B-cell receptor (BCR)-mediated signaling plays an important role in the pathogenesis of a subset of diffuse large B-cell lymphoma (DLBCL), and novel agents targeting this pathway are now in clinical use. We have previously identified a signature of active BCR signaling on formalin-fixed paraffin-embedded specimens using quantitative immunofluorescence, allowing for identification of patients who might benefit from anti-BCR therapies. We sought to characterize the clinicopathologic significance of active BCR signaling in DLBCL by correlating measures of signaling intensity with clinical features and various tumor cell characteristics. High MYC and concurrent high MYC and BCL2 double-expression was positively correlated with individual markers of active BCR signaling and cases with MYC/BCL2 double-expression showed overall greater BCR activation compared to cases lacking double-expression. Our findings suggest that the BCR signaling pathway may be more active in MYC/BCL2 double-expressor DLBCL and may represent a rational therapeutic target in this aggressive DLBCL subgroup. Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma and has a poor prognosis in approximately 50% of patients ,2. DLBCLBased on cell-of-origin (COO) gene signatures studies, DLBCL can be stratified into prognostically relevant subtypes, with the activated B-cell (ABC) type being associated with an inferior outcome compared with the germinal center B-cell (GCB) type –10. Withp = 0.004). There was no difference between any qIF variable for GCB versus non-GCB cases, nor was there any enrichment for GCB or non-GCB COO within BCR-positive or negative cases [We have previously identified a robust signature of active BCR signaling in DLBCL on formalin-fixed paraffin-embedded specimens based on quantitative immunofluorescence (qIF) of phosphorylated BCR-associated kinases SYK, LYN and BTK . We usedve cases . Our datBCL6, BCL2, as well as MYC are well documented in DLBCL, and MYC translocations are recognized to confer a worse prognosis in patients treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), with or without rituximab (R) [MYC locus in combination with another breakpoint, mainly BCL2, appears to be the result of the combination of MYC and BCL2 overexpression [MYC rearrangement, as other mechanisms of MYC activation can lead to overexpression, including copy number amplification, upstream regulation by microRNA and oncogenic mutations. However, MYC gene activation has been associated with poor outcome and thus, these alternate pathways that activate MYC may have similar biological consequences as MYC rearrangement. Indeed, some studies have shown that B-cell lymphomas with concurrent MYC and BCL2 abnormalities, other than translocations, appear to behave similarly to MYC/BCL2 double-hit lymphomas [N = 93) by correlating signaling intensity with clinical features and tumor cell characteristics. We demonstrate that concurrent high expression of MYC and BCL2 by IHC is positively correlated with previously studied markers of activated BCR signaling. These findings imply that the BCR signaling pathway is more active in DEL compared to other DLBCL subgroups and may represent a rational therapeutic target in this aggressive subgroup of DLBCL.Rearrangements of the oncogenes imab (R) –20. The pression –23. Whilpression ,24,25. Dpression . MYC oveymphomas –29. In aymphomas , althougymphomas . Currentymphomas . In thisTo analyze clinicopathologic features associated with activated BCR signaling in a cohort of primary DLBCL samples, we obtained clinical follow-up data from electronic medical records of patients from the original validation cohort from the previously published study . This coTMA construction was conducted as described previously . BrieflyAll immunohistochemical stains were evaluated via consensus review by two hematopathologists (AEK and ARS), who were blinded to the results of qIF and to patient outcome. Stains for MYC, BCL2, Ki67, CD30 and p53 were scored on tumor cells in increments of 10%. Based on prior data examining optimum survival cutoffs for dichotomizing levels of expression, 40% positivity was used as a cut-off for MYC and 50% positivity for BCL2 overexpression . TherefoMYC/8q24 , BCL2/18q21 , and BCL6/3q27 . Fifty to 100 nuclei were scored per case, and a case was considered positive for the rearrangement if 20% or more nuclei exhibited a break-apart signal.FISH was performed on 4μm thick TMA sections as described previously . Rearrancyt), a surrogate marker of AKT activation, were calculated. Results from untransformed data are based upon arithmetic means, and transformed data are based upon medians, of two to three samples per clinical specimen. Details regarding transformation and reanalysis of BCR signaling classification are provided in t-test was used to compare qIF markers , adjusted where noted by the Holm-Bonferroni correction. Pearson’s χ2 test with Yates’ correction was used to compare nominal variables. Overall survival (OS) was defined as the time from diagnosis to death from any cause. OS was analyzed using the Kaplan-Meier method with censoring. The log-rank test used to compare differences in survival between groups.The initial statistical analysis of qIF data has been previously described . The meaFcyt and were evaluable for BCR signaling by qIF. Among the immunohistochemical markers studied, high MYC expression  and DEL  were found to be positively correlated with markers of activated BCR signaling by qIF (p = 0.08), to 〈pSYK,pBTK〉 (p = 0.02), to Fcyt (p = 4×10−5). Examples of BCR+ and BCR- cases and their differences in MYC and BCL2 expression are illustrated in Of 144 clinical specimens examined, 93 fulfilled criteria for measurement of pLYN, 〈pSYK,pBTK〉 and g by qIF . The sigMYC, BCL2 or BCL6 rearrangement status as determined by FISH. Of note, only 8% of cases harbored a MYC rearrangement and no cases of MYC/BCL2 or MYC/BCL6 double-hit lymphoma were identified.No correlation was observed between BCR signaling markers and the other clinical or pathological parameters studied, including age; sex; COO as assessed by the Hans classifier; expression of BCL2 alone, BCL6, Ki67, CD30 or p53 by IHC; or Fcyt  of cases in the current cohort.Based on previously established criteria , we initFcyt . We therAccordingly, we returned to the original qIF data for BCR signaling markers in activated DLBCL cell lines  and applt-test. Following logit transformation, DEL cases showed significantly higher levels for each individual BCR signaling marker compared to non-DEL cases, including after Holm-Bonferroni correction for multiple hypotheses testing , the largest proportion of whom received R-CHOP  . SurvivaIn this study, we demonstrate that among several pathological features assessed by IHC and FISH, MYC expression by IHC was the only one positively correlated with markers of active BCR signaling as determined by qIF. In addition, DEL showed greater BCR activation as compared to non-DEL based on individual phosphomarker expression, FOXO1 cytoplasmic localization, and overall BCR activity classification. Overall, our findings suggest that the BCR signaling pathway is more active in DEL compared with other DLBCL subgroups and raise the possibility for targeting the BCR signaling pathway with agents, such as ibrutinib, in this inferior prognostic subgroup of DLBCL.Although DEL correlated with both OS and activated BCR signaling, we were unable to demonstrate a direct association between active BCR signaling and OS, which may be related to the retrospective nature of our study, the relatively small number of patients within different immunohistochemical subgroups, and the non-uniform treatment received (as treatment was not a selection criterion for inclusion in our TMA), all important limitations of our analysis. However, our findings suggest that study of a larger number of uniformly treated patients may help to clarify the prognostic and predictive implications, if any, of activated BCR signaling in DLBCL and DEL. In addition, as with immunohistochemical studies of a similar nature, pre-analytical variables and inter- and intraobserver variability in scoring tissue sections may confound the results. Therefore, validation of these findings is warranted following the development of multiplex quantitative IHC assays for simultaneous and objective scoring of multiple markers.MYC oncogene is a transcription factor that has a broad effect on gene expression: it regulates more than 15% of human genes, including a number of microRNA (miRNAs), and contributes to the pathogenesis of many human cancers [MYC gene and enrichment of MYC-repressed genes, show evidence for the role of miR-17~92, a MYC-regulated miRNA, as a major regulator of BCR pathway components [MYC transcript and MIR17HG, the precursor transcript derived from the miR-17~92 gene, compared to other subtypes [The exact mechanisms linking BCR signaling to MYC expression have yet to be elucidated. The  cancers ,36. Actimponents . The miRmponents –41. In tmponents . Interessubtypes . Other dsubtypes ,44. Thersubtypes . Interessubtypes .DEL is characterized by an aggressive clinical course and inferior response to R-CHOP therapy, indicating the need for more individualized therapeutic approaches targeting particular signaling pathways –26. ActiS1 Text(PDF)Click here for additional data file.S1 Figp) vs. p.Plot of logit((TIF)Click here for additional data file.S2 Fig(A) Pairwise scatterplots of untransformed data for %pLYN+, %pSYK+, %pBTK+ in ten DLBCL cell lines. Uneven distribution with crowding at the origin is evident. (B) Pairwise scatterplots of transformed (logit) data for %pLYN+, %pSYK+, %pBTK+. (C) Pairwise scatterplots of transformed (logit) data for %pLYN+, %pSYK+, %pBTK+, with centroids for two clusters generated by unsupervised normal mixture modeling. BCR+ (blue), BCR- (red). (D) Pairwise scatterplots of aggregated (median) transformed data for %pLYN+, %pSYK+, %pBTK+, with centroids for two clusters generated by unsupervised normal mixture modeling. BCR+ (blue), BCR- (red).(TIF)Click here for additional data file.S3 FigPairwise scatterplots of untransformed data for %pLYN+CD20+, %pSYK+CD20+, %pBTK+CD20+ in tissue microarray of primary DLBCL specimens. Uneven distribution with crowding at the origin and near 100% is evident.(TIF)Click here for additional data file.S4 FigDecision tree based on unsupervised recursive partitioning of DLBCL primary specimens according to BCR signaling markers. Each intersection within the tree is labeled based on the majority of cases, and the number of DEL cases (left) and non-DEL cases (right) given below and the percent (%) of all cases within the cohort. Below the tree are one- and two-dimensional scatterplots of cases based on single or pairwise classification by the four BCR signaling markers pLYN, pSYK, pBTK and Fcyt. Cases are assigned as either DEL (red) or non-DEL (blue), and cut-offs determined by recursive partitioning indicated by step function (1-D) or black/white masking (2D). Note: logistic transform for generation of plots used ε = 1×10–3, resulting in slight reduction in overall data range compared to other Figs.(TIF)Click here for additional data file.S5 FigOS for DEL/BCR- (solid black), OS for DEL/BCR+ (dashed black line), non-DEL/BCR- (red solid) and non-DEL/BCR+ (dashed red).(TIF)Click here for additional data file.


Giardia duodenalis is a flagellated parasite that causes diarrhea in humans and other animals. Although G. duodenalis is found in companion animals worldwide, information regarding the prevalence and genetic characteristics of G. duodenalis in pet chipmunks in China is limited. The present study therefore aimed to investigate the prevalence and genotypes of G. duodenalis in pet chipmunks in Sichuan province, southwestern China, as well as to assess zoonotic potential of revealed assemblages.G. duodenalis was 8.6% (24/279), as determined by nested PCR detection of the beta giardin (bg) gene. Giardia duodenalis assemblages and subtypes were determined using multilocus genotyping of the bg, triosephosphate isomerase (tpi), and glutamate dehydrogenase (gdh) loci. Two assemblages were identified: potentially zoonotic assemblage A  and rodent-specific assemblage G . A total of 24, 17 and 17 sequences of the bg, gdh and tpi loci, respectively, were successfully obtained, which formed four, four and three subtypes, respectively. Moreover, four assemblage A (MLGs A1-A4) and three assemblage G (MLGs G1-G3) multilocus genotypes were identified.A total of 279 fecal samples were collected from pet chipmunks in seven pet shops and one breeding facility in Sichuan province, southwestern China. The prevalence of G. duodenalis in pet chipmunks in China. Detection of assemblage A in pet chipmunks and in previous studies in humans suggests a possible role of chipmunks as a reservoir for human giardiasis in Sichuan Province, China.To our knowledge, this is the first study that investigated The online version of this article (10.1186/s13071-018-2790-z) contains supplementary material, which is available to authorized users. Giardia duodenalis is a common enteric parasite that infects a broad spectrum of vertebrate hosts, including humans, livestock, companion animals and wildlife worldwide [G. duodenalis via fecal-oral route by ingesting infective stage cysts in contaminated food or water [G. duodenalis causes self-limiting diarrhea and malabsorption, whereas in infants, elderly persons, and individuals with deficient immune system, it also can cause chronic or life-threatening diarrhea and weight loss [orldwide . Usuallyor water . Clinicaor water . In immught loss , 5.G. duodenalis comprises at least eight genetically distinct assemblages (A-H) [Giardia in pet rodents in countries such as Spain, Italy, Australia, Belgium and China [Molecular studies have demonstrated that es (A-H) . Assembles (A-H) . Assembles (A-H) , 9. So fnd China –12. In and China .Giardia prevalence have been reported in domestic dogs and cats (assemblages C and D) [Eutamias asiaticus) have become popular in China as companion animals. As such, they have been commercially bred in significant numbers in many breeding facilities. However, information regarding G. duodenalis infection in pet chipmunks in China is rare, and the zoonotic potential of this parasite in chipmunks remains unknown. Therefore, the purpose of this study was to identify the prevalence and assemblages of G. duodenalis in pet chipmunks and to determine the genetic characteristics of G. duodenalis-positive samples.In China, epidemiological data about C and D) , raccoonC and D) , cattle C and D) , horses C and D) , sheep, C and D) , 19, petC and D) , as wellC and D) . RecentlFrom March 2016 to April 2017, 279 fecal specimens were collected from chipmunks in seven pet shops and one breeding facility located in Sichuan province, southwestern China  according to the manufacturer’s instructions. Extracted DNA was stored at -20 °C until molecular analysis.All fecal specimens were washed three times by centrifugation at 1500× Giardia duodenalis prevalence and assemblages were first determined by nested PCR amplification of approximately 550 bp fragment of the beta giardin (bg) gene. Furthermore, bg-positive specimens were also analyzed by PCR amplification of the glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes. PCR amplification primers and annealing temperatures in this study .All amplified products were sent to Life Technologies  for sequencing on an ABI 3730 DNA Analyzer  using the BigDye® Terminator v3.1 cycle sequencing kit. Nucleotide sequence accuracy was confirmed by sequencing two separate PCR products. Assemblages and subtypes were identified by aligning nucleotide sequences with known reference A phylogenetic analysis was performed by constructing a neighbor-joining tree using Mega 6 software , based oG. duodenalis-positive by PCR analysis of the bg gene. In China, the prevalence of giardiasis varies from 1.9% [G. duodenalis in pet cats and dogs were 1.9 and 4.5%, respectively [G. duodenalis [G. duodenalis in pet dogs in Henan Province was 12.4% [G. duodenalis in farm dogs was 13.2% [G. duodenalis was found in 26.67 and 9.26% of samples from pets and zoo animals, respectively. The infection rate of G. duodenalis in pet chinchillas was 27.1% in four different cities in China [G. duodenalis prevalence in China may be explained by the differences in geographical location, climate, age and gender of the animals, management system, and the diagnostic method.The present study identified 24  fecal samples that were rom 1.9%  to 27.1%ectively . In Guanodenalis . The preas 12.4% . In Niaoas 13.2%  reportedG. duodenalis-positive samples was observed by sequencing the bg, tpi and gdh genes. A total of 24, 17 and 17 sequences were obtained for the three gene loci, respectively (Table bg gene identified 13 sequences as belonging to assemblage A (9 subtype AI and 4 subtype AI-1) and 11 to assemblage G (7 subtype GI and 4 subtype GII). The subtype AI sequence (MF671918) was identical to the known assemblage AI sequences: accession no. KR051224 (from tortoise in China) and accession no. GQ329671 (from human in Sweden [Rattus norvegicus in Sweden), whereas the subtype GII sequence had two SNPs  in comparison with sequence EU769221. Among 17 samples positive for the tpi gene, 13 sequences were identified as assemblage A (5 subtype AI and 8 subtype AV) and 4 were identified as assemblage G (2 subtype GI and 2 subtype GII). The genetic characterization is presented in Table Rattus norvegicus in Sweden), whereas subtype GI sequence had one SNP (at position 184 C/T) in comparison with sequence EU781013. Out of 17 samples positive for the gdh gene, 10 sequences were identified as belonging to assemblage A (7 subtype AI and 3 novel subtypes AI-1) and 7 were identified as belonging to assemblage G. The novel subtype AI-1 sequence had not been reported previously; it had 5 SNPs when compared with sequence KR075940 . The suin China ) Additional file 2:Figure S1. Phylogenetic relationships of Giardia duodenalis for the bg, tpi, and gdh loci . The relationships between G. duodenalis genotypes identified in this study and other known genotypes deposited in GenBank were inferred by a neighbor-joining analysis of three genetic loci using the Kimura 2-parameter model. Bootstrap values greater than 50% from 1000 replicates are shown. Sequences obtained in this study are marked with a triangle. (TIFF 529 kb)


Cervical cancer is the fourth most common cancer among women worldwide, many of who are still within their reproductive lifespan. Advances in screening and treatment have increased the 5-year survival for early stage disease to over 90 % in developed countries. The focus is now shifting to reducing morbidity and improving fertility outcomes for cervical cancer patients. Radical trachelectomy with lymph node assessment became the standard of care for selected women with lesions <2 cm who desire fertility preservation. However, several questions still remain regarding the degree of surgical radicality required for tumors <2 cm, and fertility-sparing options for women with early-stage disesase ≥2 cm, and those with more advanced disease. Here, we compile a narrative review of the evidence for oncologic and pregnancy outcomes following radical trachelectomy, non-radical fertility-sparing surgery, and the use of neoadjuvant chemotherapy prior to surgery for larger lesions. We also review the literature for assisted reproductive technologies in women with more advanced disease.Available literature suggests that the crude recurrence and mortality rates after radical trachelectomy are <5 and <2 %, respectively (approx. 11 and 4 % for tumors ≥ 2 cm). Among 1238 patients who underwent fertility-sparing surgery for early cervical cancer there were 469 pregnancies with a 67 % live birth rate. Among 134 cases with lesions ≥ 2 cm, there were ten conceptions with a live birth rate of 70 %. Outcomes after non-radical surgery  are similar, although only applicable among a highly selected patient population. For patients ineligible for fertility-preserving surgery or who require adjuvant radiation therapy, current options include ovarian transposition and cryopreservation of oocytes or embryos but other techniques are under investigation.Today, many cervical cancer survivors have successful pregnancies. For those with early-stage disease, minimally invasive and fertility sparing techniques have resulted in improved obstetrical outcomes without compromising oncologic safety. Results from three ongoing trials on non-radical surgery for low-risk tumors <2 cm will further inform the need for radical surgery in such patients. For those in whom natural childbearing is unachievable, advances in assisted reproductive technologies provide reproductive options. Despite our advances, the effects of cervical cancer survivorship on quality of life are not fully elucidated. Cervical cancer is the fourth most common cancer in women worldwide, with over half a million new cases diagnosed annually . It affeLoss of fertility, regardless of cause, is a source of significant psychological distress among women . SeveralRadical trachelectomy (RT) was first described by Eugen Aburel for the treatment of early-stage cervical cancers in the 1950s . This teHowever, several questions still remain about the degree of radicality required, as well as the optimal management of lesions greater than 2 cm. There continues to be a push towards less invasive procedures to reduce peri-operative morbidity, and reduce preterm delivery and perinatal morbidity without compromising oncologic safety. The purpose of this review is to examine the current state of fertility sparing management of cervical cancer, including management of ≥2 cm early stage disease and novel technologies in assisted reproduction for women with locally advanced disease.We searched Ovid EMBASE (from 1974 to 2016 week 13) and Ovid MEDLINE in process & other non-indexed citations (from inception to March 2016) for relevant citations. We performed key-word searches combining various disease-specific terms  with treatment specific terms . We limited our search to English language studies. To identify ongoing planned or unpublished trials, we searched the US National Institute of Health’s clinical trial registry at ClinicalTrials.gov. All searches were supplemented by hand searching the reference lists of key papers for relevant citations. Articles were organized based on topics deemed to be relevant by the authors. Crude recurrence, mortality, and birth rates were calculated from large reports of radical trachelectomy overall, radical trachelectomy for lesions ≥2 cm, non-radical fertility sparing procedures, and fertility-sparing procedures after neoadjuvant chemotherapy. Where follow-up studies were available, we included only the most recent and complete series to avoid double counting patients. A narrative review of these topics is presented here.Early-stage cervical cancer includes disease that is confined to the cervix, measuring ≤ 4 cm, with no apparent spread to adjacent structures or distant organs  . Given tExcellent oncological outcomes have been obtained with radical hysterectomy accompanied by bilateral pelvic lymph node dissection for early-stage disease. Five-year overall survival rates range from 73 to 98 % –14. HoweTo be eligible for fertility sparing management of cervical cancer, two main criteria must be met; 1) the patient’s desire for, and likelihood of, fertility must be sufficiently high, and 2) oncologic safety must be acceptable. A pre-operative fertility workup may be considered, particularly in those with a history of infertility . The patOncologic safety can be assessed in terms of clinico-pathologic risk factors for recurrence. These include tumour size, depth of stromal invasion, presence of lymphovascular space invasion (LVSI), lymph node and parametrial involvement and the feasibility of achieving tumour-free margins –27.Clinical stage, including vaginal and parametrial spread, should be assessed by an expert (e.g. Gynecologic Oncologist) . AlthougFor early stage disease, the difference in recurrence and mortality between squamous carcinomas and adenocarcinomas seems negligible, and both may be considered as candidates for fertility-preservation , 34. A rHowever, non-squamous, non-adenocarcinomas have significantly worse prognosis . In a feLittle prospective data exists as to the optimal surgical margin for trachelectomy specimen. A retrospective review by McCann et al. found that for patients with stage 1A2–2A cervical cancer undergoing radical hysterectomy, close surgical margins (defined as margins ≤5 mm), while not an independent risk factor for recurrence, were associated with other intermediate and high risk features, including lymph node positivity, parametrial involvement, increased size of primary lesion, increased depth of stromal invasion and LVSI . CoincidAssessment for lymph node metastases is critical for any patient with greater than 3 mm depth of invasion (i.e. > stage IA1) or other high risk features  on a biopsy specimen. Suspicious nodes should be sought on pre-operative CT or MRI but the sensitivity and specificity of these modalities in early cervical cancer is modest, given the low prevalence of enlarged nodes in this population . CombineIn the absence of grossly positive nodes on imaging, definitive nodal assessment must be made operatively. Frozen section may be utilized to assess for nodal metastases and positive surgical margins, in which case fertility-sparing surgery may be aborted, or the surgical procedure altered . However, frozen section is not universally practiced due to the concerns regarding false negatives, and loss of tissue for permanent pathological processing. A range of false negative rates for intra-operative frozen section in early cervical cancer has been reported. For stage 1A2–1B1, Panici et al. report a false negative rate of 4.2 % ; for staIt is recommended that both intraoperative, and final pathology be reviewed by a pathologist specializing in gynecology and that ultrastaging be performed for sentinel lymph nodes. Ultimately, the clinician needs to evaluate all available information, and come to a decision regarding further surgery to define the extent of disease (staging), versus adjuvant therapy be it chemo, radiation, or both- the former not necessarily precluding fertility sparing.Radical vaginal trachelectomy (VRT) accompanied by laparoscopic pelvic lymph node dissection has become an accepted treatment modality for fertility preservation in early cervical cancer measuring <2 cm. In 2007, a review of 520 cases found a recurrence and mortality rate of 4.2 and 2.8 %, respectively .Radical trachelectomy can also be performed abdominally (ART), laparoscopically (LRT), and robotically (RRT). An advantage to these alternative approaches is that they more closely resemble the radical hysterectomy familiar to gynecologic oncologists, and do not require special skills in vaginal surgery . AdditioOnce tumour-free margins (>5 mm) have been achieved, many surgeons insert a cerclage suture around the lower uterine segment, in anticipation of future pregnancy . In an aRegardless of the approach, higher recurrence rates have been found in patients with larger tumours . Our revParametrectomy is responsible for the majority of complications related to radical surgery . Among aOur review of the literature identified 203 cases of early-stage cervical cancer eligible for non-radical, fertility-sparing surgery Table . All patWe identified three ongoing prospective trials designed to assess the efficacy of non-radical surgery in the treatment of low-risk early-stage cervical cancer. The SHAPE trial (NCT01658930) is a randomized trial comparing simple hysterectomy to radical hysterectomy  in addition to pelvic lymph node assessment for cases of early-stage (IA2–IB1 < 2 cm), low-risk  cervical cancer . The goaIt is hoped that these trials will help to define a select group of patients for whom non-radical surgical management is oncologically safe.For 2–4 cm FIGO stage IB1 and IIA disease, neoadjuvant chemotherapy (NACT) has been shown to reduce nodal metastases, parametrial infiltration, and overall tumour size, theoretically making otherwise unresectable (for fertility preserving purposes) disease amenable to surgical management –66. AlthOur literature review identified 80 cases of ≥2 cm stage IB1–IIA disease eligible for NACT prior to fertility-sparing surgery Table .Table 8OThe timing of nodal assessment with respect to NACT is not standardized. A study by Vercillino et al. in 2012 showed higher rates of recurrence among a subset of women with positive nodes, in whom fertility-sparing surgery was aborted, compared to women with negative nodes that went on to have NACT and VRT. They suggested that nodal assessment prior to NACT identifies a high-risk group in whom fertility preservation should be avoided . ConversFor patients who require hysterectomy and/or pelvic radiotherapy, fertility preservation depends on assisted reproductive technologies. Recognized options include oocyte or embryo cryopreservation prior to cancer therapy and ovarian transposition , 67. TheThe estimated lethal radiation dose to destroy 50 % of oocytes is ≤2 Gy  and depeOvarian transposition was developed as a method to protect the ovaries from the effects of radiation. Due to the theoretical risk of remigration of the ovaries, the ASCO recommendations on fertility preservation suggest performing the transposition as close to the radiation treatment date as possible . Even afOvarian tissue cryopreservation is an experimental technique for fertility preservation. It is sometimes offered to patients who require immediate gonadotoxic treatment of malignancies where there is insufficient time to offer ovulation induction and cryopreservation of oocytes or embryos. Ovarian tissue can be cryopreserved as cortical biopsies, cortical strips or as whole ovaries, but only cortical biopsies and strips have been successfully transplanted after cryopreservation . The ovaAnother experimental procedure that has had recent media attention is uterine transplantation. A 1-year follow-up report of the first uterine transplant trial was published in 2015 . The triWhile we have made impressive technological advances in early cervical cancer management, the long-term effects of cancer survivorship on quality of life are still not fully understood . SeveralIn addition to GOG 278, a prospective questionnaire-based study is currently assessing quality of life and sexual function in women who have undergone radical abdominal trachelectomy . It is eToday, many cervical cancer survivors have the option of becoming a parent. For those with early-stage disease, minimally invasive and fertility sparing techniques have resulted in improved obstetrical outcomes without compromising oncologic safety. For others, natural fertility and childbearing may be unachievable. However, advances in assisted reproductive technologies continue to make pregnancy and/or parenthood a possibility for those who desire it.Several questions still remain. The safety of non-radical, fertility-sparing surgery has mainly been demonstrated in the context of small, non-randomized comparisons that are fraught with selection bias. The appropriate timing of NACT with respect to nodal assessment, in this context, has yet to be elucidated. The significance of nodal micrometastases remains unclear. For those fortunate enough to undergo fertility preservation and achieve pregnancy, management should be standardized. Centres of excellence should be established, involving gynecologic oncologists, reproductive endocrinologists, maternal fetal medicine specialists, and psychologists specializing in sexual and reproductive health. Ultimately, as we continue to seek answers to our objective questions, the patient-centered purpose of this quest should not be forgotten.


Uric acid (UA) is the end product of purine metabolism and can reportedly act as an antioxidant. However, recently, numerous clinical and basic research approaches have revealed close associations of hyperuricemia with several disorders, particularly those comprising the metabolic syndrome. In this review, we first outline the two molecular mechanisms underlying inflammation occurrence in relation to UA metabolism; one is inflammasome activation by UA crystallization and the other involves superoxide free radicals generated by xanthine oxidase (XO). Importantly, recent studies have demonstrated the therapeutic or preventive effects of XO inhibitors against atherosclerosis and nonalcoholic steatohepatitis, which were not previously considered to be related, at least not directly, to hyperuricemia. Such beneficial effects of XO inhibitors have been reported for other organs including the kidneys and the heart. Thus, a major portion of this review focuses on the relationships between UA metabolism and the development of atherosclerosis, nonalcoholic steatohepatitis, and related disorders. Although further studies are necessary, XO inhibitors are a potentially novel strategy for reducing the risk of many forms of organ failure characteristic of the metabolic syndrome. Uric acid (UA) is the end product of the metabolic pathway for purines, the main constituents of nucleotides. The pathway of UA generation is shown in 2−•, with constants being low against CH3• and t-BuOO• [Since humans are unable to catabolize UA to the more soluble compound allantoin due to lack of urate oxidase or uricase , the ser t-BuOO• . UA dire t-BuOO• . In vitr t-BuOO• . Allanto t-BuOO• .It has been suggested that this antioxidant effect of the high UA concentrations in humans contributes to neuroprotection in several neurodegenerative and neuroinflammatory diseases –14.However, despite the potential antioxidant effect of UA itself, numerous studies have revealed close associations of serum UA concentrations and various disorders, most of which are included in the metabolic syndrome category. Thus, UA metabolism may be a so-called double-edged sword as regards the inflammatory and/or oxidative responses in many organs, though on the whole, its harmful effects appear to outweigh the benefits of UA in most cases.In this review, we first explain the two putative molecular mechanisms underlying inflammation occurrence in relation to UA metabolism; one is inflammasome activation via UA crystallization and the other involves superoxide free radicals generated by XO. While the UA crystallization mechanism would be dependent on a high serum UA concentration, the latter may not necessarily reflect the serum UA concentration though XO activity does lead to the production of reactive oxygen species (ROS).Subsequently, lines of research showing relationships between UA metabolism and the development of various disorders are introduced and discussed. Importantly, recent studies have demonstrated beneficial effects of XO inhibitors against the occurrence and/or progression of several disorders, particularly atherosclerosis and nonalcoholic steatohepatitis (NASH), both of which are associated with insulin resistance, hyperlipidemia, and/or obesity. In this review, atherosclerosis and NASH are discussed extensively, while studies of gout and chronic kidney diseases (CKD) are mentioned briefly. In conclusion, we propose that such XO inhibitors may be more useful for preventing a variety of disorders, such as atherosclerosis and NASH, than previously believed, probably via an anti-inflammatory effect.Among the disorders related to hyperuricemia, gout is the most representative and well known. Features of gout include painful arthritis affecting the limbs, caused by reduced UA crystals in the joints. While symptoms of a gout attack are typical of an acute inflammatory response, as indicated by the presence of swelling, heat, rubescence, and pain, there are many disorders with mild but chronic inflammation which are very likely to be related to UA metabolism. In the latter case, superoxide free radicals generated by XO are key players leading to chronic inflammatory processes eventually resulting in impaired organ functions. Thus, we introduce two independent mechanisms underlying UA metabolism-induced inflammation.In 2002, the inflammasome concept was proposed to involve multiple proteins and to control the cleavage of prointerleukin 1 (IL-1) . InitialκB pathway and upregulate the expression levels of interleukins, including pro-IL-1β and pro-IL-18. Subsequently, the inflammasome complex activated by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) cleaves pro-IL-1β or pro-IL-18, resulting in the production of mature interleukins [Inflammasomes are known to be divided into discernible patterns, depending on component proteins . Among trleukins –17.β through the NF-κB pathway [β release by MUC. The other is the generation of ROS, because an antioxidant, N-acetyl-cysteine, abolished IL-1β secretion by MUC [+ level changes and ROS generation remains unknown, and future studies are expected to resolve this issue [MUC also reportedly serve as a danger signal and trigger the activation of inflammasomes . Althoug pathway . It is tn by MUC . Other sis issue , 28. Eleis issue , 30. ThuWhen mammalian xanthine dehydrogenase (XDH) is converted to XO under stressed conditions such as tissue damage and ischemia , superoxβ1-adrenergic stimulation and impairs the fetal α1-adrenergic vasoreflex response involving NO [ROS from XO might play physiological roles, especially in development. Treatment during pregnancy with allopurinol alters maternal vascular function involving lving NO . Fetal Xlving NO . XO depllving NO . Indeed,The tissue and cellular distributions of XO in mammals are highest in the liver and intestines due to XO-rich parenchymal cells . Xanthinβ [2 for oxidation of these compounds [XO activation is induced by LPS, angiotensin II, NADPH oxidase, hypoxia, hypoxia-inducible factor 1, and inflammatory cytokines such as IL-1β –49. The β –54. Aginβ . It appeβ , 57. XO β . The assβ , forebraβ , skin [6β , liver [β , 62, andβ , as wellβ . XO actiβ . IschemiSuperoxide production by XO may also be enhanced by increasing the amount of its substrate, purine bodies. Excess fructose metabolism results in ATP depletion which is associated with degradation of AMP to hypoxanthine, followed by conversion to UA by XO . Indeed,γ in inflammatory cells [Superoxide produced by XO is an important messenger inducing inflammation and signal transduction, leading to tissue damage. We found inflammatory cytokines to be induced via XO when foam cells form with lipid accumulation . XO regury cells . ROS frory cells .As noted above, superoxide from XO has been suggested to play roles in various forms of inflammatory or ischemic pathophysiology , not necWhile gout is a disorder well known to be caused by the precipitation of UA crystals, the involvement of hyperuricemia in CKD is also widely recognized. The major causes of CKD have been regarded as diabetes mellitus and hypertension, and thus, hyperuricemia was long viewed as a consequence of CKD. In fact, loss of kidney function reduces the excretion of UA into urine, resulting in hyperuricemia. In contrast, recent studies demonstrated a significant association between serum UA and the development of CKD. While each metabolic syndrome component, including hyperglycemia, hyperlipidemia, and hypertension, was associated with an increased CKD risk, hyperuricemia was apparently an independent risk factor not influenced by the others. Therefore, hyperuricemia is both a cause and a consequence of CKD and is frequently associated with other metabolic syndrome features.In terms of CKD pathogenesis, serum UA is likely to activate the renin-angiotensin system resulting in vascular smooth muscle cell proliferation  and to iAs mentioned in Although the relationships between serum UA levels and atherosclerotic diseases, including hypertension , 86, havDespite the association between hyperuricemia and hypertension having been recognized since the 19th century , it was Besides the association with hypertension, hyperuricemia or gout has been confirmed to be related to the morbidity and the mortality of CVD –106. Acc−/− mice [What are the mechanisms underlying the aforementioned association between hyperuricemia and atherosclerotic diseases? First, the role of XO in the pathogenesis of atherosclerosis merits attention. As described above, XO produces ROS when converting hypoxanthine into xanthine and then UA. XO is also expressed in endothelial cells  and was −/− mice , an esta−/− mice , endotheβ, IL-6, IL-12, and TNFα, and the expressions of VCAM1, MCP-1, and MMP2, which were upregulated in J774.1 cells transformed into foam cells by atherosclerogenic serum. Subsequently, febuxostat, another XO inhibitor, was also demonstrated to attenuate the development of atherosclerotic lesions in ApoE−/− mice [−/− mice. The authors demonstrated that cholesterol crystals (CCs) increased endogenous XO activity and ROS production in macrophages and that CCs enhanced not only IL-1β release via NLRP3 inflammasome activation but also secretions of other inflammatory cytokines such as IL-1α, IL-6, and MCP-1 from macrophages, processes which in turn were suppressed by febuxostat or ROS inhibitors. The significance of NLRP3 inflammasome activation in macrophages by CCs was verified by the observation that atherosclerosis in high-cholesterol diet fed LDL receptor- (LDLR-) deficient mice was alleviated by transplanting bone marrow from NLRP3-deficient, ASC-deficient, or IL-1α/β-deficient mice [Recently, we established that XO activity in macrophages also plays a key role in the development of atherosclerosis . During −/− mice . That stent mice . Taking ent mice , 114–116Independently of XO, UA itself is widely recognized to exert direct effects on vascular functions. Vascular endothelial cells express several UA transporters  and incoThe number of nonalcoholic fatty liver disease (NAFLD) patients including those with NASH has been increasing worldwide and a portion of NASH patients will progress to hepatocarcinoma onset –123. TheNASH is characterized by fat deposition, inflammation and fibrosis in the liver, and a two-hit mechanism of onset has been proposed –126. ThiMany clinical studies have been carried out to investigate the relationship between serum UA levels and NAFLD/NASH progression. For example, a cohort study in Korea found the serum UA level to be a useful marker for predicting NAFLD development because the serum UA concentration correlated positively with the 5-year incidence rate of NAFLD . Their cConsistent with these observations, hepatic XO activities and serum UA levels are reportedly increased in murine NAFLD/NASH models , 134. MoAs described above, increasing serum UA or XO activity apparently plays important roles in NAFLD/NASH onset and progression. Interestingly, UA was reported to induce fat depositions by enhancing lipogenesis in hepatocytes. Fructose treatment of HepG2 cells reportedly increased both the intracellular UA concentration and triglyceride (TG) accumulation, while allopurinol, an XO inhibitor, suppressed this fructose-mediated TG deposition. Moreover, the application of UA alone was demonstrated to increase intracellular TG contents as well as ROS generation in mitochondria . As a meROS generation by UA is considered to depend on NADPH oxidase activation , 139. FoCollectively, these observations indicate that UA enhances fatty acid synthesis by regulating lipogenesis and induces ROS generation by regulating NADPH oxidase activity and upregulating fatty acid synthesis, thereby contributing to NASH development.As described elsewhere, UA is involved in inflammasome activation. Recent investigations have provided convincing evidence that inflammasomes are key players in NASH development. An initial study revealed that inflammasome impairment exacerbated the NASH progression induced by feeding a methionine-choline deficient diet for 4 weeks to ASC or IL-1 KO mice . SubsequTaking these lines of evidence together, in the initial stage of NASH, inflammasomes appear to exert a protective effect, but continuous inflammasome activation appears to cause excessive productions of inflammatory cytokines, ultimately resulting in liver injury. Although, to date, numerous factors playing important roles in NASH progression have been identified, UA also appears to be a key participant in the onset of NAFLD/NASH.Hyperuricemia was reportedly found to be related to insulin resistance in several clinical analyses –152. In β-hydroxybutyrate levels, dose-dependently, while allopurinol, a XO inhibitor, blocked it. Because UA is the downstream product of AMPD and allopurinol abolished fructose-induced lipid accumulation, AMPD effects on AMPK appeared to depend on UA [Furthermore, excess fructose intake is one of the major causes of the development of obesity with hyperuricemia, fatty liver, and metabolic syndrome. Fructose is metabolized by fructokinase to fructose-1-phosphate and results in a drop in both intracellular phosphate and ATP levels . The intnd on UA . UA actind on UA . Via theUA is considered to be an antioxidant in human blood, though UA induces oxidative stress in cells . UA raisInvolvement of increased XO catalyst activity in pathophysiological processes  suggestsFebuxostat preserved renal function in 5/6 nephrectomized rats with and without coexisting hyperuricemia and prevented diabetic renal injury in streptozotocin-treated rats , 167. FeOn the other hand, beneficial effects of XO inhibitors on atherosclerosis and NASH constitute an evolving concept that has yet to be proven. In rats with fructose-induced metabolic syndrome, febuxostat treatment reversed hyperuricemia, hypertension, dyslipidemia, and insulin resistance . The benAtherosclerosis has been far more extensively investigated than NASH, both clinically and experimentally. Tungsten, acting as an XO inhibitor, has an inhibitory effect on both atherosclerosis and oxidative stress . We repo2− radical dot production and improves endothelial function in blood vessels from hyperlipidemic experimental animals [2− generation [XO inhibitors also improve endothelial function and prevent vascular remodeling. Oxypurinol reduces O animals . XO inhi animals . Allopur animals . However animals . Pretrea animals , in the  animals , 62, the animals , kidneys animals , and cor animals . XO inhineration .Inflammation related to UA metabolism is induced via either inflammasome activation by UA crystal precipitation or free radical production in response to XO activity. In addition to gout, many disorders are known to be related to UA metabolism and XO inhibitor treatments have been shown to be effective for preventing the onset and/or the progression of such disorders. In particular, atherosclerosis and NASH are diseases for which relationships with UA metabolism were not immediately recognized, but rodent model studies revealed the importance of UA metabolism maintenance for managing these disorders. We believe the impact of UA metabolism on many diseases accompanying chronic inflammation to have been underestimated. Future studies are anticipated to reveal the pathological contribution of serum UA and/or XO activity to the specific processes underlying various disorders. Further study of the detailed molecular mechanisms is clearly warranted.


Human T cell leukemia virus type 1 (HTLV-1) is an oncogenic human retrovirus that has infected 10–15 million people worldwide. After a long latency, 3–5% of infected individuals will develop either a severe malignancy of CD4+ T cells, known as Adult T-cell Leukemia (ATL) or a chronic and progressive inflammatory disease of the nervous system designated Tropical Spastic Paraparesis/HTLV-1-Associated Myelopathy (HAM/TSP). The precise mechanism behind HTLV-1 pathogenesis still remains elusive. Two viral regulatory proteins, Tax-1 and HTLV-1 bZIP factor (HBZ) are thought to play a critical role in HTLV-1-associated diseases. Tax-1 is mainly involved in the onset of neoplastic transformation and in elicitation of the host’s inflammatory responses; its expression may be lost during cell clonal proliferation and oncogenesis. Conversely, HBZ remains constantly expressed in all patients with ATL, playing a role in the proliferation and maintenance of leukemic cells. Recent studies have shown that the subcellular distribution of HBZ protein differs in the two pathologies: it is nuclear with a speckled-like pattern in leukemic cells and is cytoplasmic in cells from HAM/TSP patients. Thus, HBZ expression and distribution could be critical in the progression of HTLV-1 infection versus the leukemic state or the inflammatory disease. Here, we reviewed recent findings on the role of HBZ in HTLV-1 related diseases, highlighting the new perspectives open by the possibility of studying the physiologic expression of endogenous protein in primary infected cells. Human T-cell leukemia virus type 1 (HTLV-1) was the first oncogenic retrovirus identified in humans . It is cFour clinical subtypes of HTLV-1 associated ATL are described: smoldering, chronic, lymphoma and acute . DependiThe pathogenetic mechanisms at the basis of HTLV-1 infection progressing toward ATL or HAM/TSP are not clearly understood although there are sedimented indications that interactions between the viral encoded proteins and a variety of cellular targets are crucial. Two viral proteins, Tax-1 and HTLV-1 bZIP factor (HBZ), encoded by the sense and antisense viral transcripts, respectively, are largely responsible for the malignant transformation and immortalization of the infected T cells. Tax-1 is a potent activator of viral transcription and is involved in neoplastic transformation through modulation of the expression of cellular genes and deregulation of diverse cell signaling pathways involved in cell proliferation, DNA damage repair, and apoptosis  The oncoAt variance with Tax-1, HBZ mRNA  and HBZ Interestingly, several studies have demonstrated that HBZ exerts opposite effects with respect to Tax-1 on signaling pathways. Tax-1 activates while HBZ selectively inhibits the classical NF-κB pathway by affecting the binding of p65-RelA to its consensus DNA sequence and promoting its degradation. This inhibition leads to the activation of the alternative NF-κB pathway . It suppTaken together, all these data emphasize the critical role of HBZ in promoting cellular proliferation and the persistence of the viral infection.Since its discovery in 2002, HBZ has become a crucial hotspot in HTLV-1 research. HBZ gene is encoded by the minus strand of the HTLV-1 RNA genome  and tranHBZ transcripts are translated into polypeptides of 206 and 209 amino acids, respectively, and they have almost identical sequences except for a stretch of seven amino acids at the N-terminus of the protein (MAAS for SP1 HBZ and MVNFVSA for usHBZ). However, the half-life of usHBZ protein is much shorter than that of SP1 HBZ  form and two alternative spliced form (SP1 and SP2) . Regardi SP1 HBZ  and the  SP1 HBZ . Neverth SP1 HBZ , as they SP1 HBZ  and two  SP1 HBZ . Most of SP1 HBZ . Similar SP1 HBZ . Convers SP1 HBZ .in vivo in HTLV-1 infected and in ATL patient  and thus it has been hard to extrapolate these results to the real situation encountered in infected cells or in leukemic cells from patients. Only recently, the availability of an anti-HBZ monoclonal antibody (mAb), 4D4-F3, isolated in our laboratory, has made it possible to assess endogenous HBZ expression, localization and interaction  patient . Indeed, patient . By usiner group . Intereser group .These studies have also demonstrated that endogenous HBZ protein is localized in the nucleus of ATL cells  with a sFigure 1). This cytoplasmic localization was not affected by the presence of leptomycin B, a nuclear export inhibitor, indicating that the viral factor did not shuttle in and out of the nucleus . We may think that primary HTLV-1 infection is characterized by the expression of Tax-1 (this stage may be observed in PBMC of still asymptomatic patients), followed by the possible transient coexpression of HBZ (yet to be demonstrated). As we have no specific demonstration of this intermediate step, we do not know whether HBZ protein can localize in the nucleus, cytoplasm or both cellular compartments at these early stages of infection. Immediately after, a compartmentalization of HBZ in the cytoplasm can take place in case of evolution of the infection toward HAM/TSP. At this stage, the evolving pathology can either present a concomitant Tax-1 expression in the same cells (rare), or a mutually exclusive expression of Tax-1 and HBZ in different cells, as we observed in our patients. Conversely, in case of evolution toward the leukemic stage, HBZ will be expressed always and only in the nucleus with or without the coexpression of Tax-1. This hypothetical scheme is useful to test several additional hypotheses and investigate previously unforeseen mechanisms. For example, can we pinpoint a stage in which HBZ is compartmentalized in the cytoplasm still without clinical signs of neurological disease and use this parameter also as a prognostic marker of future development to HAM/TSP? Moreover, what are the molecular mechanisms that drive the distinct nuclear or cytoplasmic localization of the HBZ protein? Are there specific interactors that retain HBZ in the two different cellular compartments? Are these mechanism related to the onset and/or the persistence of the distinct pathologies? These and other fascinating questions’ are now, we believe, experimentally amenable to scrutiny.A new paradigm is emerging that suggests a possible roadmap in the evolution of infection based on these new findings (MB, GF, and RA participated in the conception and design of the review and revised the manuscript. All the authors read, critiqued, and approved the final manuscript.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.


Giardia duodenalis is an important protozoan parasite. It is an established zoonotic pathogen and dairy calves have been implicated as one of the most important sources of human infection. This study was conducted to assess the prevalence and multilocus genotyping of G. duodenalis in dairy calves in the Xinjiang Uyghur Autonomous Region, northwestern China.G. duodenalis by polymerase chain reaction (PCR) detection of the small subunit ribosomal RNA (SSU rRNA) gene, with the prevalence being 9.7 % (23/237) and 16.6 % (46/277) in pre- and post-weaned calves, respectively. Sequence analysis of the SSU rRNA gene predominantly detected G. duodenalis assemblage E , whereas assemblage A was identified in five samples . All G. duodenalis-positive samples were assayed with PCR followed by sequencing at β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes, and 29, 37 and 33 sequences were obtained, respectively. The presence of mixed G. duodenalis assemblage A and E was detected in only one sample. Multilocus genotyping yielded 15 multilocus genotypes (MLGs), one new assemblage A MLG, and 14 assemblage E MLGs. All assemblage E MLGs identified here differed genetically from those of cattle from Henan Province, Central China.A total of 514 fresh fecal samples were randomly collected from dairy calves in 15 farms in Xinjiang, 13.4 % (69/514) tested positive for G. duodenalis is a common parasite in dairy calves in Xinjiang, China, and calves appear to be a reservoir of G. duodenalis that is infectious to humans. The differences in the distribution of G. duodenalis assemblage E MLGs from cattle were likely to be because of geographical segregation.Our data indicate that  Giardia duodenalis  is one of the most frequent enteroparasites worldwide with a broad host range that includes humans, livestock, companion animals and wildlife [Giardia cysts commonly occur in the aquatic environment and transmission of Giardia cysts to humans occurs mainly through indirect routes such as contaminated surface water or foods or through direct contact with infected individuals [G. duodenalis infection combined with the large output of feces, potentially leading to contamination of surface and ground water [wildlife . Giardiaividuals . Cattle nd water , 4.Giardia duodenalis consists of at least eight genetically different assemblages, A–H, of which assemblages A and B infect both humans and other mammals, while the remaining assemblages (C–H) appear to be host-specific [Giardia duodenalis infections in adult dairy cattle are generally lower than in calves, but calves were more frequently infected with zoonotic assemblages A and B compared with assemblage E [G. duodenalis isolates from calves and from 30 humans living in the same region; the isolates were collected over a similar period, suggesting that calves posed a great risk of G. duodenalis infection to humans [specific . Surveysspecific , 6. Recespecific . Giardiamblage E –11. A sto humans .G. duodenalis in calves in China, and current data on the assemblage distribution and multilocus genotyping of G. duodenalis in dairy calves remain unclear. In the present study, fecal samples from dairy calves were collected in the Xinjiang Uyghur Autonomous Region were analyzed for the presence of G. duodenalis using polymerase chain reaction (PCR) of the small subunit ribosomal RNA (SSU rRNA) gene. All G. duodenalis-positive samples were characterized for β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes to elucidate G. duodenalis genotypes.Little is known about the prevalence of From August to September 2013, a total of 514 fecal samples consisting of 237 from pre-weaned calves (0–60 days) and 277 from post-weaned calves (61–150 days) were randomly collected from 15 intensively reared dairy cattle farms near the cities of Wujiaqu, Changji, Urumqi, Korla, Tacheng, Zhaosu and Aksu in Xinjiang Uyghur Autonomous Region , northwestern China  according to the manufacturer’s instructions. For screening G. duodenalis, previously described nested PCR assays were used to amplify the SSU rRNA gene [G. duodenalis assemblages, we analyzed multilocus sequence polymorphisms based on β-giardin (bg) [gdh) [tpi) [G. duodenalis subtypes. DNA from all G. duodenalis-positive samples were subjected to further PCR analysis of the bg, gdh and tpi genes according to previously described nested PCR protocols [Genomic DNA was extracted from all fecal samples using the E.Z.N.A.RRNA gene . Becausedin (bg) , glutamag) [gdh)  and trioh) [tpi)  genes torotocols –17.http://www.megasoftware.net/). To study the relationship between different isolates in more detail, phylogenetic analyses were performed using a concatenated dataset of bg, gdh and tpi gene sequences with the multilocus genotypes (MLGs) of G. duodenalis. The reference MLGs of G. duodenalis in cattle from Henan Province, Central China originate from a previous study [PCR amplicons were sent to Beijing Nuosai Biological Engineering Biotechnology Company for bi-directional sequencing on an ABI PRISM™ 3730 XL DNA Analyzer using the BigDye Terminator v3.1 Cycle Sequencing Kit . Sequences were identified by alignment with reference sequences downloaded from GenBank using MEGA 5 software  [G. duodenalis was 13.4 % (69/514)  , 17–27. G. duodenalis infection with the age of animals. In the present study, the prevalence in post-weaned calves (16.6 %) was higher than the prevalence in pre-weaned calves (9.7 %) . This is similar to reports from Canada [There was a significant association of %) Table . Chi-squm Canada , Norway m Canada , United m Canada , 20 and m Canada . In contm Canada , 26–28. G. duodenalis assemblage A  and the rest were assemblage E   Table . The gened Table . Only onm Canada , China [m Canada , 26, 27,m Canada , 20, 22,m Canada  and Germs Region  and Heils Region .G. duodenalis assemblages between the age groups is presented in Table G. duodenalis were detected in pre- and post-weaned calves in the present study, which is consistent with other studies in Henan and Shaanxi Province, China [G. duodenalis were detected in pre-weaned calves, only assemblage E was detected in post-weaned calves in Ningxia Hui Autonomous Region, China [A comparison of the e, China , 27, Unie, China , 20 and e, China . Howevern, China .G. duodenalis in humans and animals from different geographic regions, which is helpful for unveiling zoonotic potential and dynamic transmission [bg gene, two were identified as one assemblage A sequence, while 27 were identified as seven assemblage E sequences (Table gdh gene, of the 37 G. duodenalis isolates successfully sequenced, three were identified as one assemblage A sequence, while 34 were identified as 11 assemblage E sequences (Table tpi gene, of the 33 G. duodenalis isolates successfully sequenced, five were identified as two assemblage A sequences, while 28 were identified as eight assemblage E sequences (Table Based on multilocus genotyping, the MLG model was used to better understand the characteristics of smission , 30. In G. duodenalis infections are common in dairy calves. The livestock-specific G. duodenalis assemblage E was the predominant assemblage, but the zoonotic assemblage A was also present in Xinjiang, China. The differences in the distribution of G. duodenalis assemblage E MLGs from cattle likely indicate a geographical segregation. Moreover, more multilocus genotyping studies are needed, which may help to identify polymorphisms and to elucidate the zoonotic potential of G. duodenalis.The results of the present study confirm previous findings in other areas of China that


Screening of blood for human T-cell lymphotropic virus type 1 and type 2  is important to diagnose and prevent infection and ensure the safety of blood supplies. The Elecsys HTLV-I/II assay is a newly developed, electrochemiluminescence screening assay for the detection of HTLV-1/2 infection. The sensitivity and specificity of the Elecsys HTLV-I/II assay were determined using well-characterized HTLV-1/2-positive serum and plasma samples and routine diagnostic and blood donor samples expected to be HTLV negative, respectively. These results were compared with those for at least one of the following CE-marked assays at seven independent laboratories and the Roche Diagnostics facility in Penzberg, Germany: Abbott Architect rHTLV-I/II, Ortho Avioq HTLV-I/II Microelisa system, Abbott Prism HTLV-I/HTLV-II, and DiaSorin Murex HTLV I+II. Fujirebio INNO-LIA HTLV-I/II Score was used as a confirmatory assay. The Elecsys HTLV-I/II, Abbott Architect rHTLV-I/II, and Abbott Prism HTLV-I/HTLV-II assays detected all HTLV-1/2-positive samples . Sensitivity for Ortho Avioq HTLV-I/II was 98.63%. The Elecsys HTLV-I/II assay had a specificity of 99.95% in blood donor samples, which was comparable to results for the other assays . In routine diagnostic samples, the specificity of the Elecsys HTLV-I/II assay was 99.83%, compared with 99.70% for Abbott Architect rHTLV-I/II. Specificity for the Elecsys HTLV-I/II assay in potentially cross-reactive samples was 100%, compared with 99.0% for Ortho Avioq HTLV-I/II and 99.2% for DiaSorin Murex HTLV I+II. The Elecsys HTLV-I/II assay has the sensitivity and specificity to support its use as a routine screening assay for detecting HTLV infection. Human T-cell lymphotropic virus (HTLV) is a cell-associated retrovirus, and four types have been identified to date. Type 1 (HTLV-1) is the only type that has been linked conclusively to disease. Infection with HTLV-1 is associated with the development of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), with the former occurring in up to 5% and the latter in up to 3% of infected individuals . HTLV-1 HTLV-1 and HTLV-2 are the most prevalent types, but their global distribution is unusual and their prevalence is poorly understood. The most recent estimate suggests that 5 to 10 million people worldwide are infected with HTLV-1, but the actual number of infected individuals is likely to be much higher , 12.HTLV is transmitted through sexual intercourse, blood transfusions, organ transplantation, and needle reuse or from mother to child, predominantly during breastfeeding , 14. HTLLaboratory screening for HTLV-1/2 infection has become routine for blood donors in developed and some developing countries where the virus is endemic or where blood donors are considered to be at risk of infection , 18, 29.The Elecsys HTLV-I/II assay is a newly launched, double-antigen sandwich screening assay. The assay detects the immune response against the gp21 and p24 antigens . The immn = 10) , while Ortho Avioq HTLV-I/II missed three HTLV-1-positive samples .The overall sensitivity of each assay is shown in The overall specificity in blood donor samples was 99.91% for the Abbott Architect rHTLV-I/II, 99.95% for the Elecsys HTLV-I/II (based on results for serum and plasma samples) and Ortho Avioq HTLV-I/II assays, and 100% for Abbott Prism HTLV-I/HTLV-II . One samThe overall specificity in routine diagnostic samples was 99.70% for Abbott Architect rHTLV-I/II (the only comparator assay used in this part of the study) and 99.83% for the Elecsys HTLV-I/II assay . The speThere were no notable differences in specificity results across the individual laboratories.The Elecsys HTLV-I/II assay was shown to be unaffected by icterus , hemolysis , lipemia , and biotin (≤246 nmol/liter or ≤60 ng/ml) during development (data not shown).In the investigation of possible cross-reacting factors at the laboratories in Paris and Penzberg, no false-positive result was found with the Elecsys HTLV-I/II assay . At the Paris laboratory, the Ortho Avioq HTLV-I/II assay showed one false-positive result for a precharacterized hepatitis B virus (HBV)-positive sample . At the Penzberg laboratory, the DiaSorin Murex HTLV I+II assay showed one false-positive result for a precharacterized rubella virus-positive sample .This study demonstrates that the Elecsys HTLV-I/II assay has the sensitivity (100%) and specificity , regardless of the geographic origin of the samples, virus type, or location of the testing laboratory, to support its use as a routine screening assay worldwide.The Abbott Architect rHTLV-I/II and Abbott Prism HTLV-I/HTLV-II assays also identified all precharacterized HTLV-1/2-positive samples correctly . These findings are consistent with previously reported results. In an earlier study of 406 precharacterized HTLV-1/2-positive samples, Abbott Architect rHTLV-I/II had a sensitivity of 100% , 22. SimOverall, the Elecsys HTLV-I/II assay (99.95%) had specificity comparable to that of Ortho Avioq HTLV-I/II (99.95%), numerically lower specificity than Abbott Prism HTLV-I/HTLV-II (100%), and numerically higher specificity than Abbott Architect rHTLV-I/II (99.91%) when tested in blood donor samples. The specificity of the Elecsys HTLV-I/II assay (99.83%) was numerically higher than that of Abbott Architect rHTLV-I/II (99.70%) when tested in routine diagnostic samples. The confidence intervals of the specificity data for all four screening assays were overlapping, and therefore, these numerical differences were not statistically significant. All assays showed consistent specificity regardless of the location of the testing laboratory. The results for the comparator assays were consistent with previous data. In a study of 5,646 blood donor samples, Abbott Architect rHTLV-I/II had a specificity of 99.95%, compared with 99.86% when tested in 692 routine diagnostic samples , 22. OrtIn certain geographic regions, particularly in Africa, which represents the largest area where HTLV-1 infection is endemic, there is a wide genetic variability of HTLV-1 genotypes and a large pattern of indeterminate serology exists , 26. UnfThe Elecsys HTLV-I/II assay is a quick , one-step (double-antigen sandwich) assay, and the simplicity of the methodology and speed of the assay offer clear benefits in terms of the number of samples that can be processed by a testing facility within a given time. Furthermore, the antigen combination in the Elecsys HTLV-I/II assay was not associated with any cross-reactivity in our study.In summary, the Elecsys HTLV-I/II assay is a reliable test for screening for HTLV-I/II infection in the blood and organ donation settings and for diagnosis where there is suspicion of HTLV-associated diseases, such as ATL, HAM/TSP, and undiagnosed myelopathy.Seven independent laboratories from six countries  and the Roche Diagnostics facility at Penzberg in Germany participated in the study . The stuThe Elecsys HTLV-I/II assay  is an electrochemiluminescence immunoassay for use on the cobas e analyzers. The three principal comparator screening assays were Ortho Avioq HTLV-I/II Microelisa system , which is an enzyme-linked immunosorbent assay, and Abbott Prism HTLV-I/HTLV-II  and Abbott Architect rHTLV-I/II , which are chemiluminescent immunoassays.Each laboratory evaluated the Elecsys HTLV-I/II assay and at least one comparator assay . All assn = 1,149) were previously characterized as positive for HTLV-1/2 . Assay sensitivity was calculated as the percentage of precharacterized HTLV-1/2-positive samples identified as positive by the assay.Assay sensitivity was evaluated at the Roche Diagnostics facility at Penzberg using frozen serum samples provided by Slieagen, USA; the American Red Cross, USA; Universidad Peruana Cayetano Heredia, Peru; Laboratoire CERBA, France; ProMedDx LLC, USA; Trina Bioreactives AG, Switzerland; and the University of Tokyo, Japan (906 samples). The national reference center for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) in transfusion (INTS), Paris, France (219 samples), and the laboratory in Barcelona, Spain (24 samples), also analyzed samples. All samples . All were leftover samples from routine diagnostic requests and blood donations. Assay specificity was tested in 11,575 samples from blood donors and 2,399 routine diagnostic samples that had previously been identified as negative for HTLV-1/2. Assay specificity was evaluated using blood donor samples at four laboratories  and routine diagnostic samples at two laboratories . Assay specificity was calculated as the percentage of true HTLV-negative samples that tested negative with the assay.Escherichia coli, elevated rheumatoid factor (>200 IU/ml), and various autoimmune disorders. Testing for potential cross-reacting factors was conducted at the Roche Diagnostics facility at Penzberg (122 samples) and at the INTS laboratory in Paris (100 samples).Assays were also tested for interference using 222 frozen serum samples containing potentially cross-reacting factors. These samples had been characterized previously as positive for hepatitis A virus (HAV), HBV, HCV, HIV, herpes simplex virus, rubella virus, Epstein-Barr virus, For all screening assays, determinations were performed as single measurements. In the evaluation of specificity, samples giving an initial reactive (IR) result were retested in duplicate and were considered to be repeatedly reactive (RR) if either of the retest results had a signal/cutoff (s/co) ratio of ≥1.00. The exception to this was the laboratory at Hagen in Germany, where IR samples were retested with a single determination. All positive samples in the specificity evaluation were subjected to confirmatory testing with HTLV immunoblotting. A sample was considered to be true positive, indeterminate, or true negative depending on the result of the HTLV immunoblot assay.


Tissue patterning, through the concerted activity of a small number of signaling pathways, is critical to embryonic development. While patterning can involve signaling between neighbouring cells, in other contexts signals act over greater distances by traversing complex cellular landscapes to instruct the fate of distant cells. In this review, we explore different strategies adopted by cells to modulate signaling molecule range to allow correct patterning. We describe mechanisms for restricting signaling range and highlight how such short-range signaling can be exploited to not only control the fate of adjacent cells, but also to generate graded signaling within a field of cells. Other strategies include modulation of signaling molecule action by tissue architectural properties and the use of cellular membranous structures, such as signaling filopodia and exosomes, to actively deliver signaling ligands to target cells. Signaling filopodia can also be deployed to reach out and collect particular signals, thereby precisely controlling their site of action. Drosophila finally enabled the visualization and manipulation of the graded Bicoid and Dorsal proteins that pattern cell fates along the anterior–posterior and dorsal–ventral axes, respectively [Drosophila wing and embryo, and gradients of all major classes of signals have now been described [The ability to pattern fields of cells into distinct fates underpins multicellularity. Classical embryology experiments dating back to the early 1900s initially gave rise to the ideas of cell fate induction by other cells or tissues and the existence of gradients of substances that could generate pattern . Spemannectively . AlthougWhile the simplest mechanism for regulating signaling range is diffusion of a signaling molecule from its source, studies in many contexts have revealed more elaborate mechanisms. In this review, we highlight common themes that have emerged in relation to signaling molecule distribution based on recent studies of different types of signaling molecules in diverse contexts.In this section, we describe different mechanisms used to generate short-range signaling, showing how local signaling can generate pattern either across a single cell diameter or even within a cellular field.Drosophila ovary is a bundle of ~15 ovarioles, with a germarium structure at the anterior tip of each ovariole. Within the germarium, typically two germline stem cells (GSCs) reside within a niche comprised of somatic cells  is expressed by niche cap cells and acts within the somatic niche to promote short-range Dpp signaling within GSCs [dally transcription in escort cells (ECs) and escort stem cells that lie posterior to the niche and enclose the germline cells. dally repression in these cells is mediated by EGF signaling, with EGF ligands released by germline cells, including GSCs [dally in ECs increases GSC number [dally expression and presentation by niche cells, rather than by GSCs, may facilitate the required loss of Dpp signaling upon cells exiting the niche [dally, Dpp could remain associated with the cell upon division, which is not compatible with the sharp on–off distinction in Dpp signaling required for the GSC-CB fate change.Glypicans are a family of heparin sulfate proteoglycans (HSPGs), bound to the outer surface of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor . The Drohin GSCs , 17 , via an interaction that leads to the internalization and degradation of both Pent and Dally . Pent is element  that is  element , 23. Thetkv expression. Tkv protein is present on EC membranes and projections that extend around GSCs and cysts. Tkv, via its extracellular domain, sequesters Dpp released from the niche to limit the number of GSCs, with GSC expansion detected upon tkv knockdown in ECs. In this way the niche has a self-restraining property in that it not only produces the Dpp self-renewal signal but also secretes Wg/Wnt6 that signal to ECs to ultimately restrict Dpp distribution to the niche, thus facilitating differentiation of cells upon niche exit [dpp transcription. In turn BMPs released from the cap cells appear to attenuate Wnt-responsiveness in anterior ECs, suggesting mutual antagonism between the BMP and Wnt pathways. Interestingly, there is evidence that this BMP-Wnt signaling balance is perturbed as females age, which contributes to the decline in niche function in older females [In addition to cap cell expressed Dally, expression of the Dpp receptor, Thickveins (Tkv), in ECs appears to restrict Dpp distribution. Wg and Wnt6 expressed by cap cells signal to ECs to directly activate che exit . In addi females . As desc+ stem cells are maintained in between terminally differentiated niche Paneth cells [+ stem cells from mouse crypts can grow into self-organising “mini-gut” epithelial organoids when cultured in vitro in the presence of EGF, Noggin, and the Wnt agonist R-spondin. Lgr5+ stem cells initially form symmetric cyst structures, then budding structures that resemble crypts, followed by further expansion to generate the complete organoid. In the organoid multiple crypts, with Paneth cells and stem cells at the crypt base .A similar process has been suggested to establish a long-range Wnt3a gradient in the mouse paraxial mesoderm . Here, Wndr1 expression, initiates in the dorsal-most cells at the sphere stage before Nodal signaling is then activated within all cells at the margin by zygotic ndr1/2 expression in the yolk syncytial layer. Ndr1/2 autoactivates so that, over time, Nodal signaling is progressively transcriptionally activated in the next tier of adjacent cells through this positive feedback, which results in spreading of the Nodal signaling domain in the direction of the animal pole  and Ndr2 . Here, a temporal window for signal activation defines the spatial dimensions of the Nodal signaling domain in a mechanism involving differences in timing of the production of Ndr1/2 and their antagonists Lefty1 and 2 (Lft1/2), primarily due to miRNA repression of the latter . Nodal signaling . The miRnot ndr2 .miR-430 repression, through an as yet unknown mechanism, at the 50% epiboly stage. Lft1/2 proteins inhibit Ndr1/2 signaling to prevent further expansion of the signaling domain, which is limited spatially to 5–6 tiers of marginal cells. However, signaling within the domain of cells that received Ndr1/2 ligands persists for several hours, potentially due to signaling from internalized receptors. Moreover, within the Nodal signaling domain, dorsal margin cells that have received Ndr1/2 signals for a longer duration have higher pSmad2 levels resulting in graded signaling across the active domain [Lft1/2 proteins eventually accumulate following a loss of e domain . There ie domain . Ndr2 ise domain , the insOverall, this graded Ndr1/2 signaling results in the highest levels of pSmad2 and downstream transcriptional targets in a dorsal-to-ventral gradient at the blastula stage, then in a vegetal-to-animal gradient in late blastula stage embryos. Restriction of Ndr1/2 signaling to the six cell layers can be reconciled with the previous reports suggesting the expression of long-range Nodal targets beyond this domain, as these genes were instead found to be activated by FGF signaling, which itself is activated by Ndr1/2 signaling . FinallyThe examples in this section describe how tissue architecture can modify signaling molecule distribution and activity at various levels, from composition of the local extracellular matrix environment through to tissue macro-structure.Drosophila, collagen IV is encoded by the viking and Dcg1 genes [viking hypomorphic mutant females with reduced collagen IV levels [Dad-LacZ and the extra GSC phenotype can be rescued by removal of a single copy of dpp [As described above, Dally and Tkv, expressed in the germarial niche and ECs, respectively, play a role in restricting Dpp distribution and signaling to GSCs. However, another mechanism, involving the collagen IV extracellular matrix protein, also limits Dpp protein to the ovarian GSC niche , 39. In g1 genes . Collageg1 genes , 39. NicV levels , 39, upoV levels , 41; theV levels , 39. In dally mutants to retain GSCs. While the focus here has been on regulation of Dpp distribution, multiple mechanisms also exist in the cystoblast to extinguish transduction of the self-renewal Dpp signal within this cell that is destined for differentiation [It will be interesting to investigate the interplay between Dally, collagen IV, and EC-expressed Tkv in regulating Dpp distribution/signaling through analysis of double mutants, as currently it is unclear, for example, why collagen IV and Tkv cannot concentrate Dpp in ntiation .Drosophila early embryo, collagen IV proteins play a key role in the generation of the BMP gradient that patterns cell fates in the dorsal ectoderm, but in this case collagen IV has a scaffold rather than barrier function [dpp mRNA is uniformly expressed in the dorsal ectoderm and scw expression is ubiquitous in the embryo, formation of the Dpp–Scw gradient involves redistributing the heterodimer across a field of dorsal ectoderm cells that uniformly express it. This is achieved by two extracellular BMP-binding proteins, Short Gastrulation (Sog) and Twisted Gastrulation (Tsg), that form an inhibitory complex with Dpp–Scw, as well as a protease, Tolloid (Tld), which cleaves Sog within this complex to liberate Dpp–Scw. tsg and tld, like dpp, are expressed in the dorsal ectoderm, whereas sog is expressed in the neuroectoderm underlying the dorsal ectoderm [In the function , 41, 42.ectoderm .Embryos with lower collagen IV levels show disrupted Dpp–Scw signaling with a reduced gradient. As Dpp, Sog and Tld bind to collagen IV, it has been proposed that collagen IV acts as a scaffold for assembly of a Dpp–Scw–Sog–Tld–Tsg shuttling complex , 41, 42 Overall, this model provides an explanation for the observation that Dpp cannot diffuse in the embryo in the absence of Sog and Tsg , as hereXenopus embryos where the activity of BMPs is counteracted by antagonists such as Chordin that are released from Spemann’s organizer. One minor difference is that Tolloid proteases, predominantly BMP-1, degrade free Chordin, unlike Tld cleavage of Drosophila Sog that only occurs when Sog is bound to BMP [Xenopus, the BMP gradient is stabilized by the dorsally expressed ONT-1 extracellular matrix protein. ONT-1 binds Chordin and BMP-1/Tolloid proteases, which promotes their association and, therefore, Chordin degradation [Drosophila embryo.The mechanism of DV patterning is conserved in vertebrates, including d to BMP , 47. In radation . In addiradation . As bothradation , it willcxcr4b and -7b) expression [Distinct from this role of the local extracellular environment in modulating BMP distribution, recent studies highlight the role of tissue architecture and its dynamic changes throughout development in regulating cell fate decisions. Development of the zebrafish posterior lateral line system involves the assembly and sequential deposition of mechanosensory organs by a collectively migrating epithelial primordium along the flanks of the embryo . FGF ligpression  and contpression , 54. HerTime-lapse imaging of developing embryos reveals that the pattern of organ spacing is determined by the timing of organ deposition in response to Fgf3, rather than the speed of the collective cell migration or embryonic growth. By inhibiting FGF ligand activity, the timing of organ deposition is delayed and organs are widely spaced. Conversely ectopic expression of Fgf3-GFP expression results in early organ deposition and organ spacing is reduced in a concentration-dependent manner .Imaging of uniformly overexpressed Fgf3-GFP reveals strong localization within spherical microlumina that form through the apical constriction of organ epithelial cells . All celSox9 [A similar method of coupling tissue morphogenesis with the regulation of cell fate was recently described in the mouse and chick developing gut . The priSox9 –60. DuriBmp4 in the underlying mesenchyme [Bmp4 expression being necessary for the repression of endodermal proliferation. In addition, cyclopamine treatment also results in the maintenance of uniform Lgr5 expression throughout the gut showing that this reciprocal signaling cascade is necessary for the restriction of ISC numbers. This is achieved by the BMP-dependent repression of Wnt signaling, illustrated by the repression of Sox9 expression in gut explants cultured in the presence of Shh or BMP4. This model describes how adult ISCs are specified but how do uniformly secreted morphogens define discrete stem cell populations? The answer comes from the tissue architectural changes that occur in the developing gut. Due to sequential compressive forces generated by the underlying smooth muscle, the primitive gut epithelium begins to buckle and form ridges that eventually form into villi. The authors illustrate this idea through computational modeling of an epithelium uniformly expressing a ligand. As the epithelium becomes increasingly folded, as is seen in the developing gut, ligand concentration can be seen to increase significantly within the folds as the number of ligand-producing cells concentrates at the tip of the fold  nanotubes (further reviewed in ) have bea testis . While ta testis . In thisThese protrusions were visualized through the expression of GFP-tagged α-tubulin and were found to be generated by GSCs and extend in a largely uniform orientation into the stem cell niche hub cells . In contTo determine the role of MT-nanotubes in hub-GSC signaling, the localization of Dpp signaling components was visualized, as Dpp acts as a critical GSC maintenance signal . Live imdpp mutant or upon germline knockdown of tkv, overexpression of tkv resulted in longer MT-nanotubes. Expression of a dominant negative form of Tkv carrying the extracellular domain but lacking the intracellular domains resulted in the thickening of MT-nanotubes, demonstrating that ligand–receptor interaction, and not downstream signaling is important for MT-nanotube formation [dpp in somatic cyst cells resulted in the formation of ectopic MT-nanotubes. Overall, it was proposed that MT-nanotubes contribute to short-range Dpp signaling by forming a specialized cell surface area for productive Dpp–Tkv interactions that selectively allow GSCs, but not gonialblasts, to access the high concentration of the self-renewal Dpp signal in the niche [Furthermore, manipulation of the Dpp pathway reveals Dpp–Tkv interaction to be both necessary and sufficient for MT-nanotube formation. While shorter, less frequent MT-nanotubes were observed in a ormation . In addihe niche .Cytonemes (‘cell threads’) are specialized actin-based signaling filopodia, ~0.2 µm in diameter, and of varying lengths reaching up to 80 µm  that areReciprocal signaling between the cells of the wing disc epithelium; overlying flight muscle progenitors (myoblasts), and tracheal epithelial cells directs the formation of the Air Sac Primordium (ASP). This epithelial monolayer surrounds an air-filled lumen and will eventually form the dorsal air sac –72. Buddprickle mutant clones and loss of the integrin subunits αPS1, αPS2, and βPS, and Integrin-linked kinase resulted in abnormal ASPs and a reduced number of cytonemes. Furthermore, while flies heterozygous for mutations in a laminin subunit or αPS1 develop normal ASPs, those of the double heterozygous flies developed abnormally with fewer cytonemes and reduced Dpp and FGF signal transduction. Together these results suggest that laminin-activated integrins are also necessary for cytoneme function, while Dally and Dlp act as substrates for cytoneme growth [Various factors were identified as regulators of cytoneme formation. Perturbation of the function of the formin Diaphanous, the adhesion molecule Neuroglian or Dynamin (Shibire) caused a reduction in cytoneme number and length. This resulted in a decrease in ASP-Dpp signaling and caused malformation of the ASP. In addition, loss-of-function of the adhesion molecule Capricious also reduced the ability of cytonemes to contact Dpp-producing cells and caused abnormal ASP development . The aute growth .Cytonemes have also been suggested to provide a means for the indirect effects of Wingless (Wg) on ASP development through myoblast-mediated Notch signaling . Notch iNotch loss of function, causing a severe inhibition of ASP development despite the absence of Wg signaling within the ASP [Interestingly, Wg overexpression in the wing disc phenocopies  the ASP . The use the ASP , reveale the ASP .Similar but distinct structures have also been described in vertebrates. Airinemes are cellular projections that were identified in zebrafish xanthoblasts, non-terminally differentiated neural crest cells, and predicted to have a role in Notch signaling during pigmentation. Xanthoblasts are suggested to extend Dl positive airinemes that contact Notch-expressing melanophores, promoting Notch signaling to increase melanophore stripe formation. Although airinemes show some similarities to actin-based cytonemes, airinemes are also dependent on microtubules and exhibit more convoluted trajectories, move faster and are associated with larger exosome-like vesicular particles .In the chick limb bud, Filopodia-Like Cellular Protrusions (FiLiPs) are produced by the epithelial cells of the dermomyotome and are proposed to play a role in somite development . They coDrosophila ovarian stem cell niche highlights a different functionality of filopodia-like protrusions. As previously described, niche somatic cells maintain GSC self-renewal through the secretion of the BMP ligands Dpp and Gbb [dpp and gbb in ECs is regulated by niche Cap cells (CpCs) that express hh and have been shown to transport Hh to ECs along short cytonemes [The  and Gbb . The expytonemes , 84. Theytonemes , 84. TheThis same mechanism of ligand delivery has also been visualized in a number of developmental contexts. In the chick limb bud, Shh is produced by mesenchymal cells at the Zone of Polarizing Activity (ZPA). From there it acts over a long-range to specify digit identity and is able to act far beyond its site of production , 86. ActTransport of Wnts by cytoplasmic filopodia has also been observed in multiple situations. During patterning of the zebrafish neural ectoderm, Wnt8a, produced at the blastoderm margin, acts as a posteriorising factor for the distant midbrain–hindbrain boundary . Live imSome extracellular ligands appear paradoxically ill-suited to free extracellular diffusion due to post-translational lipid-modifications that drive their membrane association. Therefore, one mechanism for transporting lipophilic ligands is to load them onto lipid-based transporters.Drosophila wing disc migrated away from the expressing cells in small particles. Labelling both the inner and outer membrane leaflets revealed that these particles, termed argosomes, were composed of a membrane bilayer and when GPI-GFP expression was driven in Wg-expressing cells, Wg was found to co-localize within these migrating GFP positive particles [It was first suggested that lipophilic ligands could ‘hitch a ride’ on and/or within extracellular membranous vesicles resembling exosomes that had originally been described in haematopoietic cellular communication . Exosomearticles . ArgosomDrosophila WntD [Xenopus Wnt8 in complex with murine FZD8 reveals that one of the two binding interfaces is dominated by a palmitoleate moiety and a hydrophobic groove on the FZD8 cysteine-rich domain [Members of the Wnt protein family, with the exception of ila WntD , 96, undila WntD . The forila WntD , 100 as ila WntD –104. In ila WntD , 106 andh domain . LipidatDrosophila nervous system and in cell culture. Wg and Wls are trafficked between synapses or cells in exosome-like vesicles [+ vesicles revealed that they contain many key classes of exosome associated proteins, including membrane trafficking components , V-ATPase subunits and proteins involved in lipid raft (Flotillin-1) and MVB formation  [Similar structures were later identified in the vesicles . Electrolathrin) .Drosophila cells have been shown to secrete exosome-like vesicular structures that contain exosomal markers, such as human CD63 and murine TSG101. A portion of Wnt3A, in murine fibroblast cells, and Wnt5A, in human Caco-2 cells was found to co-fractionate with their respective exosomal markers and EM analysis of Drosophila Kc167 cell culture supernatant reveals small vesicles (40–100 nm) with Wg localized to the outer surface. Importantly, the exosome-bound pool of Wnts is biologically relevant as they are able to activate Wnt responsive reporters in cell culture [A number of studies have now revealed the conserved role of exosomes in Wnt signaling. Human, mouse and  culture , 111.Drosophila nervous system, Wls-GFP also co-localizes with Wnts and exosomal markers within MVBs in the wing disc and the culture media of HEK293 cells and S2 cells suggesting a role in the shuttling of Wnts in these intraluminal vesicles. However, Wls is not found to significantly co-localize with Wg on exosomes from the wing imaginal disc, suggesting that Wls is dispensable following trafficking to the MVB [Drosophila larval neuromuscular junction where Rab11 RNAi knockdown depletes the Wg-exosomal localization without affecting its overall secretion [As was seen in the  the MVB . The sma the MVB , as had ecretion . In vivoDrosophila embryo and wing imaginal disc, cholesterol-modified Hh is largely localized to the plasma membrane of producing cells, although it does also move away [Another morphogen was also identified around the same time as Wg to use this cellular shuttle bus service. The Hh family is lipid-modified through the addition of N-terminal cholesterol and C-terminal palmitic acid, both of which are necessary for controlling extracellular diffusion and signaling in a number of contexts . In the ove away , 114. Inove away . GPI-ancove away .C. elegans during cuticle formation [vha-5 (a V-ATPase subunit), drives severe cuticle malformation. Collagen secretion was found to be unaffected; however, the secretion of the GFP-tagged Hh-related peptides, WRT-2 and -8 [che-4, the dispatched (disp) homologue, partially resembles the abnormal cuticle phenotype observed in vha-5 mutants [Drosophila ectodermal epithelium [The nature of these plasma membrane-associated puncta was later clarified by multiple studies in diverse biological contexts. Shh and retinoic acid were found to associate with similar punctate structures, termed ‘nodal vesicular parcels’, secreted by cells of the chick node. These extracellular vesicles associate with microvilli and migrate along the stream of the nodal flow to transport Shh and retinoic acid during left–right patterning . Again, ormation . Perturb2 and -8 , was inh mutants . Disp isithelium , 119. ToDrosophila wing disc now suggests a role for these MVB-derived extracellular vesicles in long-range Hh signaling. Cells of the posterior wing disc secrete Hh that localizes to basal cytonemes with its co-receptor Interference hedgehog (Ihog) in punctate structures (0.2–0.6 µm) that exceed the diameter of the cytonemes [ptc reporter and phosphorylation of Fused, while in vivo RNAi against exosome associated proteins, such as Rab11 and AnxB11, all reduce the length of the Hh signaling gradient [Recent work in the ytonemes –122  compartment boundary, by dpp enhancer driven expression of the Flp recombinase, revealed only minor growth defects in third instar larvae. Evidence is presented that growth is instead supported by earlier dpp expression in the anterior compartment. However, Dpp target gene expression was lost upon removal of the anterior–posterior stripe of dpp expression, suggesting that the Dpp gradient is required for patterning but not growth [The role of the classical wing disc Dpp gradient has also been revisited by genome engineering. Many studies support this Dpp gradient, emanating from a stripe of the wing . Genome t growth .dpp stripe expression [dpp exon deletion experiments that found the stripe of expression to be dispensable for growth [dpp in the AP stripe  that is sufficient to promote growth in the medial region.A separate study manipulated Dpp–GFP spreading in the wing disc through the localized expression of an anti-GFP nanobody, a single domain camelid antibody, which immobilizes Dpp–GFP. Results from this study indicate that Dpp spreading is required for cell patterning , consistpression . Howeverpression , in contr growth . FurtherDrosophila embryo. Here, maternally deposited mRNA is anteriorly localized and Bicoid protein diffuses within the syncytial cytoplasm from this source generating an anterior–posterior gradient [The different strategies used to control signaling range that we have described here highlight how few rely on passive diffusion of the signaling molecule. A classic example of free diffusion is the formation of the Bicoid gradient in the gradient . Similargradient , 152. HoGiven that diffusion is likely to only be appropriate in limited contexts, cells have adapted to increasing organismal complexity by evolving specialized structures and proteins that facilitate ligand movement and drive long-range signaling. Indeed, membranous protrusions and extracellular vesicles are highly conserved phenomena that have been shown to drive lipophilic ligand diffusion in a variety of contexts. Given the multitude of roles that signaling ligands play in development and tissue homeostasis, it is possible that these methods of active morphogen movement may represent common mechanisms for driving long-range movement, even if long-range signaling may in fact be dispensable in some contexts. However, the individual contribution of membranous protrusions or extracellular vesicles to cell fate patterning remains unclear, due to the potentially pleiotropic nature of the methods used to manipulate these structures to date. Advances in this area will rely on determining how these structures are formed, how ligands are targeted to them, and how they actively drive movement, to manipulate each specific mechanism.Drosophila wg, given the opportunities it affords for manipulating endogenous signaling molecules with precise spatial and temporal control [In terms of future advances, genome engineering is likely to make a big impact, as for the study of  control . Similar control , 153. In control . Conform control . Despite control  will aid