The inflammatory infiltrate plays a pivotal role in classical Hodgkin lymphoma (cHL). Here, we focussed on the role of macrophages (MΦ) and dendritic cells (DC).GM-CSF, pro-inflammatory MΦ-1-model) or M-CSF  were incubated with cHL cell line  supernatants (SN). DC maturation or MΦ polarization were investigated by flow cytometry. Furthermore, the impact of DC or MΦ on cHL cell proliferation was analyzed by BrdU/CFSE assay.MΦ and DC infiltration was investigated in 106 cHL specimens using immunohistochemistry and cytokine expression was analyzed in a subset by real-time PCR. Human peripheral blood-derived monocytes, DC, MΦ stimulated with GM-CSF (MΦIn cHL tissues mature myeloid (m)DC and MΦ predominated. High numbers of CD83+ mDC and low numbers of CD163+ MΦ were associated with improved disease specific survival. In numerous cHL specimens increased levels of both pro- and anti-inflammatory cytokines and of IL13 and GM-CSF were observed compared to reactive lymphadenopathies. Maturation of DC and induction and maintenance of an immunomodulatory MΦ phenotype were promoted by SN derived from cHL cell lines. TNFα neutralization in SN resulted in a significant inhibition of mDC maturation. DC and pro-inflammatory MΦ inhibited the proliferation of cHL cells.Adopting an immunomodulatory phenotype is a potential mechanism for how MΦ promote immune evasion in cHL. Mature DC, in contrast, might participate in antitumoral immunity. Intense attention has been paid to antigen-presenting cells (APC) as part of the tumor stroma. Myeloid dendritic cells (mDC) are found in different malignancies In classical Hodgkin lymphoma (cHL) few malignant Hodgkin-Reed-Sternberg (HRS) cells are embedded in a prominent inflammatory infiltrate Immune evasion might also be achieved by modulating the polarization of intratumoral macrophages (MΦ), being numerous in HL In the present study we aimed to investigate the role of mDC and MΦ in cHL cases by analyzing their distribution and influence on outcome as well as the intratumoral cytokine profile. Furthermore, in a cell culture system the mutual effect of cHL cell lines on monocytes, monocyte-derived (mo)DC and MΦ and vice versa was investigated.Formalin-fixed paraffin-embedded (FFPE) specimens of 106 cHL cases and of 10 reactive lymphadenopathies were retrieved from the Archives of the Institute of Pathology, University Hospital, Erlangen, Germany. Most cases have been included in a previous study ethic@zuv.uni-erlangen.de) on 24th January 2005, waiving the need for retrospective consent for the use of archived rest material.The use of FFPE material from the Archive of the Institute of Pathology was approved by the Ethics Committee of the Friedrich-Alexander-University of Erlangen-Nuremberg  antigen-retrieval was performed in a steam cooker  for up to 5 min at 120°C using a target retrieval solution pH 6  or 0.01 M Na-Citrate buffer pH 6. CD163 and CD83 stains were performed on a Ventana BenchMark Ultra stainer  using CC1 buffer  for antigen retrieval. Primary antibodies specific for CD1a , CD83 , CD123 , CD68  and CD163  were applied. In manual stains an alkaline phosphatase-labeled polymer kit was used  or the biotin-streptavidin method. Fast Red  or aminoethyl carbazole  served as chromogens.in situ hybridization (ISH) or latent membrane protein 1 (LMP1) specific immunohistochemistry was performed as described before To assess the EBV state of CHL cases EBER-2 in the area with the densest infiltration were evaluated using the image analysis software “Count” . In CD83 stains positive cells showing the morphology of neoplastic HRS cells were excluded from the analysis. For evaluation of CD123 only cells with plasmacytoid morphology were counted.A computer assisted hot spot analysis was performed on whole block sections. Images were acquired with a standard light microscope and a CCD camera or a Mirax Midi system  for digital slide scanning and Mirax viewer software (Zeiss). Positive cells were manually selected by a pathologist. Numbers of positive cells per mmTNF, IL10, TGFβ1 and CALM2 (reference gene). For the analysis of IL4, IL13, GM-CSF, M-CSF and CALM2 (reference gene) RNA was isolated using Maxwell 16 LEV RNA FFPE Kit, according to the manufacturer's instructions. A probe of L1236-RNA was used as positive control and for comparison of the PCR-runs. Standard and no-template controls were included. ΔΔCT qRT-PCR with probes for CALM2 (reference gene), TNF, IL10 and TGFβ1 as well as for IL4, IL13, GM-CSF and M-CSF was performed with 40 cycles of nucleic acid amplification using QuantiFast Probe Assays for one-step qRT-PCR . Assays were analyzed by the Applied Biosystems 7500 Fast Real-Time PCR System and the 7500 Software Version 2.0.6 .From 10 µm FFPE sections total RNA isolation and DNAse digestion were performed, using a standardized fully automated isolation method based on germanium-coated magnetic beads  in combination with the liquid handling robot XTRAKT SL (STRATIFYER) as previously described 2 in RPMI-1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 50 µg/ml streptomycin . PBMCs were isolated by Ficoll-Paque  gradient centrifugation of peripheral blood from 40 healthy volunteers (mean age 35 years).The cHL cell lines L1236 and HDLM2 were obtained in 2009 and 2002 from “Deutsche Sammlung von Mikroorganismen und Zellkulturen”, Braunschweig, Germany. The cells showed heterogeneity in cell size and typical morphology including mono-, bi-, and also multi-nucleated cells. HDLM2 were suspension cells and L1236 semi-adherent as stated by the “Deutsche Sammlung von Mikroorganismen und Zellkulturen” and showed typical growth characteristics. Cells were negative for mycoplasma at the onset of the study and were re-tested negative at the end of the study. L1236 and HDLM2 and blood cells were maintained at 37°C and 5% COGM-CSF, a model of MΦ-1) or 50 ng/ml rhM-CSF . All other cytokines were also obtained from R&D Systems, Minneapolis, USA. moDC and MΦ purity and differentiation was confirmed by FACS analysis  for 6 days with cytokine renewal at day 2 and 5. To generate MΦ, adherent cells were incubated for 5 days with either 50 ng/ml rhGM-CSF , APC-conjuagted mouse anti-CD83 mAB  and PE-conjugated mouse anti-CD14 mAB  or isotype controls  for 15 min at 4°C and fixed in 1% PFA. MΦ were fixed and permeabilized using the Cytofix/Cytosperm kit (BD Pharmingen) and were then incubated with FITC-conjugated mouse anit-CD68 mAB , PE-conjugated mouse anit-CD163 mAB , PerCP-conjugated mouse anti-HLA-DR mAB , and APC-conjugated mouse anti-CD206 mAB  or isotype controls  for 30 min at 4°C. Flow cytometric analysis was performed on a FACSCanto II flow cytometer using the FACSDiva software v6.1.3 (BD Pharmingen). Data were analyzed using FlowJo software .6 cells/ml and with either 10 µg/ml neutralizing anti-TNFα mAB  or Infliximab  for 24 h.moDC were incubated with L1236-SN, generated after 48 h incubation of 106 L1236 cells were stained with 3.5 µM CFSE (Sigma-Aldrich) for 4 min at RT. 1.5×105 CFSE-labeled L1236 cells/ml were incubated for 96 h with MΦGM-CSF-SN, MΦM-CSF-SN or moDC-SN generated from 5×105 cells/ml after 48 h incubation. FACS analysis was performed to measure the CFSE signal/cell with a reduction of intensity as a marker of proliferation.4–8×10GM-CSF and MΦM-CSF were co-cultured with L1236 cells in 96-well plates at a ratio of 6∶1 for 48 h with 50% of MΦGM-CSF or MΦM-CSF conditioned medium (medium generated after 5 days of monocyte incubation with either rhGM-CSF or rhM-CSF) and 100 µM BrdU (Sigma-Aldrich). Alternatively 3×105 L1236 cells/ml were treated with 90% of MΦGM-CSF-SN or MΦM-CSF-SN alone. After centrifugation cells were washed with PBS, air dried, fixed in ice cold methanol for 20 min and then a blocking solution  was added for 1 h. The mouse anti-BrdU mAB  was added over night, followed by an Alexa Fluor 488 coupled secondary antibody  for 1 h. Fluorescence intensity was measured at an excitation of 485/20 and an emission of 528/20 using Synergy 2 (BioTek).MΦContinuous variables with normal distribution and homogenous variances were analyzed by unpaired or paired t-test. Mann-Whitney U-tests or Wilcoxon signed rank test were used for data with skewed distributions. Disease-specific survival (DSS) of cHL was defined from the receipt of tissue until the last follow-up or death caused by cHL. DSS rates of cHL patients were calculated according to Kaplan-Meier. DSS curves were compared with log-rank test. In all tests p<0.05 was accepted as statistically significant. Statistical analyses were performed using SPSS for Windows software  or GraphPad Prism 5 for Windows software .CD1a and CD83 were used as markers of immature and mature mDC as described elsewhere In Kaplan-Meier analysis the numbers of CD1a+, CD68+ and CD123+ cells did not influence DSS of cHL . DSS of patients with high numbers of CD83+ mature mDC was significantly improved . Convers2 of CD83+ or CD163+ cells below (low) or above (high) the 25th or 75th percentile, respectively. TNF, IL10, TGFβ1 were investigated, which were reported in cHL tissue and can influence DC maturation/development and MΦ polarization GM-CSF, M-CSF, IL4 and IL13 were analyzed, which might also partake in the polarization of MΦ IL10 and TGFβ1 compared to the other cHL cases  expressed high levels of HLA-DR and lacked CD163 expression whereas MΦM-CSF (MΦ-2 model) presented with high CD163 and low HLA-DR expression, as expected  with cHL cases with an expected better prognosis (CD83 high or CD163 low) anti-inflammatory  cytokines were significantly upregulated in the adverse group. The upregulation of immunoregulatory cytokines IL-10 and TGFβ1 in cHL and especially in the prognostically adverse group goes well in line with immune evasion. At first glance, in contrast, it appears paradoxical that TNF and GM-CSF are excessively produced in cHL. However, persistent inflammation has been connected to tumorigenesis IL13 and M-CSF in cHL cases might be responsible for the induction of MΦ-2-like CD163+ tumor-associated macrophages in cHL In therapy-refractory cHL a TAM-specific gene signature Going in line with the dominance of mature mDC in cHL specimens, SN of cHL cell lines promoted the maturation and/or activation of moDC in our study. This effect was stronger for L1236 cells producing higher TNFα concentrations than HDLM2. When combining IL10 and TGFβ that hamper DC maturation and maturation-inducing TNFα GM-CSF or MΦM-CSF depending on the used stimulus. One has, however, to keep in mind that depending on the stimulus used, many different activation states can be achieved far beyond the MΦ-1 and MΦ-2 model M-CSF, as also demonstrated by the upregulation of CD163 known to promote an anti-inflammatory microenvironment in vitro finding, that MΦ-2 markers were enhanced by cHL cell SN, together with the predominance and prognostic role of CD163+ cell in cHL specimens, might therefore indicate that immunoregulatory CD163+ MΦ might play an important role in the tumor site and are actively promoted and maintained by the tumor cells. The excess of pro- as well as anti-inflammatory cytokines in the tumor tissue, however, indicates, that not a typical MΦ-2 milieu is found in the tumor, going in line with the idea that TAM cannot be assigned to one of the extreme polarization patterns of MΦ-1 or MΦ-2.For the investigation of MΦ, we used a published model representing two extreme polarization states of pro-inflammatory MΦ-1 and immunoregulatory MΦ-2 obtained by stimulation with M-CSF and GM-CSF M-CSF on L1236 growth, whereas both moDC and MΦGM-CSF inhibited cHL cell proliferation. This effect was not depending on cell-cell contact. Hence, soluble factors appeared sufficient to mediate some inhibitory properties. Going in line with our observations, Dufresne et al reported that MΦ-1 inhibited the proliferation of human bladder carcinoma cells 2O2, another potentially cytotoxic agent, from mouse macrophages When investigating the influence of moDC and MΦ on the proliferation of the L1236 cHL cell line, we found no effect of MΦIn conclusion, mature mDC, which associated with improved outcome in cHL patients, might partake in the antitumoral immunity either by antigen presentation or by direct cytotoxicity. CD163+ MΦ, in contrast, were associated with inferior survival and might actively be induced and maintained by the tumor cells. However, no typical immunosuppressive profile but rather an excess of anti- and pro-inflammatory cytokines was observed in cHL patients with adverse prognostic markers.Figure S1GM-CSF and anti-inflammatory MΦM-CSF.Generation of moDC, pro-inflammatory MΦ (A) Human peripheral blood monocytes were differentiated into moDC, MΦGM-CSF or MΦM-CSF in the presence of IL4 + GM-CSF (6 days), GM-CSF (5 days) or M-CSF (5 days), respectively. Depicted are monocytes one day after isolation (left), moDC with branching projections, MΦGM-CSF with a fried-egg morphology and MΦM-CSF with a spindle-shaped morphology. All images were acquired with an Olympus IX81  using cellSens dimension software. (B) Purity analysis of moDC, MΦGM-CSF and MΦM-CSF. moDC purity was shown by FACS analysis of CD11c, CD14, CD3 and CD20 (BD Pharmingen) expression, being 98%, 12%, 0% and <1%, respectively. MΦ purity was proven by FACS analysis of CD68, CD11b, CD3 and CD20 (BD Pharmingen). MΦ-1 were positive in 95%, 92%, <1% and <1%, MΦ-2 in 98%, 86%, <1% and <1%, respectively. Data are indicated as mean with SEM of 4 independent experiments. (C) Analysis of cytokine secretion. For the determination of IL10 and IL12 secretion DuoSet ELISAs (R&D Systems) were applied according to the manufacturer's instruction. For generation of supernatants (SN), cells were seeded at a concentration of 1×106/ml+/−100 ng/ml LPS (Sigma-Aldrich) and incubated for 24 h. Absorbance was measured at 450 nm and 540 nm as reference using Synergy 2 . Data are shown as mean with SEM of 3–4 independent experiments each.(PDF)Click here for additional data file.Figure S2Characterization of the maturation and activation profile of moDC and cytokine profile of cHL cell lines L1236 and HDLM2. (A) moDC were treated for 24 h with 10 ng/ml TNFα, IL10 or TGFβ (each 10 ng/ml) or with 50% B-cell supernatants. B-cells were isolated from human blood PBMC by MACS separation (positive selection) using CD19 MicroBeads, FcR Blocking reagent and MS columns for an OctoMACS separator . Supernatants were generated at a concentration of 1×106 cells/ml medium after 48 h incubation. The expression of the DC activation marker CD40, the monocyte marker CD14 and the DC maturation marker CD83 was determined by flow cytometry. As anticipated, TNFα induced the activation marker CD40 and the maturation marker CD83, IL10 counter-acted moDC development as shown by the up-regulation of the monocyte marker CD14, and TGFβ blocked moDC maturation by down-regulation of CD83. SN generated from B-cells from PBMC showed no effect on moDC when compared to medium controls Mean fluorescence intensity (MFI) is indicated on the bar charts. Isotype controls are depicted as dotted lines. Paired student's t-tests or Wilcoxon signed-rank tests were conducted with *P<0.05, **P<0.01 and ***P<0.001. (B) Cytokine profile of cHL cell lines L1236 and HDLM2. The expression of TNFα, TGFβ and IL10 was analyzed by ELISA (R&D Systems). L1236 showed higher TNFα expression than HDLM2. Data are indicated as mean with SEM of at least 3 independent experiments.(PDF)Click here for additional data file.Figure S3Effect of GM-CSF and M-CSF on L1236 proliferation and proliferative activity of MΦ in co-culture with L1236. (A) Co-cultures of L1236 and MΦ were kept in 50% MΦ-conditioned medium containing GM-CSF or M-CSF. Therefore we analyzed the effect of GM-CSF and M-CSF on L1236 growth. L1236 were treated with 25 ng/ml GM-CSF or M-CSF in the presence of BrdU for 48 h. BrdU was stained by an AlexaFluor 488 secondary antibody and fluorescence intensity (FI) was measured by a fluorescence reader. No significant influence of GM-CSF or M-CSF on L1236 proliferation was observed. Mann-Whitney-U-tests were conducted. Data are indicated as mean with SEM of 3 independent experiments. (B) L1236 cells were co-incubated wit MΦGM-CSF or MΦM-CSF for 48 h in a ratio of 1∶6 and immunocytochemical doublestainings with Ki67 and CD68 antibodies was conducted. No difference in Ki67 expression in MΦGM-CSF and MΦM-CSF was observed. Wilcoxon signed-rank test was conducted. Data are indicated as mean with SEM of 8 independent experiments.(PDF)Click here for additional data file.


There are no previous reports comparing CSF biochemistry and its flow dynamics. As suggested by Milhorat, the complex system of CSF circulation through the brain may help to maintain a balance between the CSF flow rate and the CSF biochemical composition between different compartments. We hypothesize that this equilibrium may be disturbed in hydrocephalus patients and by using flow-MRI and CSF biological assessment, we could determine if alterations of CSF flow found in hydrocephalus patients affects their CSF biochemistry.9 hydrocephalus patients, 73 ± 8 years old, underwent a morphological MRI in which two CSF flow acquisitions were added to quantify CSF stroke volume in the aqueduct and in the spinal canal. A dynamic CSF index (Dynindex) was calculated equal to the product of the two CSF stroke volumes and to the cardiac cycle duration.All patients had a CSF tap test insertion of a shunt. During these operations, CSF was collected from both the ventricular and spinal compartments. From these CSF samples, standard biochemical measurements were performed including chlorine, glucose and protein levels. For each CSF analysis, a biochemical ratio (Biochratio) was defined by divided the ventricular concentration by the spinal concentration.The ventricular and spinal CSF stroke volumes were heterogeneous among the 9 patients. In comparison with previous normal aging investigations, stroke volumes were respectively in the aqueduct and the spinal compartments: diminished ; normal  and increased  . The Dynindex varied from 0 to 100 with a mean value equal to 35. No difference between the ventricular and the spinal compartment was found for the concentrations of chlorine and glucose, and their Biochratio were respectively: 0,99±0.01 and 0,91±0.10. In contrast, the Biochratio of the protein demonstrated very different values amongst the population (from 1.21 and 5.58) and for each patient, Biochratios were strongly correlated  with the corresponding Dynindex.In the this small hydrocephalus population, CSF flow varied between the ventricles and the spinal compartments. Chlorine and glucose concentrations were equal in the ventricles and the spine, but protein concentration was different between the ventricles and spine and was highly correlated with the CSF flow oscillations.


A repeat LP (RLP) may be done 1–3 days after the initial DLP, if the patient shows no improvement. If a patient with ABM is not doing well after three days, adequacy of antimicrobial therapy is the main concern. Other reasons for RLP is to detect possible intracranial complications of ABM unrelated to adequacy of therapy [A common clinical problem concerns the utility of repeat lumbar puncture (LP) in adults with acute bacterial meningitis (ABM), e.g., pneumococcal meningitis . An LP i therapy . S. pneumoniae ABM are highly elevated (>12 mmol/L). Equally important, should the CSF lactic acid levels obtained on RLP have greatly decreased, this is predictive of cure. In the case of ABM, the most important CSF as predictor of therapeutic efficacy and good prognosis are CSF glucose and CSF lactic acid levels [Cerebrospinal fluid (CSF) Gram stain and culture aside, the single most important CSF diagnostic parameter of ABM is the CSF lactic acid level ,4,5,6, ei.e., the higher the CSF albumin, the greater BBB penetrability [S. pneumoniae (PRSP), and are often empirically treated with “high dose” vancomycin. In spite of the “high dose” vancomycin, CSF penetration is not assured as only ~15% of vancomycin simultaneous serum levels penetrate the CSF in the presence of inflammation, i.e., ABM and <1% penetrates the BBB in the absence of inflammation, i.e., no ABM. Therefore, increasing the dose of an inflammation dependent antibiotic, e.g., vancomycin, is likely to be ineffective in the case of PRSP ABM. In contrast, “meningeal doses” of third generation cephalosporins or meropenem penetrate the CSF if PRSP is administered in therapeutic concentrations [Antibiotic penetration into CSF depends on the antibiotic’s molecular size, lipid solubility and degree of blood brain barrier (BBB) permeability. The best index of BBB penetrability is CSF albumin, rability . This istrations .In ABM, RLP with a marked decrease in CSF lactic acid levels, is predictive of cure. Clinically this is important since the CSF Gram stains may remain positive for RLP . A persiUnless the patient is clearly not improving or has even deteriorated, RLP is usually not necessary. CSF lactic acid levels are helpful both diagnostically in the DLP and prognostically in the RLP, and helpful in confirming whether antimicrobial therapy is effective and predictive of cure. The treatment for “penicillin resistant” pneumococci (PRSP) remains as penicillin . In contrast to vancomycin, in “meningeal doses” of third generation cephalosporins, e.g., ceftriaxone 2 g (IV) q12 h, the penicillin non-susceptibility and resistance breakpoints for PRSP are well below achievable CSF concentrations . CSF lev


Darunavir/r (DRV/r) is currently used at a dose of 800/100 mg once daily (OD) in a high proportion of patients. Pharmacokinetic data suggest that 600/100 OD may be effective, reducing toxicity and cost. However, drug concentrations in reservoirs such as cerebrospinal fluid (CSF) might not be adequate to inhibit viral replication. We aimed to evaluate concentrations of DRV and HIV-1 viral load (VL) in CSF patients receiving DRV 600/100 mg OD.DRV600 is an ongoing randomized open study comparing DRV/r 800/100 mg (DRV800) vs 600/100 mg (DRV600) OD plus TDF/FTC or ABC/3TC in 100 virologically suppressed patients (eudraCT 2011-006272-39). Here we present the results of a CSF sub-study. A lumbar puncture (LP) was performed in participating patients after at least six months of inclusion in the study, 20–28 hours after a dose of DRV/r. VL  was determined in CSF and in plasma. DRV concentrations were quantified in CSF by liquid chromatography mass spectrometry (LC/MS/MS) and in plasma using high-performance liquid chromatography (HPLC).Sixteen patients were included (eight in each arm). All DRV600 patients and four out of eight DRV800 patients received TDF/FTC, and the other four ABC/3TC. 75% were males, median (range) age was 48 (17–71) years, CD4 cell count 532 cells/mL . Median total time on DRV/r was 30 (11–57) months, and since the beginning of the study 8 (6–12) months in DRV800 and 10 (7–12) months in DRV600 patients. LP was performed a median of 26 (24–28) hours after the last DRV/r+TVD or KVX dose. In DRV600 patients the median DRV plasma levels were 1,674  ng/mL, CSF levels 17.08 (5.79–30.19) ng/mL and DRV CSF:plasma ratio 0.0084 (0.0028–0.0388), while in the DRV800 arm, median DRV plasma levels were 1,707  ng/mL, CSF levels 13.23 (3.47–32.98) ng/mL and DRV CSF:plasma ratio 0.0104 (0.0018–0.0262). All patients had VL<40 copies/mL in plasma and 14 patients VL<40 copies/mL in CSF. Two patients  had detectable VL in CSF (280 and 159 c/mL). These patients had the lowest CSF DRV concentrations (5.47 and 3.47 ng/mL), with plasma DRV concentrations of 802 and 958 ng/mL respectively.CSF DRV concentrations and CSF VL were similar between patients receiving DRV/r 800/100 mg or 600/100 mg OD. Low CSF DRV concentrations might be associated with viral escape in CNS. This may be taken into account in patients receiving OD DRV/r. Larger studies should confirm these findings.


Cerebral infarction is an ischemic stroke resulting from a disturbance in the blood vessels supplying blood to the brain, being the leading cause of physical and cognitive disabilities in adults . The curMus musculus) males (n = 16), weighing approximately 30 g, underwent global cerebral ischemia. Was occluded common carotid arteries for 80 minutes, and after this period the blood flow of the common carotid artery was released, while the arterial blood supply to the left remained interrupted. The stroke animals received vehicle (5% glucose solution) or were treated with G-CSF at a dose of 100 mg /kg /day, administering it after 24 hours of treatment. All the experimental procedures were performed in accordance withNational Institutes of Health (NIH) guidelines, and study protocols were previously approved by the Institutional Animal Care and Use Committee (CEUA Protocol # 011/2011). The measurement of the strength of the mice was performed in the pre and post surgery through software coupled to a force transducer. The quantification of the area of cerebral infarction using 2,3,5-triphenil tetrazolium chloride was established by morphometric analysis using Image J program (NIH). The data are presented as means ± SEM. Statistical analysis was performed using Student's t test for comparison of groups using the software Prism® 5.0 . p values <0.05 were considered to be statistically significant.Swiss webster mice  when compared with vehicle group (71 ± 5 g). The areal extent of cerebral infarction was significantly lower (p <0.05) in animals treated with G-CSF  compared to the control group (0.401 ± 0.07 cm2).A significant increase in the number of circulating leukocytes in the animals treated with G-CSF  was observed. The strength after surgery was significantly higher (p <0.05), in the group treated with G-CSF (88 ± 4 g; Our results demonstrate the neuroprotective effect of G-CSF in mice undergoing ischemic brain, thereby contributing to the reduction of neurofunctional impairment caused by stroke, as the maintenance of strength in the treated group.


Background: Pollination has a great effect on the yield of fruit trees. Blow flies are considered as an effective pollinator compared to hand pollination in fruit orchards. Therefore, this study was designed to evaluate the effect of different pollination methods in mango orchards.Methodology: The impact of pollination on quantity and quality of mango yield by blow flies was estimated by using three treatments, i.e., open pollinated trees, trees were covered by a net in the presence of blow flies for pollination, and trees were covered with a net but without insects.Results: The maximum number of flowers was recorded in irregular types of inflorescence, i.e., 434.80 flowers/inflorescence. Fruit setting (bud) was higher in open pollinated mango trees (i.e. 37.00/inflorescence) than enclosed pollination by blow flies (i.e. 22.34/inflorescence). The size of the mango fruit was the highest (5.06 mm) in open pollinated tree than those pollinated by blow flies (3.93 mm) and followed by without any pollinator (3.18 mm) at marble stage. We found that the maximum weight of mango fruit (201.19 g) was in open pollinated trees.Discussion: The results demonstrated that blow flies can be used as effective mango pollinators along with other flies and bees. The blow flies have shown a positive impact on the quality and quantity of mango. This study will be helpful in future and also applicable at farm level to use blow flies as pollinators that are cheap and easy to rear. Mangifera indica L., is very popular and economically important fruit. It is widely cultivated in the tropical and subtropical areas of the world  favor yellow and white colored models over brown and purple ones in the presence of a sweet scent, but the opposite in the presence of a carrion scent while unscented models were ignored. The survey by Unfortunately, research regarding pollinators usually focuses on hymenopterans, syrphids and butterflies. Therefore, all the ecology conservation schemes and other strategies are predominantly aimed to conserve these insects . Very liApis dorsata F., Apis florea F., Episyrphis balteatus De Geer., Ischiodon scutellaris F. and lucilia spp , Bahauddin Zakariya University, Multan. A total of three trees and 10 branches from each tree were selected for recording the data. The following treatments were used: (1) open pollinated trees; (2) trees were covered by net and blow flies were used for pollination; (3) trees were covered by nets and no insect was kept inside the net for pollination. Three replications were used for each treatment.Calliphora spp.) were collected from the different poultry farms of Multan, Pakistan. Mass culture of blow flies was reared in the Bio-Ecology Laboratory of Faculty of Agricultural Science and Technology, Bahauddin Zakariya University, Multan Pakistan. Adults were released into the plastic cage (18 cm in diameter and 24 cm in height) with diet (10% honey solution), and chicken livers were also placed in the plastic trays for egg laying. The six plastic cages were used for rearing blow flies. Then hatched larvae were separated into the plastic pots (4 cm in diameter and 8 cm in height) that were half filled with sterilized sand and 50 g chicken liver. In each plastic pot, 20 larvae were released and maximum adults of blow flies were reared in the laboratory for field application.Adults of blow fly . Mango trees with a height of 2.1 m and width of 2.4 m at the emergence of inflorescence were selected for the installation of cages. The cages, made by muslin cloths, were used for the covering of mango trees (3.35 × 3.35 × 3.35 m). A total of 100 adult blow flies were released for pollination efficacy in the covered mango trees  and contTotal number of flowers and their types of inflorescences were counted in each treatment. Data as number of flowers on each type of inflorescence, size (mm) and weight (g) of fruits at marble stage (30 days after the fruit set and have no stone (stage of seed) inside the fruit) of mangos was recorded by tagging ten twigs in each repeating unit.The data regarding type of the inflorescence  in three different treatments , numbers of flowers, buds, fruits, size and weight of fruits were subjected to statistical analysis using analysis of variance (ANOVA). Means were compared by use of least significance difference test at P = 0.05. Data were analyzed using SAS program .Number of inflorescences was initially counted before pollination/treatment among each type of inflorescence  . The numThe highest number of buds/inflorescence were found in open mango pollinated trees (2.67 ± 0.51/inflorescence) than the blow fly cage and the cage without pollinators, in case of conical inflorescence . Data regarding pyramid type of inflorescence depicted that maximum numbers of buds (1.96 ± 0.45/inflorescence) were observed in open pollinated trees than blow fly and without pollinators cage trees . Similar pattern of results was found in case of irregular inflorescence where higher number of buds i.e. 1.93 ± 0.47/inflorescence were found as compared to rest of two treatments  . A signiA significant difference was recorded in mango fruit formation at marble stage among three tested treatments in each type of inflorescence . The number of fruits was significantly higher with conical inflorescence (1.20 ± 0.20/inflorescence) than in blow fly and close cage/without pollinators trees . Number of fruits (1.24 ± 0.21/inflorescence) at marble stage on pyramid inflorescence was more in open pollinated trees than other two treatments . A higher number of fruits/inflorescences was found in open pollinated mango trees, at marble stage, on irregular inflorescence (1.36 ± 0.20/inflorescence) than in blow fly pollinated and unpollinated cages of trees  .The average size and weight of mango fruits at marble stage varied significantly among the treatments. The average size of mango fruits, i.e. 5.06 ± 0.29 mm, was statistically higher in open pollinated trees than in blow fly pollinated cages and closed cages trees . A similar pattern was also observed in the case of weight of mango fruits where average weight of each mango i.e. 210.20 ± 13.92 g, was significantly higher as compared to that in blow fly pollinated cages and closed cages trees  . Data shA huge number of economically nutritive plants depend on different types of pollinators for pollination . Modern A mango panicle contains around 200–4,000 flowers, and a mature tree may have approximately 600–1,000 panicles . About 4Apis dorsata F., Apis florea F., Episyrphis balteatus De Geer., Ischiodon scutellaris F. and lucilia spp at the farm (Pollinators are one of the main components of the ecosystem . There athe farm . The oveM. indica. Therefore, this research will be helpful in the future and will be a applicable at the farm level where keeping honey in the orchard is difficult for pollination because of the environment and the high cost. We speculated that blow flies are the best, cheapest source of pollination as a replacement for honey bees and other pollinators which are expensive to purchase and to maintain in the orchards for pollination. Blow flies would be easily maintained in orchards by providing a diet of organic matter, dead birds etc, in one side of the mango orchard. This study also showed that irregular types of inflorescence lead to the maximum number of flowers, buds and fruits, so breeders could focus on developing the varieties of M. indica by having a greater number of irregular types of inflorescence.The results revealed that fruit setting was better in open trees than blow flies and without pollinated trees, respectively. Mango weight and size of was significantly better in open trees than blow flies and without pollinators. However, we detected fruits with maximum weight and size in the open pollinated mango trees where a greater number of pollinators visit the trees for pollination and resulted in the better quality and quantity of mango fruit. We concluded that blow flies have the potential for pollination in 10.7717/peerj.2076/supp-1Supplemental Information 1Click here for additional data file.


Arabidopsis thaliana (Brassicaceae) has led to a richer understanding of the molecular genetics underlying this diversity. The character of individual meristems, a combination of the number (determinacy) and nature (identity) of the products a meristem produces, is key in the development of plant form. A framework that describes inflorescence development in terms of shifting meristem identities has emerged and garnered empirical support in a number of model systems. We discuss this framework and highlight one important aspect of meristem identity that is often considered in isolation, phyllotaxis. Phyllotaxis refers to the arrangement of lateral organs around a central axis. The development and evolution of phyllotaxis in the inflorescence remains underexplored, but recent work analyzing early inflorescence development in the grasses identified an evolutionary shift in primary branch phyllotaxis in the Pooideae. We discuss the evidence for an intimate connection between meristem identity and phyllotaxis in both the inflorescence and vegetative shoot, and touch on what is known about the establishment of phyllotactic patterns in the meristem. Localized auxin maxima are instrumental in determining the position of lateral primordia. Upstream factors that regulate the position of these maxima remain unclear, and how phyllotactic patterns change over the course of a plant's lifetime and evolutionary time, is largely unknown. A more complete understanding of the molecular underpinnings of phyllotaxis and architectural diversity in inflorescences will require capitalizing on the extensive resources available in existing genetic systems, and developing new model systems that more fully represent the diversity of plant morphology.Inflorescence morphology is incredibly diverse. This diversity of form has been a fruitful source of inquiry for plant morphologists for more than a century. Work in the grasses (Poaceae), the tomato family (Solanaceae), and  Inflorescence morphology is an important determinant of yield in agricultural settings, and fitness in natural ones Wyatt, . Inflorephyllotaxis . This paper highlights two phyllotactic shifts, one at the evolutionary level  and another at the developmental level (from vegetative to reproductive development).Although all grass inflorescences are termed “panicles,” this descriptor belies the immense diversity within the family Box . A recenichy Box  in the lBox 1KEY CONCEPT 1The geometric arrangement of organs around a central axis. Phyllotactic patterns are determined largely by auxin and cytokinin gradients in the meristem. Altered hormone activity likely underlies developmental shifts in phyllotaxis.Meristem identity refers to the type of primordia produced, whereas meristem determinacy refers to the number of primordia produced and whether or not the meristem is consumed in the production of primordia. For example, a meristem has floral meristem identity if it initiates floral organs and floral meristems are determinate because they are consumed in the production of a limited number of organ primordia. Each one of these factors can vary across both developmental and evolutionary time, leading to adult inflorescence architectures that show vast morphological diversity.The development of inflorescence morphology involves a complex interplay between a number of interacting processes: establishment of phyllotaxis and meristem identity, as well as variable patterns of cell division and expansion. As in all plant development, meristems and meristematic cells are of key importance in inflorescence development. Meristems are pools of undifferentiated cells that generate both more meristematic cells, as well as differentiated cells that form lateral organs. The particular character of a meristem can be described using the interrelated concepts of meristem identity and determinacy. KEY CONCEPT 2Meristem identity refers to the type of primordia produced by a meristem, whereas meristem determinacy refers to the number of primordia produced and whether or not the meristem is consumed in the production of primordia.KEY CONCEPT 3The three dimensional arrangement of branches, leaves, flowers, and floral buds in an inflorescence Box . InfloreArabidopsis, has lead to greater understanding of the regulatory logic that underlies inflorescence development , petunia (Petunia hybrida), Arabidopsis thaliana, and Pisum sativum (Fabaceae) have revealed a framework of progressive changes in meristem identity underlying morphology.Meristem identity changes during plant development. One key change in meristem identity is at the transition to flowering, where the vegetative meristem (VM) or shoot apical meristem (SAM) transitions to an inflorescence meristem (IM). A conceptual framework is emerging for understanding inflorescence development after this point in terms of further transitions between meristem identities. Computational modeling and molecular analyses of inflorescence development in the grasses, tomato (vegetativeness (veg). For a meristem to transition to floral meristem fate, veg must drop below a threshold. veg in individual meristems could be specified by two major regulators of inflorescence development in Arabidopsis: LEAFY (LFY) and TERMINAL FLOWER1 (TFL1). LFY and TFL1 are transcription factors with roughly opposite roles. LFY promotes the transition from inflorescence to determinate floral meristem fate, while TFL1 acts to repress floral meristem fate, effectively maintaining indeterminacy of the IM  and its rice ortholog, FRIZZY PANICLE1 (FZP1) regulate SM identity and determinacy. In bd1 and fzp1 mutants, SMs take on a more BM-like identity  genes are key regulators of SPM  and SM (RA3) determinacy , several genetic mutants that disrupt inflorescence architecture have been characterized and cloned  plays a role in specifying secondary inflorescence meristem identity. In veg1 mutants, VMs form in the position of secondary inflorescence meristems, which never produce flowers. VEG1 encodes an AGL79-like MADS-box gene that appears to be eudicot-specific , occupying distinct regions of the inflorescence Troll, . The conTitanotrichum oldhamii (Gesneriaceae), for example, later “floral” meristems are replaced with bulbils under short day conditions. A LFY homolog is downregulated in these bulbils, as expected for a transition to VM identity , where vegetative growth continues after flowering  . As researchers sample further there may be more examples of genetic conservation, but we need more genetic models, throughout the angiosperms, to find the genes. For example, thyrses   and squamosa (squ) mutants, floral meristems are converted to IMs, and whorled floral phyllotaxis is replaced by spiral phyllotaxis  to revert to an indeterminate branch-like meristem, and have BM-like phyllotaxis, rather than FM-like whorled phyllotaxis  to floral meristem identity  is often accompanied by a shift to whorled phyllotaxis Figure  Bell an. EarlierCorngrass1 (Cg1) overexpresses miR156 and extends the juvenile phase. Cg1 inflorescences, while still spiral, have abnormal phyllotaxis and do not produce ordered rows of meristems   in Arabidopsis, bearded-ear1 (bde) in maize, and OsMads3/53 in rice), albeit these mutants usually do not exhibit a discrete shift from one phyllotactic pattern to another  mutant. tsh4 suppresses bract outgrowth in the inflorescence and in tsh4 mutants, cells that would normally be partitioned to the axillary meristem are partitioned to the bract. This reallocation of cells results in increased meristem determinacy , as well as disruption of phyllotaxis in the ear  coincides with primordium initiation  auxin efflux carriers, of which PIN1 appears to be most critical for initiation of lateral primordia during reproductive development  in the central zone of the SAM  transcription factors are required for spiral phyllotaxis in the Auxin promotes organogenesis at least in part by affecting cell wall rigidity. Demethylesterification of pectin, which increases elasticity in the meristem, is necessary and sufficient for primordium initiation. Overexpression of pectin methylesterase 5 results in increased numbers of lateral primordia, with aberrant phyllotaxis  and the rice decussate (dec) mutants stably change leaf phyllotaxis from distichous  to opposite decussate  that negatively regulates cytokinin signaling  controls the size of the stem cell niche by negatively regulating stem cell number and directly represses ARR genes  mutant of maize occasionally leads to a shift to opposite decussate phyllotaxis , ARF5/MP, directly modulates the expression of two ARR genes, ARR7 and ARR15 and the cytokinin inhibitor, AHP6  and timing determinates (time between subsequent primordium initiation events), this interplay is a key determinate of phyllotaxis. In maize PLT or IDD genes), which can differ depending on meristem identity. Thus, phyllotaxis is intimately linked to and one aspect of meristem identity.Molecular analyses clearly indicate that the position of auxin maxima determines the position of lateral primordia and thereby phyllotactic pattern. Cytokinin also appears to play a key role in generating robust phyllotactic patterns. How then do phyllotactic patterns change, both over the course of development, and across evolutionary time? The positioning of auxin maxima, modulated by cytokinin signaling, appears to be key. This suggests that regulation of auxin synthesis and transport is different in meristems with different phyllotactic patterns. Differences in auxin transport and regulation are likely due to differential expression or regulation of PIN proteins or auxin biosynthesis genes  or between species (over evolutionary time). One of the first steps to understanding evolutionary shifts in phyllotaxis is to continue to trace phyllotactic patterns across the tree  are direct targets of RA1  signaling within the meristem, meristems with different identities (and phyllotaxes) must also differentially regulate auxin accumulation and signaling. Indeed, in ChIP-Seq and RNA-Seq experiments, transcriptional regulators of meristem identity  directly regulate genes in the auxin regulatory network (Kaufmann et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Star, Hsd3b1, and Hsd17b3) and testis-descent related gene Insl3 as well as protein levels of 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and insulin-like 3 (INSL3) at ≥10 mg/kg. DCHP significantly inhibited testicular testosterone levels at ≥100 mg/kg. The results indicate that in utero exposure to DCHP affects the expression levels of fetal Leydig cell steroidogenic genes and results in the occurrence of multinucleated gonocytes and Leydig cell aggregation.Dicyclohexyl phthalate (DCHP) is one of the phthalate plasticizers. The objective of the present study was to investigate the effects of DCHP on fetal Leydig cell distribution and function as well as testis development. Female pregnant Sprague Dawley dams orally received vehicle  or DCHP  from gestational day (GD) 12 to GD 21. At GD 21.5, testicular testosterone production, fetal Leydig cell number and distribution, testicular gene and protein expression levels were examined. DCHP administration produced a dose-dependent increase of the incidence of multinucleated gonocytes at ≥100 mg/kg. DCHP dose-dependently increased abnormal fetal Leydig cell aggregation and decreased fetal Leydig cell size, cytoplasmic size, and nuclear size at ≥10 mg/kg. DCHP reduced the expression levels of steroidogenesis-related genes (including  Dicyclohexyl phthalate (DCHP) is one of a family of synthetic compounds known as phthalates. It is a diester formed by reaction of a molecule of phthalic acid with two molecules of cyclohexyl alcohol. It is widely applied to stabilize rubbers, resins, and polymers, including nitrocellulose, polyvinyl acetate, and polyvinyl chloride. In polyvinyl chloride products it is mainly used as a plasticizer, which increases polyvinyl chloride flexibility, transparency, durability, and longevity.DCHP is not covalently bound to the polymers ,2. Insteet al. investigated the two-generation effects of DCHP exposure at doses of 240, 1200, and 6000 ppm  per day in the diet from gestational day (GD) 1–21 of F0 females in Sprague Dawley rats [in utero to DCHP at 250, 500, and 750 mg/kg per day by gavage from GD 6 to GD 20, male fetuses at GD 21 had low body weight at 750 mg/kg and reduced anogenital distance (AGD) at 500 and 750 mg/kg doses, suggesting that the anti-androgenic effect of DCHP, since AGD is the biomarker of androgen-dependent action [Hsd3b1) and 17β-hydroxysteroid dehydrogenase 3  [Insl3) mRNA/protein (INSL3) expression in the fetal testis have been well documented in some phthalates including di(2-ethylhexyl) phthalate (DEHP) and di(2-butyl) phthalate (DBP) [Several reports about the effects of DCHP on mammalian development and reproductive system were published ,6,7. HosHsd17b3) ,11). TheHsd17b3) . TesticuHsd17b3) . The occte (DBP) . Therefote (DBP) .Testosterone and INSL3 are secreted by fetal Leydig cells in the male embryo. Fetal Leydig cells are important testicular endocrine cells, which are arranged usually in clusters with each cluster containing two or more fetal Leydig cells surrounded by several lawyers of collagen-rich membrane in the interstitial space between seminiferous tubules. Fetal Leydig cells are typically different from adult Leydig cells, the second generation of Leydig cells that emerge during pubertal period and do not appear in clusters. Fetal Leydig cells play important roles in the development of the male reproductive tract and the testis descent . The numn = 6 for each group). The choice of maternal exposure window from GD12 to GD21 is based on the fact that the fetal Leydig cells emerge at GD12 and a dam gives birth at GD21.5 [2 at GD 21.5 . Three sets of randomly selected fetal testes (at least one testis per dam) were immediately removed, frozen in liquid nitrogen, and stored at −80 °C for further analysis of cell distribution (six testes per group), Leydig cell-specific mRNA levels (six testes per group), and testicular testosterone (six testes per group). Other remaining testes were fixed by Bouin’s solution for one day for histochemical staining of testes, which were used for analysis of testis dysgenesis (six testes per group), Leydig cell morphological changes (six testes per group), and semiquantation of Leydig cell specific protein levels (6 testes per group).Adult male and female Sprague-Dawley rats were purchased from the Shanghai Laboratory Animal Center . After acclimation for one week, male and female rats were mated. When the pregnancy was confirmed, the female rat was individually housed , in a 12-h light-dark cycle environment. The animals were housed in individually ventilated caging (IVC) cages (one rat per cage) on soft chip bedding and provided pellet chow . This study was approved by the Wenzhou Medical University Laboratory Animal Ethics Committee , and all procedures were performed in accordance with the policies. The investigation conformed to the procedures described in the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health . DCHP was purchased from Sigma  and suspended in corn oil (vehicle control) for gavage. The pregnant dams were gavaged with DCHP (Sigma) at 0 , 10, 100, or 500 mg/kg body weight daily from GD 12 to GD 21  as described [Testicular steroids were extracted from the testes of control and DCHP-exposed pups  diluted 1:200 for 1 h at room temperature. The antibody-antigen complexes were visualized with diaminobenzidine alone, resulting in brown cytoplasmic staining in positively labeled peritubular cells. The sections were counterstained with Mayer hematoxylin, dehydrated in graded concentrations of alcohol, and cover-slipped with resin . The testis showing at least one area of focal testis dysgenesis was referred as the focal dysgenic testis. The incidence of focal testis dysgenesis was calculated as the number of the focal dysgenic testis divided by the number of testes counted.In the fetal testis, the seminiferous tubules were surrounded by peritubular myoid cells. The peritubular myoid cell layer was brightly positive for desmin . In the Sections were stained using hematoxylin and eosin stain. To analyze the occurrence of MNGs, we used an Olympus BH-2 microscope  to capture image. One complete testis cross-section from each animal was analyzed, and the percentage of seminiferous cord cross-sections that contained one or more MNGs was recorded and calculated as the percentage in total tubules counted.+ as a cofactor. In addition, the incubation medium contained tetranitroblue tetrazolium as the H+ acceptor, and 0.1 M phosphate buffer at pH 7.2. The sections were incubated for 30 min at 37 °C in a humidified chamber in darkness. Additional sections were incubated without substrate as negative controls in order to determine nonspecific dehydrogenase effect. The sections were washed using phosphate buffered saline twice, and fixed using 4% phosphate saline buffered paraformaldehyde solution. After twice washes, the sections were counterstained using 4′6-diamidino-2-phenylindole . The sections were covered with 50% glycerol and covered with a coverslip. The morphology of the testis was observed using a fluorescence microscope (Olympus BH Series) at excitation wavelength 350 nm combined with bright-field photography. The number of fetal Leydig cell was counted by a stereological method as described [3β-HSD is the biomarker of the fetal Leydig cell. Enzymological staining for 3β-HSD in the frozen section of the fetal testis was performed as described previously with a little bit modification . In brieescribed . In brieescribed .The immunohistochemical detection of 3β-HSD was performed as above. The sections were then incubated with a 3β-HSD polyclonal antibody diluted 1:200 for 1 h at the room temperature. The antibody-antigen complexes were visualized with diaminobenzidine alone, resulting in brown cytoplasmic staining in positively labeled Leydig cells. The sections were counterstained with Mayer hematoxylin, dehydrated in graded concentrations of alcohol, and cover-slipped with resin . Cell size and nuclear size were measured using image analysis software as below.2 of testis. Cell size and nuclear size were estimated using image analysis software . Perimeters of the fetal Leydig cell and nucleus were drawn and calculated by the image analysis software. The cytoplasmic area was calculated by the area of fetal Leydig cell minus its nuclear area. More than 50 fetal Leydig cells were evaluated in each testis and the cell size and nuclear size and their ratio were averaged.Eight randomly selected fields in each of three nonadjacent sections per testis were captured using a BX53 Olympus microscope  equipped with digital camera interfaced to a computer. The images that were displayed on the monitor represented areas of 0.9 mmRps16) mRNA level was assayed in each sample as the internal control. The primers of seven other testicular genes were provided in Lhcgr); cholesterol transporting genes including cholesterol LDL receptor (Scarb1), steroidogenic acute regulatory protein (Star) and steroidogenic enzyme genes including CYP11A1 (Cyp11a1), 3β-HSD1 (Hsd3b1), P450c17 (Cyp17a1), 17β-hydroxysteroid dehydrogenase 3 (Hsd17b3) and INSL3 (Insl3). The relative mRNA levels of targeted genes were normalized to Rps16  using double standard curve method as described [Total RNA was extracted from rat testes in TRIzol according to the manufacturer’s instruction . First strand synthesis and qPCR were performed as described . RibosomInsulin-like 3 (INSL3) is an important hormone secreted by fetal Leydig cells to induce the testis descent . 3β-HSD Values are expressed as mean ± SEM, and data were analyzed by using one-way ANOVA and then ad hoc Dunnett’s test to compare values from DCHP treated rats to control values. GraphPad Prism  was used.p < 0.001) at ≥10 mg/kg doses of DCHP (p < 0.05).The birth rate was defined as the number of pregnant rats that gave birth to pups/the number of female rats being pregnant as identified by the formation of the vaginal plug. The litter size each dam was defined as the number of pups that a dam gave birth to. When compared with the control, DCHP exposure did not affect the birth rate, the litter size, and the male-to-female sex ratio at GD 21.5 . The bod of DCHP . The AGDPhotomicrographs of testis showed that MNGs barely occurred in the testis of control group A, and leFetal Leydig cell numbers per testis did not change in all DCHP exposure groups A. The feFetal Leydig cells are not uniformly distributed in the interstitial space of the fetal testis but rather are found in discrete clusters . This waIntratesticular testosterone concentrations at GD 21.5 were measured. As shown in Insl3 , Lhcgr , Scarb1 , Star , and androgen biosynthetic enzymes . DCHP dose-dependently inhibited the expression of Insl3, Star, Hsd3b1, and Hsd17b3 at ≥10 mg/kg. At 500 mg/kg, DCHP also inhibited the expression of Lhcgr, Scarb1 and Cyp17a1  as well as testis decent related gene Insl3.In the present study, Sprague Dawley rats were administered orally with daily doses of DCHP from GD 12 to 21 with 10, 100, and 500 mg/kg. It remarkably induced testis dysgenesis syndrome, including the formation of focal testis dysgenesis, multinucleated gonocytes, and abnormal cluster of fetal Leydig cells, and the decreases of the expression levels of steroidogenesis-related genes  elicited fetal Leydig cell aggregation in the DCHP treated testis. The physiological consequences of fetal Leydig cell aggregation per se are uncertain, although there is evidence that it is associated with reduced fertility [DCHP increased the incidence of MNGs and focal testis dysgenesis after  and DBP ,20. FetaP or DBP ,13,21. WP or DBP ,13,21, Dertility  and incrertility . The hugertility .in utero exposure also significantly reduced testicular testosterone levels  in the gubernaculum. INSL3 acts on the gubernaculum to cause its shortening, thus inducing fetal testis descent ,27. Thus and DBP ,23,28,29P [Insl3 A.These results, taken together, indicate that fetal exposures to DCHP have affected on fetal Leydig cell number, distribution, and most importantly, steroidogenic capacity.


Rana sylvatica, survives whole-body freezing and thawing each winter. The extensive adaptations required at the biochemical level are facilitated by alterations to signaling pathways, including the insulin/Akt and AMPK pathways. Past studies investigating changing tissue-specific patterns of the second messenger IP3 in adapted frogs have suggested important roles for protein kinase C (PKC) in response to stress. In addition to their dependence on second messengers, phosphorylation of three PKC sites by upstream kinases (most notably PDK1) is needed for full PKC activation, according to widely-accepted models. The present study uses phospho-specific immunoblotting to investigate phosphorylation states of PKC—as they relate to distinct tissues, PKC isozymes, and phosphorylation sites—in control and frozen frogs. In contrast to past studies where second messengers of PKC increased during the freezing process, phosphorylation of PKC tended to generally decline in most tissues of frozen frogs. All PKC isozymes and specific phosphorylation sites detected by immunoblotting decreased in phosphorylation levels in hind leg skeletal muscle and hearts of frozen frogs. Most PKC isozymes and specific phosphorylation sites detected in livers and kidneys also declined; the only exceptions were the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641) antibody, which remained unchanged from control to frozen frogs. Changes in brains of frozen frogs were unique; no decreases were observed in the phosphorylation levels of any of the PKC isozymes and/or specific phosphorylation sites detected by immunoblotting. Rather, increases were observed for the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641), phospho-PKCδ (Thr505), and phospho-PKCθ (Thr538) antibodies; all other isozymes/phosphorylation sites detected in brain remained unchanged from control to frozen frogs. The results of this study indicate a potential important role for PKC in cerebral protection during wood frog freezing. Our findings also call for a reassessment of the previously-inferred importance of PKC in other tissues, particularly in liver; a more thorough investigation is required to determine whether PKC activity in this physiological situation is indeed dependent on phosphorylation, or whether it deviates from the generally-accepted model and can be “overridden” by exceedingly high levels of second messengers, as has been demonstrated with certain PKC isozymes .The wood frog,  Rana sylvatica , a second messenger associated with cytosolic calcium increases and a co-product of diacylglycerol , additional kinases and phosphatases have been shown to play a role in wood frog freeze-tolerance. For instance, mitogen activated protein kinases (MAPKs) are activated in various tissues and are suggested as having a role in regulating metabolic or gene expression responses that would facilitate survival in the freezing and/or thawing processes . Past strol DAG; . IncreasXenopus), the NCBI gene database contains entries for sequences identified as PKCα, PKCβ, PKCγ, PKCδ, PKCε, PKCζ, PKCη, PKCθ, PKCι, PKD/PKCμ. Our lab has previously demonstrated in vivo roles for PKC in various forms of animal stress physiology, including: (1) reptilian anaerobiosis  mamrobiosis , and;  in accordance with guidelines set down by the Canadian Council on Animal Care. Male wood frogs were captured from spring breeding ponds in the Ottawa, Ontario area. Animals were washed in a tetracycline bath, and placed in plastic containers with damp sphagnum moss at 5 °C for two weeks prior to experimentation. Control frogs were sampled from this condition. For freezing exposure, frogs were placed in closed plastic containers with damp paper toweling on the bottom, and put in an incubator set at −3 °C. A 45 min cooling period was allowed during which body temperature of the frogs cooled to below −0.5 °C and nucleation was triggered due to skin contact with ice crystals formed on the paper toweling . Subsequ3V O4, and 10 mM ß-glycerophosphate). Protease and phosphatase inhibitors were added just prior to homogenization: 1:1,000 v:v protease inhibitor cocktail , 1:1,000 v:v phosphatase inhibitor cocktail 1 , and a few crystals of phenylmethylsulfonyl fluoride (PMSF). Samples were centrifuged at 10,000 × g for 15 min at 4 °C and then supernatants were removed and held on ice.Soluble protein extracts were prepared from tissues that had been previously stored at −80 °C. Briefly, samples of frozen tissues were weighed and then quickly homogenized using a Polytron PT1000 homogenizer  at 50% of full power in a 1:5 w:v ratio with ice-cold buffer A , along with Kaleidoscope prestained markers  as a guide for the approximate molecular weight of PKC isozymes. On a typical 12-laned gel, 5 independently-prepared protein extracts from control frogs, and 5 independently-prepared protein extracts from frozen frogs, were loaded in parallel ; thus, for any given PKC isozyme or specific phosphorylation site being probed, all immunoreactive bands from both control and experimental animals detected at the chemiluminescence stage will have been treated identically through all electrophoretic, transfer, immunoblotting, and chemiluminescence/exposure steps. Samples were electrophoresed at 180 V in a Mini-PROTEAN III apparatus  using 1x running buffer . Proteins were then wet-transferred to polyvinylidene difluoride (PVDF) membrane  using a current of 300 mA for 1.5 h at 4 °C in a Bio-Rad Mini Trans-Blot Cell apparatus . Transfer buffer contained 25 mM Tris-base pH 8.8, 192 mM glycine, and 20% v:v methanol, chilled to 4 °C.βII Ser660) antibody (9371), which detects all of PKCα, βI, βII, δ, ε and η isoforms only when phosphorylated at a carboxy-terminal residue homologous to Ser660 of PKCβII; phospho-PKCδ/θ (Ser643/676) antibody (9376), which detects both PKCδ when phosphorylated at Ser643 and PKCθ when phosphorylated at Ser676; phospho-PKCα/βII (Thr638/641) antibody (9375); phospho-PKCδ (Thr505) antibody ; phospho-PKCθ (Thr538) antibody (9377); phospho-PKCζ/λ (Thr410/403) antibody (9378); PKD/PKCμ antibody (2052); phospho-PKD/PKCμ (Ser916) antibody (2051); phospho-PKD/PKCμ (Ser744/748) antibody (2054). All primary IgG antibodies were raised in rabbit. These were purchased together as the Phospho-PKC Antibody Sampler Kit . Stock primary antibodies were diluted between 1:5,000 and 1:10,000 in Tris-buffered saline supplemented with Tween-20 . Secondary antibody used was the anti-rabbit IgG, HRP-linked antibody . Stock secondary antibodies were diluted 1:2,000 in TBST. We opted to use these antibodies, focusing on phospho-PKC and not unphosphorylated forms of PKC, for two main reasons. Firstly, given that these antibodies were specifically distributed as an assembled kit (at that time), we were hesitant to introduce additional antibodies that had possibly been developed, raised, and purified differently  from those provided in the kit. Secondly, as will be further detailed in the Discussion section, the scope of this study followed the widely-accepted model that only phosphorylated PKC is catalytically active; we therefore were especially interested in phospho-specific forms, so as to relate our previous forays into PKC second messengers  were the following: phospho-PKC (pan) (ssengers  to resulRana sylvatica stress–tolerance studies conducted by our group) frogs will only be sacrificed and dissected once they have lost ∼40% of their total body water  using multiple antibodies but with a single starting tissue extract and PVDF membrane . This prdy water . These PTotal protein was then visualized on the PVDF membrane by staining for 30 min with Coomassie blue staining solution  followed by destaining with destain solution . Three Coomassie-stained bands that did not differ in intensity between active and frozen conditions were used to normalize the corresponding intensity of the immunoreactive band in each lane to correct for any unequal protein loading, as described previously . Our grop < 0.05 or smaller.Intensities of ECL-visualized and Coomassie-stained bands were quantified using the associated Gene Tools program (v. 3.00.02). Data were analyzed by one-way ANOVA followed by Tukey’s test; a statistically-significant difference was accepted with values of 3; IP3 will in turn trigger calcium efflux from the endoplasmic reticulum, which then propagates further calcium influx from the extracellular environment. Conventional PKC isozymes  bind to the membrane via two specific bridging interactions: C1 domains that bind to DAG and C2 domains that bind to calcium-phospholipid complexes. Once bound, cPKCs unfold such that their hydrophobic motifs interact with and activate 3-phosphoinositide dependent protein kinase-1 (PDK1), and the PKC pseudosubstrate motif is withdrawn from its catalytic core. PDK1, currently the single conclusive upstream kinase responsible for phosphorylation of the PKC activation loop  and atypical PKC isozymes . nPKCs are calcium-independent, but still rely on DAG for activity; aPKCs rely on neither calcium nor DAG, or any other phospholipids, though they possess other unique domains such as the phox-bem1 domain, which suggests that protein–protein interactions with cytosolic partners may be necessary for activity . A summary of changes between control and frozen frogs is presented in μ) tended to globally decrease during wood frog freezing in hind leg skeletal muscle, liver, kidney, and heart; the only tissue in which increases in phospho-PKC were observed was the brain.Extracts of hind leg skeletal muscle, liver, heart, kidney, and brain from control and frozen frogs were probed with all 9 primary antibodies of the Phospho-PKC Antibody Sampler kit; however, not all antibodies revealed the presence of immunoreactive bands in each tissue extract . In each case where antibodies detected bands, only a single and distinct band appeared in the area of our cut PVDF membrane section; immunoreactive bands were confirmed to be PKC isozymes by comparing their approximate molecular weights to those listed on the manufacturer’s datasheet provided  decrease in muscle and heart during freezing, suggesting decreased action of PDK1 in these tissues. By contrast, levels of both phospho-Ser241-PDK1 and phospho-Thr308-Akt increase in liver, suggesting increased PDK1 action in livers of frozen frogs . For optα/βII (Thr638/641), and phospho-PKCθ (Thr538). Bands detected by both these antibodies decreased in intensity in frozen frogs  antibody failed to reveal any bands. Overall, band intensities again tended to decrease in frozen frogs  decreased by over 50%. While the role of this turn motif phosphorylation is inconclusive in PKCδ or PKCθ , Ser744/748 is in fact phosphorylated by other PKC isozymes upstream of PKD/PKCμ, most notably PKCδ , which is in turn a substrate of PKC isozymes  (βII Ser660) antibody decreased in frozen frogs. This antibody is specific for PKCα, βI, βII, δ, ε and η when autophosphorylated at a carboxy-terminal residue homologous to Ser660 in the hydrophobic motif of PKCβII, following initial phosphorylation of Thr500 by PDK1; phospho-Ser660 plays important roles in correct folding, as well as the binding of protein substrate, ATP, and calcium  (βII Ser660) antibodies. Moreover, decreases were also observed in the intensities of the bands detected by the phospho-PKCζ/λ (Thr410/403) antibody. Thr410 is an activation loop residue in PKCζ, as is Thr403 in PKCλ , and these are directly phosphorylated by PDK1 and are critical to activity  (βII Ser660), phospho-Thr538-PKCθ, and phospho-Thr410/403-PKCζ/λ antibodies all decreased significantly; indeed, phospho-Thr676/643-PKCδ/θ, non-phosphorylated PKD/PKCμ, and phospho-Thr410/403-PKCζ/λ levels all decreased to an extent where they were no longer detectable in frozen frogs.6 antibodies revealed bands in frog kidney extracts, and all of these decreased in frozen frogs ; Table 13 levels in response to freezing  (βII Ser660) antibodies; each of these were detectable in both control and frozen frogs to approximately the same extent. Interestingly, phospho-Thr638/641-PKCα/βII levels increased by 121.3% in brains of frozen frogs. Moreover, phospho-Thr505-PKCδ and phospho-Thr538-PKCθ, which were not detectable in control frogs, were detectable  in frozen frogs. Overall, whereas phospho-PKC levels generally tended to decline in other organs of frozen frogs, phospho-PKC levels largely remained unchanged or even increased in brain.8 antibodies revealed bands in frog brain extracts. Of the five tissues investigated in the present study, the results obtained in the brain were the most unique; changes (or lack thereof) in phospho-PKC levels in brain during frog freezing contrasted strongly with those observed in muscle, liver, heart, and kidney ; Table 13 levels rose significantly after 4 h of freezing  moderately-increased expression of fr10  in brain, contrasted against the decreased or unchanged phosphorylation of PKC in other tissues, suggests a unique and vital role for brain PKC during freezing. Indeed, the importance of PKC in cerebral protection has been demonstrated well-beyond the niche of wood frog freezing . Future μ/PKD. Broadly, phospho-PKC levels and non-phosphorylated PKCμ/PKD decreased in muscle, livers, kidneys, and hearts of frozen frogs; the only exception was protein detected by the phospho-Thr638/641-PKCα/βII (turn motif), which showed no change in livers or kidneys between the control and frozen states. This isozyme alone would partially support the findings of past studies, where IP3 was shown to dramatically increase in livers of frozen frogs and thus an important role was suggested for PKC in the freezing process; however, even the steady phosphorylation state of Thr638/641-PKCα/βII is seemingly contradicted by the decreased phosphorylation of Ser660-PKC (pan) (hydrophobic motif) in both livers and kidneys of frozen frogs. A particularly interesting finding in this study is PKC would seem to play an important role in the brains of frozen frogs, as the phospho-levels of all isozymes detected remain either remain unchanged or even increase in the frozen state.The present study investigated the phosphorylation state of conventional, novel, and atypical PKC isozymes in five tissues of freeze-tolerant frogs, as well as non-phosphorylated PKCδ). To clarify the role of PKC in wood frog freezing, it is now evident that catalytic assays are necessary in order to unequivocally establish the actual activities of each of these isozymes in the control and frozen states. Moreover, there is an additional difficulty in contextualizing the role of PKC due to the fact that very few downstream targets of PKC have been assessed in wood frogs. Those that were discussed in this report are either only speculative , or are several degrees removed from being direct PKC substrates and/or are under the control of multiple kinases  There is a clear need to assess the phosphorylation state of direct PKC substrates  in order to better determine the activity and role of PKC in freeze-tolerance.The results of this study succeed in answering some questions of past studies pertaining to the state of PKC in freeze-tolerance, but raise many others and indeed pose some contradictions. Whereas our lab has previously asserted that based on second messenger patterns, PKC would play an important role (particularly in liver and muscle), our present findings would seem to instead suggest a diminished role for PKC in most tissues, based on our current understanding of PKC isozymes and their generally-accepted activation model. This in itself, however, can be debated by pointing to studies which demonstrate that activation loop phosphorylation is, in fact, not needed for PKC activity . 5 independently-prepared protein extracts from control frogs (left) and 5 independently-prepared protein extracts from frozen frogs (right) were electrophoresed, transferred, immunoblotted, and exposed in parallel.Click here for additional data file.10.7717/peerj.558/supp-2Supplemental Information 2A full blot is presented here for kidney, but in other instances PVDF membranes were typically cut at the approximate molecular weight of PKC so that membranes could be used for multiple antibodies . 5 independently-prepared protein extracts from control frogs (left) and 5 independently-prepared protein extracts from frozen frogs (right) were electrophoresed, transferred, immunoblotted, and exposed in parallel.Click here for additional data file.10.7717/peerj.558/supp-3Supplemental Information 3A full blot is presented here for liver, but in other instances PVDF membranes were typically cut at the approximate molecular weight of PKC so that membranes could be used for multiple antibodies . 5 independently-prepared protein extracts from control frogs (left) and 5 independently-prepared protein extracts from frozen frogs (right) were electrophoresed, transferred, immunoblotted, and exposed in parallel.Click here for additional data file.10.7717/peerj.558/supp-4Supplemental Information 4A full blot is presented here for liver, but in other instances PVDF membranes were typically cut at the approximate molecular weight of PKC so that membranes could be used for multiple antibodies . 5 independently-prepared protein extracts from control frogs (left) and 5 independently-prepared protein extracts from frozen frogs (right) were electrophoresed, transferred, immunoblotted, and exposed in parallel.Click here for additional data file.10.7717/peerj.558/supp-5Supplemental Information 5A full blot is presented here for liver, but in other instances PVDF membranes were typically cut at the approximate molecular weight of PKC so that membranes could be used for multiple antibodies . 5 independently-prepared protein extracts from control frogs (left) and 5 independently-prepared protein extracts from frozen frogs (right) were electrophoresed, transferred, immunoblotted, and exposed in parallel.Click here for additional data file.10.7717/peerj.558/supp-6Supplemental Information 6A full blot is presented here for kidney, but in other instances PVDF membranes were typically cut at the approximate molecular weight of PKC so that membranes could be used for multiple antibodies . 5 independently-prepared protein extracts from control frogs (left) and 5 independently-prepared protein extracts from frozen frogs (right) were electrophoresed, transferred, immunoblotted, and exposed in parallel.Click here for additional data file.10.7717/peerj.558/supp-7Supplemental Information 7A full blot is presented here for liver, but in other instances PVDF membranes were typically cut at the approximate molecular weight of PKC so that membranes could be used for multiple antibodies . 5 independently-prepared protein extracts from control frogs (left) and 5 independently-prepared protein extracts from frozen frogs (right) were electrophoresed, transferred, immunoblotted, and exposed in parallel.Click here for additional data file.10.7717/peerj.558/supp-8Supplemental Information 8A full blot is presented here for liver, but in other instances PVDF membranes were typically cut at the approximate molecular weight of PKC so that membranes could be used for multiple antibodies . 5 independently-prepared protein extracts from control frogs (left) and 5 independently-prepared protein extracts from frozen frogs (right) were electrophoresed, transferred, immunoblotted, and exposed in parallel.Click here for additional data file.10.7717/peerj.558/supp-9Supplemental Information 9A full blot is presented here for liver, but in other instances PVDF membranes were typically cut at the approximate molecular weight of PKC so that membranes could be used for multiple antibodies . 5 independently-prepared protein extracts from control frogs (left) and 5 independently-prepared protein extracts from frozen frogs (right) were electrophoresed, transferred, immunoblotted, and exposed in parallel.Click here for additional data file.


The purpose of the present study was to determine whether the image levels of the distal femur affected the measurement of the tibial tubercle-trochlear groove (TT-TG) distance.Thirty sets of computer tomography (CT) images and 30 sets of MR images of the knee were evaluated. The TT-TG distance was quantified at multiple image levels in 1.5-mm increments, covering the proximodistal range of the trochlear groove. The CT measurement was based on osseous landmarks; the magnetic resonance imaging (MRI) measurement was based on cartilaginous and osseous landmarks.P = 0.64, P = 0.11); yet, the difference was significant in the MRI measurement upon cartilaginous landmarks (P < 0.01). Large deviation was found between levels in individual subjects in all the three sorts of measurement. The proximal levels were the most variable, while the mid levels were the least variable.The average TT-TG distances measured with CT, with MRI based on cartilaginous landmarks, and with MRI based on osseous landmarks were 15.74 mm (SD 3.83 mm), 12.8 mm (SD 5.67 mm), and 12.36 mm (SD 5.58 mm), respectively. No significant difference was found across image levels in the CT measurement and the MRI measurement upon osseous landmarks (Measurements of the TT-TG distance are not identical across the levels of the distal femur. Cautions should be taken when specific image slices were selected for evaluation. The tibial tubercle-trochlear groove (TT-TG) distance is a widely used parameter to evaluate the lateral offset of the distal extensor mechanism. Increased TT-TG distance is usually deemed as a predisposing factor to patellar instability and anterior knee pain , 2. For The TT-TG distance is generally defined as the distance between the tip of the tibial tubercle and the bottom of the trochlear groove along the posterior tangent of the femoral condyles, measured on superimposed axial images of the knee . HoweverThe objective of the current study was to quantify the TT-TG distances with respect to different levels of the distal femur with both CT and magnetic resonance imaging (MRI) measurements. We hypothesized that the TT-TG distance might not be identically measured throughout the proximodistal range of the femoral trochlea.http://www.gpower.hhu.de), which suggested a sample size of 20  to provide a power of 95 %. The current study has been approved by the ethic committee of the authors’ institution . All subjects in the imaging repository had been informed that their image data would be utilized for scientific research as well as publication and had provided informed consent prior to the image collection.From a knee imaging repository in our laboratory, we retrospectively selected 30 subjects (30 knees) with CT scans and another 30 subjects (30 knees) with axial MRI scans in the knee. These subjects were recruited for image collection between 2009 and 2012 for research purpose. All subjects were healthy and had been excluded for deformity, injury, or surgery history in the lower limbs. The CT group consisted of 18 males and 12 females, 15 left knees and 15 right knees, with an average age of 26.4 years (SD 4.8 years); the MRI group consisted of 11 males and 19 females, 7 left knees and 23 right knees, with an average age of 27.3 years (SD 5.3 years). The sample size was estimated by a priori power analysis using G-Power  with the knee in extension. The range of the distal femur through the proximal tibia was covered. Images were obtained with use of 0.625 mm of slice increment, 110 kV 230 mAs, and in-plane resolution of 512 × 512. The MRI scans were performed on a 3.0T scanner  with the knee in extension, using an eight-element knee coil and a 3D fat-suppressed fast spoiled gradient-recalled echo sequence .The original CT and MRI data were imported into Mimics . The imported images were first reconstructed into volume and then resliced to create a new axial image set with the slice increment of 1.5 mm.For each resliced CT or MRI image set, the consecutive image sequence from the level showing the most proximal groove point of the trochlea (referred to as the trochlear entrance hereafter) to the most distal level on which a complete trochlea remained identifiable was picked and exported, named the trochlear range. The numbers of the image slices within the trochlear range varied from 10 to 14, depending on the individual knee size , the deepest point in the trochlear groove as well as the two points of tangency on the posterior femoral condyles were identified and marked on each image within the trochlear range. Similarly, the most prominent point of the tibial tubercle was also marked  were picked for repeated measurements. These three slices, respectively, represented the proximal, mid, and distal levels of the trochlear range. For each subject, two orthopedic surgeons (LY and RX) with 7 and 8 years of experience, respectively, independently marked the three selected images with a 2-week interval. As the reliability tests primarily focused on the systematic error induced by the image levels on the femoral side, all measurements of one subject used the same image with marked tibial tubercle to eliminate noises from the tibial side. The images underwent the same processes mentioned above to calculate the TT-TG distances. Intra- and inter-rater reliability was evaluated using the intra-class correlation coefficient (ICC), with the absolute agreement checked by a two-way random model.t tests. All statistical calculations in the current study were performed in SPSS 18.0 . The significance level was set as P < 0.05.For each subject, the TT-TG distance measured at every level of the distal femur was quantified; the mean value across all levels was then calculated. Based on these data, two sorts of deviations were subsequently calculated in each subject: the deviation between the maximal and the minimal TT-TG distances across levels, which indicated the range of the variance derived from levels, and the deviation of the TT-TG distance at each level to the mean value across levels, which indicated the level-specific variance. After data normality was checked using the Shapiro-Wilk method, the influence of the image levels on the TT-TG distance was characterized with one-way repeated measures ANOVA. Furthermore, the TT-TG distances of the cartilaginous-MRI group and osseous-MRI group at the same level were compared utilizing paired-sample The TT-TG distances at all levels throughout the trochlear range were successfully quantified for all subjects in the CT, cartilaginous-MRI, and osseous-MRI groups Table . RepeateP = 0.37)  due to individual knee size, only the first 10 levels counted in the proximal-to-distal direction which were common across subjects were selected for the repeated measures ANOVA. In the CT group, the levels of the distal femur did not have a significant effect on the TT-TG distance (P < 0.01). The value tended to decline with the distalization of levels   Fig. . Similar11) Fig. .Fig. 6Ret tests between the osseous-MRI group and cartilaginous-MRI group. Except for the 8th, the 9th, and the 10th levels , significant differences were observed between groups (P < 0.05)  Fig. .Fig. 7CoBased on reliable measurements of the TT-TG distance across multiple image levels of the distal femur, we found that the levels had a significant influence on the results when cartilaginous landmarks were used. The influence was not significant when the values were based on osseous landmarks, with both MRI and CT measurements. However, large deviations of the TT-TG distance between levels could still be observed in some individual subjects. These findings supported our hypothesis that the image levels of the distal femur might affect the results of the TT-TG measurements.The examination as well as the measurement techniques of the TT-TG distance has been evolving over the past decades. The TT-TG distance was initially described by Goutallier et al., who suggested the use of an axial radiograph taken at 30° of knee flexion with neutral axial rotation for the measurement . With thP < 0.001). Although no significant difference in the measurements across levels was found when osseous landmarks were used, notable deviations between values as high as 5 mm could still be observed in some individual subjects. Therefore, a standardized and consistent criterion for the selection of image levels would be beneficial for the TT-TG measurements. According to our results, the interval of 0 through 4.5 mm below the trochlear entrance  had the greatest deviation to the mean TT-TG distance; in contrast, the interval of 6 through 9 mm below the trochlear entrance (approximating the mid levels) was the least variable. From this point of view, the latter may be a favorable range for the TT-TG measurements; yet, cautions should be taken when the former is employed.As currently documented in the literature, various criterions have been held for the selection of image levels. These differing criterions may induce errors to the measurements, leading to less comparable results between studies. For example, according to our data, the most proximal and the most distal slices showing a complete cartilaginous trochlea had an average vertical distance of 16 mm; the average TT-TG distances measured based on cartilaginous landmarks at these two levels were 13.95 and 11.95 mm, respectively, which were significantly different  of the femoral trochlea may be a favorable range.


Saccharosydne procerus (Matsumura) were examined. Most S. procerus have a black spot on their forewings at high temperatures in the field and in the laboratory. In PO activity assay, a positive association between PO level and temperature was found. Our results showed that a diversification of melanism occurred under different temperatures and that melanism in S. procerus presented an opposite pattern to the one proposed by the thermal hypothesis.Melanism is a common polymorphism in many insect species that also influences immune function. According to the thermal melanin hypothesis, ectothermic individuals from cooler environments have darker cuticles and higher polyphenol oxidase (PO) levels, which represent a better immunocompetence. In this study, the links among environmental temperature, melanism, and PO activity of  Biston betularia Linn). During the nineteenth century, the frequency of melanic phenotypes increased rapidly and was considered to be a response to environmental change [Melanism is a polymorphism in the same species or in related species in which dark color pigments develop in individuals . Melanisl change .Chaerodes trachyscelides) closely matched the color of the sand in their environment [Spodoptera littoralis) developed darker cuticles when reared under high density [Tetranychus spider mites became conspicuously orange when exposed to short-day and low-temperature conditions [Allonemobius was affected by the thermal environment [Color polymorphism not only reflects gene variation but also the plasticity of the environment , and theironment , and the density . Female nditions , and theironment .Because insects are ectothermic, melanism may affect the absorption of heat. The thermal hypothesis states that dark individuals absorb heat faster under low-temperature conditions compared with individuals lighter in color . Hence, Moreover, cuticular pigmentation requires the oxidation of cuticular catechols to ortho-quinones by polyphenol oxidase (PO) . There aThe melanin-based immune system, in a number of insect species, is regulated by environmental factors such as the thermal environment because temperature influences the host-pathogen interaction by regulating pathogen growth and host disease resistance ,23. A hoSaccharosydne procerus Matsumura), which is the primary pest of water bamboo (Zizania latifolia) and rice (Oryza sativa) in East Asian countries, including China and Vietnam. The species has two morph forms. The melanic morph has a black spot on the terminus of the forewing , China. The greenhouse was only covered by gauze net to provide living conditions similar to the natural habitat. The plant hoppers were not exposed to any pesticides, and therefore, they did not face any selection pressure from pesticides.The The proportion of melanic morphs was investigated at five major water bamboo producing areas: Wuhan (N30°28’ and E114°21’), Anqing (N30°35’ and E117°07’), Suzhou (N31°19’ and E120°37’), Changsha (N28°13’ and E112°56’), and Jinhua (N29°04’ and E119°38’). In Wuhan, the melanism proportion was recorded once a month from June to October in 2012 because the population density was sufficient in the field during this period. At the other locations, the investigations were conducted from July to September 2012. In each area, five plots were selected in a rectangular field (the area was approximately 30 m × 40 m), with one in the center and the other four in the corners. Leaves with adults were cut off and stored in plastic bags. In total, 600 individuals  were collected in each area. The proportion of melanic morphs was recorded in the field.To compare the relationship between environmental temperature and melanism, temperature was recorded three times a day  in Wuhan by a thermometer that was in the experimental field. At the other four locations, the weather information was downloaded from the Internet . AccordiThe eggs of green slender plant hoppers were collected from the field by cutting the water bamboo leaves with spawning marks in Wuhan. These eggs were placed in an artificial climate box at 26°C and L: D 16:8 h photoperiod. The nymphs were divided evenly and were maintained at seven temperature treatments . For each treatment, the plant hoppers were fed with water bamboo leaf wrapped with wet cotton at the bottom in a glass tube (14 cm × 1.5 cm). Each tube contained one water bamboo leaf and 5 nymphs, and 100 individuals were used in each treatment. The leaves were changed daily. If mortality of nymphs occurred during the experiment, new first instar nymphs were added to keep the population density constant. After emergence, the adults were placed on a tissue paper to serve as a background, and the proportion of melanic morphs was recorded.The circulating PO activity was compared in nymphs near emergence  that weChi-squared test was employed to analyze (1) the variation of melanism in the field (2) the effect of temperature on melanization. We used one-way ANOVAs and pair-wise assessment was done using the Multiple Range Test method to analyze the PO activity of the two morphs. All analyses were conducted by SPSS Statistics 17.0.S. procerus in all four primary water bamboo producing areas; however, the proportion of melanic morphs was different in the four areas. The proportion of melanic morphs in Suzhou was significantly higher than the proportions found in Anqing , Jinhua  and Changsha . And the mean temperature of Suzhou was the highest when the investigation was conducted (The field investigation found melanism in onducted .P<0.001), July , September  and October . The mean temperature in the field was also highest in August 2012 , and the plant hoppers at 26°C showed a significantly higher proportion of melanism than those at 22°C , whereas no significant differences in PO activity were found at 26°C compared with that at 30°C (P = 0.1709) and 22°C  showed a higher PO level [Spodoptera littoralis) positively correlated with its PO activity [The present study was conducted to determine the associations among melanism of PO level  and the activity .As discussed in the Introduction part, the PO has two types and they have different functions. Considering that melanism shares some same biochemical pathway with other physiological processes, the effect of ambient temperature on these processes such as immunity may indirectly shape the body color .Interestingly our results are opposite to those predicted by the thermal hypothesis. The thermal hypothesis is widely accepted, and the expectation would be the proportion of melanic morphs would increase in cool conditions to absorb more heat from the environment . HoweverS. procerus was changed in the same way. High temperature may influence the host-pathogen interaction [A previous study showed that individuals invest more in the immune system when they have a greater risk of infection . As we deraction , 23, theeraction  and finaEphestia kuhuiella were better able to inhibit the oviposition and larval development of parasitic wasps [Spodoptera exempta showed a significantly higher resistance to baculovirus than non-melanic morphs[Tenebrio molitor showed lower mortality when exposed to a generalist entomopathogenic fungus [S. procerus may increase if climate become warmer in future, due to the advantages of melanic morphs when faced with increased risk of infection caused by globle warming [Considering the body color variation under high temperature, we are interested in the effect of climate warming on the melanism. Organisms can adapt to this alteration through phenotypic plasticity including melanism which can deal with the increase in infectious diseases associated with global warming . When faic wasps , and theic morphs. Additioc fungus . Therefo warming .Harmonia axyridis prefer typical (succinea form) males to melanic ones in the spring generation due to the thermal disadvantages of melanism during summer [S. procerus may be different from that of H. axyridis due to the opposite pattern to conventional thermal hypothesis, thus we think it is interesting to study the variation in the fitness of two morphs including mating behavior in the future.Melanin-based coloration can be costly to produce, maintain or wear , thus ing summer . While wS. Procerus. Therefore, we think it is worthwhile to explore it in the future.In summary, we suggest that more melanic morphs and a high level of PO will appear in hot conditions. Although we did not simulate an immune challenge, PO activity, as an important component of the immune system, represented immunocompetence . However