problem. This can be further amplified if the sample has a low intrinsic X-ray absorbance, as is the case for most non lignified tissues , and scanning, in FAA. When precise quantitative datasets are aimed for , such a procedure can be used to minimize shrinking artefacts.One of the most powerful aspects of computed tomography is the production of quantitative 3D data, which can be correlated with other datasets. If plant material is most commonly fixed in FAA, it is usually stored in 70% EtOH. During storage in 70% EtOH, plant material tends to shrink; however, FAA causes little shrinkage of the material Leaching of phosphotungstate out of the sample can be prevented by scanning the sample in the same solution as it was infiltrated in. However, leaching of phosphotungstate out the sample could also be turned into an advantage, because it could allow removal of most of the heavy metal from the sample after scanning by washing the sample with solvent. Loss of contrast up to the level of untreated samples was observed, but complete removal of phosphotungstate from a sample was not tested. This could allow to borrow and to return, intact, rare samples from spirit collections.via phosphotungstate infiltration allows grayscale thresholding of the scan data . The latter does not only allow to separate the sample from the background, but also to separate the different parts of the sample. Therefore, it is possible, to some extent, to virtually dissect a well infiltrated sample by choosing different selections of grayscales to be displayed, e.g. gynoecium, ovules and vasculature  via tables of values of linearly transformed grayscale values called Hounsfield units Arabidopsis flower in i.e. a 2.2% error  and osmium tetroxide (with or without ferrocyanide). Given the penetration limitations of osmium, it is suggested to restrict its use to open and thin material . Furthermore, phosphotungstate (in EtOH or in FAA) and osmium tetroxide (with or without ferrocyanide) clearly outperform previously described infiltration agents based on iodine via the setting of morphometric landmarks, in order to advance our understanding of phenotypic evolution and developmental trajectories vs. ctrl) in plants in which the young reproductive organs are difficult to access (inside stems or covered by many bracts). In silico phenotyping of plant inner parts, e.g., ovules, vasculature, pollen, and cell nuclei, is now possible via grayscale thresholding.Although our results allow the straightforward use of CT for qualitative morphological work Figure S1Literature statistics: percentage of paper containing “computed tomography” per Subject Area vs. time.(TIF)Click here for additional data file.Figure S2Contrast improvement over time for different floral tissues.(TIF)Click here for additional data file.Figure S3Reproducibility and speed: distribution of the half-saturation times estimated by permutations of data points.(TIF)Click here for additional data file.Figure S4Grayscale thresholding on A. thaliana (wt ecotype Col-0) stigma reveal germinating pollen and cell nuclei.(TIF)Click here for additional data file.Figure S5Total pollen grain number estimates from volumetric assessment after thresholding.(TIF)Click here for additional data file.Table S1Literature statistics: comparative underuse of computed tomography in plant sciences.(DOCX)Click here for additional data file.Video S1Longitudinal section series played as a video though the inflorescence in .(MPG)Click here for additional data file.Video S2Animation of a 3D model of the inflorescence in A. thaliana flower meristems of double line ap1 cal pAP1::AP1-GR mutant; courtesy Toshiro Ito research group, Temasek Life Sciences Laboratory, National University of Singapore, unpublished data), virtually cut at the level of the section in  (.(MPG)Click here for additional data file.Video S3Stigma of Arabidopsis thaliana (wt ecotype Col-0) with grayscale thresholding set to remove solvent signal.(MPG)Click here for additional data file.Video S4Stigma of Arabidopsis thaliana (wt ecotype Col-0). Grayscale thresholding set to remove most of papillae.(MPG)Click here for additional data file.


P > 0.05). In addition, CT and IT similarly increased functional capillary density of skeletal muscle  and the capillary-to-fiber ratio in skeletal muscle  and in the left ventricle . In conclusion, at equivalent total work volumes, interval exercise training induced similar functional and structural alterations in the microcirculation of skeletal muscle and myocardium in healthy rats compared to continuous exercise training.Interval training (IT), consisting of alternated periods of high and low intensity exercise, has been proposed as a strategy to induce more marked biological adaptations than continuous exercise training (CT). The purpose of this study was to assess the effects of IT and CT with equivalent total energy expenditure on capillary skeletal and cardiac muscles in rats. Wistar rats ran on a treadmill for 30 min per day with no slope (0%), 4 times/week for 13 weeks. CT has constant load of 70% max; IT has cycles of 90% max for 1 min followed by 1 min at 50% max. CT and IT increased endurance and muscle oxidative capacity and attenuated body weight gain to a similar extent ( Regular practice of moderate aerobic exercise is widely recognized to reduce cardiovascular risk . Since aAlong with metabolic and biochemical myocardial adaptations, major vascular growth and remodeling occur in coronary circulation after exercise training. Enhanced angiogenesis, that is, capillary growth, and vascularization along with altered regulation of coronary blood flow are well-known features of physiological adaptations to chronic exercise and have been extensively revised –7. The bThe increased capillary network and coronary blood flow play an important role in improving aerobic capacity and facilitating oxygen transport, conductance, and muscle extraction, therefore contributing to increase of maximal oxygen uptake and physical performance , 9, 10. intensity itself. On the other hand, a series of well-planned and well-conducted studies have matched the total amount of work in order to investigate the influence of exercise intensity itself on a variety of different myocardial measures such as substrate utilization, oxygen consumption ) was calculated as follows: Vv[cap] = Pp/PT (%), where Pp is the number of points that are coincident with a capillary and PT is the total number of test points (PT = 56 in the present case). The fiber volume density (Vv[fib]) was similarly calculated. The ratio of the capillary volume density to the fiber volume density (Vv[cap]/Vv[fib]) was calculated to negate any influences of cardiac hypertrophy on myocardial capillary density.The tissue samples were dehydrated in a graded series of ethanol  and embedded in paraffin. The left ventricular structural capillary density was determined according to the orientator method as previously described . BrieflyTissue samples from the gracilis muscle were obtained to biochemically analyze citrate synthase activity to determine the effectiveness of the exercise training. The muscle samples were immediately frozen in liquid nitrogen and stored at −80°C until processing. Citrate synthase activity was measured from whole muscle homogenate using the spectrophotometric method published by Srere .Griffonia simplicifolia I lectin .The following drugs were used: sodium pentobarbital, pancuronium bromide, FITC-labeled dextran, and FITC-conjugated P value less than 0.05 were considered significant. All calculations were made by computer-assisted analyses with a commercially available statistical package .The results are expressed as the mean ± SD for each group, and comparisons between the different groups were made with one-way analysis of variance (ANOVA). If a significant difference was detected by ANOVA, the Bonferroni test was used to identify the statistically significant differences. Differences with a P > 0.05) and total oxygen consumption during the exercise session  were similar between training protocols.Total running distance  in the trained animals than in the SED animals (P < 0.001) than in the SED animals (189 ± 100 nmol/mg/min). The percentage increase in citrate synthase activity was of 69.0 ± 38.3 and 81.2 ± 32.9 for the CT and IT groups, respectively  induced several changes indicative of a trained state in rats . The max animals ; the obsectively . These r2, resp.; P < 0.001), compared with that of the sedentary animals (SED: 216 ± 7 capillaries/mm2). The percentage increase in muscle functional capillary density was of 30.6 ± 11.7 and 28.7 ± 11.9 for the CT and IT groups, respectively . The percentage increase in muscle structural capillary density was of 28.7 ± 14.4 and 40.1 ± 17.2 for the CT and IT groups, respectively (Vv[cap]/Vv[fib]) in the left ventricle of both exercise groups was increased to a similar magnitude, when compared to that in the control group . The percentage increase in left ventricle structural capillary density was of 57.3 ± 53.1 and 54.3 ± 40.5 for the CT and IT groups, respectively  CT and IT increased exercise endurance and muscle oxidative capacity and attenuated body weight gain to the same extent; (2) CT and IT increased similarly functional and structural alterations in the microcirculation of locomotor skeletal muscle and of the myocardium of rats. These results suggest that IT mode is equivalent to CT in increasing exercise capacity and in inducing microvascular adaptations in both skeletal and cardiac muscles, when total training volume is matched supporting the concept that total energy expenditure, and not exercise intensity per se, is the major physiological stimulus, at least for chronic changes in skeletal and myocardium microcirculation.The large increase in oxidative enzyme citrate synthase, performance, and capillary density produced by both IT and CT in the present study is consistent with numerous studies of endurance trained athletes and animals , 20, 21.V2peakO have met criticism because of the insufficient equation between intensity and duration [Previous studies investigating the influence of different exercise intensities on improvements of microcirculation and duration –28. ThusPrevious studies combined provide evidence that the adaptations to exercise training may vary depending on training protocols, exercise intensity, and total training volume regarding the effect on microcirculation and oxidative capacity –32. The V2peakO, the continuous training group cycled at only 50% of V2peakO, a much lower training intensity. This may have contributed to their findings of greater improvements in V2peakO after high intensity interval training than in moderate intensity continuous training.Despite the established concept that exercise training adaptations are primarily dependent on training volume, at least two previous studies have reported greater improvements in aerobic fitness in healthy humans after interval training compared with continuous training, regardless of total work being matched , 36. NeiV2peakO: 73 ± 4 and 58 ± 2 mL/kg/min for the males and females, resp.) and trained over a 5-month off-season period, thus encompassing a much larger total training volume than the present study.The magnitude of improvement in performance after interval and continuous training programs may also depend on other training variables, such as the length of the training regimen and prior fitness status of the subjects. The study by Eversten et al.  reportedOther earlier reports , 37–40 hThe present study reveals that in normal rats, IT induces significant changes in the structure and function of the gracilis muscle. These changes are similar to those induced by CT. The structural and functional adaptations within our rats gracilis muscle are consistent with those in earlier reports , 41, 42.The present results revealed a marked increase in the number of spontaneously perfused capillaries in locomotor skeletal muscle in both the CT and IT groups as assessed by intravital video microscopy. In contrast, there was no change in the functional capillary density of the ear skin. Thus, these results suggest that both CT and IT increase perfusion only in tissues that are actively participating in exercise and are subjected to repeated increases in metabolic demand, blood flow, and vascular shear stress.The mechanisms underlying exercise-induced angiogenesis and vasculogenesis in skeletal muscle are well defined , 44. By Finally, it is worth mentioning that the vascular adaptations, as well as their underlying mechanisms, induced by exercise training, are not consistently distributed along the arterial tree . MoreoveIn conclusion, we found that when total exercise volume is matched, interval training that included periods of high intensity exercise produces similar adaptations to moderate and constant exercise with regard to effects on exercise capacity, skeletal muscle citrate synthase effects, and microcirculatory beds of skeletal muscle and myocardium.


Background: Coal tar and bitumen have been historically used to coat the insides of cast iron drinking water mains. Polycyclic aromatic hydrocarbons (PAHs) may leach from these coatings into the drinking water and form a potential health risk for humans.Objective: We estimated the potential human cancer risk from PAHs in coated cast iron water mains.Method: In a Dutch nationwide study, we collected drinking water samples at 120 locations over a period of 17 days under various operational conditions, such as undisturbed operation, during flushing of pipes, and after a mains repair, and analyzed these samples for PAHs. We then estimated the health risk associated with an exposure scenario over a lifetime.Results: During flushing, PAH levels frequently exceeded drinking water quality standards; after flushing, these levels dropped rapidly. After the repair of cast iron water mains, PAH levels exceeded the drinking water standards for up to 40 days in some locations.Conclusions: The estimated margin of exposure for PAH exposure through drinking water was > 10,000 for all 120 measurement locations, which suggests that PAH exposure through drinking water is of low concern for consumer health. However, factors that differ among water systems, such as the use of chlorination for disinfection, may influence PAH levels in other locations. Several polycyclic aromatic hydrocarbons (PAHs) have been associated with the development of a wide range of cancers . For thiCast iron water mains are susceptible to corrosion , and theBoth coal tar and bitumen are highly viscous liquids that include a complex mixture of polycyclic aromatic compounds . The exaAlthough the purpose of coating cast iron water mains is to protect the cast iron from corrosion, the linings themselves can deteriorate over time. As a result, PAHs can leach into water during low-flow conditions when the water stays in the mains for longer periods . In addiTo summarize, PAHs may leach from bitumen coatings into drinking water, and the erosion of bitumen coatings may release particles that contain PAHs. PAHs may be increased when PAH-contaminated sediment particles are resuspended. Thus, PAHs might be expected to increase during periods of stagnant water (resulting in increased leaching due to long exposure times), during flushing of pipes (because of sediment resuspension and biofilm destruction), and during maintenance (invasive work on pipes).The literature on PAHs in drinking water distribution systems is limited, and there is little quantitative information on the presence of PAHs in drinking water. Therefore, in 2010 we carried out an extensive research project in collaboration with all Dutch water companies, which serve a total population of almost 17 million people. PAHs were measured in samples collected at 120 locations under different operational conditions to identify the conditions associated with increased PAHs in drinking water, and to estimate the potential human health impact of the resulting PAH exposures.Measurement locations. The Dutch drinking water distribution system has a total length of 112,000 km, of which approximately 10% is cast iron mains installed before 1980. We selected 120 measurement locations throughout the Netherlands with cast iron and steel mains 80–120 mm in diameter that were likely to be coated with coal tar or bitumen because they were constructed before 1980. This resulted in approximately 1 sampling location per 100 km of mains. All 10 Dutch water companies participated in the study. The number of measurement locations for each water company was determined based on the length of suspect mains in their networks. We made no further distinctions between water companies.Measurement protocol. Drinking water companies collected water samples from cast iron mains in their own districts under well-defined conditions: during undisturbed operations, during and directly after flushing of pipes, and after the removal of a section of pipe and repair of the water main , and effective remaining diameter with calipers and visually assessed the coating. When the visual assessment was inconclusive (in approximately 35% of samples), we used a burner to heat the main and performed a scent-based assessment to determine the presence and type of coating. We categorized the type of coating ; the thickness of the coating [thin (< 1 mm) or thick (≥ 1 mm)]; and the coverage of the main . The drinking water companies provided information on the years of installation. We examined whether the year of installation was indicative for the coating coverage.Health risk assessment. Cancer is the main outcome of concern for PAH exposure. Risk assessment followed the method used by the European Food Safety Authority (EFSA) to evaluate the risk of PAH contamination in food , that is, the lower 95% confidence limit estimated for the dose that causes no more than a 10% increase in cancer incidence in rodents. The 10 from an animal study, would be of “low concern from a public health point of view and might be considered as a low priority for risk management actions.” The a]pyrene (BaP), chrysene, benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[ghi]perylene, dibenz[ah]anthracene, and indenopyrene) can be used as indicators of the carcinogenic potency of PAH mixtures. The EFSA also uses the sum of four PAHs , the sum of two PAHs , and the concentration of BaP alone, to assess carcinogenicity /day for BaP, Σ2PAH, Σ4PAH, and Σ8PAH, respectively] derived by the g,h,i]perylene), which is more stringent than the TDI of 40 μg/kg bw/day for noncarcinogenic PAHs with 17–35 hydrogen atoms . The TDI represents an estimate of the daily intake level to which humans can be exposed during their entire lifetime without causing adverse health effects.We calculated the MOEs for each of the summed PAH combinations by dividing the specific BMDLa) data on dermal absorption of PAHs from aqueous matrices are scarce, b) absorption of PAHs is highly dependent on the vehicle used in the tests, and c) available information suggests that exposure from dermal absorption would be negligible compared with oral ingestion from drinking water. Specifically, 14C-labeled phenanthrene in vitro in human skin and observed 24% absorption after exposure of 0.64 cm2 skin for 24 hr to a 0.1 mL solution of 1 µg/mL phenanthrene. From this, an equilibrium constant in terms of partial pressure (Kp) was determined (1.6 × 10–3 cm/hr) , taking into consideration both aerosol formation and evaporation of PAHs. We did not estimate dermal exposure during bathing or showering because 3 cm/hr) . On the 3 cm/hr)  and a suanthrene .Water PAH levels and different measurement circumstances. We observed each of the 16 PAHs in at least 1 of the 960 samples (8 samples for each of 120 sampling locations). Of the PAHs measured, naphthalene, phenanthrene, pyrene, and fluoranthene were the most prevalent. Fluoranthene was detected more often than any other PAH in the low- and high-flush samples, and phenanthrene was detected more often in samples collected after main repair (repair 1 and repair 2) (epair 2) .During normal operation , PAHs were undetectable or were present only at very low levels , which iFlushing samples collected at the hydrants contained the highest PAH levels , which sR2 < 0.2; data not shown).Because PAHs are hydrophobic, we expected PAHs to adsorb to sediment particles; high PAH levels in the flushing samples were consistent with this expectation. However, PAH levels were not correlated with turbidity (an indication of the amount of suspended particles in the sample) or with estimated flushing velocities (which are associated with sediment resuspension) (data not shown). In addition, PAH levels were not correlated with total organic carbon, pH, electric conductivity, or temperature  and using the maximum concentration measured in any sample for each individual PAH, indicated that inhalation exposure by aerosol formation during showering was negligible compared with estimated oral intakes . However, inhalation exposure due to avaporation of volatile PAHs (2.0 µg/kg bw/day) was comparable to oral exposure (2.1 µg/kg bw/day) for Σ16PAH. However, only 0.2%, 10%, 11%, and 6.9% of the total exposure to the carcinogenic PAHs BaP, Σ2PAH, Σ4PAH, and Σ8PAH, respectively, were attributed to inhalation during showering, which suggests that inhalation exposure primarily involves PAHs classified as noncarcinogens or as low potency carcinogens. Therefore, we did not include inhalation exposure during showering when assessing risk for carcinogenic effects of PAHs in drinking water.We determined that oral and inhalation exposures to the maximum concentration of Σ16PAH totaled 4.1 µg/kg bw/day , whereasGeneral discussion. Exceedance of PAH standards can occur after invasive repairs in cast iron water mains, but our estimates suggest that human health risks related to coated cast iron water mains are low. To prevent increased exposures during flushing operations, drinking water networks should be flushed in a systematic way to ensure that all particles containing PAH are removed ( removed .Water quality aspects, such as hardness , chlorinPAH levels are likely to be increased in cast iron water mains as a consequence of invasive work and raised flow velocities in water distribution networks in coutries other than the Netherlands. However, the specific circumstances associated with elevated PAH levels may differ, for example, in countries where chlorine is used as a disinfectant, and additional field measurements would be needed to determine whether our findings apply elsewhere.PAHs from coal tar and bitumen coatings in cast iron water mains are a potential health risk for humans. Drinking water samples were collected from 120 measurement locations in cast iron water distribution networks in the Netherlands over a period of 17 days under various operational conditions: undisturbed operation, during flushing, and after repair of water mains. We analyzed these samples for PAHs and estimated the health risk using the MOE approach. During flushing, high PAH levels may occur, but PAH concentrations dropped rapidly after flushing. After coated cast iron mains were cut and repaired, we observed PAH levels that exceeded the water quality standard up to 40 days after the repair. However, even after accounting for such exceedances, estimated MOEs were > 10,000 for all 120 measurement locations. Therefore, we conclude that PAH exposure through Dutch drinking water is of low concern for consumer health.


Delivering early nutrition support therapy, primarily using the enteral route, is seen as a strategy that may reduce disease severity, diminish complications, decrease length of stay in the ICU, and favorably impact patient outcome. SCCM and ASPEN guidelines support that after the initiation of enteral feeding we have 10 days to meet 100% of predicted energy requirements before we consider supplementation with parenteral nutrition (PN). There are scarce data about the clinical effects of using a more accelerated approach to reach full caloric adequacy with enteral nutrition (EN).The aim of this observational study is to evaluate whether a diminished time to target caloric goal is associated with more patient intolerance and clinical benefits in ICU patients receiving EN.t test and Pearson's correlation coefficient.From January 2010 to June 2010 we prospectively followed all consecutive ICU patients receiving EN. We collected epidemiological data, APACHE II score, LOS , need for mechanical ventilation, incidence of nosocomial infection and hospital mortality. We also collected data on nutrition therapy as the time to target caloric goal (120 hours), total time on nutrition therapy, incidence of diarrhea and other signs of EN intolerance . For statistical analysis we used the Kolmogorov-Smirnov test, Student's n = 3) of patients. Comparing the different groups  we were unable to detect any difference with statistical significance regarding incidence of diarrhea, EN intolerance, need for MV, total time on nutrition therapy, incidence of nosocomial infection, ICU and hospital LOS and hospital mortality.We enrolled 32 patients  in the study. The mean age was 66 ± 18 years, mean APACHE II score 21 ± 9, mean ICU and hospital LOS were 21.3 and 35 days respectively, incidence of nosocomial infection was 21.8%, mean total time in nutrition therapy was 18.3 ± 14 days and hospital mortality was 28%. There was need for mechanical ventilation in 56%. There was need for PN supplementation in 9.4% (These preliminary data have shown no correlation of a diminished time to meet energy requirements in EN with patient tolerance to nutrition therapy and clinical benefits.


The rising incidence of diabetes and its negative impact on quality of life highlights the urgent need to develop biomarkers of early nerve damage. Measurement of total vitamin B12 has some limitations. We want to determine the levels of urinary methylmalonic acid and its relationships with serum vitamin B12 and polyneuropathy. The 176 Chinese patients with Type 2 diabetes mellitus were divided into 3 groups according to the levels of vitamin B12. A gas chromatography mass spectrometric technique was used to determine blood methylmalonic acid and urinary methylmalonic acid. The diagnosis of distal diabetic polyneuropathy was based on the determination of bilateral limb sensory and motor nerve conduction velocity and amplitude with electromyogram. Multiple regression analysis revealed that urinary methylmalonic acid/creatinine, blood methylmalonic acid, and so forth were variables that influenced diabetic polyneuropathy significantly. Nerve sensory conduction velocity and nerve amplitude in the group of urinary methylmalonic acid/creatinine >3.5 mmol/mol decreased significantly. Superficial peroneal nerve sensory and motor conduction velocity and ulnar nerve compound motor active potential amplitude were inversely correlated with urinary methylmalonic acid/creatinine. Urinary methylmalonic acid correlates with serum vitamin B12 levels in person with diabetes and is a sensitive marker of early polyneuropathy. Diabetes mellitus (DM) is caused by genetic and environmental interactions, along with changing lifestyles and an aging population, increasing incidence of diabetes. Diabetic neuropathy is one of the major complications of diabetes with both Type 1 and Type 2. Up to 50% of all diabetes have polyneuropathy which is a major cause of morbidity and associated with increased mortality, and up to 26% of diabetics develop painful diabetic neuropathy with debilitating effects on quality of life .The rising incidence of diabetes and its negative impact on quality of life highlights the urgent need to develop biomarkers of early nerve damage. The gold-standard method to evaluate morphological change in small nerve fibres was the skin biopsy ; howeverVitamin B12 (Vit B12) is a cofactor for methylmalonylCoA mutase, which converts methylmalonyl CoA to succinyl CoA. Measurement of total Vit B12 suffers from some limitations; in particular, most of the cobalamin measured is that bound to haptocorrin (HC) , 6. MethBased on the above considerations, we believed that uMMA was an intermediate metabolite during the course of neuropathy, and there was an important relationship between uMMA and the formation of neuropathy. The aim of the present study was to assess uMMA levels in patients presenting with and without diabetic polyneuropathy (DPN) and to determine the role of uMMA in DPN.The study population consisted of 176 Chinese Han patients  with Type 2 diabetes mellitus aged 38–70 y recruited upon admission to the Second Hospital of Shandong University between June in 2009 and June in 2011 who were eligible for the study.Exclusion criteria were as follows: history of serious heart and lung disease, diabetic kidney disease or other kidney diseases, pernicious anemia, intestinal surgery, and gastrointestinal disease, excluding antibiotics, colchicine, aminosalicylic acid, H2 receptor antagonists and proton pump inhibitors and other effects of gastrointestinal motility drugs, and the patients with the Vit B12 or methylcobalamin treatment. Long-term vegetarians (more than six months) and those refused concurrent electrophysiological or laboratory testing were excluded too.n = 58), group B , and group C .According to the level of Vit B12, the patients were divided into 3 groups: group A  via venipuncture into EDTA-coated tubes (5 mL) for a full blood were counted to check glycosylated hemoglobin A1c (HbA1c), triglycerides (TG), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), serum creatinine (sCr), Vit B12, folic acid, ferritin, homocysteine (Hcy), hemoglobin (HGB), and mean corpuscular volume (MCV). Holotranscobalamin (holoTC) level was determined by microparticle enzyme immunoassay on Axsym analyzer . Fasting morning urine samples were for use. Serum samples were stored at −80°C. Approximately 10 mL fasting urine samples were stored at −80°C untreated.The application of stable isotope dilution method for rapid extraction of the solid phase sample by gas chromatography mass spectrometry was used for the determination of blood methylmalonic acid (bMMA) and uMMA. Urinary MMA was expressed relative to urinary creatinine as the uMMA/creatinine.Diabetic polyneuropathy: the diagnosis of bilateral limb sensory and motor nerve conduction velocity (SNCV/MNCV) and amplitude was performed, according to standard techniques protocols, by means of Keypoint, Dantec equipment, for all individuals. Tests were carried out in a warm room to minimize the effects of temperature on nerve conduction velocity (CV), and the limbs were warmed throughout with an infrared lamp to maintain a skin temperature over 35°C. Suprathreshold stimulation was used, the frequency was l Hz (feeling) and 1 Hz (movement), respectively, and course was both for 0.2 ms. Nerve conduction and electromyographic studies were performed in four limbs, which included 2 nerves of upper limbs and 2 nerves of lower limbs, by means of standard techniques. We measured the amplitude and velocity of motor nerve conduction and sensor nerve conduction in 4 nerves: superficial peroneal, sural, median, and ulnar.χ2 test, and logistic regression analysis was used for an independent risk factor of diabetic polyneuropathy. P < 0.05 was considered statistically significant.All experimental data were analysed by SPSS software 16.0 . Measurement data were presented as mean ± standard deviation (mean ± SD). Single factor and multifactor ANOVA analysis were for the mean difference among the groups. Correlation was analysed using Pearson correlation. The count data were analyzed by Patients in the two groups with normal Vit B12 had decreased serum concentrations of HDL, ferritin, folic acid, holoTC, and HGB and increased uMMA/creatinine and bMMA compared to those lack Vit B12. In addition, DM Type 2 patients with Vit B12 over 400 ng/L had significantly increased ferritin, holoTC, and HGB and decreased uMMA/creatinine compared to those with Vit B12 180–400 ng/L. There was no significant difference in LDL, MCV, duration, sCr, TG, BMI, and HbA1c among the three groups. Polyneuropathy was present in 24.7% of our diabetic patients using electromyogram.P = 0.001), bMMA (P = 0.02), diabetes duration (P = 0.045), and holoTC (P = 0.003) were variables that influenced DPN significantly and independently while the serum concentration of Vit B12 did not have an independent significant influence on DPN  and ulnar nerve sensory active nerve potential amplitude  were inversely correlated with the uMMA/creatinine. Sural nerve compound motor active potential amplitude , sural nerve motor conduction velocity , superficial peroneal nerve compound motor active potential amplitude , and ulnar nerve sensory conduction velocity  showed modest inverse correlations with uMMA/creatinine.Univariate analysis was carried out with the uMMA/creatinine as the outcome variable. Some significant correlations were revealed between the uMMA/creatinine and other electrophysiological markers of neuropathy severity . SuperfiOur data showed polyneuropathy was present in 24.7% of our diabetic patients using electromyogram (EMG), which was higher than the previous report , maybe bThe bMMA level in the patients with Vit B12 deficiency was significantly higher than in the people with normal Vit B12, while there was no significant difference between the two groups with normal Vit B12, which meant bMMA was not sensitive for early vitamin B12 deficiency. It is not considered the gold standard for the determination of Vit B12 deficiency , becauseIn the study, holoTC gradually reduced accompanied with Vit B12 decrease. There was great difference among the three groups, and it was closely related to DPN. We thought that holoTC is a sensitive marker for Vit B12 deficiency, as previously reported . HoloTC The present results demonstrated that when part of Vit B12 was in normal range, uMMA had obviously exceeded the normal level, and there was great difference among the three groups, so we thought that uMMA was sensitive for the early deficiency of cobalamin, consistent with the previous reports , 19. TheThe nerve conduction velocity and amplitude of four limbs decreased obviously as uMMA/creatinine was above normal. There was no significant difference between the decreased conduction velocity of median and ulnar nerves and the increased uMMA/creatinine. The upper limb sensory nerves appeared to be preferably affected by axonal damage as demonstrated by the finding that the sensory active nerve potential amplitude of the median and ulnar nerves was reduced in a remarkably high percentage of study patients, versus the conduction velocity of the two nerves . The abnOur findings were presented with some limitations. Although we identified patients prospectively, they were not randomly selected from a population with Type 2 diabetes with or without DPN. We underwent EMG for each patient. Being an invasive operation, EMG could not reflect early neuropathy screening, so some early neuropathy patients might be missed. We excluded the patients with Type 1 diabetes because of their expected metformin restriction and the distinct pathophysiological mechanisms, and further study would be conducted in depth.In conclusion, uMMA and holoTC are significantly associated with cobalamin deficiency and polyneuropathy. The relationships among cobalamin deficiency, decline holoTC, elevated uMMA levels, and the polyneuropathy are controversial, and further work is needed to prove a direct causal relationship and the mechanism, but both cobalamin deficiency and elevation of its serum metabolites are associated with the presence of a sensorimotor peripheral neuropathy . uMMA ma


When reaching 80–90% confluence, the cells were passaged. Cells of the second and eighth passages were cultured in 24-well plates (4 × 104 cells/well) for 6 days and three wells per day were counted. The average cell counts at each time point were then plotted against time and the population doubling time (PDT) was determined. Then, vials of cells (2 × 106 cells/mL) were cryopreserved for 1 month and after thawing, the cell viability was evaluated. The PDT of the second passage was about 23 h and for the eighth passage was about 30 h. The viability of the cultures was 95% in the second passage and 74.5% in the eighth passage. It was shown that the Guinea pig fetal fibroblast cell culture can be established using the adherent culture method while, after freezing, the viability indices of these cells were favorable.Establishment of Guinea pig fetal fibroblast cells and their biological evaluation before and after cryopreservation were the main purposes of this study. After determination of the proper age of pregnancy by ultrasonography, 30 days old fetuses of Guinea pigs were recovered. Their skins were cut into small pieces (1 mm At present, preservation of individual animals, semen, embryos, and genomic and cDNA libraries are all practical options. Traditionally, in vitro conservation includes materials like semen, oocytes, and embryos even for a few numbers of species that require species-specific techniques . CryopreThe establishment of fibroblast banks especially for endangered species can provide an excellent resource for biological research and preserve precious genetic materials . FibroblTo preserve a valuable genetic resource, establishing fibroblast banks has been proposed as a practical approach; not only does it preserve precious genetic material, but it also provides an excellent resource for biological research. The purpose of this study was to establish and biologically evaluate the fibroblast cultures provided from fetal skin tissue of Guinea pigs.Cavia porcellus) were selected and housed in the Laboratory Animal Center of Shiraz University of Medical Sciences, Shiraz, Iran. The animals were housed in standard cages, three per cage , under controlled temperature (22°C), with a 12 h light and 12 h dark cycle. Standard laboratory chow and tap water were available ad libitum. Four pregnant female Guinea pigs (Cavia porcellus) were enrolled and the gestational age was determined ultrasonographically by measuring the fetal biparietal diameter [Y = −0.00043X2 + 0.06881X − 0.75941; where Y = gestation day and X = biparietal diameter (cm).Mature female Guinea pigs (diameter  using a 2) and cultured in 88% Dulbecco's Modified Eagle Medium , 10% fetal bovine serum , 1% penicillin and streptomycin, and 1% L-glutamine (Sigma cat. number G5840) in 25 cm2 flasks and were cultured at 37°C in an incubator with 5% CO2 and saturated humidity. The medium was replaced after 48 h. When fibroblast cells reached 80–90% confluence, the cells were harvested using 0.25% trypsin (Gibco cat. number 15090-046) and were then passaged until reaching the eight passages.The Guinea pigs were anesthetized by an intraperitoneal injection of ketamine  and xylazine . They were decapitated and their uteruses were removed immediately and washed in sterile phosphate buffered saline  containing 1% penicillin and streptomycin . The skin of fetuses was cut into small pieces . The skin of fetuses was cultured as described before and the time intervals between the first and second passages (when the cell cultures reached 80–90% confluence) in the three different ages were determined.4 and 5.5 × 104 cells per well, cultured for eight days, and counted every day (3 wells each time per group) to determine the number of cells and the best population doubling time (PDT). In the third step, the cells of the second and the eighth passages were seeded in two 24-well plates at a density of approximately 4 × 104 cells per well, cultured for 6 days, and counted every day (3 wells each time).In the second step, the cells of the second passage of the three 30 days old fetuses were seeded in two 24-well plates at a density of approximately 4 × 10The mean cell numbers at each time point were then plotted to time using GraphPad Prism version 5.01 for Windows . The PDT was determined based on this curve .6 viable cells/mL. The cell suspension was aliquoted into sterile plastic cryovials that were labeled with the fetus number, freezing serial number, and the date. The vials were sealed and kept at −20°C for 60 min to equilibrate the DMSO and then they were transferred into −70°C for 24 h, and finally transferred into liquid nitrogen for long-term storage [2.In each passage, cells at the logarithmic growth phase were collected and counted with a hemocytometer and then resuspended in frozen solution including 10% dimethyl sulfoxide  and 90% FBS, at a density of 2 × 10 storage . The cryViabilities before freezing and after recovery were determined using the trypan blue exclusion test (0.4% trypan blue in PBS). The number of nonviable cells was determined in a visual field of 1000 cells after one month of cryopreservation .The chromosomes were prepared, fixed, and stained following standard method . The celt-test .The mean and SE of counted cells in growth curve analysis were subjected to the test of normality and then were compared using independent sample The time interval between the first and second passages for Guinea pig fetal fibroblast cells was 30 days for day 20, 10 days for day 30, and 15 days for day 45 of pregnancy. The proper pregnancy age for collection and culture of Guinea pig fetal fibroblast cells was at the 30th day of pregnancy which made the shortest interval for reaching the maximum confluence in the fibroblast cell culture.At about 4-5 days after the tissue explants adhered to the flasks, fibroblast cells were observed sprouting from the margins of these tissue pieces. The cells showed typical fusiform morphology with centrally located oval nuclei . The fet4 per well (The proper number of cells for growth curve analysis was 4 × 10per well . The groper well . In two n = 64, comprising 62 autosomal and 2 sex chromosomes (The chromosome number of the Guinea pig fetal fibroblast cells was 2omosomes . The chrThe fetal fibroblast cell culture of the Guinea pigs was established using the adherent culture method and these cells were frozen within 8 passages. Morphology, as the most important qualitative parameter of epidermal tissue, was evaluated by light microscopy. In our study, the cells had fibrous characteristics with turgor vitalis cytoplasm, and during growth, they showed typical fibroblast-like morphology as radiating, flame-like, or whirlpool migrating shapes. Using phase-contrast microscopy, fibroblast-like, or epithelial-like cells were observed that could be seen as migrating ones from the tissue pieces 5 days after explanting . When ex6 cells/mL. The viability was 95% and 74.5% after the second and eighth passages, respectively, indicating the cells were healthy with good viability under these culture conditions, while freezing resulted in damage in the cell viability after the eighth passage. The growth curve of the Guinea pig fetal fibroblast cells showed a typical “S” shape, consisting of the latent phase, exponential phase, and plateau phase. The cells after passage had a latent phase of about 24 h. Thereafter, the cells experienced exponential proliferation, followed by a plateau phase. Finally, cell growth ceased as a result of contact inhibition from the day 5. These results indicated that the second and the eighth passages of Guinea pig fetal cells cultured in vitro had the same biological characteristics of normal fibroblast cells.The Guinea pig fibroblast cells were cryopreserved within 8 passages, at a density of about 2 × 10n = 64 frequency of 98% indicated that the Guinea pig fetal fibroblast cultures were stably diploid in accordance with the previous reports in Guinea pigs [Maintaining the same diploid character of fibroblast cells is critical to preserve the genetic resources in vivo. The in vitro cultured cells that kept the division capability and after successive cell divisions present any cell differentiation or alteration cannot be used in breeding conservation. A 2nea pigs . In addinea pigs , the incIt is not uncommon for cells to cease growth and show changes in biological characteristics or lose their diplont properties with time in cultures due to a variety of stimuli and factors. Effective measures are thus required to ensure diploid stability in cultures of cells that are used to preserve valuable genetic resources. The pH must therefore be controlled strictly, the culture medium must be changed at appropriate intervals, and a split ratio of between 1 : 2 and 1 : 3 should be used. Importantly, also, the passage intervals should not be too great and the cells should be maintained at subconfluence to avoid deleterious effects from contact inhibition. Explanted cells should be passaged quickly and with care and high quality reagents should always be used. Finally, cells should be cryopreserved with the utmost care as required to maintain a high quality cell bank that is viable over a long-term. The biological characteristics, especially the genetic characteristics, of the cells may be changed by in vitro culture conditions after many passages, so a minimal number of passages are recommended to conserve them .In conclusion, a fibroblast cell culture was established from explanted fetal skin tissue of the Guinea pig standard tissue adherent culture and continuous passaging following trypsinization. In order to ensure the cell quality, cells of each individual were tested using the same method. We concluded that the cell quality was identical among all cell cultures. So our work made a valuable contribution to the preservation of the genetic resources of the Guinea pig and provided useful biomaterial for future studies in cell biology, medicine, genomics, postgenomics, and both genetic and embryonic engineering.