No statistically significant differences in Hg vapor measurements were observed between neighborhoods. This study demonstrates that building materials used widely in developing communities, such as adobe bricks, may be a substantial source of residential Hg exposure in silver or gold refining communities where Hg is produced or used for amalgamation in artisanal gold production. This is the first study of adobe brick contamination anywhere in the world. Huancavelica, Peru is the site of historic cinnabar refining and one of the most mercury (Hg) contaminated urban areas in the world. Over 80% of homes in Huancavelica are constructed with adobe bricks made from Hg contaminated soil. In this study we measured total Hg concentrations in adobe brick, dirt floor, surface dust, and air samples from the interior of 60 adobe brick houses located in four neighborhoods. Concentrations of total Hg in adobe bricks, dirt floors, and surface dust ranged from 8.00 to 1070 µg/g, 3.06 to 926 µg/g, and 0.02 to 9.69 µg/wipe, respectively, with statistically significant differences between the four neighborhoods. Concentrations of Hg in adobe brick and dirt floor samples in Huancavelica were orders of magnitude higher than in Ayacucho, a non-mining town in Peru. A strong correlation exists between total Hg concentrations in adobe bricks and dirt floors which confirms that adobe bricks were being made on-site and not purchased from an off-site source. A strong correlation between surface dust and adobe bricks and dirt floors indicates that walls and floors serve as indoor sources of Hg contamination. Elemental Hg vapor concentrations were below detection (<0.5 µg/m Legacy mercury (Hg) contamination resulting from cinnabar mining and refining, particularly in ambient soils, has been studied in various parts of the world, most notably Almadén, Spain ,2 and IdHuancavelica, Peru is located at an elevation of 3660 m in the Andes, where for over 350 years it served as the primary source of Hg used for amalgamation-based silver production in South America . Most ofIn Huancavelica, exposure to Hg contamination may be intensified compared to other cinnabar refining areas like Almadén and Idrija. Huancavelica is the capital of the most impoverished department of Peru where its 42,000 inhabitants live with the toxic legacy of over 400 years of Hg contamination. Most residents are not aware of the high levels of Hg to which they may be exposed or the health effects of Hg contamination; those who are informed often lack the resources to alleviate the problem. Health effects may be exacerbated as more than 80% of homes in the city are constructed with adobe bricks fabricated on-site from contaminated soil [The results of this study have broad application to other areas in the world that use Hg for artisanal silver and gold refining and that construct buildings and homes from adobe brick, such as Madre de Dios and Puerto Maldonado, both also in Peru. It is important to identify the primary indoor sources of Hg and the distribution of contamination across the city with respect to historic sources, both to characterize exposure and potential health outcomes, as well as to develop and evaluate mitigation and remediation efforts.Samples of interior adobe brick, dirt floor, surface dust, and vapor were collected from 60 residences in Huancavelica, Peru in August 2010. These samples were collected from 15 homes in each of the four neighborhoods, Ascención, San Cristóbal, Santa Ana and Yananaco, shown in 2 areas of a hard, flat surface  with three moistened smear tabs . Each smear tab was folded with the sample side inward and stored in a separate specimen bag. Once transported, all samples were stored at 4 °C in the laboratory until analysis.In each home, samples were collected from the room where residents spent the most time. Triplicate samples were collected from the same room of each house. Adobe brick samples were obtained by scraping approximately 20 g of surface material to a depth of 2.5 cm from the interior adobe brick walls from three locations in the room. Dirt floor samples were collected by removing approximately 20 g of surface soil from the floor to a depth of 2.5 cm from three locations in the same room. In homes that had solid sealants on floors , floor samples were obtained from the dirt patio immediately outside of the home (less than 10% of homes in the study). Triplicate samples of the adobe brick walls and dirt floors were not pooled; each sample was stored individually in a specimen bag. Surface dust samples were obtained by wiping three separate 100 cmControl samples were collected in Ayacucho, a non-mining town in Peru, located about 150 km southeast of Huancavelica. Triplicate samples of exterior adobe brick scrapings were collected from three homes and duplicate samples of ambient soil were collected from five homes using the same sampling and storage methods used in Huancavelica.3) were set to LOD divided by the square root of 2 (resulting in 0.35 µg/m3 used for below LOD samples) when averaging the data.In situ mercury vapor measurements were recorded using a Jerome J405 Mercury Vapor Analyzer . The instrument was purged outside each home prior to collecting measurements indoors. Three measurements were recorded for each household. Measurements below the limit of detection  [Adobe brick, dirt floor, and surface dust wipe samples were digested by hot block extraction in 4:1 HCl:HNOod 1631)  using a Total Hg concentrations were tested for normality and were found to be non-normally distributed; therefore, the data were log-transformed prior to statistical analyses. Diagnostic tests for fit  indicated that there were no true outliers in the data set following log-transformation. Results of a three-way analysis of variance (ANOVA) suggested that differences in Hg concentrations for all sample types were driven by neighborhood and household variability. Statistically significant differences were not found between the triplicate samples; therefore, concentrations of Hg in all triplicate samples were averaged for each sample type within a home.Results shown in figures and tables are arithmetic means. Descriptive statistics were computed and boxplots were used to summarize Hg concentrations. The values represented in the boxplots are the first quartile, median, and third quartile. The whiskers are the minimum and maximum values and the diamonds represent the arithmetic mean.Exploratory analyses were performed where log-transformed total Hg concentrations in adobe brick, dirt floor, surface dust, and vapor samples were compared between neighborhoods using one-way ANOVA. Tukey’s Studentized Range (HSD) test was used to identify all pairwise comparisons that were statistically significantly different controlling for the experiment wise error rate. In addition, Pearson correlation coefficients, denoted by r, were calculated to estimate the dependence of sample types on a neighborhood level and Huancavelica-wide. No adjustment was made for multiple comparisons in testing the correlations between sample types. All statistical analyses were completed using SAS 9.2 software .th and 90th percentile) total Hg concentrations in adobe bricks and dirt floors were 85.5 µg/g  and 63.3 µg/g . In comparison, the total Hg concentrations in exterior adobe brick and ambient soil samples from the reference site, Ayacucho, ranged from 0.03 to 0.57 µg/g and 0.07 to 0.15 µg/g, respectively. The total Hg concentrations in surface dust wipes across the 60 households are shown in th and 90th percentile) total Hg concentrations in surface dust were 2.25 µg/g .The total Hg concentrations in the adobe bricks and dirt floors from 15 homes in each of the four neighborhoods are shown in 3.Results of the one-way ANOVA for adobe bricks, dirt floors, and surface dust suggest that a significant difference exists in the total Hg concentrations according to the neighborhood (p<0.0001). Results of the Tukey’s HSD test for adobe brick samples, dirt floor samples and for surface dust samples indicate that Ascensión (A), San Cristóbal (B), and Yananaco (D) were not significantly different from one another; however, Santa Ana (C) had significantly lower Hg concentrations in adobe bricks, dirt floors, and surface dust than the other three neighborhoods (p<0.0001).As shown in Pearson correlation coefficients were calculated for total Hg concentrations in adobe bricks, dirt floors, and surface dust on a neighborhood and Huancavelica-wide basis, as shown in Pearson correlation coefficients were also calculated city-wide after excluding Santa Ana . The Tukey’s HSD test for elemental Hg vapor confirms that there are not significant differences between neighborhoods at a significance level of α=0.05. Hg vapor is relatively mobile, so may move more readily than dust or dirt. This finding may also be a reflection of the large number of measurements that were below the limit of detection of the instrument (39 of 60 homes) and suggests that, while some individual households appear to have elevated elemental Hg vapor concentrations, these variations cannot be attributed to differences in characteristics of the four neighborhoods.The semi-arid climate of Huancavelica results in a very dusty ambient environment stemming from many unpaved roads and outdoor surfaces. Results of a previous study have identified ambient total Hg concentrations in surface soil in Huancavelica ranging between 1.75 and 698 µg/g . Results-1 day-1) [In the dusty, widely contaminated community of Huancavelica, it is important to recognize that ingestion of particle-bound Hg may affect health. A previous study demonstrated that children are especially vulnerable due to their hand-to-mouth activity . An 11 k1 day-1)  if the sThis study demonstrates that building materials used widely in the developing world, such as adobe bricks, may be a substantial source of residential Hg exposure in contaminated communities. In Huancavelica, Hg in adobe brick homes is a result of legacy contamination that primarily occurred hundreds of years ago during the Spanish colonial period. Understanding exposure to Hg from such building materials, particularly adobe bricks made in the community, has broader relevance to evaluating risks in communities that currently produce or refine silver and gold, such as Madre de Dios and Puerto Maldonado, both also in Peru. In these communities, gold shops and other buildings used for refining are often constructed from adobe brick that has the potential to act as a sink for elemental Hg vapor released from current refining processes and may be a source of Hg exposure in the future.The purpose of this study was to determine the current levels of mercury to which residents of adobe brick homes in Huancavelica are exposed. Results from this study suggest that total Hg concentrations in residential samples are similar to or greater than ambient soil samples from previous studies conducted in Huancavelica, and that widespread residential exposure to Hg is presently occurring there. Total Hg concentrations ranged from 8.00 to 1070 µg/g in adobe bricks, 3.06 to 926 µg/g in dirt floors, and 0.02 to 9.69 µg/wipe in surface dust. These results indicate that total Hg concentrations vary by neighborhood, with Santa Ana (C) having consistently lower Hg concentrations than the other three neighborhoods. This finding may suggest that the present-day spatial distribution of total Hg contamination in residences is influenced by the location of historical cinnabar smelters.These results also point to a strong relationship between total Hg concentrations in adobe bricks and dirt floors. This result confirms that residents of Huancavelica are using materials on their property to construct adobe bricks for their homes, rather than purchasing manufactured adobe bricks. Additionally, the strong relationship between total Hg concentrations in surface dust and adobe bricks and surface dust and dirt floors suggests that adobe bricks and dirt floors are the primary source of Hg in indoor dust, rather than an outdoor source.3. While individual homes vary in Hg vapor concentrations, no significant difference in these concentrations was found between neighborhoods.Elemental Hg vapor concentrations from 39 of the 60 residences in Huancavelica were below the limit of detection. For residences with measurements above the limit of detection, concentrations of elemental Hg vapor ranged up to 5.1 µg/mThe results of this study demonstrate that adobe bricks and other building materials used in developing communities may be a substantial source of residential Hg exposure, particularly in communities with past or present mercury production and where silver or gold refining utilizes Hg for amalgamation. Adobe bricks are used to construct buildings around the world, from the southwest United States to South America to Africa. As the first study of adobe brick contamination anywhere in the world, the present study draws attention to a previously unidentified source of potential exposure to Hg and possibly to other contaminants. To better understand potential health effects from exposure, it is important to quantify levels of contaminants, identify the chemical species and bioaccessibility of contaminants in the building materials, and to identify possible mitigation strategies to reduce exposure within these homes.


Extreme swing of phosphor from severe hyperphosphatemia to severe hypophosphatemia in a patient with blast crisis of myeloid origin was the result of imbalance between massive apoptosis of leukemic cells in the context of spontaneous tumor lysis syndrome and massive production of leukemic cells with only 1% of blast in peripheral blood. The mutated p53 protein suggested acting as oncogene in the presented case and possibly affecting phosphor status. Severe symptomatic hypophsopahatemia was reported in a small number of case reports in patients with acute myelomonocytic leukemia with extLeukemic blast cells of myeloid origin fail to show inhibition of glycolysis by oxygen and causSevere hypophosphatemia may adversely affect major organ systems including severe generalized muscle weakness as observed in our patient. Reduction in the erythrocyte content of 2,3-diphosphoglycerate may impair release of oxygen resulting in hypoxia. On the An 80-year-old white Ashkenazi Jewish woman presented at hematology out-patient clinic with persistent leukocytosis for the last 10 months. A peripheral blood count revealed white-cell count (WBC) of 36,000/μL with 58% neutrophils, 14% lymphocytes, 4% monocytes, 1% blasts, 2% metamyelocytes, 6% myelocytes and 10% segments. The hemoglobin was 12gr/dL, platelet counts 476,000/μL and blood chemistry was unremarkable. Her physical examination was normal as well as whole body computerized tomography scanning (CT). Bone marrow (BM) aspiration at that time was hyperplastic, enriched with white cells with normal maturation and no blasts. Megakaryocytes had dysplastic features with single nucleous without lobulations. The BM findings were compatible with a myeloproliferative disorder with some dysplastic changes. Cytogenetic analysis revealed 47XX in 75% of the cells with trisomy 8 (+8) detected also by fluorescent in situ hybridization. Janus Kinase 2 gene V617F mutation and Bcr/Abl fusion gene were not detected.neg) chronic myelogenous leukemia (CML) with+8. Of note that Bcr/Abl negative CML is clinically distinct from Bcr/Abl positive CML, myelodysplastic syndrome and chronic myelomonocytic leukemia[At that point, the patient's diagnosis was Philadelphia negative  Doppler revealed splenomegaly with several peripheral hypoechogenic areas compatible with spleen infarcts .A rise in creatinine at the time of presentation from 0.65 to 1.9 mg/dL, uric acid up to 13.5 mg/dL, phosphate up to 5.9 with calcium 9.6 mg/dL, LDH 403 IU/L and alkaline phosphatase 57 IU/L support a diagnosis of acute urate nephropathy. The latter is the outcome of precipitation of uric acid in distal tubules and collecting ducts because of increased filtered load of urate due to excessive production of purines as occurs in tumor lysis syndrome (TLS), but it The patient's creatinine continued to rise, and the phosphor reached a value of 9.6 mg/dL. At that point the patient was started on hemodialysis and received 5 treatments, before further deterioration occurred. The patient was also treated with aluminum hydroxide (950 mg tid).. The use of oral sodium phosphate solution preparations for bowel preparation before colonoscopy could cause acute renal failure because of high phosphorus content that potentially can cause chronic kidney damage[She had hyperuricemia and severe hyperphosphatemia when neither chemotherapy nor biological agents or steroids were administered. Furthermore, there was no evidence of a bulky disease. Patient's counts remained stable. Although the patient's ultrasound study revealed a kidney stone, there was no evidence of any ureteral obstruction to explain the onset of renal failurey damage but she At this point, monocytosis in peripheral blood counts became significant and anemia was evident. Histopathology of the BM showed hypercellularity with sheets of immature-looking cells comprising at least 90% of the BM spaces. The red cell line and the megakaryocytes were inconspicuous. Extensive apoptosis of the cells with phagocytosis and a “starry sky” appearance, reminding Burkitt’s-like lymphoma was a prominent feature Figure, a+b. OnConstitutional activation of the MYC proto-oncogene resulting from a t has been demonstrated in approximately 80% of patients with Burkitt lymphoma, but only in two cases with acute myeloid leukemia (AML). Search The acute myelomonocytic leukemia that developed in our patient might explain the relative hypokalemia in spite of tumor lysis and renal failure. Hypokalemia is known to develop in patients with acute monoblastic leukemia and significantly correlates with hyperleukocytosis , increased blood and urine lysozyme. The lack of hyperkalemia initially raised questions regarding the diagnosis of TLS and prompted the aforementioned investigation for other etiologies of renal failure.In a three week period the patient developed weakness and fatigue which led to diagnosis of severe hypophosphatemia <0.5 mg% Figurec while pSeveral potential causes that could contribute to hypophosphatemia in our patient were ruled out, such as nutritional causes, antacids ingestion, sepsis or diabetic ketoacidosis. Urine output was low, and parathyroid hormone and 1,25(OH)2 dihydroxyvitamin D levels were within normal limits, thereby excluding overt hyperparathyroidism and oncogenic hypophosphatemic osteomalacia even though, urine phosphor levels were not measured.In addition the patient was also never treated with chemotherapeutic agents beside hydroxyurea or thyrosine kinase inhibitor such as imatinib, known to cause mild hypophosphatemia (around 2 mg%), possibly through inhibition of bone turnover, which in turn, triggered a secondary hyperparathyroidism in an attempt to maintain calcium homeostasis. AnotherIn our patient the shift of phosphor from severe hyperphosphatemia of 9.6 mg/dL to <0.5 mg/dL phosphor six days later even when WBC counts solely tripled in number Figurec and anaThe presence of massive apoptosis was noted aside to tumor genesis in the patient's BM Figure and it ineg CML. Hyperphosphatemia was due to massive apoptosis of leukemic cells in the context of TLS and then inverted to severe hypophosphatemia despite end stage renal failure when tumor genesis prevailed and resulted in the patient's death with peripheral WBC of 132,000 /uL, of those only 1% were blasts. Since neither the occurrence nor the severity of this hyper-hypophosphatemic shift can be predicted from the patient’s baseline disease, and therefore cannot be treated ahead of time, it is very important to be aware of this possible complication, carefully follow-up serum phosphor and provide immediate treatment when even mild changed occur.The presented case demonstrates the extreme shift from severe hyperphsosphatemia to severe hypophosphatemia during blast crisis of a patient with PhInstitutional Helsinki approval, no. 9829-12-SMC.The authors declare that they have no competing interest.OS – designed and wrote the manuscript. EH – treated patients, designed and wrote the manuscript. PB – was involved in writing the manuscript. CA – performed the immunohistochemical stains of all pathology sections. LT – performed the FISH analysis. AK – treated the patients and analyzed the results. TT – performed the PCR work. YK – performed the ultrasound study and analyzed the results. SA – performed the CT scan and analyzed the results. NA – involved in PCR and FISH tests and analysed the results. EG designed the study, participated in the sequence alignment and in writing the manuscript. RS – performed all pathology sections, analysed the data and was involved in writing the manuscript. All authors read and approved the final manuscript.


Illegal hunting is one of the major threats to vertebrate populations in tropical regions. This unsustainable practice has serious consequences not only for the target populations, but also for the dynamics and structure of tropical ecosystems. Generally, in cases of suspected illegal hunting, the only evidence available is pieces of meat, skin or bone. In these cases, species identification can only be reliably determined using molecular technologies. Here, we reported an investigative study of three cases of suspected wildlife poaching in which molecular biology techniques were employed to identify the hunted species from remains of meat.b (cyt-b) and cytochrome oxidase subunit I (COI) molecular markers, the suspected illegal poaching was confirmed by the identification of three wild species, capybara (Hydrochoerus hydrochaeris), Chaco Chachalaca  and Pampas deer (Ozotoceros bezoarticus). In Brazil, hunting is a criminal offense, and based on this evidence, the defendants were found guilty and punished with fines; they may still be sentenced to prison for a period of 6 to 12 months.By applying cytochrome The genetic analysis used in this investigative study was suitable to diagnose the species killed and solve these criminal investigations. Molecular forensic techniques can therefore provide an important tool that enables local law enforcement agencies to apprehend illegal poachers. It is estimated that illegal hunting kills millions of vertebrates per year in tropical rainforests . A numbeEven when hunters are captured, the precise identification of bush meat is often questionable. Moreover, the remains of meat, fur, skin and bone are the only evidence recovered from the crime scene. In such cases, species identification can only be resolved with molecular tools -5.b[b was amplified using the universal primers L14841 (5’AAAAAGCTTCCATCCAACATCTCAGCATGAAA3’) [2, 2.5 U Taq DNA polymerase (Invitrogen) and 8 pmol of each primer. The PCR conditions were initiated by a denaturation at 94°C for 5 min, followed by 40 cycles at 94°C/30 s, 50°C/45 s, 72°C/45 s and a final extension at 72°C for 10 min. PCR contamination controls were performed. Each sample was sequenced twice on both strands using the Big Dye v3.1 Terminator Cycle Sequencing Kit (Applied Biosystems) in an ABI 3730 DNA Analyzer (Applied Biosystems). Here, we reported an investigative study of three suspected offenses of wildlife poaching. In July 2010, based on suspected illegal hunting, a wildlife inspector of the Brazilian Environmental Agency (IBAMA) seized and sent us biltong samples of a mammal species (MAM1)   and H159TGAAA3’) . The ampThe sequences were manually edited using the software BioEdit Sequence Alignment Editor 7.0.9.0 . This soThe sequences of the seized samples were compared against those of species that were likely to be hunted or consumed at the seizure region Table , which wb sequencing of BIRD and mammal species (MAM1 and MAM2) produced readable sequences of approximately 550 and 1100 bp, respectively, which did not present insertions, deletions or stop codons. BLAST analysis of cyt-b indicated that the MAM1 meat sample was highly similar (99%) to the capybara (Hydrochoerus hydrochaeris) reference sequence, the MAM2 was similar (98%) to Pampas deer (Ozotoceros bezoarticus), and the BIRD sample was similar (98%) to Chaco chachalaca .The cyt-b reference sequence available in GenBank. MAM2 differed by 0.32% from the five Pampas deer specimens. The genetic divergence between the BIRD sample and the Chaco chachalaca was 0.9%. These genetic distance values were as low as those for the intraspecific genetic distances obtained for the species represented for more than one specimen reference sequence , and lower than the cyt b-genetic distances (<2%) typical of population and intraspecific variation observed in mammal and bird species [The comparative analysis using reference sequences of species that were likely to be hunted or consumed at the seizure region Table  confirme species ,18. The b, species identification was not possible using COI. The amplification of COI in BIRD, which could be solved using primers designed for bird species, was not successful. For the mammal species, the BLAST analysis did not match a reference sequence based in our threshold (≥98%). At the time of the manuscript writing, Genbank and BOLD did not contain COI reference sequences for capybara or for Pampas deer. Although the COI gene is considered the DNA barcoding gene [b gene for species identifications [Contrary to results with cyt-ing gene , referenications ,19 sinceications ,21.The three species killed are widely reported in hunting studies ,22,23. Tb molecular marker was suitable to diagnose the species killed and solve these criminal investigations. The suspected poaching in all three cases was confirmed with the identification of three wild species, capybara, Pampas deer and Chaco Chachalaca. In Brazil, hunting is a criminal offense, and based on the results of our molecular analysis, the defendants were found guilty and punished with fines; they may still be sentenced to prison for a period of 6 to 12 months.In this study the cyt-c oxidase subunit I; cyt-b: cytochrome b; BOLD: Barcode of Life Database; mtDNA: mitochondrial DNA; IBAMA: Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis; MAM1: sample meat of the mammal seized in case 1; BIRD: sample meat of the bird species seized in case 2; MAM2: sample meat of the mammal seized in case 2; PCR: polymerase chain reaction.COI: cytochrome The authors declare that they have no competing interests.AS coordinated and participated in the study design, carried out procedures in the laboratory, and drafted the manuscript. PMT, MSTG, CSC, CP and TGG helped in the DNA extractions, PCRs, data analysis and manuscript drafting. WAMP and FLN participated in the study design and helped to draft the manuscript. MG participated of the study conception and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.


Satellite cells, normally quiescent underneath the myofibre basal lamina, are skeletal muscle stem cells responsible for postnatal muscle growth, repair and regeneration. Since their scarcity and small size have limited study on transverse muscle sections, techniques to isolate individual myofibres, bearing their attendant satellite cells, were developed. Studies on mouse myofibres have generated much information on satellite cells, but the limited availability and small size of human muscle biopsies have hampered equivalent studies of satellite cells on human myofibres. Here, we identified satellite cells on fragments of human and mouse myofibres, using a method applicable to small muscle biopsies. Satellite cells are the principal stem cells of skeletal muscle, responsible for its homeostasis, maintenance, growth and regeneration through adulthood Quiescent satellite cells on isolated mouse muscle fibres express Pax7 Unfortunately, due to the difficulty in obtaining human muscle in a size and state adequate to allow the preparation of viable fibres, there is a gap in our knowledge of human, compared to mouse, satellite cells  Act 1986. extensor digitorum brevis (EDB) muscle of a DMD adolescent patient  were collected and fixed in 4% paraformaldehyde  at room temperature for 15 minutes. After sequential washes with phosphate buffer saline , the sample was embedded at 4◦ C, first in 30% glycerol/PBS (Sigma) overnight, and then sequentially in 50%, 80% glycerol/PBS and finally absolute glycerol until the sample sank to the bottom of the vial. The sample was then kept in glycerol at 4◦  C until dissection of fibre fragments. Biopsies of paraspinal muscle of a healthy adolescent patient  and of Under a stereomicroscope (Leica), fine forceps were used to dissect out fragments of fibres and exclude any other tissues, e.g. connective tissue.tibialis anterior (TA) muscles from 2 transgenic 3F-nLacZ-2E mice , in which myonuclei, but not satellite cells express the nLacZ reporter gene As a control, ◦C with the following primary antibodies: mouse monoclonal anti-Pax7 , rabbit polyclonal anti-laminin (Sigma), mouse monoclonal lamin A/C (Vector Laboratories), rabbit polyclonal caveolin-1 , mouse monoclonal M-cadherin Dissected fibre fragments were washed in PBS at room temperature, permeabilized with 0.5% Triton X-100 (Sigma) for 6 minutes, blocked for 30 minutes with 10% goat serum, and incubated overnight at 4Fluorescence detection, bright-field microscopy and image capturing were done using a Zeiss Axiophot microscope (Carl Zeiss), and Metamorph software (Metamorph Productions). Fibre length was calculated by using SigmaScan software in order to allow satellite cell quantification per 100µm fibre length. All nuclei (both myonuclei and satellite cell nuclei) on isolated fibres harvested from normal human paraspinal muscle biopsies expressed, as expected, human-specific lamin A/C in their nuclear membrane Pax7 antibody clearly marked both human  and mouse  satellite cells; co-staining with laminin antibody confirmed that satellite cells were retained under the basal lamina of human fibres following the preparation procedure . The mean number of human Pax7+ve satellite cells per 100µm length of fibre was 0.9 (±0.05 SEM) . Considering that the paraspinal muscle is composed of fascicles of different lengths, As Pax7 characterizes the majority of satellite cells in mouse We were not able to prepare fibre fragments derived from DMD muscles using the method of myofibre isolation that we successfully applied to normal human muscle . The fragility of fibres from DMD muscle caused them to hyper-contract before the isolation and to break during the isolation process. Furthermore, the interference of connective tissue prevented single fibre isolation and only rarely could bundles of fibres be isolated for immunostaining . Although our technique did not allow a clear identification of human satellite cells, Pax7 is expressed by a few nuclei in this preparation of DMD muscle .Studies on human single myofibres can be performed when muscles can be harvested from tendon to tendon, which is possible for very small muscles like abdominal, intercostals and gluteux maximus To date, most of our knowledge about human satellite cells derives from immunostaining muscle cryosections Mouse studies have shown that satellite cells express a mesenchymal marker, the integrin CD 29 We cannot exclude the possibility that satellite cells in different human muscles are characterized by different markers and future investigations are needed in order to establish whether satellite cells in human muscles are heterogeneous, as has been shown in mouse  Although the technique that we used was not applicable to DMD muscles, we suspect that, were a DMD biopsy available in the early stage of the pathology, when fibres are not so fragile, we would be able to obtain fibre fragments for study. Moreover, for disorders such as polymyositis, DMD and myotonic dystrophy, where satellite cell number is known to be altered In summary, we have shown that a clear qualitative and quantitative identification of satellite cells can be performed by analysis of human muscle fibre fragments. Further investigations are needed in order to explore the cell biology of human satellite cells. Luisa Boldrin, UCL, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom.We would like to thank the Biobank of the MRC Centre for Neuromuscular Diseases that provides human muscle cells and tissue for research. We would also like to thank Dr Geraldine Edge, Department of Anaesthetics, Stanmore Hospital, London and Dr Jan Lehovsky, Department of Orthopaedics, Stanmore Hospital, London for providing us with human muscle samples. The Pax7 antibody developed by A. Kawakami was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.LB is founded by the Muscular Dystrophy Campaign (MDC) and JM holds a Wellcome Trust University award.The authors have declared that no competing interests exist.


Fifty healthy subjects (23 men and 27 women) who intended tofast Ramadan were recruited. Urine and serum sampling and anthropometric and dietary assessments were conducted one week before Ramadan (T0), at the end of the third week of Ramadan (T1), and one month after Ramadan (T2). Biochemical measurements included urinary 15FIP, creatinine, and hematological indices. Results revealed that the urinary level of 15FIP measured at T0 was normal, while they showed a significantly (P < 0.05) higher level when measured at T1 concomitant with a significant (P < 0.05) increase in the body weight and total body fat percent. In conclusion, results suggest that increased body weight is associated with increased lipid peroxidation and oxidative stress, and the impact of RIF on oxidative stress is mediated by the changes in body weight at the end of the month.Fasting and caloric restriction have been associated with reduced incidence of chronic diseases and cancers. These effects have been attributed to reduced oxidative stress. Since Ramadan intermittent fasting (RIF) has been associated with reduced caloric intake, it was hypothesized that RIF would alleviate oxidative stress in healthy volunteers. The study was designed to elucidate the impact of RIF on oxidative stress measured by 15-F It is r2-like compounds, termed isoprostanes, provided a major advance in assessing oxidative stress and lipid peroxidation [2-Isoprostanes are considered to be sensitive and specific markers for oxidative stress in experimental animals and human diseases [2t-isoprostane (15FIP), one class of isoprostanes, has been recognized as a specific, quantitative, and chemically stable marker of oxidative stress. It can be measured in urine and peripheral blood [Prostaglandin Fxidation . F2-Isopdiseases . 15-F2t-al blood . Measureal blood , 8. It hal blood . For insal blood .Intermittent fasting and caloric restriction, or alternate-day feeding, had been shown to increase resistance to toxicity and stress and to prolong maximum life span in experimental animals and humans , 12. A sBased on the established relationship between body weight, caloric intake, and oxidative stress , we hypoThis study was carried out over three time points: one week before Ramadan (T0), after the end of the third week of Ramadan fasting (on the 22nd day of Ramadan month) (T1), and one month after the end of Ramadan fasting month (30 days from the cessation of Ramadan month) (T2). Before fasting (T0) represents the time point before practicing fasting, during which the body presents in its normal routine conditions for the majority of the year time. However, T1 represents the time point after practicing RIF for three consecutive weeks and is expected to show the impacts of fasting, while T2 is the time point after cessation of RIF by one month and shows to which extent the effect of RIF would extend and become long standing after the end of Ramadan month. The range of fasting hours during the month was about 14-15 hours a day. The female subjects did not fast during their menstrual period as Islamic rules do not permit fasting during the menstrual period. Accordingly, the fasting period in female subjects ranged from 25 to 23 days, while for men it was 30 days.To avoid different confounding socioeconomic and cultural disparities and to maintain sample homogeneity, the enrolled subjects for this cross-sectional study were recruited on voluntary basis from the surrounding community of medium-to-low economic status . The inclusion criteria necessitated that subjects were fasting at least 21 days of Ramadan month and were not taking any medication or receiving any medical treatment immediately before or during the study. Exclusion criteria included excluding subjects who have diabetes, hypertension, asthma, or metabolic disorders and who take any supplements including antioxidant vitamins. Female subjects were not pregnant, lactating or receiving contraceptive medications.Fifty healthy subjects  were eligible for this study and voluntary completed the study protocol upon a written informed consent. The study protocol was approved by Petra University Committee on Human Research Ethics. A questionnaire was administered, in which medical history and socioeconomic information were obtained through a personal interview with each subject by a well-trained staff member; the study questionnaire was tested for validity, reliability, and reproducibility before conducting the study.Venous blood and urine samples were collected from the enrolled volunteers during the three time points. Each subject served as his own control by comparing his/her values before Ramadan (T0) with those during (T1) and after Ramadan (T2). Blood and urine sampling was conducted in the private medical laboratory once a time at each time point. Blood and urine sampling were performed early  during the period of investigation (between 10 am and 4 pm) at each time point. To narrow the time interval for blood and urine sampling and thus to eliminate the effect of day-time on blood and urine variables, volunteers were asked to come early (at 10 am), and blood was first drawn from most of the volunteers according to their arrivals to the laboratory, and then the less time-sensitive assessments (anthropometric and dietary) were subsequently performed during the rest of investigation period. The obtained sera were separated within an hour by centrifuging the blood samples at room temperature for 5 minutes at 4000 rpm . Aliquots of the blood samples were directly analyzed for complete blood count cells using Auto Hematology Analyzer . Urine samples were immediately frozen in special sampling containers at −80°C.2) was calculated, where volunteers with BMI <25 Kg/m2 were considered having normal weight and those with BMI ≥25 were considered having overweight [During each of the three visits, the anthropometric measurements including body weight and height  and body fat percent  were performed by a well-trained staff member. Body weight was measured at the same time to the nearest 0.1 kg and body height to the nearest 0.01 m. Body mass index . Urinary creatinine was measured by colorimetric Jaffe reaction by using Spectroscan 80 D UV-Visible Spectrophotometers (UK) [Urinary 15FIP levels were analyzed using competitive enzyme-linked immunosorbent assay (ELISA) kits form US Biological  according to the manufacturer's directions and using Biotek Micro plate reader (USA). Samples were analyzed in triplicates. According to the manufacturer, the lower limit of reliable detection is 0.2 ng/mL. A quality control sample consisting of urine pooled from five controls was analyzed with each batch of test samples. The coefficient of variation was <20% (ers (UK) .Blood samples were analyzed directly for hemoglobin (Hb), hematocrit (Hct), mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) using Mindray BC-3000 Auto Hematology Analyzer (China). Serum samples were stored at −80°C for the analysis of total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL).t test was used to test oxidative stress biomarker with gender, BMI, and age differences among volunteers and to compare baseline volunteer characteristics based on gender differences. The paired t test was used to compare levels of isoprostane, HDL, LDL, Hb, Hct, MCV, MCH, and MCHC. The data were analyzed with SPSS version 16.0.1. . Statistical significance was defined as P < 0.05.Data are presented as means ± standard deviation (SD) unless stated elsewhere. Continuous variables are presented as means ± SD and categorical variables are presented as frequencies and percentages. Unpaired Fifty healthy volunteers were included in this study, with 23 men (46%) with a mean age of 31.8 ± 9.0 years and 27 women (54%) with a mean age of 36.0 ± 10.0 years. The baseline characteristics of the subjects are presented in P < 0.005) higher among males as compared to female subjects. Other hematological markers , lipid profile  and urinary oxidative stress marker (15FIP) were not significantly different. The hematological makers (Hb and Hct) as well as urine creatinine were significantly (As shown in P < 0.05) when compared to T0 values. The levels of Hb, Hct, MCV at T2, however, showed significant reductions as compared to T0.P = 0.411). At T2 urinary 15FIP ranged from 0.2 to 10.84 ng/mg creatinine, with a mean of 3.0 ng/mg creatinine, which was significantly higher (P = 0.029) in comparison to baseline levels.At T0 urinary levels of 15FIP varied from 0.27 to 7.28 ng/mg creatinine with a mean of 2.33 ± 1.45 ng/mg creatinine. No significant change was observed during T1 compared to baseline values (P = 0.006) at T2 as compared to T0, with a 33% increment over the baseline level, and persisted for more than one month after the last test during Ramadan.Changes in serum lipid profile are shown in Interestingly, volunteers who lost weight during RIF showed a decrease in the level of 15FIP during Ramadan, with a reduction value of about 11% in reference to the baseline value. However, those who gained or maintained body weight during RIF exhibited an increase in urinary 15FIP levels during Ramadan, with an increment with about 19% . NonetheP = 0.026) in their urinary 15FIP levels at T2 as compared to T1, while those who lost or gained body weights did not exhibit significant changes in their 15FIP levels (The majority of subjects (66%) who maintained their body weights showed a significant increase . It is also important to note that the high variation in the level of urinary 15FIP among the study volunteers was also reported by other published studies , which iGreater oxidative stress in men has been reported by many studies , 38. Howα (8-iso-PGF2α) than nonobese ones. They also observed a statistically significant reduction of urinary 8-iso-PGF2α in obese subjects who successfully achieved weight loss.Although not significantly correlated, our study demonstrated a positive association between levels of 15FIP and BMI, which comes in agreement with the findings of several researchers , 42–45 iIn order to explain this association, urinary 15FIP could be viewed as a reflection of compensatory metabolic changes that act to oppose further weight changes . This coFat oxidation plays an essential role in the change of body weight. For example, the physiological response to weight gain is an increase in fat oxidation, while weight loss is associated with a decrease in fat oxidation level , 47. On Based on these studies and the metabolic adaptation theory, it can be concluded that the decrease in levels of 15FIP during RIF among individuals was due to the significant decrease in body weight and fat mass%. However, after Ramadan, individuals maintained a stable weight despite the significant increase in fat mass%. This was accomplished by activation of the adaptive increase in fat oxidation with corresponding significant increase in 15FIP in order to oppose weight gain.Although previous studies did not correlate the change in weight with isoprostane level, reviewing their results indicates that they agree with the adaptation theory. For example the studies that showed an increase in level of oxidative stress during or after Ramadan also showed increased or stable weight , 16, whiThe main limitations of the study include the small sample size, which implies that the effect of RIF on 15FIP might be confined to the selected participants. The lack of a control nonfasting group deprived the study from the ability to compare the effect of fasting to nonfasting, and thus making it difficult to attribute the changes in oxidative stress to the absolute effect of fasting. In these studies, the control group of nonfasting subjects is difficult to be obtained, because Ramadan fasting is a religious worship; therefore nonfasting people are very scarce and could not be found due to social, psychosocial and religious limitations. In such cases, the baseline values are considered as reference control values for which different changes are compared. Further, the samples before and after Ramadan were nonfasting which limit the ability to compare pre- and postlipid profiles with lipid profile during Ramadan. Another limitation of this study was the use of ELISA rather than GC/MS, the golden standard for isoprostane analysis, due to limitations in time and cost. Furthermore, this study represents a healthy population and it is not representative for diabetic, hypertensive, or dyslipidemic patients.In conclusion, this study indicated that Ramadan fasting activates metabolic changes such as increasing fat oxidation, in order for the body to adapt to the alteration in feeding pattern during the month of Ramadan, and to maintain the body composition within the physiological range. The main findings of this study are that the body weight and BMI remained unchanged after Ramadan, with the significant increase in fat mass%, which may be strongly related to the marked variation in qualitative food intake. These changes in eating patterns during Ramadan may be responsible also for the decrease in LDL and the increase in HDL levels and may contribute to lowering the risks of coronary heart disease. Finally 15FIP was found to be positively associated with obesity and to increase significantly after Ramadan which was parallel to the increase in fat mass%.


The latter scale was dealt with by comparing the data here obtained with that of birds of prey from the same locality and with those published previously on owls and birds of prey from Galicia (Spain). A total of 19 helminth taxa were found in owls from Calabria. Statistical comparison showed only marginal differences between scops owls (Otus scops) and little owls (Athene noctua) and tawny owls . It would indicate that all owl species are exposed to a common pool of ‘owl generalist’ helminth taxa, with quantitative differences being determined by differences in diet within a range of prey relatively narrow. In contrast, birds of prey from the same region exhibited strong differences because they feed on different and wider spectra of prey. In Calabria, owls can be separated as a whole from birds of prey with regard to the structure of their helminth communities while in Galicia helminths of owls represent a subset of those of birds of prey. This difference is related to the occurrence in Calabria, but not Galicia, of a pool of ‘owl specialist’ species. The wide geographical occurrence of these taxa suggest that local conditions may determine fundamental differences in the composition of local communities. Finally, in both Calabria and Galicia, helminth communities from owls were species-poor compared to those from sympatric birds of prey. However, birds of prey appear to share a greater pool of specific helmith taxa derived from cospeciation processes, and a greater potential exchange of parasites between them than with owls because of phylogenetic closeness.We compared the helminth communities of 5 owl species from Calabria  and evaluated the effect of phylogenetic and ecological factors on community structure. Two host taxonomic scales were considered, i.e., owl species, and owls  In the last 30 years a number of papers on helminths of European owls (Strigiformes) have been published Sanmartín et al. In southern Italy, Strigiformes include at least 7 species, displaying a wide variety of ecological and life-history patterns, including biological, environmental and dietary requirements Here we studied the helminth community of 5 owl species in Italy investigating the factors which may influence their community structure and comparing patterns of diversity and composition with those obtained among birds of prey from the same geographical area Athene noctua, 31 tawny owls Strix aluco, 41 barn owls Tyto alba, 10 long-eared owls Asio otus, and 10 scops owls Otus scops were all from the Calabria region. All owls included in the present study had a clinical course less than 7 days to minimize parasite losses; and no anthelmintic treatments were used in these birds A total of 122 owls that died between January 2004 and December 2011 at the Wildlife Rescue Centre in Rende, Cosenza , in southern Italy, were examined for helminth parasites. The birds belonging to 5 species of strigiforms including 30 little owls All owl individuals were weighed prior to parasitological analysis. During necropsy examination, the trachea, lungs, air sacs, kidneys, spleen, liver, gallbladder, and the whole digestive tract of birds including oesophagus, stomach and intestines  were examined and helminths were collected, counted and identified following the techniques by Krone Trichinella spp. by artificial pepsin digestion The whole pectoral muscles (depending by owl species approximately from 2 to 15 grams) and an aliquot of leg muscles (approximately from 2 to 5 grams) from each bird were examined for Total abundance, species richness, Brillouin’s index of diversity, and the Berger-Parker dominance index were used as overall descriptors of infracommunities. Total abundance is the number of individuals of all helminth species, and species richness the number of helminth species, harbored by each individual owl. The 95% confidence interval (CI) for prevalence was calculated with Sterne’s exact method For each owl species, differences of total abundance, species richness, Brillouin’s index, and Berger-Parker index between genders were compared with Mann–Whitney U- tests, respectively. These parameters were also compared between owl species with Kruskal-Wallis tests using post hoc comparisons Buteo buteo; 20 European sparrow hawks Accipiter nisus; 21 western honey buzzards Pernis apivorus; 17 marsh harriers Circus aeruginosus; 25 common kestrels Falco tinnunculus, and 17 peregrine falcons Falco peregrinusHelminth data from the owl species analyzed in this study were compared with those obtained from 6 species of birds of prey examined by us in the same recovery centre between January 2000 and December 2010, i.e., 35 Eurasian buzzards Accipiter gentilis; 12 common kestrels; 5 Montagu's harrier Circus pygargus; 3 western honey buzzards, 3 Eurasian hobbies Falco subbuteo; 2 peregrine falcons; 1 red kite Milvus milvus and 1 black kite Milvus migrans) and 4 owl species  that were collected in four recovery centers from 1991 to 1996. This dataset provide a unique opportunity to replicate the above comparisons between owls and birds of prey in another geographical region.In Galicia, Sanmartín et al. To examine compositional differences between owls and birds of prey, prevalence values for each helminth species scaled to unity were used to obtain a matrix of similarities between raptor species using the Bray-Curtis coefficient. The resulting matrix was used to perform a group average hierarchical cluster of owl and birds of prey species To interpret differences in overall composition of helminth faunas of owls compared to birds of prey we derived a measure of specificity for each helminth species found in the samples of owls from both Calabria and Galicia based on records on each species worldwide. For each helminth species we checked all references for synonymies and looked for taxonomic updates, assuming that original identifications were correct. Then, we established the following categories: a helminth species was considered ‘specialist’ if it had been reported in single host species; ‘owl specialist’ if it had been reported mainly, or only, in owls (Strigiformes); ‘birds of prey specialist’, if it had been reported mainly, or only, in birds of prey ; ‘raptor generalist’, if it had extensively been reported in both owls and birds of prey, and ‘bird generalists’, if it also occurred extensively in birds other than raptors. The use of host-parasite lists may suffer from well-known problems of representativity (10-transformed) as a covariate that correct for differences in sampling effort At the component community level , we compared differences of species richness between owls and birds of prey from Calabria with an ANCOVA, considering the number of helminth taxa in each host species as the dependent variable, ‘raptor group’ as a fixed factor and ‘sample size’  to 12 (in the tawny owl)  and 758 helminth individuals were found in the total sample of owls . All helwny owl) . No helmops owls . Four heops owls . However species , and pre species .2 = 8.64, 4 d.f., p = 0.071), mean total abundance  and Brillouin’s diversity index  of helminths between owl species. The Berger-Parker index did not differ also between owl species  and the most abundant helminth species in infracommunities accounted for a very high proportion of total helminth abundance , whereas S. affinis was dominant in scops owls and Synhimantus laticeps in long-eared owls (shared with E. excisiformis)  and 38.9% (scops owls vs. tawny owls) of mean dissimilarity. We found no statistically significant effects of host weight  or sex  on any 4 infracommunity parameters of The proportion of individual hosts that were infected in the sample differed significantly among host species , ranging from 2 out of 10 (10%) in long-eared owls to 7 out of 10 (70%) in scops owls . Infracoiformis) . Mean siiformis)  and, theiformis) . StatistThe group-average hierarchical cluster of raptor species based on prevalence of their helminth fauna is shown in P. candelabria, was found only in a single species  = 6.124, p = 0.038, Galicia: F = 26.532, p<0.001); differences between raptor groups were also significant in both regions  = 8.568, p = 0.019, Galicia: F = 8.602, p = 0.014), with birds of prey having higher values of species richness in their helminth communities  = 0.001, p = 0.995, Galicia: F = 2.694, p = 0.162), although sample size in Galicia was very low  = 5.518, p = 0.045). In Galicia, this pattern was less marked  = 1.771, p = 0.241).At the component community level, the ANCOVA for species richness indicated that the interaction between host sample size and raptor group was significant neither in Calabria nor in Galicia and, therefore, interactions were removed from the models. Host sample size had an overall significant positive effect on species richness  = 11.861, p = 0.011). Apparently, host sample size influenced MMI only in birds of prey  = 5.445, p = 0.048), and birds of prey tended to exhibit higher values of MMI. In Galicia, none of the predictors was significant (results not shown), and MMI was similar between owls and birds of prey  also offered contrasting pattern between geographical regions. In Calabria, the interaction between host sample size and raptor group was significant , species shared also with birds of prey, and generalist parasites common to other birds. It is not possible to determine whether all owl species are equally suitable hosts for each of these parasites, but patterns of specificity, and the absence of significant subdivisions of owls in the cluster analysis, strongly suggest that there are not fundamental barriers for exchange of helminth taxa among owl species. Therefore, factors driving the contact between owls and parasites Ecological determinants are important when considering the similarities and differences of helminth communities between owls in Calabria. Three types of helminth species can be recognized, namely, species typical from owls  and S. affinis (higher in scops owls). Centrorhynchus aluconis is known to use a wide range of micro-mammals and reptiles as paratenic hosts S. affinis is not known, but data from allied species indicates that insects and terrestrial isopods act as intermediate hosts S. affinis, and less of C. aluconis, compared to the other owl species.Statistical comparison of helminth communities in owls from Calabria showed only marginal differences between scops owls and little owls and tawny owls. These differences are largely accounted for variability of infection levels of 2 helminth species which account for over 80% of total helminth abundance, i.e., , having a more diverse diet. In contrast, the diet of the little owl was more variable, in two of the study areas the main prey were mammals but other prey involved resulted in relatively high diversity. In the other three areas the species took mainly insects, thus showing a more restricted diet based on small-sized prey The otherwise strong similarities in community structure of helminth communities of owls from Calabria are in contrast to the strong differences observed in birds of prey from the same region. Santoro et al. C. globocaudatus, C. falconis, S. laticeps, and B. fuscatum  showing different infection levels; shared parasites included atum see . The firP. candelabraria has extensively been reported only in owl species from Europe and North America  included in the present study were also examined for helminths in Galicia cialist’ . The rolDispharynx spp., Excisa spp., Synhimantus spp., Skrjabinura spp., and Subulura spp. use a wide range of insects as intermediate hosts, and Capillaria falconis and Heterakis gallinarum use earthworms; Porrocaecum spp. use insectivorous mammals Choanotaenia spp. use coleopterans and dipterans, Passerilepis spp. use insects, and P. candelabraria uses micro-mammals Neodiplostomum spp. use amphibians, B. fuscatum uses terrestrial snails Skrjabinus spp. use terrestrial mollusks and arthropods Centrorhynchus spp. use orthopteran insects as intermediate hosts and mammals, reptiles and anurans as paratenic hosts Of the helminths species found here, the nematodes including The statistical comparison of diversity of helminth communities between owls and birds of prey assume that observations are independent. This is not the case because species within each bird group are related through phylogenetic relationships A number of host-related factors have been put forward to account for differences in species richness of parasites among vertebrates, of which factors related to host body size often play a prominent role Otus asio) harbouring fewer species than euryphagic species like barred owls (Strix varia). However, at a larger taxonomic scale, Sanmartín et al. We suspect that other factors probably blur the expected influence of host body size upon helminth communities. One potential candidate is trophic niche breath A factor missing in latter explanation is the influence of parasite specificity, which is much more apparent in birds from Calabria. In the previous section we pointed out that, regardless of contacts between parasites and hosts, the compatibility filter prevents some parasites from being established in certain hosts, but the filter should be more relaxed insofar as hosts species are phylogenetically closer. Accipitriformes plus Falconiformes represent a more speciose group than Strigiformes (ca. 58 vs. 19 spp., respectively, in the western Palaearctic, see Snow et al.


Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) is documented. Egg hatching success and offspring survival of inbred mating pairs are significantly lower than that of outbred mating pairs. Two possible mechanisms for avoiding selfing are examined: avoidance of partners of identical genetic makeup and avoidance of partners of the same body color (as a proxy for genetic similarity). Mating between males and females of the same color was as successful as mating between partners of different colors. In contrast, the success of mating between close kin was consistently reduced compared to that of mating between genetically unrelated partners. Interestingly, mating between close kin proceeded normally until the very last stage of the mating process. Thus, inbreeding avoidance appears to take place sometime between copulation and sperm transfer, suggesting that cryptic female choice may play a role in the process.Inbreeding depression has detrimental effects on many organisms, but its effects are potentially greater in organisms that have at least one asexually reproducing life stage. Here, the existence of severe inbreeding depression upon selfing (r = 1) in the cyclic parthenogenetic aphid  Inbreeding depression occurs when there is a decrease in fitness of offspring as a result of consistent breeding between closely related individuals . The offAs a consequence, many organisms have evolved three main ways of avoiding inbreeding . TownsenSitobion avenae, the fitness consequences of selfing are magnified because males and females of almost identical genomes can often be found on the same plant  often face a very high level of inbreeding. Aphids are especially susceptible to inbreeding because approximately three quarters of their life cycle involves asexual reproduction . All offme plant .Acyrthosiphon pisum (Harris) (Hemiptera: Sternorrhyncha). First, reproductive success in inbred mating pairs and outbred mating pairs is compared. Inbreeding depression upon selfing has been suspected  in five different locations in Tompkins County  in 1998 . These genotypes have reproduced parthenogenetically  on alfalfa plants (cv. Oneida) since their collection.All aphid genotypes considered in this study were collected in alfalfa fields . Male and oviparae aphids can be easily recognized by the presence of two black claspers close to the tip of the abdomen  and thickened hind tibia (oviparae) . When soTwo types of crosses were set up. First, males and oviparae of the same genotype were allowed to mate, thus generating inbred progeny (r = 1). Nine genotypes were subjected to selfing: L9, LSR1, LSR2, LSR3, PBR7, PBG7, PBR2, FVR1, and FVG1. Second, virgin oviparae from each of these genotypes were collected and mated with males of one of the other genotypes, thus generating outbred progeny: L9 × LSR1, LSR1 × LSR2, LSR2 × LSR3, LSR3 × FVG1, PBR7 × PBG7, PBG7 × LSR1, PBR2 × FVG1, FVR1 × LSR2, and FVG1 × PBR2.Crosses were performed as described in Via . All ferReproductive success was measured as (1) the percentage of eggs laid by females that successfully hatched into live fundatrices, and (2) the percentage of fundatrices hatched that matured into adults. For each variable, inbred and outbred mating pairs were compared using one-way ANOVA as implemented by the statistical package SPSS 10.0.The following genotypes were used for behavioral experiments: four red genotypes  and four green genotypes . Fully matured females ranging from six to 10 days old were used, since that is their most sexually active age, while mature males of any age were used, since the level of male sexual activity does not vary with age . Experiments were always performed approximately four to seven hours after onset of daylight because pea aphids are most sexually active at that time of the day .The behavioral arena was designed to limit background stimuli which are known to disturb aphids. The base of the behavioral arena was 81 × 50 cm and the walls were 48 cm high. Each wall was covered with off-white paper. Four 10 × 10 cm pots, each containing four well-separated stems of alfalfa, were used in the experiments. Each stem of alfalfa was trimmed so that only three distinctly isolated sets of leaves remained. Each pot was placed in alternating forward and back positions to assure that the pots were not too close together. Doing this helped minimize the effects of activities in one pot influencing the activities in neighboring pots. A set of florescent lamps hanging over the experimental arena was used to facilitate the making of observations and to simulate daylight.Experiment #1: Do pea aphids avoid inbreeding by recognition of close kin? The mating success of outbred and inbred mating pairs was compared. The mating pairs that were categorized as “inbred” consisted of oviparae and males that belonged to the same genotype . We tested eight oviparae × male mating pairs: four outbred pairs  and four inbred pairs .During each day of observation, each mating pair was assigned to one of the four available pots in the experimental arena. The positions assigned were rotated every observation day. This rotation helped minimize the unwanted effects of plant position on the results of the experiment. Approximately half an hour before the observations started, 12 virgin oviparae (three per mating pair) and 20 males (five per mating pair) were isolated into separate Petri dishes with a moistened piece of filter paper and one leaf of alfalfa. Oviparae were placed individually on each leaf of three of the four alfalfa stems in each pot. The egg-laying females were allowed five min to settle on the leaves before the males were released. Then, two to three males were placed on the tip of the stems of alfalfa and the time of introduction was noted. Hand-held stop watches were used to record the duration of various stages of the mating process. Since multiple mating can create inaccuracy in the results, oviparae were collected immediately after their first mating so that other males would not get a chance to mate with them. At the end of each observation day, the oviparae that mated were dissected (details below), and the ones that did not mate were discarded. The percentage of females successfully mated was calculated as the number of females from a particular mating pair that received sperm transfer divided by the total number of females used for that mating pair that day.The mating success of outbred mating  and inbred mating  were compared using one-way ANOVA for each of the six variables measured  at which rejection of close kin occurred  see .F6,98 = 7.69,p < 0.01, R2 = 0.32). However, there was no significant relationship between oviparae weight and any of the six variables tested: (1) Time Between Introduction and Physical Contact , (2) Time Between Physical Contact and Attempt , (3) Time Between Attempt and Copulation , (4) Number of Attempts , (5) Mating Duration , and (6) Number of Actual Successful Matings . A regression analysis was used for variables (1), (2), (3), and (5); a log transformation was performed on variables (2) and (3) since the data points were not spread evenly in their respective residual plots. Since more than half of the males only made one attempt during variable (4), the data remained non-parametric even after transformation. Thus, a Kendall's Tau test (non-parametric regression) was used to test for a correlation between female weight and number of attempts. A logistic regression test was used for variable (6).The difference between weights of males and oviparae could cause differences in mating success that are independent of the degree of genetic relatedness or body color. This factor was controlled for by the random selection of both oviparae and males for experiments. However, there was greater potential for female body weight to affect our results since oviparae had a greater variation in size. This possible effect was investigated further by weighing each of the egg-laying females that mated during observations using a Cahn Instruments Inc. C-31 Microbalance (accurate up to the thousandth milligram). Oviparae from a total of seven different female clones were weighed. A one-way ANOVA analysis using the statistical package JMP 5.1 showed that there was significant difference in oviparae weight across all clones used   and 73 batches of inbred eggs . The percentage of egg hatching of inbred crosses was significantly lower than that of outbred crosses (= 0.236) . The aveF1,148 = 13.6, p < 0.01, R2= 0.084)  . The aveExperiment #1: Do pea aphids avoid inbreeding by recognition of close kin?F1,105 = 0.127, p = 0.723, R2 = 0.0012), Time Between Physical Contact and Attempt , Time Between Attempt and Copulation , Number of Attempts , and Mating Duration . However, the outbreds had a significantly higher Percentage of Females Successfully Mated than the inbreds . The outbreds had a mean of 40.28%  while the inbreds had a mean of only 14.58% .Experiment #2: Do pea aphids avoid inbreeding by recognition of body color?F1,117 = 0.010,p = 0.905, R2 = 0.00012), Time Between Physical Contact and Attempt , Time Between Attempt and Copulation , Number of Attempts , Mating Duration , or Percentage of Females Successfully Mated .The main objective of this study was to test the hypothesis that pea aphids avoid selfing. To examine inbreeding avoidance in pea aphids, their general mating behavior was first characterized. Observations revealed that females initiate the mating process by waiving one of their hind tibia in an up-and-down motion, releasing sex pheromones  Figure . Within After revealing that inbred matings indeed result in lower fitness in pea aphids , we testSitobion avenae and Dysaphis plantaginea, showed similarly strong responses by males to pheromone released by oviparae of the same species before making physical contact. The fact that inbred and outbred mating pairs did not differ in variables (2) - (5) shows that inbreeding avoidance does not occur before a prolonged copulation event. This suggests that cues perceived at a close range  are not used to assess the genetic relatedness of individuals. Instead, inbred and outbred mating pairs differ only in variable (6)  time between introduction and physical contact, (2) time between physical contact and attempt, (3) time between attempt and copulation, (4) number of attempts, (5) mating duration, and (6) percentage of females successfully mated. Inbred and outbred mating pairs did not differ in variable (1) . Almost able (6) , suggestable (6) . These rChorthippus parallelus (Orthoptera)  ). In thehoptera) . This sta anilis .Recognition of genetically identical individuals using very specific cues within the oviparae during copulation may be the most suitable mechanism for pea aphids. Recognizing different body colors to avoid inbreeding is not very effective because it gives information that is too vague; individuals of the same body color can either be a genetically identical clone, close relative, or non-relative. Since mating with an identical clone has greater detrimental effects , recogniAn alternative mechanism commonly used by mammals and birds for inbreeding avoidance is dispersal. However, this mechanism is unlikely to be effective in pea aphids. As mentioned in the introduction, pea aphid oviparae are always wingless and relatively slow walkers. Therefore, it would be very difficult for them to travel very far from natal habitats without being preyed upon or parasitized. Oviparae also cannot travel long distances for long periods of time because they constantly need to feed in order to obtain enough nourishment to produce eggs and survive. Given the short period for sexual reproduction, the pea aphid oviparae would not have enough time to disperse a short distance, feed, and then disperse further. Although some males do have wings , they arAnother possible mechanism of avoiding inbreeding not examined in this study is the use of extra-pair matings to neutralize the effects of inbreeding . A studyMetaplastes ornatus (Orthoptera)  ). The maMales can also physically plug the cavity of a female's external genitalia with a mass of material after insemination occurs . Sperm


Zinc deficiency can lead to clinically relevant disturbances in tissue functions and particularly important for birth weight of neonates. The aim of this study was to determine the relationship between serum zinc in pregnant women and the incidence of low birth weight (<2500 g) in their newborns.This case-control study was done on women who delivered low birth weight infants (Cases), and normal birth weight infants (Controls). Blood samples got in all women within 5 minutes of delivery, and assessed the concentration of zinc using electrothermal atomic absorption spectrometry. Serum concentration of zinc was compared.One hundred and seventeen mothers were enrolled, of them, 65 cases were low birth weight infants (1845±472 g) and 52 were controls (3166± 435 g). Mothers in the case and control groups did not differ in age, body mass index, and socioeconomic or demographic factors. Maternal zinc concentration differed between cases and controls; 753.34±15 µg/l vs. 654.76±12 µg/l respectively. Maternal zinc differed between premature and full term deliveries.Maternal zinc concentration was shown to affect birth weight and prematurity. Low birth weight (LBW) is a crucial and a substantial factor contributing to infant mortality.2] Infan Infan2] Pregnant women in developing countries were shown to consume diets with a lower density of minerals and vitamins. Suboptimal zinc intake may be relatively common throughout the world. Low zincEvidences suggests that zinc deficiency is one of the important problems within the developed and developing countries. In 1991,Pregnant women are facing zinc deficiency more than the other groups, due to having fetus which need zinc for its poor growth. A separaWe conducted this prospective case-control study at the maternity ward of Imam Reza Hospital and the Neonatal Research Center of Mashhad University for Medical Sciences in Iran. The study protocol was approved by the research office at the university, and written informed consent was obtained from each subject before enrollment. The study was done between May 2006 and February 2007. Mother-infants pairs were included if (i) maternal age was 17-35 years at the time of delivery, and (ii) She had uncomplicated singleton pregnancy.“Cases” were considered if the birth weight of the newborn was <2500 g without any known underlying risk factors that could explain the reason for the LBW. A “Control” subject was enrolled when a subsequent mother delivered an infant with birth weight =2500 g.Mothers were not considered for the study if any of the following exclusion criteria were present: A previous history of LBW delivery, twin and multiple pregnancies, preeclampsia and eclampsia, uterine cervical abnormalities, antenatal bleeding, oligo- and polyhydramnios, abnormal maternal conditions prior pregnancies; such as lupus, chronic hypertension, diabetes mellitus, seizure disorders, malignancies, and drug or alcohol abuse.The investigator did a thorough physical examination of every infant recruited into the study. The infant's weight was taken to the nearest 10 g and the length was recorded to the nearest 0.1 cm. The gestational age was assessed clinically using the Dubowitz method. Medical For zinc assays in the first, 5 minutes after delivery, immediately after collection of blood, it was centrifuged at 3000 rpm for 15 min. Plasma was separated and kept frozen at -20°C. The samples were analyzed after all of them collected. Serum zinc concentration was determined by flame atomic absorption spectroscopy (AAS) in a Perkin-Elmer 1100B apparatus (Atomic Absorption Spectrophotometer). All samples were analyzed in duplicate. Our laboratory reference range for serum zinc was ≥500 µg/L, which is similar to that of other laboratories.[15]16]16]All specimens were de-identified before processing, therefore, laboratory staff was masked to clinical conditions. The data were analyzed using the SPSS software . For quantitative data with normal distribution t-test and with abnormal distribution, Kruskal-Wallis test were used. For qualitative data, X2 test was used.Multiple linear regression analysis was performed to control potential confounding variables. Statistical significance was considered when p was less than 0.05.The study included 117 mother-infant pairs, of them, 65 were cases and 52 were controls. The demographic and clinical characteristics of the study population are listed in On regression analysis, zinc concentrations did not differ between cases and controls (p>0.05) after we controlled for various confounders such as maternal age, body mass index (BMI), educational level, income, neonatal gender, birth weight, gestational age, and Apgar scores.This study demonstrated zinc concentrations in the serum of mothers who delivered LBW infants differ from mothers who delivered infants with a normal birth weight, and serum zinc level in case group was significantly higher than mothers with normal birth weight and mature infants (p<0.01).Our results showed that zinc level decreased with increasing gestational period. The maximum decrease in zinc level was observed during weeks of 35-36 of gestational age. These results are similar to other studies.18]19][[19][18][[[19][18]A study carried out by Diet Health and Knowledge Survey of Iran Ministry of Health in 2001 about micronutrient consumption among the pregnant women in eleven regions of Iran was representative of all geographical areas. Analysis showed that prevalence of maternal zinc deficiency in Mashhad was 43.3% and among others areas of Iran, it was a third grade maternal zinc deficiency. Although significant correlation was found between the age of pregnancy and maternal zinc concentration, so during the third trimester, 44% of pregnant women were zinc deficient.A total of 12 randomized, controlled intervention trials of supplemental zinc and pregnancy outcome have been reported. Of the 1Although plasma zinc concentration is commonly used as an indicator of zinc status, interpretation of the measurement is difficult because plasma zinc concentration decreased with infection, vigorous exercise, and food intake. Assessment is more difficult during pregnancy because plasma zinc concentration declines in proportion to the increase in plasma volume.It can be concluded that, when mothers receive zinc supplementation during pregnancy, zinc concentration in the blood plays a role in determining the birth weight of term and preterm infants. Our study determined if such conclusion can apply to zinc depleted mothers.It is accomplished that an early and progressive decline in serum zinc occurs during gestation and a poor maternal zinc status may limit the metabolic adaptation capacity of women especially during pregnancy. Some previous studies demonstrated that zinc deficiency during pregnancy in early weeks can produce abortion and congenital malformation.27] If t If t27]


Anopheles gambiae, and discovered a novel insect immune tissue, herein named periostial hemocytes, that sequesters pathogens as they flow with the hemolymph. Specifically, we show that there are two types of endocytic cells that flank the heart: periostial hemocytes and pericardial cells. Resident periostial hemocytes engage in the rapid phagocytosis of pathogens, and during the course of a bacterial or Plasmodium infection, circulating hemocytes migrate to the periostial regions where they bind the cardiac musculature and each other, and continue the phagocytosis of invaders. Periostial hemocyte aggregation occurs in a time- and infection dose-dependent manner, and once this immune process is triggered, the number of periostial hemocytes remains elevated for the lifetime of the mosquito. Finally, the soluble immune elicitors peptidoglycan and β-1,3-glucan also induce periostial hemocyte aggregation, indicating that this is a generalized and basal immune response that is induced by diverse immune stimuli. These data describe a novel insect cellular immune response that fundamentally relies on the physiological interaction between the insect circulatory and immune systems.Insects counter infection with innate immune responses that rely on cells called hemocytes. Hemocytes exist in association with the insect's open circulatory system and this mode of existence has likely influenced the organization and control of anti-pathogen immune responses. Previous studies reported that pathogens in the mosquito body cavity (hemocoel) accumulate on the surface of the heart. Using novel cell staining, microdissection and intravital imaging techniques, we investigated the mechanism of pathogen accumulation in the pericardium of the malaria mosquito,  Mosquitoes transmit diseases such as malaria, dengue fever, West Nile virus and lymphatic filariasis. A mosquito initially acquires a pathogen when she ingests a blood meal from an infected person or animal. Then, after a period of development and/or replication in the mosquito gut, the pathogen enters the hemocoel (body cavity) and undergoes an obligate migration to the salivary glands (the destination for viruses and protozoans) or the mouthparts (the destination for larger worms). During this migration, pathogens are subject to two potentially antagonistic mosquito forces: immune responses and circulatory currents. In this study, we examined the physiological interactions between the mosquito immune and circulatory systems. We show that when mosquitoes are infected with bacteria or malaria parasites, mosquito immune cells (hemocytes) migrate to the areas surrounding the valves of the heart. At these areas of rapid and dynamic hemolymph (mosquito blood) flow, hemocytes swiftly phagocytose and kill pathogens. These experiments describe a novel and basal insect immune response that fundamentally relies on the physiological interaction between the mosquito circulatory and immune system. Furthermore, because traversal of the hemocoel is required for pathogen transmission, this new knowledge could be used in the development of novel disease control strategies. Pathogens transmitted by mosquitoes must traverse the insect's open body cavity (hemocoel) during their journey from the midgut to the salivary glands, and this obligate migration places them in direct contact with the insect's circulatory and immune systems. The insect circulatory system consists of hemolymph (blood), the hemocoel, and pulsatile organs, of which the dorsal vessel is the most important Drosophila melanogaster provides a unique perspective on the evolution of the insect immune and circulatory systems, and (4) it has been proposed that mosquito immune responses could be harnessed for the control of mosquito borne disease While insect circulatory processes have been primarily studied for their role in transporting nutrients, wastes and signaling molecules, one aspect that has been overlooked is the relationship between hemolymph circulation and immune responses. The insect immune system relies on innate reactions to fight pathogens and involves both cellular and humoral components Anopheles gambiae, pathogens accumulate in discrete foci along the surface of the mosquito heart We have reported that during the course of an infection in the malaria mosquito, The initial goal of this study was to investigate whether pathogen accumulation around the dorsal vessel is due to a physical barrier or due to an active immune response  [11]. HeCo-staining of PCs and the abdominal musculature showed that PCs are binucleated cells that flank the mosquito heart . PCs occEscherichia coli showed that pathogens are not phagocytosed by PCs. Instead, pathogens accumulate at the periostial regions between the four PCs that flank the ostia  mosquitoes . Intrathoracic injection of CM-DiI into live mosquitoes, followed by hemolymph perfusion and ex vivo analysis of cell staining efficiencies showed that CM-DiI stains greater than 97% of the hemocytes collected from naïve (99%), injured  and squitoes . QuantitIn addition to hemocytes, hemolymph perfusion results in the collection of small amounts of fat body as well as free nuclei from lysed cells E. coli-infected mosquitoes ial pair .E. coli overlap in all three dimensions, supporting the phagocytic activity of heart-associated hemocytes , the number of hemocytes in the periostial regions roughly doubles. This increase is due to the movement of hemocytes into the periostial regions and not hemocyte replication at the pericardium. As seen in Movie S2, some hemocytes flowing into the pericardium adhere to the heart-associated musculature. These hemocytes then slowly glide into the periostial regions, where they settle and phagocytose pathogens. While hemocytes exist both in circulation and attached to tissues (sessile), the relatively low number of sessile hemocytes observed in non-periostial areas of the dorsal cuticular epithelium, together with the relatively rapid arrival of hemocytes into the pericardium suggests that the recruited cells originate from the circulating hemocyte population. The molecular trigger for their arrival is unclear, but we hypothesize that hemolymph flow brings circulating hemocytes to the pericardium, where they bind the alary muscles and then slowly undergo a directed migration into the periostial regions. This migration is facilitated by hemolymph flow but is not exclusively driven by it; the median velocity of the hemocytes tracked in Movie S2 was 1.2 µm/sec . Quantitative analysis of periostial hemocyte numbers during the course of an infection revealed that hemocyte numbers in the periostial regions approximately double within the first hour, and plateau at 4 h post infection  bacteria in the periostial regions steadily increases until 12 h post-infection and then begins to decline  revealed that hemocytes are recruited to the periostial regions within the first 24 h post-infection, but that the number of periostial hemocytes decreases by day 3 post-infection (not shown). Infection of mosquitoes with fewer E. coli (OD600 = 2) revealed a similar trend: the number of periostial hemocytes decreases significantly by 3 days post-infection, but remains elevated for the lifetime of the mosquito, relative to similarly aged naïve controls . While the large number of sporozoites released by each oocyst makes it unlikely that phagocytosis is the primary component of the anti-Plasmodium response in the hemocoel, increases in melanization and periostial hemocyte aggregation suggest that hemocyte activation leads to the production of humoral factors that target Plasmodium via lytic and melanization pathways. Evidence from others supports this idea, as Plasmodium development in mosquitoes induces the transcriptional regulation of immune genes in hemocytes Plasmodium interactions are in agreement with studies on the anti-sporozoite response Although it is known that most Drosophila hemocyte markers are not encoded in the mosquito genome , the CM-DiI approach described here for the first time allows the study of mosquito hemocyte cell biology in vivo and in real time. We expect that this procedure could be adapted for the study of hemocyte biology in a broad range of insects.In most insects, one problem foiling hemocyte research is that no effective means of specifically staining hemocytes in vivo exists In conclusion, there remain deficits in our current knowledge of hemocyte biology in adult insects, as well as in our understanding of the direct interactions between the insect circulatory and immune systems. Here, we developed new methods for the in vivo study of mosquito hemocytes and pericardial cells (nephrocytes), and applied these methods to discover a novel mosquito immune response. Namely, we uncovered periostial hemocyte aggregates, an immune tissue that is located on the surface of the mosquito heart and represents a basal component of the cellular immune response against bacteria and malaria parasites.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, U.S.A. The protocol was approved by Vanderbilt University's Institutional Animal Care and Use Committee . Animals were maintained in a certified animal room and were cared for by trained personnel and veterinarians.Anopheles gambiae (G3 strain) were reared and maintained in an environmental chamber as described For injections, mosquitoes were cold anesthetized and a finely pulled glass needle was inserted through the thoracic anepisternal cleft. A volume of 0.2 µl was slowly injected into the hemocoel and mosquitoes were then placed back in an environmental chamber until assayed.E. coli (modified DH5α) were grown overnight in a shaking incubator at 37°C in Luria-Bertani's rich nutrient medium (LB broth) and, unless otherwise stated, cultures were normalized to OD600 = 2 or OD600 = 4 using a BioPhotometer plus spectrophotometer  prior to injection. To determine the absolute infection dose, dilutions of each OD600 = 2 and OD600 = 4 E. coli culture were plated on LB agar with tetracycline, incubated at 37°C, and the resultant colony forming units were counted 18 h later. On average, OD600 = 2 and OD600 = 4 represented infection doses of 38,000 and 131,000 E. coli per mosquito, respectively. As a non-living phagocytosis elicitor For bacterial infections, tetracycline-resistant GFP-expressing E. coli, PGN purified from Gram(+) Bacillus thuringiensis, and β-1,3-glucan. For PGN purification, E. coli and B. thuringiensis were grown overnight in LB broth at 37°C. A volume of 2 ml of each bacterial culture was independently centrifuged for 1 min at 10,000 rcf, and the resulting pellets were suspended in 1 ml of PBS. Bacteria were then lysed, while on ice, by sonication for 1 min using a Branson Sonifier 450  equipped with a 3 mm tip that was set to 20% power and 30% duty cycle. Trichloroacetic acid (TCA) was added to the lysed bacteria to a final concentration of 10% v/v, the resultant solutions were incubated for 10 min at 90°C, and the PGNs were extracted by centrifugation for 1 min at 12,000 rcf 600 = 1 prior to injection.Three soluble immune elicitors were used in this study: peptidoglycan (PGN) purified from Gram(−) β-1,3-glucan (microparticulate curdlan), a fungal immune elicitor, was prepared by sonicating 1% w/v curdlan  in PBS for 5 min at 20% power and 20% duty, while on ice. Particulates remaining in the solution were then allowed to settle for 30 min while on ice, and the clear top phase was removed and used for injections P. berghei-infected mouse with approximately 10% blood-stage parasitemia and a 1–2% gametocytemia. A control group of mosquitoes originating from the same cohort was allowed to feed, concurrently, on an uninfected mouse. Both groups were then housed in a humidified chamber at 20.5°C for 20 days prior to the assessment of the periostial cellular immune response (see below). The PbGFPCONP. berghei strain Five-day-old female adult mosquitoes were starved for 6 h and then allowed to feed on a To stain hemocytes inside live mosquitoes, 0.2 µl of a solution consisting of 75 µM CM-DiI  and 0.75 mM Hoechst 33342 (Invitrogen) in PBS was injected into mosquitoes. It was crucial that this solution be injected within minutes of its preparation, as once the CM-DiI is placed in an aqueous environment its hemocyte-staining effectiveness rapidly decreases, approaching 0% after 10–15 min of mixing. After CM-DiI injection, mosquitoes were immediately returned to 27°C and 75% relative humidity for an incubation period of 20 min.2 digital camera . Cells were counted at 1000× magnification by scanning the slides from the far left to the far right until 50 hemocytes from each individual mosquito were visualized, also keeping track of the number of fat body cells observed. Intact cells were first identified as either hemocytes or fat body by confirming the presence of a nucleus using fluorescence microscopy (Hoechst 33342) and comparing cell morphology by differential interference contrast (DIC) microscopy to the descriptions of previous authors E. coli, and all cell types were examined for their incorporation of CM-DiI. Three treatments were performed , and for each treatment, hemocytes from 15 individual mosquitoes that originated from 5 independent but paired cohorts were examined .Circulating hemocytes were collected by perfusing the hemolymph onto the center of 1 cm diameter etched rings on Rite-On  glass slides The dorsal portion of mosquito abdomens were analyzed after the labeling of PCs, hemocytes and heart muscle. These tissues were labeled in the presence or absence of an immune challenge, and were labeled in the following combinations: (1) PCs and heart muscle, (2) PCs and hemocytes, and (3) hemocytes and heart muscle. In all experiments, Hoechst 33342 was used as a nuclear stain.Depending on the experiment, PCs were stained using one of two novel methods. In the first, more permanent method, 0.2 µl of 0.2 mg/ml Alexa Fluor conjugated IgG  in PBS was intrathoracically injected and the mosquitoes were placed at 27°C for 1 h to allow the labeled proteins to be pinocytosed by the PCs. The mosquitoes were then placed in PBS and their abdomens were bisected along a coronal plane. The dorsal half of each abdomen, sans any internal organs, was isolated, rinsed, fixed in 4% formaldehyde in PBS for 10 min, washed 3×5 min in 0.1% Tween 20 in PBS (PBST), and mounted on a glass slide using Aqua Poly/Mount. For the second PC staining method, 0.2 µl of a 0.1 mM mixture of LysoTracker Red (Molecular Probes) in PBS was intrathoracically injected and allowed to incubate for 10 min. Abdomens were then bisected along a coronal plane, rinsed in PBS, and mounted using Aqua Poly/Mount. Because LysoTracker Red stains any region that contains high lysosomal activity, it also serves as a marker for hemocyte-mediated phagocytosis of bacteria. However, LysoTracker Red cannot be aldehyde-fixed, so this method stains the PCs briefly before the dye diffuses out, and is not useful in combination with most other staining techniques.To examine and quantify hemocytes that were adhered to tissues, 0.2 µl of a solution consisting of 75 µM CM-DiI and 0.75 mM Hoechst 33342 in PBS was injected into mosquitoes. After allowing this solution to incubate in live mosquitoes for 20 min at 27°C, mosquitoes were injected with 0.2 µl of 16% formaldehyde. Tissues were then allowed to fix for 5 min, the mosquitoes were bisected along a coronal plane, and the dorsal halves were mounted on glass slides using Aqua Poly/Mount. Immediately following mounting, CM-DiI stained hemocytes in the periostial regions were counted through the 90i's oculars at 400× or 1000× total magnification. Hemocytes were only counted if their presence was supported by both CM-DiI and Hoechst 33342 staining, so a small number of cells (<5%) might have been excluded from our counts using this conservative method. Cells were counted as periostial hemocytes only if they were attached to the dorsal vessel at the ostia, or formed part of a contiguous mass of hemocytes that were attached to this region. Numbers of periostial hemocytes were recorded on a per mosquito basis, and for each treatment cell counts were conducted on at least 12 mosquitoes that originated from no fewer than 4 independently-reared cohorts. In these experiments, trials were excluded when background staining interfered with the cellular boundaries of hemocyte aggregates in any of the treatment groups.For the co-staining of PCs and heart muscle, 1 h after the injection of Alexa Fluor-conjugated IgG (568 nm) muscle was stained by injecting a formaldehyde-phalloidin-Hoechst-Triton×100 mixture as described Perfused hemocytes and abdominal whole mounts were imaged between 200× and 1000× magnification depending on which phenomenon was being captured. Specimens were viewed under differential interference contrast microscopy (DIC) and/or fluorescence illumination using the Nikon 90i microscope ensemble described above, and Z-stack images were captured using a linear encoded Z-motor and Nikon's Advanced Research NIS-Elements software. To display 2 dimensional images, all images within a stack were combined to form a focused image using the Extended Depth of Focus (EDF) module of NIS Elements. For 3 dimensional rendering, Z-stacks were quantitatively deconvolved using the AQ 3D Blind Deconvolution module of NIS Elements and rendered using the volume view feature.E. coli fluorescence in the abdomen following infection, a series of images were captured and analyzed using the fixed thresholding feature in NIS-Elements. All images were acquired under non-saturating conditions using identical settings, and were identically thresholded to isolate only the portions containing GFP-E. coli fluorescence. For each specimen, fluorescence intensity data was collected for the dorsal abdomen, the entire abdomen , and the periostial regions.To quantify relative levels of E. coli were injected and fluorescence-based intravital video recording of the heart region was initiated within 10 sec of treatment. Time-lapse image sequences were captured for green (GFP-E. coli) and red (CM-DiI-stained hemocytes) channels at 5 sec intervals using the ND-experiment capture module of NIS Elements. Hemocyte tracking was then done using the manual feature of the Object Tracker module of NIS-Elements, and hemocyte velocity was calculated by dividing the path length by the total time of tracking. To reduce the potential for damage to the mosquito, light shutters were only open while each image was being acquired, and light intensity was greatly reduced by using a neutral density filter of 16.For intravital time-lapse video recording of the interactions between hemocytes, pathogens and the mosquito heart, hemocytes were labeled in vivo using the CM-DiI method described above. Mosquitoes were then restrained on glass slides using small strips of Parafilm “M”  to gently adhere the proboscis, legs and wings (extended) to glass slides, and spheres of Parafilm were placed on either side of the abdomen to restrict side-to-side movement. Once mosquitoes were restrained, GFP-expressing Cell count data was separated by treatment group and tested for normality using the Kolmogorov-Smirnov test. After confirming normality, datasets with one variable and two groups were analyzed by the t-test. Datasets with one variable and more than two groups were analyzed by one-way ANOVA, and multiple comparisons were done using Tukey's post hoc test. Differences were deemed significant at P<0.05.E. coli fluorescence (GFP) and hemocyte fluorescence (CM-DiI) within perfused hemocyte samples or whole mount abdomens, images were analyzed using the Mander's overlap or Pearson's correlation coefficient (PCC) features in NIS-Elements. These measures are similar in that they describe the amount of spatial overlap between two fluorescence channels, with the main difference being that Mander's overlap corrects for differences in signal intensity while PCC does not For statistical evaluation of the co-localization of Movie S1E. coli by periostial hemocytes.Phagocytosis of (MOV)Click here for additional data file.Movie S2E. coli infection.Hemocyte migration to the periostial regions in response to (MOV)Click here for additional data file.