To dissect the regulatory network of cardiac Ca2+ handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca2+ extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca2+ uptake and accelerates the transfer of Ca2+ from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca2+ sparks and thereby inhibits Ca2+ overload-induced erratic Ca2+ waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca2+ uptake in the regulation of cardiac rhythmicity.Tightly regulated CaDOI:http://dx.doi.org/10.7554/eLife.04801.001 The heart is a large muscle that pumps blood around the body by maintaining a regular rhythm of contraction and relaxation. If the heart loses this regular rhythm it works less efficiently, which can lead to life-threatening conditions.Regular heart rhythms are maintained by changes in the concentration of calcium ions in the cytoplasm of the heart muscle cells. These changes are synchronised so that the heart cells contract in a controlled manner. In each cell, a contraction begins when calcium ions from outside the cell enter the cytoplasm by passing through a channel protein in the membrane that surrounds the cell. This triggers the release of even more calcium ions into the cytoplasm from stores within the cell. For the cells to relax, the calcium ions must then be pumped out of the cytoplasm to lower the calcium ion concentration back to the original level.tremblor—that has irregular heart rhythms because its heart muscle cells are unable to efficiently remove calcium ions from the cytoplasm. Embryos of the tremblor mutant were treated with a wide variety of chemical compounds with the aim of finding some that could correct the heart defect.Shimizu et al. studied a zebrafish mutant—called tremblor mutants. Efsevin binds to a pump protein called VDAC2, which is found in compartments called mitochondria within the cell. Although mitochondria are best known for their role in supplying energy for the cell, they also act as internal stores for calcium. By binding to VDAC2, efsevin increases the rate at which calcium ions are pumped from the cytoplasm into the mitochondria. This restores rhythmic calcium ion cycling in the cytoplasm and enables the heart muscle cells to develop regular rhythms of contraction and relaxation. Increasing the levels of VDAC2 or another similar calcium ion pump protein in the heart cells can also restore a regular heart rhythm.A compound called efsevin restores regular heart rhythms in Efsevin can also correct irregular heart rhythms in human and mouse heart muscle cells, therefore the new role for mitochondria in controlling heart rhythms found by Shimizu et al. appears to be shared in other animals. The experiments have also identified the VDAC family of proteins as potential new targets for drug therapies to treat people with irregular heart rhythms.DOI:http://dx.doi.org/10.7554/eLife.04801.002 Ca2+ influx after activation of the L-type Ca2+ channel in the plasma membrane induces the release of Ca2+ from the SR via ryanodine receptor (RyR) channels, which leads to an increase of the intracellular Ca2+ concentration and cardiac contraction. During diastolic relaxation, Ca2+ is transferred back into the SR by the SR Ca2+ pump or extruded from the cell through NCX1. Defects in cardiac Ca2+ handling and Ca2+ overload, for example during cardiac ischemia/reperfusion or in long QT syndrome, are well known causes of contractile dysfunction and many types of arrhythmias including early and delayed afterdepolarizations and Torsade des pointes  and the mitochondrial Ca2+ uniporter (MCU) serve as primary routes for Ca2+ entry through the outer and inner mitochondrial membranes, respectively  locus encodes a cardiac-specific isoform of the Na+/Ca2+ exchanger 1, NCX1h   . The treillation . In thistre mutant embryos suffer from Ca2+ extrusion defects and manifest chaotic cardiac contractions resembling fibrillation  establish a regular contraction pattern with rhythmic Ca2+ transients  (ncx1h deficient embryos (LB). A 32kD protein species was detected from zebrafish lysate due to its binding ability to efsevinLB and OK-C125LB, an active efsevin derivative conjugated to beads, but not to beads capped with ethanolamine alone or beads conjugated with an inactive efsevin analog (OK-C19LB)  . This mo embryos  and was K-C19LB) . FurtherK-C125LB . Mass spK-C125LB .10.7554/2+ handling. To examine this possibility, we injected in vitro synthesized VDAC2 RNA into tre embryos and found that the majority of these embryos had coordinated cardiac contractions similar to those subjected to efsevin treatment (myl7:VDAC2 transgenic fish in which VDAC2 expression can be induced in the heart by tebufenozide (TBF)  . KnockinO hearts . Convers efsevin . Further efsevin . Similar embryos . These f2+ flux  MEFs where VDAC2 is the only VDAC isoform being expressed  were maintained and bred as described previously  and cloned into pCS2+ or pCS2+3XFLAG. Full length cDNA fragments of zebrafish MCU (Accession number: JX424822) and MICU1 (JX42823) were amplified from 2 dpf embryos and cloned into pCS2+. For mRNA synthesis, plasmids were linearized and mRNA was synthesized using the SP6 mMESSAGE mMachine kit according to the manufacturers manual .tretc318 heterozygotes. Cardiac performance was analyzed by visual inspection on 1 dpf. The tre mutant embryos were identified either by observing the fibrillation phenotype at 2–3 dpf or by genotyping as previously described (VDAC2 mRNA and morpholino antisense oligos (5′-GGGAACGGCCATTTTATCTGTTAAA-3′)  were injected into one-cell stage embryos collected from crosses of escribed .tre embryos. 12 embryos collected from crosses of tretc318 heterozygotes were raised in the presence of individual compounds at a concentration of 10 µM from 4 hpf (tretc318 embryos manifest a chaotic movement resembling cardiac fibrillation with intermittent contractions in rare occasion (tre mutant embryos and NCX1h morphants (>500 embryos).Chemicals from a synthetic library  and fromom 4 hpf . Cardiacoccasion . Compounmyl7:GFP hearts were taken at 30 frames per second. Line-scan analysis was performed along a line through the atria or the ventricles of these hearts  treatment. Human EBs were differentiated for 15 days and treated with 5 mM CaCl2 for 10 min before DMSO or efsevin (5 μM) treatment. Images of beating EBs were acquired at a rate of 30 frames/s and analyzed by motion-detection software. For calcium recording, the EBs were loaded with 10 μM fluo-4 AM in culture media for 30 min at 37°C. Line-scan analysis was performed and fluorescent signals were acquired by a Zeiss LSM510 confocal microscope.The mouse E14Tg2a ESC and human H9 ESC line were cultured and differentiated as previously described . At day tre, and efsevin-treated tre embryos were placed on uncoated, microelectrode arrays (MEAs) containing 120 integrated TiN electrodes . Local field potentials (LFPs) at each electrode were collected for three trials per embryo type over a period of three minutes at a sampling rate of 1 kHz using the MEA2100-HS120 system . Raw data was low-pass filtered at a cutoff frequency of 10 Hz using a third-order Butterworth filter. Data analysis was carried out using the MC_DataTool (Multichannel Systems) and Matlab (MathWorks).2-day-old wild type, 2. For the recording of Ca2+ sparks and transients, the external solution contained 2 mM CaCl2. For Ca2+ transients, cells were field stimulated at 0.5 Hz with a 5 ms pulse at a voltage of 20% above contraction threshold. For all measurements, efsevin was added 2 hr prior to the actual experiment. Images were recorded on a Zeiss LSM 5 Pascal confocal microscope. Data analysis was carried out using the Zeiss LSM Image Browser and ImageJ with the SparkMaster plugin (Murine ventricular cardiomyocytes were isolated as previously described . Cells wr plugin . Cells w4HCO3 (30 min at 56°C) and 100 mM iodoacetamide (45 min in dark), respectively. Gel plugs were washed with 50 mM NH4HCO3, dehydrated with ACN, and dried down in a Speedvac. Gel pieces were then swollen in digestion buffer containing 50 mM NH4HCO3, and 20.0 ng/μl of chymotrypsin . Peptides were extracted with 0.1% TFA in 50% ACN solution, dried down and resuspended in LC buffer A .For pull down assays mono-N-Boc protected 2,2'-(ethylenedioxy)bis(ethylamine) was attached to the carboxylic ester of efsevin and its derivatives through the amide bond. After removal of the Boc group using TFA, the primary amine was coupled to the carboxylic acid of Affi-Gel 10 Gel . 2-day-old zebrafish embryos were deyolked by centrifugation before being lysed with Rubinfeld's lysis buffer . The lysDanio rerio IPI database v3.45 using the SEQUEST algorithm in the BioWorks software program version 3.3.1 SP1. All spectra used for identification had deltaCN>0.1 and met the following Xcorr criteria: >2 (+1), >3 (+2), >4 (+3), and >5 (+4). Searches required full cleavage with the enzyme, <4 missed cleavages and were performed with the differential modifications of carbamidomethylation on cysteine and methionine oxidation.Extracted peptides were analyzed by nano-flow LC/MS/MS on a Thermo Orbitrap with dedicated Eksigent nanopump using a reversed phase column . The flow rate was 200 nl/min for separation: mobile phase A contained 0.1% formic acid, 2% ACN in water, and mobile phase B contained 0.1% formic acid, 20% water in ACN. The gradient used for analyses was linear from 5% B to 50% B over 60 min, then to 95% B over 15 min, and finally keeping constant 95% B for 10 min. Spectra were acquired in data-dependent mode with dynamic exclusion where the instrument selects the top six most abundant ions in the parent spectra for fragmentation. Data were searched against the In situ hybridization was performed as previously described . DIG-labHeLa cells were transfected with a C-terminally flag-tagged zebrafish VDAC1 or VDAC2 in plasmid pCS2+ using Lipofectamine 2000 (Invitrogen). After staining with MitoTracker Orange (Invitrogen) cells were fixed in 3.7% formaldehyde and permeabilized with acetone. Immunostaining was performed using primary antibody ANTI-FLAG M2  at 1:100 and secondary antibody Anti-Mouse IgG1-FITC  at 1:200. Cells were mounted and counterstained using Vectashield Hard Set with DAPI .2+ indicator preferentially localized in mitochondria, for 1 hr at 15°C followed by a 30 min de-esterification period at 37°C. Subsequently, cells were permeabilized with 100 µM digitonin for 1 min at room temperature. Fluorescence changes in Rhod2  immediately after the addition of Ca2+  were monitored in internal buffer  using a FLUOSTAR plate reader .HeLa cells were transfected with zebrafish VDAC2 using Lipofectamine 2000 . 36 hrs after transfection, cells were loaded with 5 µM Rhod2-AM (Invitrogen), a Ca2+]c in suspensions of permeabilized cells or imaging of [Ca2+]m simultaneously with [Ca2+]c in intact single cells. Permeabilization of the plasma membrane was performed by digitonin (40 μM/ml). Changes in [Ca2+] in the cytoplasmic buffer upon IP3 (7.5 μM) addition in the presence or absence of ruthenium red (3 μM) was measured by fura2 in a fluorometer  and subsequently transferred to the microscope stage. Stimulation with 1 μM ATP was carried out in a norminally Ca2+ free buffer. Changes in [Ca2+]c and [Ca2+]m were imaged using fura2 (ratio of ex:340 nm–380 nm) and mitochondria-targeted inverse pericam (ex: 495 nm), respectively . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and 2 reviewers.Thank you for sending your work entitled “Activation of VDAC2 regulates mitochondrial Ca2+ uptake and cardiac rhythmicity” for consideration at The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Jodi Nunnari (Reviewing editor); Rosario Rizzuto (peer reviewer 1). Reviewer 2 remains anonymous.The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission. All agree that the work is of high quality and represents a significant advance in our understanding of the role of VDAC in calcium handling and cardiac physiology. A majority of the comments pasted below can be addressed by a careful revision of the manuscript. However, as pointed out by reviewer 2, the experiments do not adequately test the authors' conclusion that Ca2+ is directly transferred between the ER and mitochondria. Additional experimentation, for example examining the effect of addition of a Ca2+ chelator.Reviewer #1:The authors point to mitochondrial Ca2+ buffering as a protective mechanism against erratic electrical activity, and indicates a prominent role of VDAC2 as a “facilitator” of Ca2+ transfer through MCU channels. In this respect, the authors could consider investigating whether additional, possibly isoform-specific mechanisms add to the role of VDAC2 in Ca2+ permeation across the outer mitochondrial membrane.1) Does VDAC 2 interact with MCU and/or with some of its regulators ?2) Is the expression of MCU and its regulators (“MCU complex”) altered in the tre mutants?3) Does pharmacological or molecular stimulation of VDAC2 affect the expression of the MCU complex?Reviewer #2:The manuscript reports exciting results but needs to be improved in the following ways:1) The results in 2) The authors state “... efsevin restores rhythmic cardiac contraction in tre by potentiating VDAC2 activity.” The authors do not measure VDAC2 activity and thus this conclusion is unnecessary speculation. VDAC2 is not an enzyme and thus its action is not so easily defined in this context. The first part of the last sentence on that page is the appropriate summarizing statement.3) The authors state: “VDAC is an abundant channel located on the outer mitochondrial membrane serving as a primary passageway for metabolites and ions including Ca2+  Rapizzi.“ The au4) In addition, the authors state “We examined whether Ca2+ released from intracellular stores could be directly transported into mitochondria through VDAC2 and whether this process could be modulated by efsevin...” The experiments reported do not distinguish between Ca++ release into the medium and quickly taken up my mitochondria or Ca++ directly traveling between the ER and the mitochondria. That could have been tested in 3C by having a strong chelator in the medium. Thus, with the current data the authors cannot claim evidence for direct transfer. They can claim a more effective uptake of Ca++ into mitochondria after efsevin treatment.Based on the results reported in 5B, the authors conclude that Ca++ has been transported into mitochondria in the permeabilized cells, ignoring the ER. The choice of Rhod2 was presumably based on its affinity for Ca++ and its likely saturation with Ca++ in the ER, so that observed changes are likely due to the mitochondria. The authors make no effort to point this out to the reader. It should be addressed.5) In the conclusions the authors state: “In this model, VDAC-dependent Ca2+ uptake controls the duration and the diffusion of cytosolic Ca2+ near the Ca2+ release sites...” How, physically, can VDAC control the “duration of cytosolic Ca++” and the “diffusion of cytosolic Ca++”? VDAC cannot control diffusion nor can it control the duration of diffusion. What I hope the authors want to say is that VDAC2 facilitates Ca++ uptake via MCU thus reducing the local Ca++ concentration more rapidly than otherwise. We appreciate the reviewers’ insightful and constructive suggestions to help us improve our manuscript. We have taken the reviewers’ advice and revised the manuscript accordingly. In addition to clarifying the text, we revised Reviewer #1 questioned whether the expression of MCU complex would be affected by NCX1 mutation or by the molecular stimulation of VDAC2.tremblor hearts with and without efsevin treatment. These findings are presented in We address this question by in situ hybridization. We find that the expression levels of MCU and MICU1 are comparable between wild type and Reviewer#1 also questioned whether VDAC2 interacts with MCU and its regulators.tremblor requires the activity of both VDAC2 and MCU provides genetic evidence supporting a functional interaction between VDAC2 and MCU  MICU1 is expressed in the developing heart  overal VDAC2 , and 3) al VDAC2 . These fReviewer #2 made a few suggestions toThe reviewer thought that the sample Mass Spec image presented inwas too small for inspection, and questioned the identities of peptides detected in our Mass Spec analysis.We thank the reviewer for pointing out this problem. In the original The reviewer also questioned whether we identified any other proteins interacting with efsevin.tremblor and knocking down VDAC2 abolishes the responsiveness of tremblor to efsevin. Together, these findings support VDAC2 as a target of efsevin.In the course of our study, we inspected six sets of data. VDAC2 was the only protein that was not present in the controls but was consistently identified from the efsevin affinity column. Vitellogenin 1 (vtg1) and NADPH quinine 1 (nqo1) were frequently found in both the control and efsevin affinity columns and thus were considered as contaminants. More importantly, our genetic analysis showed that overexpression of VDAC2 recapitulates the rescue effect of efsevin on Reviewer #2 raised a few questions regarding the mitochondrial Ca2+uptake analyses.The reviewer questioned the experimental detail about the mitochondrial Ca2+uptake experiment in HeLa cells.2+ into the samples to make the final Ca2+ concentration as approximately 10 μM. We have now clarified this information in the Methods and Figure Legend.We apologize for the confusing description. In this experiment, we added CaThe reviewer thought it would be helpful to point out how Rhod2 functions as a mitochondrial Ca2+indicator.We appreciate the reviewer’s concern and include this information in the Methods section.The reviewer thought that “examine whether Ca2+released from intracellular stores could be ‘locally’ transported into mitochondria through VDAC2” would be a better description of our analysis.2+ transfer through VDAC2 .We thank the reviewer for the suggestions. We have revised the manuscript accordingly.


T1 mapping is a rapidly evolving field and is emerging as a novel quantitative tissue characterisation technique with potential applications in fibrosis, oedema, fat, iron and other patho-physiology. The impact of gender, segmental analysis and image piloting is not yet fully explored and has the potential to complicate the interpretation of observed differences.9 healthy volunteers and 7 patients with no evidence/history of disease were scanned on a Siemens Avanto 1.5T scanner and a 32 channel coil using the Siemens modified look locker inversion recovery (MOLLI) WIP version 448B (5:3:3 acquisition with inline motion correction and T1 map generation). All subjects had a 4 chamber, basal and mid ventricular short axis acquisition performed. In all slices, a single, slender ROI was placed in the mid wall of the left ventricular septum with meticulous attention to avoiding partial volume of blood pool. In addition, the mid ventricular short axis slice was segmented  into 6 AHA segments to allow regional T1 analysis.There were 9 females and 7 males, mean age 47. Mean T1 times are displayed in table We present T1 times for MOLLI imaging in healthy individuals and demonstrate 1) significantly higher T1 in females than males 2) segment to segment T1 variation and 3) a trend to longer T1 in the 4 chamber compared to short axis. These results suggest that caution should be used when interpreting absolute T1 values. Differences may be observed by gender, slice orientation and segment. Whether these differences are due to true physiological  or artefactual  differences is an area for further study.No external funding.


Studies that show frequencies of different orthodontic treatment protocols can be used as valuable parameters in the interpretation of treatment tendency with time. The purpose of this retrospective study was to evaluate all orthodontic treatment planning conducted at the Orthodontic Department at Bauru Dental School, University of São Paulo, Brazil, since 1973, in order to investigate extraction and non-extraction protocol frequencies selected at each considered period.P < 0.05).The sample comprised 3,413 records of treated patients and was evaluated according to the protocol choice, divided into 10 groups: Protocol 0 (non-extraction); Protocol 1 (four first premolar extractions); Protocol 2 (two first maxillary and two second mandibular premolars); Protocol 3 (two maxillary premolar extractions); Protocol 4 (four second premolars); Protocol 5 (asymmetric premolar extractions); Protocol 6 (incisor or canine extractions); Protocol 7 (first or second molar extractions); Protocol 8  and Protocol 9 (agenesis and previously missing permanent teeth). These protocols were evaluated in seven 5-year intervals: Interval 1 (1973 to 1977); Interval 2 (1978 to 1982); Interval 3 (1983 to 1987); Interval 4 (1988 to 1992); Interval 5 (1993 to 1997); Interval 6 (1998 to 2002); Interval 7 (2003 to 2007). The frequency of each protocol was compared between the seven intervals, using the proportion test (The results showed that 10 protocol frequencies were significantly different among the 7 time intervals.The non-extraction protocol frequency increased gradually with consequent reduction of extraction treatments. The four premolar extraction protocol frequency decreased gradually while the two maxillary premolar extraction protocol has maintained the same frequency of indications throughout time. The decision to extract teeth or not and the number of teeth to be extracted can influence the final result of orthodontic treatment, including esthetics, occlusion, satisfaction of patients and their families, as well as the treatment time ,2. For mRetrospective studies –8 of extThe data was retrospectively obtained from 3,745 consecutively treated patients from the files of the Orthodontic Department at Bauru Dental School, University of São Paulo, from 1973 to 2007. Patient files  were sequentially evaluated and 332 (8.86%) were excluded, resulting in 3,413 cases in the sample. Exclusion criteria were patient transfer or treatment drop out, preventive and orthopedic treatment without following orthodontic treatment with fixed appliances and cases without complete records.The sample consisted of 1,475 males (43.21%) and 1,938 females (56.79%) treated with an initial mean age of 13.76 years , divided according to the treatment protocol: Protocol 0 (non-extraction); Protocol 1 (four first premolar extractions); Protocol 2 (two maxillary first and two mandibular second premolar or a variation to three first premolar and one mandibular second premolar extractions); Protocol 3 (two maxillary premolar extractions); Protocol 4 (four second premolar or a variation to three second premolar and one mandibular first premolar extractions); Protocol 5 (asymmetric extractions - three premolars or only one premolar); Protocol 6 (incisor or canine extractions); Protocol 7 (first or second molar extractions); Protocol 8  and Protocol 9 (patients with agenesis or previously missing permanent teeth). The frequency of these protocols were evaluated in seven 5-year intervals similar to Profitt [The study protocol was approved by the Ethics Committee on Human Research of Bauru Dental School, University of São Paulo.P < 0.05. These analyses were performed with Statistica software .The frequencies of each protocol were compared among the intervals with the proportion test . ResultsAll 30 re-evaluated patient records presented complete agreement with the first observation, confirming the high reproducibility of the methodology. Figure Because the sample consisted of all treated patients in the Orthodontic Department at Bauru Dental School, from 1973 to 2007, it would not be necessary to apply inferential statistical tests. Even though, to provide more mathematical precision, the Proportion Test was used to evaluate whether there was any significant difference in the treatment protocol frequencies between each time interval. This type of frequency distribution test is strongly influenced by the number of events observed. This explains why, in Table In this study, the treatment protocols with (Protocols 1 to 9) and without extractions (Protocol 0) showed great statistically significant variation among the considered intervals  decreased significantly, corroborating the findings of other studies ,6–8 (Tab decreaseThe two maxillary premolar extraction protocol (Protocol 3) showed a relatively stable frequency around 10% in most of the evaluated periods (Table The therapeutic choice of four second premolar extractions (Protocol 4) demonstrated a much-reduced frequency in all evaluated intervals Table . AlthougThe asymmetric extraction protocol of three premolars (Protocol 5 - two maxillary and one mandibular premolar) is indicated in Type 1 Class II subdivision malocclusion treatment . AsymmetProtocols 6, 7, and 8 exhibited low frequencies without significant differences among them Table . These fProtocol 9 included all cases with previous dental absences. It was considered that these patients should not be excluded from the study by the fact that similar circumstances happen in daily clinical routine, and should be statistically described. They were placed in a separate group because their previous condition could have influenced the treatment planning. The total mean frequency of these cases was 11.28%, and their frequencies in the several periods showed no statistically significant differences  to 54.55% (2003 to 2007), with consequent reduction of extraction treatments from 85.71% (1973 to 1977) to 45.45% (2003 to 2007).The four premolar extraction protocol frequency decreased gradually from 65.72% (1973 to 1977) to 10.72% (2003 to 2007), while the two maxillary premolar extraction protocol has shown the same frequency of indications in the same time period.The following conclusions are drawn from the study:


To the Editor: In 2011, a chicken anemia virus (CAV)–related sequence, designated avian gyrovirus 2 (AGV2), was first identified in serum samples from diseased chickens in Brazil  play a critical role in the transmission of poultry pathogens to humans, we used PCR to investigate the presence of AGV2 in chickens (54 feather shaft samples) from 4 LPMs in Yangzhou and in 178 human blood samples from healthy persons living in Yangzhou. The DNA from the feather shafts and human blood were extracted as previously described . The positive rates for samples from the 4 LPMs tested were 25%, 12.5%, 15.8%, and 20%; the positive rate for the 178 human blood samples was 1.1%. The low positive frequency of AGV2 in human blood detected in this study is consistent with that found by investigation in other countries  with MEGA6 . The 12 <15.8%, and these variants of AGV2 were mainly detected in diseased chickens (Our results demonstrate the presence of AGV2 in LPMs and human blood in mainland China. The amplification and analysis of partial AGV2 sequences was the major limitation in our method. The high homology between sequences identified in LPMs and human blood indicates the LPMs are a potential source for AGV2 in humans. Unlike our 12 conserved AGV2, AGV2 identified by Santos et al. in southern Brazil varied


The observed photo-responsivity of ~300 A/W is four orders of magnitude larger than the earlier reported results on this material. Even though the role of 2D surface states responsible for high reponsivity is unclear, the novel and simple micromechanical cleavage (exfoliation) technique for the deposition of Bi2Se3 flakes followed by nanowire fabrication using FIB milling enables the construction and designing of ultrasensitive broad spectral TIs based nanowire photodetector which can be exploited further as a promising material for optoelectronic devices.Recently, very exciting optoelectronic properties of Topological insulators (TIs) such as strong light absorption, photocurrent sensitivity to the polarization of light, layer thickness and size dependent band gap tuning have been demonstrated experimentally. Strong interaction of light with TIs has been shown theoretically along with a proposal for a TIs based broad spectral photodetector having potential to perform at the same level as that of a graphene based photodetector. Here we demonstrate that focused ion beam (FIB) fabricated nanowires of TIs could be used as ultrasensitive visible-NIR nanowire photodetector based on TIs. We have observed efficient electron hole pair generation in the studied Bi Thickness and size dependent interesting and exciting light absorption properties of Bi2Se3 has been recently studied122Se3 has shown enhanced light absorption in the visible region compared to bulk sample2Se3 nanodevices has shown that the polarization of light can control the generation of photocurrents2Se3 have been successfully employed for the photocurrent studies2Se3 nanowires fabricated from high quality exfoliated flakes are largely remained elusive and have not been studied yet.Bi2Se3 nanowires fabricated by a focused ion beam microscope. Significant photocurrent is observed when nanowires are illuminated with visible laser (532 nm) and IR laser (1064 nm). In these nanowire devices, we have observed faster rise and decay times which depend on the laser power and the applied electrical bias voltage. The best photoresponsivity measured among the nanowire devices is in the order of ~300 A/W which is ~10000 times larger than the ultrathin nanosheets of Bi2Se3Here we report the broadspectral photo response of Bi2Se3 flake where surface looks clean and smooth. Note that a thin flake of interest was first localized under FESEM but was never exposed to Ga ion imaging. Green colour rectangle shows the area selected for the milling and the small square was used for the alignment of electron beam and Ga ion beam. FIB milling was performed for the fabrication of nanowire geometry by etching out selected portion of the flake as shown in the 2Se3 nanowire device used for the photocurrent measurement in this study. The linear current voltage relationship shown by the black (light off) and the red (light on) curves in the inset of 232Se3 flakes was observed under high resolution transmission electron microscope (HRTEM) ( (HRTEM)  which is2Se3 nanowire devices were characterized for their time dependent photoresponse properties to broad spectral laser excitations using visible laser  and infrared laser . Prior to any photocurrent measurements, laser illumination was guided perpendicularly over wide area covering the entire device as shown schematically in 2 and ~32 mW/cm2, respectively. A Keithley 2634B source measure unit (SMU) was used for the electrical characterization of the fabricated photodetector devices.The BiIPh) was extracted by subtracting the dark current (Idark) from the measured current in presence of laser light (Ilight) i.e. Iph = Ilight-Idark24252Se3 nanowire devices as photo switches or high quality photodetectors.First, we have characterized the photocurrent dynamics of the nanowire photodetector for cyclic exposures of visible light  to study the stability and repeatability of the photocurrent as shown in the The response and decay times are important parameters for any photodetector. We have used following exponential equations to calculate the response and decay times, i.e., 2. These values of 2Se3 nanowire based photodetectors compared to photodetectors made from other novel layered materials like MoS2  and is shown in Evolution of photocurrent is further characterized under the illumination of IR light source (1064 nm) for various applied bias voltages as shown in  ~10 nA,  was obse ~10 nA, . Data in2 but small fluctuation in current is noticed at the photocurrent saturation plateau when the bias voltage is 150 mV. For power densities <5 mW/cm2, no noticeable measurement in photocurrent was found, above this threshold power, significant photocurrent rise is detected which is shown in Iph ~ PΘ, to fit the data of photocurrent measured at different powers. Consequently, a sub-linear increase is found in the photocurrent with increasing power density for the incident illumination systematically. This increment in linearity is the characteristic feature of a photocurrent indicating minimal contribution from the thermoelectric current as Bi2Se3 is a very good thermoelectric material. Light sensitive, polarization dependent generation and control of photocurrents originating from topological surface states in Bi2Se3 have been studied alreadyPhotocurrent detection in nanowire device due to IR light has been further characterized to check dependency of the photocurrent on the incident power density of the laser while keeping the bias at constant voltage. Photocurrent of about ~2 nA is observed for 5 mW/cmR = photoresponsivity (A/W), Iph = photocurrent, P = Light intensity and A = effective area of the nanowire. We have calculated the photoresponsivity for both visible and near infrared wavelengths. The photoresponsivity of Bi2Se3 nanowire as a function of laser power density is plotted in 2. This photosensitivity is a huge improvement  compared to the previous photoresponse reports on ultrathin Bi2Se3 nanosheetsD) and external quantum efficiencies  of it can be exfoliated on the substrate for FIB milling by a simple scotch tape  method. The fabricated Bi2Se3 nanowire device has shown broad spectral response (visible and NIR). The response, rise and decay time, of FIB fabricated Bi2Se3 nanowire device is very fast and excellent photocurrent stability and repeatability have been noticed. The photocurrent showed dependency on the laser power density and applied bias voltages. The high photoresponsivity is observed which is ~4 orders of magnitude improvement as compared to ultrathin Bi2Se3 nanosheetsIn conclusion, the photocurrent dynamics of Bi2Se3 nanowires were made by using scotch tape method and focused ion beam (FIB) microscopy. Prior to deposition of flakes on SiO2/Si, substrates were cleaned with acetone, isopropanol, methanol and treated with oxygen plasma for ~5 min. Bi2Se3 ( 99.999% CAS#12068-69-8) material was procured from company Alfa Aesar. By using micromechanical cleavage (scotch tape method), thin flakes of Bi2Se3 were exfoliated on SiO2/Si substrates having predefined gold pads. This method yields random sizes and thicknesses of the Bi2Se3 flakes which were observed under optical (Olympus) and electron microscope . The devices fabricated were loaded in the probe station setup (Cascade Microtech EPS150TRIAX) which has shield enclosure (EPS-ACC-SE750) for low signal measurements.Devices of Biga, 2Se3  were fab2Se3 samples to reveal microstructural information even up to atomic scale including the reciprocal space. In general, a thin flake-type morphology with a facetted-edge was observed in the microstructure. As an illustrative example, 2Se3 (space group: R2Se3 of 10High resolution transmission electron microscopy (HRTEM model: Tecnai G2 F30 STWIN assisted with field emission gun and with an electron accelerating voltage of 300 kV) was employed on Biinset in . The atoinset in  are dispcrograph . The inscrograph  further How to cite this article: Sharma, A. et al. High performance broadband photodetector using fabricated nanowires of bismuth selenide. Sci. Rep.6, 19138; doi: 10.1038/srep19138 (2016).


White matter disease in preterm infants comes along with focal destructions or with diffuse myelination disturbance. Recent experimental work with transgenic mice paves the way for a unifying molecular model for both types of brain injury, placing oxygen sensing by oligodendrocyte precursor cells (OPCs) at the center stage. Mice genetically altered to mimic high local oxygen tension in oligodendroglia lineage cells  develop white matter disease resembling cystic periventricular leukomalacia within the first 7 days of life. Mice in which local hypoxia is mimicked in oligodendroglial cells (via genetic inhibition of HIF decay) display arrested OPC maturation and subsequent hypomyelination, reminiscent of the diffuse white matter disease observed in preterm infants and infants with congenital heart disease. These recent experimental findings on oxygen sensing and myelination are awaiting integration into a clinical framework. Gene regulation in response to hyperoxia or hypoxia, rather than oxidative stress, may be an important mechanism underlying neonatal white matter disease. Brain injury in preterm newborn infants involves both destructive and developmental disturbances . Cystic Evidence of dWMD has been revealed by advanced magnetic resonance imaging techniques, notably diffusion tensor imaging and magnetic resonance spectroscopy imaging, in many very low birth weight preterm infants , 12–14. Changes of ambient oxygen tension cause a cellular response orchestrated by hypoxia-inducible factors (HIFs) in almost all mammalian cell types . The lac2 0.1) between day 3 and 11 of life.As intrauterine oxygen tension is difficult to manipulate in experimental animals, researchers at the University of California in San Francisco used mice with either targeted deletions of HIF1α and HIF2α, or lack of VHL-mediated HIF1α/HIF2α decay in the oligodendroglial lineage . Embryon2 0.1 for 6–72 h) has recently been shown to induce canonical (β-catenin-dependent) Wnt signaling alongside increased cell proliferation in the hippocampus of adult mice  show increased white matter vessel densities but myelination deficits are only found in rat pups (2 0.6 for 7 days) but prevented by slowly increasing FiO2 from 0.15 to 0.21 during the first 7 days of life (2 0.1) between day 3 and day 11 of life display reduced cortical gray and white matter volumes and delayed maturation of OPCs  , and theiO2 0.21 , 49. In 2 0.6), postnatal day 3 or day 6 (FiO2 0.6), but not day 10, has nevertheless a deleterious effect on the developing white matter in normal rat pups (2 0.8) at 6 days of life for 48 h continue to show signs of dWMD when examined by diffusion tensor imaging (While experimental hyperoxia in rodents is apt to reproduce several findings of human ROP, exposure to neonatal rats or mice to high oxygen does not necessarily generate the cystic white matter lesions observed with gene-mediated HIF1α/HIF2α-suppression in OPCs acting already before birth. While the brain maturation of newborn rats and mice recapitulates many features of the human situation during the last trimester of pregnancy and therefore that of preterm infants born 2–3 months early, the newborn rodents used in experiments are physiologically adjusted to the perinatal oxygen surge and thus potentially less vulnerable than human preterm babies. Experimental hyperoxia at birth (FiOrat pups , 50. Gro imaging  that ref imaging . Culture imaging  but it iIn sum, the immature white matter of both human beings and rodents is highly sensitive to altered oxygen tension during a critical developmental window prior to the onset of myelination. Both too much and too little oxygen can cause damage by profoundly altering expression of various genes, unrelated to any oxidative stress. These two conditions may occur sequentially in the same brain, as reduced vessel density after high oxygen subsequently leads to low oxygen supply at a time when demand is on the rise. Meticulous attempts to avoid extremes of oxygen tension in the brains of preterm infants and in infants with congenital heart disease undergoing surgery, possibly aided by directly monitoring cerebral oxygenation , will hoThe 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.


Sickle cell disease (SCD) is a major health burden in India. The objective of the study was to establish a neonatal screening program and to understand the clinical course of children with SCD in central India.Pregnant mothers were screened for sickle hemoglobin using the solubility test. Babies were screened by high performance liquid chromatography if the mother was positive for sickle hemoglobin. The diagnosis was confirmed by molecular analysis. They received early prophylactic treatment and vaccination. Of 2134 newborns screened, 104 were sickle homozygous (SS), seven had sickle β-thalassemia  and 978 were sickle heterozygous (AS). The other hemoglobin abnormalities detected included HbS -δβ thalassemia-1, HbSD disease-2, HbE traits-5, β-thalassemia traits-4, alpha chain variants-3 and HbH disease-1.These babies were followed up regularly for hematological and clinical evaluation. Pain, severe anemia requiring blood transfusions and acute febrile illness were the major complications with 59.7, 45.1 and 42.6 cases per 100 person years. Fetal hemoglobin (HbF) levels were inversely associated with vaso-oclussive crisis (VOC) and severe anemia while presence of alpha thalassemia increased the rate of painful events and sepsis. Six early deaths occurred among the SS babies.A systematic follow up of this first newborn SCD cohort in central India showed that 47% of babies presented within 1 year of age. In spite of the presence of the Arab-Indian haplotype many babies had severe manifestations. Studies on the natural history of sickle cell disease (SCD) from Jamaica and the USA have confirmed that the greatest morbidity and mortality occurs between 6 and 12 months and that early identification of affected infants by neonatal screening, careful follow up coupled with relatively simple measures decreased the mortality rate . The sicThis is one of the first efforts to raise a cohort of SCD babies by newborn screening and follow them regularly to record the early clinical and hematological presentation in central India.The study was approved by the Institutional Ethics Committee Review Board- “Institutional Committee for Research on Human Subjects, National Institute of Immunohaematology (ICMR) (NIIH/IEC/21-2007)”, written informed consent was obtained from all participants and all investigations were conducted according to the principles expressed in the Declaration of Helsinki.Pregnant women were screened for sickle hemoglobin (HbS) using the solubility test at Govt. Medical College, Nagpur after a written informed consent was taken from them. Babies of all the mothers who had a positive solubility test were screened by high performance liquid chromatography (HPLC). Heel prick samples were collected in EDTA after birth or within 7 days of birth after a written informed consent from the parents and all the investigations on the babies and the parents samples were conducted according to the principles expressed in the Declaration of Helsinki. A complete blood count was done on an automated cell counter . Hemoglobin (Hb) analysis was done using automated HPLC on the VARIANT™ Hemoglobin Testing System  using the sickle cell short programme and the β thal short programme during follow up. Molecular analysis was done to confirm the sickle and other genotypes . Alpha gThe gestational age at delivery, demographic details and neonatal complications were recorded. The babies with SCD were enrolled at the sickle cell clinic in Nagpur. Vaccination included conjugate vaccine for Haemophilus influenzae type B and 7-valent conjugate pneumococcal vaccine  within 4 weeks of birth. All the babies received oral penicillin V starting at 3 months of age and 23-valent polysaccharide pneumococcal vaccine (Pneumovax) was given after 2 years of age. Clinical crisis were defined according to previous published criteria –13. A foIncidence rates are presented as the number of cases per 100 person years. The estimates were based on clinical events and follow up time. Descriptive statistics are presented as percentages and mean ± standard deviation. The p values are calculated by Fischer’s exact test (two tailed). A p value of 0.05 or less was considered significant.0 thal (CD 15 (G→A), CD 41/42 (-CTTT); 5 babies had S-β+ thal (IVS 1→5 (G→C)], one baby had sickle-delta-beta thalassemia  and two babies had HbSD disease. In addition, five babies with HbE trait, four with β-thalassemia trait, three with alpha chain variants  and one case of HbH disease were also identified. The cohort of 104 SS babies  was followed up clinically every month for a year and then every 3 months for 3–4 years for hematological and clinical evaluation. However, in case of any symptoms they came earlier. 92% of the SS babies belonged to non-tribal communities while 8% belonged to tribal communities.From 2009 to 2012, 10,181 pregnant women were screened and 2134 newborn babies were tested of whom 1040 (48.7%) were normal, 978 (45.8%) were sickle cell traits, 104 (4.9%) were sickle homozygous (SS), seven had sickle β-thalassemia : [2 babies had S-β75 out of the 104 SS newborns, six of the seven S-β thal babies and the two babies with Hb SD disease could be followed up clinically while 29 (28%) were lost to follow up.Sixty two of the 75 SS babies had some clinical complications while 13 babies remained asymptomatic. Among them, 12 babies (19%) presented within 6 months of age, 17 (28%) between 6 and 12 months of age, 25 (40%) between 1 and 2 years of age while 8 babies presented after 24 months. The 2 Hb SD disease babies also presented within the first 6 months of life.The clinical events observed in the SS, S-β thal and SD babies are summarised in st clinical event observed. Acute splenic sequestration (ASS) crisis was seen in the first 2–3 years of life in two SS babies and one HbSD disease baby. One SS and one HbSD disease baby had three episodes each of sequestration crisis but splenectomy was not advised. Severe anemia requiring transfusions (3 or more/year) was the main clinical event in the two HbSD disease babies while the six sickle β-thalassemia babies had none of the above complaints. Acute chest syndrome (ACS) was not found in this cohort.Staphylococcus, E-coli and Klebsiella.Eight SS babies had sepsis with the overall incidence of 4.8 per 100 person years  and two Seven SS babies had eight episodes of siezures and three babies had five episodes of stroke at 14, 42 and 30 months with an overall incidence of 3.0 per 100 person years . One babThe hematological and hemoglobin analysis at birth and at the last follow up (3–4 years) in SS babies is illustrated in Out of the 150 SS chromosomes, 141 (94.0%) were linked to the Arab-Indian haplotype, six were linked to the Bantu A2 and three to an Atypical1 haplotype (+ - + - + + + + -). Of the six babies having the Arab-Indian/Bantu A2 haplotype, two patients showed a moderately severe clinical course and one baby died due to severe anemia. Of the three babies with the Arab-Indian/Atypical1 haplotype, one could not be followed up and two patients showed a mildly severe clinical course.3.7/αα = 12, -α4.2/αα = 6, -α3.7/-α4.2 = 1). The incidence of clinical events per 100 person years was determined in babies with and without alpha thalassemia (4.2/αα.Alpha genotyping was done in 69 of the 75 SS babies. 50 babies (72%) had a normal alpha genotype while 19 babies (28%) had alpha gene deletions (-αlassemia . InfantsHbF levels in the SS cohort varied from 3.3 to 37.2% (mean 21.4 ± 5.4%). Based on an earlier study,  a cut ofSix SS babies (8%) died during the follow up with the overall mortality rate of 3.65 per 100 person years . In threNeonatal screening helps to recognize the early signs of the disease and minimize morbidity and mortality by early comprehensive care and prophylactic treatment. Our targeted newborn screening approach in central India identified 4.9% of SS babies along with other hemoglobinopathies. The above babies were enrolled at the sickle cell clinic and were given early comprehensive care and prophylactic treatment. Few babies with HbS-β thalassemia may have been missed as universal screening was not done.Vichinsky et al demonstrated that the overall mortality rate for American patients with sickle cell anemia diagnosed in the neonatal period was 1.8% compared with 8% for children diagnosed after three months of age . In the Such established newborn screening programs have not been initiated in India although the load of SCD is very high in central India. An initial pilot effort was made in Raipur district in Chhattisgarh in India, on screening few babies , however the affected babies were not followed up . ScreeniAnother recent study on newborn screening among tribal populations in South Gujrat in Western India showed that 21.8% of SCD babies had severe clinical complications before 5 years of age . Our preSince all painful events do not lead to VOC, pain and VOC were considered as separate complications. Pain followed by acute febrile illness and severe anemia with blood transfusion requirements were the major complications observed in this study . PainfulAs noted by Miller et al “Three easily identifiable manifestations of sickle cell disease that may appear in the first two years of life  can help to predict the possibility of severe sickle cell disease later in life”. Severe Acute chest syndrome and pneumonia are very difficult to differentiate in babies and pneumonia being one of the precipitating factors for ACS, this complication in the babies was labelled as pneumonia. ACS followed by ASS and sepsis were the major cause of mortality in Jamaica . ACS wasStaphylococcus, E-coli and Klebsiella. This is in contrast to the CSSCD cohort where infections occurred most commonly due to Streptococcus pneumoniae and Hemophilus influenzae and caused 11 deaths [Infection particularly pneumococcal sepsis is the leading cause of death in children with SCD , 30. Sep1 deaths .Stroke is a feature of early childhood with an incidence of 8% by 14 years of age in the Jamaican cohort . Three oSCD patients with alpha thalassemia had a higher risk of painful events, sepsis and dactylitis and lower incidence of severe anemia . Our fin2 levels at the last follow up was observed due to the presence of HbS adducts which tend to increase the actual HbA2 levels on HPLC. The HbF levels at 3–4 years were 21.4 ± 5.4%, majority of the sickle chromosomes being linked to the Arab- Indian haplotype.The hematological profile in the SS patients showed that anemia was apparent from 6 months of age. An increase in the HbAIn conclusion, this is the first newborn SCD cohort in central India to be systematically evaluated for 3–4 years. It emphasises the importance of early diagnosis and prophylactic treatment, parental education and the importance of tertiary care centres that should be made available to allow for prompt treatment. We have also shown recently that 75.4% adult SCD patients in India with the Arab Indian haplotype also have severe manifestations . Thus SC


WT1 gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD) limited to 11p15.5-p15.2 including the IGF2 gene. The tumor carried a heterozygous p.T41A mutation in CTNNB1. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45Δ CTNNB1 mutation. Uniparental disomy (UPD) 3p21.3pter lead to the homozygous CTNNB1 mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (hTERT) in conjunction with a novel thermolabile mutant (U19dl89-97tsA58) of SV40 large T antigen (LT). This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of WT1 in tumor cells. The origin/fate of Wilms tumors with WT1 mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. FOXD1, PAX3, SIX1, OSR1, OSR2 and MEIS1 and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other WT1 mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of WT1 mutant cells.We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire  WT1 gene are found in most stromal-type tumors, often associated with mutations in the CTNNB1 gene [Wilms tumor (WT), a malignant childhood neoplasm of the kidney, is thought to arise from embryonic renal mesenchyme with impaired nephrogenic differentiation potential. Most tumors have a mixed histology, containing blastema, epithelia and stroma. In the WT variant with a predominating stromal component, heterotypic cells, such as rhabdomyoblasts, fat, cartilage and bone can be found, not normally present in the kidney and likely to be derived from abnormal mesenchymal differentiation. Constitutional or somatic mutations in the NB1 gene .WT1 mutant tumors [WT1 mutant Wilms tumors revealed that these carry biallelic WT1 mutations but no CTNNB1 mutations, whereas the associated tumor cells had CTNNB1 mutations [WT1 mutant tumors have additional mutations in CTNNB1 or WTX, highlighting the importance of activated WNT signaling in tumors with inactive WT1 [CTNNB1 or WTX suggests that the functional loss of WT1 poses a strong selection pressure for additional mutations. This is further supported by our previous description of a patient with a germ line WT1 mutation who developed four tumors with different CTNNB1 mutations, suggesting their independent origin and/or tumor heterogeneity. In addition the same tumor harbored different CTNNB1 mutations in different histological areas [WT1 mutation, the second is the loss of heterozygosity (LOH) in 11p, resulting in loss of the WT1 wild type allele and the third is a CTNNB1 mutation [Intralobar nephrogenic rests (ILNR) occurring early in kidney development can be found as precursor lesions in t tumors . Microdiutations . Most WTtive WT1 ,5,8. Theal areas   exhibits gain of function properties. The mutant protein has lost the wild type WT1 function for sequence specific DNA binding, but facilitates the expression of genes regulating the cell cycle [CTNNB1 gene; the gain of function of the mutant WT1 protein in regulating the cell cycle could explain, why these cells do not need additional mutations in CTNNB1 nor WTX [Most cell lines that we have established from ll cycle . Therefo nor WTX .WT1 mutant Wilms tumor cell lines have a limited life span, similar to normal human mesenchymal stem cells (hMSC). Under in vitro growth conditions they can be cultivated at most for 60 population doublings. Primary cells in culture as well as some tumor cells have a limited life span that limits their use for experimental manipulation. Most biochemical and genetic studies require large cell numbers. Therefore, immortalized cell lines would be very useful for such studies. It has been described that the life span of normal human cells can be extended by introduction of the catalytic subunit of telomerase (TERT) [TERT was described for many different normal cell types, for example glomerular mesangial cells, glomerular endothelial cells, podocyctes, mammary fibroblasts and endothelial cells, airway epithelial cells and hepatocytes [All our previously established e (TERT) . More reatocytes –17. All WT1 nested within a heterozygous 11p13 deletion and a CTNNB1 mutation. Genetic analyses of cells derived from the Wilms tumor (Wilms10) showed the same WT1/11p13 alteration but a different mutation in CTNNB1 as compared to the bulk tumor DNA. aSNP/CGH revealed UPD in 3p and 11p15 not extending to the WT1 gene. Through immortalization of this cell line with ts LT and hTERT, we have established the first Wilms tumor cell line with a homozygous deletion of the entire WT1 gene, resulting in a complete lack of WT1. This immortalized Wilms10 cell line should be useful for further exploration of the effect of WT1 loss in genetic and biochemical studies and to further explore the origin and cell fate of Wilms tumors with WT1 mutations.Here we describe a stromal type Wilms tumor with a homozygous deletion of A 2 year-old girl presented with an isolated left renal mass. The tumor was surgically removed upfront and the histology showed a triphasic stromal predominant Wilms tumor with focal anaplasia and p53 over-expression in these anaplastic foci . The tumWT1 gene and CTNNB1 exon3 was analyzed as described before [Tumor DNA was isolated directly from a piece of solid tumor tissue. DNA from blood and cell culture cells was isolated by standard methods. The complete d before .TP53 gene was carried out on a GS Junior System instrument (Roche Applied Science). All sequencing data were generated using the Junior Sequencer Instrument software version 2.7 (Roche Applied Science). Sequence alignment and variant detection were performed using the GS Amplicon Variant Analyzer software version 2.7 (Roche Applied Science).P53 mutation analysis was done by Next-generation pyrosequencing using 454 Titanium Amplicon chemistry  according to the manufacturer's instructions . Primer sequences and PCR conditions were kindly provided by A. Kohlmann . Sequencing run of the The tumor was finely minced and a cell culture was established using MSCG medium as described . AccordiTERT, pBABE-hygro-hTERT, Addgene 1773) [TERT from a triple transfected HEK293FT cell line were mixed and used to transduce the primary Wilms 10 cells. After infection, the cells were cultured in G418  for 1 week, followed by hygromycin  for another week to select for successfully doubly transduced cells. Single cell clones were isolated and cultivated at 33°C initially to maintain the active form of the temperature sensitive LT. After the first confluence, clones were split and cultured at 33° and 37°C. One immortalized clone was selected for further studies and was named imWilms10. The imWilms10 cells continued to grow at 37°C, whereas normal hMSC immortalized with the same genes did not continue to proliferate at 37°C. The imWilms10 cells were continuously cultured in medium with alternating hygromycin and G418 for one week each, with a break of selection for one week.To establish an immortalized cell line, the catalytic subunit of human telomerase (hne 1773)  and a none 1773) . The trine 1773) ; dl89-97ne 1773) ; and tsAne 1773) ,23. It wne 1773) . The pZine 1773) . AmphotrThe SNP/CGH array (aSNP/aCGH) analysis of the primary tumor and the tumor cell culture from patient Wilms10 was performed using a 2x400K oligonucleotide microarray . With this array format, copy number changes, as well as copy neutral aberrations, such as uniparental disomy (UPD) can be detected on the same array. The samples were prepared and labeled as described by the manufacturer (Protocol Version 7.3 March 2014). To detect uniparental disomy (UPD), a female gender reference DNA  has to be used as control. To quantify the array data the feature extraction module of Agilent's CytoGenomics software (Version 1.5.1.0) was used and, for visualization, CytoGenomics Version 2.0.6.0.WT1 and heterozygous 11p13 deletion, a high-resolution array was designed (http://earray.chem.agilent.com/earray/). The region of the homozygous WT1 alteration was covered with oligonucleotides at a distance of 100 bp, whereas the probes for the heterozygous deletion had a spacing of 300 bp.To determine the breakpoints of the homozygous For the analysis of tsLT and MEST, proteins were extracted from the cells using RIPA buffer ), separated by SDS-PAGE and transferred to a PVDF membrane. The blot was incubated with a monoclonal antibody against SV40 LT (MAb423) or a monoclonal antibody against MEST .TM antibody array was used. Proteins from Wilms10 and imWilms10 cells cultured at 33°C were extracted and incubated with the antibody arrays as described by the manufacturer (R&D systems).For the simultaneous analysis of the phosphorylation status of 49 tyrosine kinase receptors a ProteomeProfilerPPARG  was used for the analysis of adipogenesis [Adipogenic and osteogenic differentiation was analyzed using reagents from Lonza and as described by the manufacturer. hMSC cells were used as controls in the same experiments. Non-induced control cells were kept in the respective maintenance media. After a 3 week induction and a maintenance phase, cells were processed for adipogenic differentiation by staining with Oil Red O. In addition cells were cultured for 10 days in the induction and maintenance medium for adipogenesis and total RNA  was extracted at day 0 and 10 for Q-RT-PCR analyses. Assay on Demand for ogenesis . ImmortaFor osteogenic differentiation cells were harvested after 19 days of differentiation by scraping them in the presence of 0.5M HCl and a calcium Liquicolor assay was performed according to the manufacturers instructions .Myogenic differentiation was induced with DMEM/F12 (Gibco) supplemented with 2% horse serum for 12 days and HSMM cells were used as controls. For immunofluorescence analysis cells were seeded in four chamber slides (BD Biosciences) and after growth in induction or control medium for 9 days, cells were stained with a Titin-specific antibody as described before .www.bioconductor.org) was used for quantile normalization of microarrays [For gene expression analyses total RNA was isolated from the Wilms10 tumor cell line and the immortalized cells (imWilms10) cultured at 33°C using the RNeasy Mini Kit (Qiagen). RNA from two biological replicates was analyzed in all gene expression experiments. RNAs were labeled in the One-Color format as described by the manufacturer (Agilent Technologies) and hybridized to 4x44K”Whole Human Genome Oligo Microarrays " in the presence of “Spike-In positive controls” (Agilent). The microarray scans were quantified using Agilent Feature Extraction Software (V10.1.1.1). Basic statistical analyses were performed within the 'R' statistical computing environment . The R-library 'Limma'  Bioconduroarrays . To compWT1 (Hs00240913_m1) and IGF2 (Hs01005963_m1) with Brilliant II QPCR Master Mix with Low Rox (Agilent Technologies). The expression levels were normalized with RER1 (Hs00199824_m1) RNA. The reason for using RER1 as calibrator was that this gene was basically never regulated in any of our gene expression studies. The Q-RT-PCRs experiments were run on a Mx3000P Sequence Detection System (Stratagene). Statistical significance of the normalized Ct values (ΔCt) was assessed by a t-test.For Q-RT-PCR analyses cDNAs were synthesized using TaqMan Reverse Transcription Reagents (Applied Biosystems). The Q-RT-PCR experiments were performed in triplicates using the TaqMan gene expression assay probes from Applied Biosystems for WT1 was found in blood or tumor DNA. Multiple-ligation probe analysis  did not identify copy number variations in the blood DNA   and a smaller deletion at 1p31.1 with a maximal size of 203kb (74946777–75185022)   . The het5185022) . Further 12.8Mb)  and anot located .DNA from the established tumor cell line (Wilms10) was also analyzed with aCGH/aSNP and the same 11p13 heterozygous/homozygous deletion was identified . A cytogWT1 gene within a heterozygous 11p13 deletion. The derived cell culture harbored the same alterations as the tumor sample.A custom array covering the heterozygous/homozygous deleted 11p13 segment at a high density revealed the start and endpoint position of the heterozygous/homozygous deletions in the tumor and the established Wilms10 tumor cell line . Fig 1F WT1 gene it was of interest to compare the level of WT1 gene expression in the cell lines with the different WT1 mutations versus the Wilms10 cell line. The genetic characteristics of all established cell lines is shown in WT1 mRNA and that the level varies between cell lines with the lowest, almost undetectable level in Wilms1 cells and the highest level in Wilms3 (WT1 in future studies but this is not within the scope of this work.As this is the first cell line with no genomic DNA covering the n Wilms3 . It willCTNNB1 mutation (CTNNB1 mutation identified in the tumor specimen was not seen in the cell culture. As cultured tumor cells have two copies of chromosome 3 (data not shown) and UPD of 3p  enabling their long term passaging. For this purpose an expression construct encoding a novel triple mutant of LT (U19dl89-97tsA58) was used; this mutant T antigen does not bind to ori-like DNA sequences, does not bind BUB1 and is thermolabile [TERT, immortalized cells were readily established and these were termed "imWilms10". The cells were cultured so far for more than 30 passages, corresponding to 90 population doublings. Cytogenetic analyses of immortalized cells revealed a normal karyotype (not shown). The immortalized cell line remained cytogenetically normal for many passages, possibly due to the deletion of the amino acids responsible for BUB1 binding [WT1 and the two deletions on chromosome 1 (not shown).All established Wilms tumor derived cell lines from tumors with molabile ,21,23. W binding . aCGH shTERT expression in these cells was verified by RT-PCR  and 39°C (nonpermissive temperature) and protein was extracted from these cells. Although the tsLT construct encoded a ts mutant protein a low level was detected at 39°C . Sequency RT-PCR .TERT and tsLT we established gene expression profiles of cells cultivated at 33°C where both genes are active (permissive temperature for tsLT) using Agilent whole genome microarrays. It was the aim of this experiment to investigate the impact of the expression of these two genes on the transcriptome of Wilms10 tumor derived cells. This information shows whether the “biology” of immortalized (imWilms10 cells) is affected. However, it should be noted that 33°C is a nonphysiological temperature for human cells and they grow very slowly at that temperature. First we compared the expression levels of selected genes that are expressed in early kidney development and/or different compartments of the kidney [WT1 mutant cell lines [FOXD1 is typically expressed in stromal cells and also in all WT1 mutant tumor derived cell lines we have established previously. FOXD1 mRNA. Likewise PAX3 is expressed in stromal cells and ectopically in WT with a myogenic histopathology [WT1 mutant cell lines [MEIS1, OSR1, OSR2 and SIX1 [TERT expression ) are down-regulated in imWilms10 cells  and MEST . The strong down-regulation of IGF2 was confirmed by Q-RT-PCR  and an absolute fold change > 1.5. Under these conditions we detected 270 genes with higher and 212 genes with lower expression levels in imWilms10 cells. To gain insight into the function of these genes we used the MetaCore algorithm “enrichment of pathway maps”. The 10 most significant pathway maps are shown in 10 cells . A complQ-RT-PCR  and the nificant . A complHistone H1C, BARD1, CDC7, CCNE1, MCM3 and CCNA1 gene expression levels were strongly induced in imWilms10 cells . A similarity of Wilms tumor derived cell lines with mutant ur group . FurtherWT1 mutant cells have a differentiation potential for these cell fates. Osteogenic differentiation was analyzed by Alizarin red staining and Wilms10 . This indicates that only a few cells have reached a more terminal osteogenic differentiation state and deposit calcium.A number of genes involved in osteogenic, adipogenic and muscle differentiation are expressed at a low level in Wilms10 cells. Therefore, we tested whether parental Wilms10 cells as all other  Wilms10  and imWi Wilms10 . Therefo Wilms10 . This isTo analyze the cells for muscle differentiation we used immunofluorescence with a Titin antibody. Titin is only produced in different muscle cells . ExpressLPL and PPARG [PPARG but not LPL in comparison to uninduced control cells was observed in Wilms10 cells, indicating a limited adipogenic differentiation potential , 37°C (semipermissive temperature) and 39°C (nonpermissive temperature). The imWilms10 cells formed large colonies at 37° and 39°C with an efficiency of 77.2% and 80.7% respectively, whereas at 33°C only 39.3% of the cells formed small colonies . SimilarOur differentiation experiments showed that the parental Wilms10 cells have restricted potentials for muscle, osteogenic and adipogenic differentiation, while these differentiation potentials are still present in imWilms10, they are even further reduced.WT1 nested within a heterozygous 11p13 deletion. The 228kb homozygous deletion only covers the WT1 gene and the 1Mb heterozygous 11p13 deletion extends from WT1 to HIPK3. In addition, UPD restricted to 11p15.2pter and including the IGF2 gene was identified, suggesting a more distal mitotic recombination in 11p15. The high expression of IGF2 in these cells indicates that the paternal allele was duplicated, whereas the rest of chromosome 11 was derived from both parental alleles. We have thus identified a Wilms tumor with a complete lack of WT1 due to a homozygous deletion. It is highly desirable to establish tumor derived cell lines to develop cell culture model systems for Wilms tumors with deleted WT1 genes. Thus our immortalized cell line (imWilms10) represents the first cell culture model system for Wilms tumors with deleted WT1 genes. The immortalized imWilms10 cells were characterized by the expression of selected kidney marker genes, by gene expression profiling, DNA analysis and cell differentiation experiments and were compared to parental Wilms10 cells.We describe here a Wilms tumor (Wilms10) with an unusual tumor specific homozygous deletion of WT1 mutations. These cell lines harbor two genomic DNA copies of mutant WT1, and all of them have two copies of chromosome 11 (unpublished observation). In most cases, the homozygous mutation of WT1 is due to a mitotic recombination event between the last heterozygous marker in 11q11 and the first homozygous marker in 11p12 (unpublished observation). The high expression of IGF2 and low expression of H19 in these cell lines indicates that the paternal allele is duplicated . In one cell line from a WAGR case with a 11p13 deletion on one allele, the remaining copy of WT1 carries a mutation, i.e. the WT1 mutation is hemizygous; both paternal chromosome 11 alleles are present in this case except for the 11p13 deletion. These cell lines express different levels of mutant WT1 mRNAs, whereas Wilms10 cells do not express any WT1 mRNA . This indicates that the tsLT is indeed functionally temperature sensitive. The complete inactivation of the tsLT occurs at 39°C, but we did not study the cells further at that non-physiological temperature. However, a low level of the tsLT protein was detected by Western blot analysis of imWilms10 cells cultured at 39°C, suggesting that inactivation of the tsLT protein is due to a conformational effect. Hardy et al., who studied normal human mammary fibroblasts immortalized with telomerase and a tsLT (HMF3A), also detected the tsLT protein at 37° and 39°C by Western blotting . It is iFoxD1 is highly expressed in cortical and nephrogenic interstitium, in the same compartment that expresses Meis1 [Osr1 was shown to identify multi-potent cells of intermediate mesoderm and its expression becomes restricted during kidney development to an Osr1-dependent nephron progenitor compartment [Osr2, was also shown to be expressed during kidney development [Six1 gene plays a role in limb development and in the developing kidney; it is expressed in uninduced metanephric mesenchyme at E10.5 and in induced mesenchyme at E11.5 [WT1 mutant cell lines and Wilms10 cells and are unaltered in imWilms10 cells. This indicates that these WT1 mutant cells may correspond to an early stage of kidney development before specification of the different lineages and the imWilms10 cells have retained these properties. In addition other genes expressed in kidney cells such as SNPO, CD2AP and NES are not changed by the immortalization process.In order to study whether the immortalized cells retain biological properties of the parental Wilms10 cells we first studied the expression of several characteristic kidney marker genes. Laser capture microdissection of major kidney compartments at mouse embryonic day E15.5 and fluorescence-activated cell sorting of component-specific GFP transgenic mice established a kidney atlas of gene expression . FoxD1 ies Meis1 . Expresses Meis1 . Osr1 wapartment . The relelopment . The Sixat E11.5 . Furtherat E11.5 . These cTERT and the tsLT. Another difference is their state of signaling pathways. Paternal Wilms10 cells showed activation of several signaling pathways, i.e. PDGFR, EGFR, IGF and AXL as revealed by proteome profiler antibody arrays. Upon immortalization, these cells show a down-regulation as well as functional inactivation of several enzyme-linked receptors (RTK). In the case of PDGFRA and PDGFRB decreased phosphorylation is linked to a down-regulation of mRNA expression. In contrast, down-regulation of IGF1R phosphorylation was observed with unchanged IGF1R gene expression levels. As phosphorylation of this receptor is triggered by ligand binding, the almost complete down-regulation of IGF2 gene expression in imWilms10 cells might explain this finding. It is interesting that besides IGF2, expression of another fetal growth factor (MEST), is also reduced to basal levels after immortalization. The down-regulation of IGF2 in imWilms10 cells is remarkable in the context of the paternal duplication of chromosome 11p15 and expression of the gene from both alleles. The down-regulation IGF2 and MEST expression occurs at permissive and nonpermissive temperatures and is therefore independent of tsLT. It therefore likely that telomerase is linked to IGF2 and MEST gene down-regulation in imWilms10 cells, as a function of telomerase in overall chromatin structure regulation and epigenetic mechanisms has been described [IGF2 expression [IGF2 expression in immortalized Wilms tumor cells. This observation suggests that the effect of telomerase is highly cell context dependent and that the expression of fetal growth factors in embryonal Wilms tumor cells is not compatible with the establishment of immortalized cells with an unlimited life span. In summary, the immortalized Wilms tumor cells can proliferate despite a down-regulation of ligands and receptors for several signaling pathways. It will be of interest to study the mechanisms that lead to the activation of these different signaling pathways in the parental Wilms tumor cells and the effects of their down-regulation on the growth of these cells. This should be kept in mind when novel therapeutic approaches targeting of one of these signaling pathways are considered, as the others can still be activated [WT1 mutation in the kidney of mice [One major difference between the Wilms10 cells and imWilms10 cells is the up-regulation of cell cycle genes caused by the expression of hescribed . Note thpression , whereasctivated . However of mice . TherefoWT1 mutant cells. The derived immortalized cells have retained these properties although at a reduced capacity, likely due to the fact that they continue to grow at 37°C, which is not compatible with differentiation. Therefore the parental Wilms tumor cell line with a novel and unusual chromosome 11 alteration and the immortalized derivative with a homozygous deletion of the entire WT1 gene will be useful for further studies on the function of WT1 in Wilms tumor development, to further explore the origin of WT1 mutant tumors as well as for the analysis of activated signaling pathways. Last not least these cells are valuable as negative controls to test various batches of WT1 antibodies, that are known for their unspecific cross reactivity with many proteins.The primary Wilms10 tumor cells show a limited multilineage differentiation potential for osteogenesis, adipogenesis and muscle differentiation, similar to the other S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S11 Fig(PDF)Click here for additional data file.S12 Fig(PDF)Click here for additional data file.S13 Fig(PDF)Click here for additional data file.S14 Fig(PDF)Click here for additional data file.S15 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


The reported high level of mortality among patients both on clinical care and on Highly Active Antiretroviral Therapy (HAART) in Ghana in recent years has been blamed on an advance presentation of AIDS cases due to delay by patients accessing care at Hospitals. The study hypothesized that HIV and AIDS patients report at the ART clinics with very low CD4 lymphocyte count and determined the CD4 lymphocytes count of HIV positive patient at first presentation in the Ashanti Region of Ghana.This retrospective study reviewed clinical records of 2,971 patients accessing care at 19 ART Clinics in the Ashanti region of Ghana for the period January 2010 to December 2012. The date of first reporting and the level of first CD4 counts were recorded. Socio demographic information was also recorded. Data was analyzed using SPSS version 16.More than half  of all HIV patients reviewed presented with CD4 count of less than 250 cells/mm3. Of this number, 37.7% (615/1631) reported with CD4 count less than 50 cell/mm3, 17.3 %(282/1631) with CD4 count of less than 100 cells/mm3, 45.2% (737/1631) with CD4 count of less than 250 cells/mm3. Almost a quarter  presented with CD4 count between 250 and 350cells/mm3 with 21.2% (630/2971) reporting with CD4 count of 350 cells/mm3 and above of which only 11.1% ( 70/630) came with CD4 greater than 500cell/mm3. In all more than three quarter  of the patients reported with CD4 count of less than 350cell/mm3.The study shows that CD4 lymphocytes count of HIV patients accessing care at Hospitals is very low. Delay reporting might account for this development. Public education on the need to access care at the earliest possible time once tested HIV positive must be intensified. Further study is needed to determine the causes for late presenting at the HIV clinic and address them, since this may account for high mortality among HIV positive patients.


Tumor-infiltrang immune cells such as metastasis-associated macrophages (MAM), regulatory T (Tregreg) cells, and myeloid-derived suppressor cells (MDSC) are reported to promote establishment of the lethal metasta-c foci and restrict efficacy of cytotoxic drugs or tumoricidal immune responses by natural killer (NK) and CD8+ T cells. Recent studies suggest that these pro-tumor immune cells are accumulated a chemokine network established in the tumor microenvironment. Therefore, blockade of these chemokine signals could improve therapeu-c efficacy of chemotherapy and immunotherapy. Metastatic breast cancer is incurable by current therapies including chemotherapy and immunotherapy. Accumulating evidence indicates that tumor-infiltrating macrophages promote establishment of the lethal metastatic foci and contribute to therapeutic resistance. Recent studies suggest that the accumulation of these macrophages is regulated by a chemokine network established in the tumor microenvironment. In this perspective paper, we elaborate on the chemokine signals that can attract monocytes/macrophages to the site of metastasis, and discuss whether inhibition of these chemokine signals can represent a new therapeutic strategy for metastatic breast cancer. The 5-year survival of patients with metastatic disease drops to 21% whereas that of patients with early-stage breast cancer is 89–100% +CD11b +Ly6C–) that is barely found in the normal lung + macrophages reduces the metastatic tumor burden in the lung In breast cancer mouse models, lung metastatic foci show marked accumulation of a distinct macrophage population (F4/80It has been reported that the recruitment of macrophages to the primary site is promoted by various cytokines and chemokines such as colony stimulating factor-1 (CSF-1), vascular endothelial growth factor (VEGF) and CC-chemokine ligand 2 (CCL2), although the mechanisms underlying macrophage accumulation in the metastasis sites are still largely unknown. We have recently reported that the accumulation of MAMs at the metastatic lung is regulated by chemokine ligands CCL2 and CCL3 and their respective receptors CCR2 and CCR1 2+Ly6Chigh) to the tumor challenged lung. These results indicate that the CCL2-CCR2 signal recruits circulating IMs to the site of metastasis where they differentiate into MAMs and promote establishment of metastatic foci. We have further found that the MAMs isolated from the mouse lung with metastatic foci express much higher level of CCL3 compared with circulating IMs It has been reported that high levels of CCL2 in breast cancer specimens correlate with high number of macrophages in the primary tumors In our breast cancer model, another CCL3 receptor CCR5 is not necessary for the early MAM accumulation observed within 24 h after tumor injection. However, it is reported that CCR5 is required for macrophage accumulation in the lung foci after 7 days of renal cancer cell injection 3The ultimate objective of macrophage-targeting therapy is withdrawal of tumor-supporting and immunosuppressive microenvironment from the secondary sites by disrupting accumulation and/or function of MAMs. Accordingly, the above-mentioned chemokine signaling molecules are potential targets for the treatment of metastatic breast cancer.Ccl3 expression in MAMs as well as their recruitment following mammary tumor metastasis Results from our breast cancer metastasis model suggest that the inhibition of CCL3 secretion from MAMs is one of the possible strategies as they are a major source of CCL3 among other leukocytes such as neutrophils, T, B, and NK cells in the metastatic lung Ccr1Ccr1−/−−/−−/−−/− mice are healthy without any overt hematopoietic abnormalities unless challenged with specific pathogens Ccr2Ccr2−/−−/−−/−−/− mice show a reduced number of circulating monocytes Another possible strategy to suppress MAM accumulation is blockade of CCR1 and CCR2. Several companies have developed small molecule inhibitors against CCR1 or CCR2 for rheumatoid arthritis or multiple sclerosis, and most of them are well tolerated and show no adverse effects Ccr1Ccr1Ccr1 and Ccr2Ccr2Ccr2 deficiency can achieve complete elimination of metastatic tumors in the mouse model (maximum reduction rate was 60%) and model dependent reg) cells that are recruited via different chemokine signals reg cells migrate towards primary ovarian tumors via CCL22-CCR4 signal reg cells respectively Despite this optimism a treatment with single chemokine antagonist will almost certainly not be enough to suppress metastatic tumor growth since neither + T cell responses + monocytes (CD11b+Ly6C+) enhances accumulation of adoptively transferred CD8+ T cells in the primary tumor and thereby augments therapeutic effect of the adoptive T-cell therapy on the tumor growth in a melanoma model Although breast cancer cells also express chemokine receptors including CCR5, CCR7 and CXCR4 that enhance tumor cell invasiveness and metastasis 4reg cells. Interestingly, mouse breast cancer cells used in our metastasis model can promote CCL5 expression in cultured macrophages (unpublished data). Furthermore, GM-CSF produced by human breast cancer cell lines induces CCL18 secretion from cultured macrophages reg cells (Our data suggest that a gradient of CCL2 attracts monocytes/macrophages towards the metastatic tumor microenvironment where they are exposed to high levels of CCL2 and are prompted to secrete another chemokine CCL3. As mentioned above, such a chemokine-induced chemokine secretion is also reported in human monocyte culture systems, i.e., exposure to CCL5 or CCL18 induce secretion of CCL2, CCL3, CCL22, and CXCL8 eg cells . Since tDr. Pollard has patents pending on targeting CCR1 at metastatic sites.


This paper is aimed to explore the significance of plasma kisspeptin level in diagnosis and therapeutic evaluation through the detection of kisspeption level of girls diagnosed with idiopathic central precocious puberty (ICPP) before treatment and after 6-months of treatment and girls with simple premature thelarche (PT).A total of 70 girls including 24 girls diagnosed with ICPP, 21 girls with PT and 25 normal girls were enrolled. ELISA was adopted to detect plasma kisspeptin level.The kisspeptin level of ICPP group before treatment (1.80±0.13ng/ml)was higher than those of other groups with significantly statistic difference. The kisspeptin level of ICPP group after 6-months of treatment (1.49±0.21ng/ml) was significantly lower than those before treatment (P<0.05).We can conclude that plasma kisspeptin level is related with initiation of pubertal development, and it can be served as important parameter in ICPP diagnosis and therapeutic effect evaluation.