CCL3−/− mice had loss of mature myeloid populations, while myeloid progenitors and HSPCs were increased, and microenvironmental populations were unchanged. These data show that CCL3 promotes myeloid lineage differentiation and the size of the HSPC pool independent of the supportive bone marrow microenvironment. Our results demonstrate a previously unrecognized role of CCL3 in the maintenance of homeostatic hematopoiesis that should be evaluated when targeting CCL3 signaling for the treatment of hematologic malignancy.The chemokine CCL3 is frequently overexpressed in malignancies and overexpression leads to microenvironmental dysfunction. In murine models of chronic myelogenous leukemia (CML), CCL3 is critical for the maintenance of a leukemia stem cell population, and leukemia progression. With CCL3 implicated as a potentially viable therapeutic target, it is important to carefully characterize its role in normal hematopoietic homeostasis. CCL3 One mechanism that has been identified as a driver of BMME dysfunction is the inflammatory chemokine CCL3.The bone marrow microenvironment (BMME) is comprised of a complex network of cellular and molecular components that regulate hematopoiesis and hematopoietic stem cells (HSCs). The BMME contributes to the initiation and progression of hematologic malignancies such as chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and myeloproliferative neoplasms (MPN)5. In hematologic malignancies CCL3 is a well-known mechanism of BMME disruption in multiple myeloma, both activating osteoclastic bone resorption, and inhibiting bone forming osteoblastic cells6. We have previously identified CCL3 as an abundant leukemia-derived factor in a murine model of blast crisis chronic myelogenous leukemia (bcCML) that also displayed a profound loss of mature osteoblastic cells in the BMME7. CCL3 has also been implicated as a factor leading to microenvironmental dysfunction in a variety of malignancies including myelodysplastic syndrome (MDS), metastatic disease of renal cell carcinoma, and B-Cell lymphomas11.CCL3 is a chemotactic factor, signaling through the chemokine receptors CCR1 and CCR5 to recruit lymphocytes to sites of infection, and CD8+ T-cells to active lymph nodes12. In addition, when a mutant allele of the gene Ptpn11 that is associated with juvenile myelomonocytic leukemia (JMML) is homozygously expressed in murine osteoprogenitors mice develop a myeloproliferative neoplasm reminiscent of JMML. The murine disease correlates with high levels of CCL3 expression and is reversed with the administration of CCL3 receptor antagonists13. In human AML approximately 75% of patients exhibit elevated expression of CCL37, and overexpression of CCL3 in Nup98/HoxD13 expressing pre-leukemic cells leads to transformation to AML14. These studies confirm an essential role for CCL3 in the emergence and maintenance of LSCs in these murine models of myelogenous leukemias. Therefore, CCL3 is an appealing therapeutic target in hematologic malignancies.In a murine model of CML, malignant cells that do not express CCL3 cannot maintain a leukemia stem cell (LSC) population and consequently do not give rise to leukemic disease15. Further, the CCL3 receptor CCR5 is a coreceptor for the cellular entry of human immunodeficiency virus (HIV)16, and the FDA has approved the drug maraviroc, a small molecule CCR5 antagonist, for the treatment of HIV infection15. Since leukemia initiating cells share a number of characteristics with normal stem cells, such as increased self-renewal and a more quiescent state, targeting of LIC may also impact HSCs. In fact, the role of CCL3 in support of hematopoiesis is controversial. CCL3 was previously demonstrated to inhibit hematopoietic progenitor proliferation in colony forming assays18, to enhance myelopoiesis in vitro19, and to be dispensable for HSC maintenance in vivo20. To determine the necessity of CCL3 for maintenance of normal hematopoiesis we performed a comprehensive in vivo analysis of homeostatic hematopoiesis and the BMME in CCL3 knockout (CCL3−/−) mice21.The potential for translation of CCL3 targeting is further supported by the prior development of small molecule inhibitors of the CCL3 receptors CCR1 and CCR5. Multiple CCR1 inhibitors have been developed for the treatment of a variety of diseases such as rheumatoid arthritis and multiple sclerosis, and, having proceeded through phase 1 and 2 clinical trials, have been found safe for use in humans22. However, while previous reports have shown no overt hematopoietic abnormalities in CCL3−/− mice a detailed analysis of HSC function in the absence of CCL3 signaling has not been performed. To perform an unbiased multidimensional analysis of the bone marrow we generated CyTOF data to phenotype bone marrow cells based on 31 antigens , and positive for Sca1 and cKit , short-term repopulating HSCs (ST-HSC), multipotent progenitor (MPP) 2, and MPP3, while the lymphoid biased MPP4 population was not increased 7. Under homeostatic conditions CCL3−/− mice show no difference from controls in the number of Lineage-CD45-CD31-CD51+ Sca1- osteoblastic cells as measured by flow cytometric analysis  of primary AML samples that were analyzed7. Therefore there is potential for translation of CCL3 pathway inhibition to the clinic. Additional support for the feasibility of CCL3 inhibition as a therapy for hematologic malignancies is provided by the availability of inhibitors for CCL3 receptors. CCL3 signals through the chemokine receptors CCR1 and CCR55, and several small molecule antagonists have been developed for both of these receptors for a variety of diseases and with varying degrees of success27. CCR1 antagonism has been seen as a potential strategy for diseases such as rheumatoid arthritis and multiple sclerosis; however, clinical trials for the antagonists developed have generally failed in phase 2 after being found safe, but not efficacious for treatment27. Antagonists of CCR5 have also been developed for the treatment of HIV infection, as CCR5 is a co-receptor for cellular entry of HIV. One such antagonist, Maraviroc, was developed by Pfizer and is currently FDA approved for the treatment of HIV infection28. Notably, though CCL3 is emerging as a potential therapeutic target, its role in homeostatic regulation of HSCs has not been carefully studied.The chemokine CCL3 is relevant to human disease as a therapeutic target and previous findings in murine models, including our own, have demonstrated a role in hematologic malignancies−/− mice. This is consistent with previous reports of CCL3 acting as an inhibitor of stem cell proliferation18. A careful analysis of the heterogeneous HSPC compartment in the bone marrow of CCL3−/− and control mice revealed that in addition to HSCs, myeloid biased MPP2 and MPP3 populations were significantly increased, while lymphoid biased MPP4 populations were unchanged. Further, the levels of monocytes and granulocytes were lower in CCL3−/− mice compared to WT. This indicates that CCL3 is involved in the differentiation process of these cell types, leading to a decrease of mature cells in the periphery and an increase in their progenitors in the bone marrow. Together these data suggest that CCL3 regulates the self-renewal capacity of the most immature hematopoietic cells, as well as the differentiation of progenitors of the myeloid lineage.To establish the role that CCL3 plays in regulation of homeostatic hematopoiesis and the BMME we characterized a global CCL3 knockout murine model. Results from flow cytometric analysis of HSPC populations demonstrate that lack of CCL3 signaling results in an expansion of the HSC pool in the bone marrow of CCL3−/− HSPCs repopulate peripheral blood B cells, T cells, and myeloid cells with a reduced contribution compared to WT controls. These results suggest that increased proliferation and accumulation of HSPCs in the bone marrow is caused by a defect in myeloid differentiation and a relative loss of functional HSPCs in CCL3−/− mice.Increased numbers of phenotypic HSC populations in the bone marrow do not necessarily correlate with increased functional repopulating ability. For this reason we analyzed the ability of sorted HSPCs to repopulate the hematopoietic system of myeloablated recipients. Following competitive transplantation, CCL37, loss of CCL3 expression has no effect on the osteoblastic populations, including MSCs. However, in female mice there is a loss of osteoclastic cells in the bone marrow, suggesting that the change in osteoclastic activity is the cause of altered trabecular architecture.The differences that were observed can be caused either by direct signaling to HSPCs, or indirect signaling through alterations in the BMME. Micro-CT analysis of trabecular and cortical bone in the femur revealed no change in overall bone volume, though some minor differences in microarchitecture were observed in female mice, suggesting that CCL3 may regulate trabecular bone remodeling in a sexually dimorphic manner. Though it has previously been shown that CCL3 overexpression in malignant conditions leads to loss of mature osteoblastic cells, and over-activation of osteoclasts leading ultimately to a loss of boneOverall, our data demonstrate that CCL3 plays an active role in the homeostatic maintenance of myeloid differentiation. Since CCL3 has previously been shown to be critical for the maintenance and emergence of leukemia initiating cells in multiple murine models, it will be important to evaluate any impact of CCL3 inhibition on the normal hematopoietic system as well as the malignancy being treated. However, these results, coupled with the availability of CCR1 and CCR5 antagonists suggest that the CCL3 signaling pathway may be an attractive therapeutic target in myelogenous leukemias.Ccl3tm1Unc/J (CCL3−/−) mice fully backcrossed onto a C57BL/6J were purchased from Jackson Laboratory. B6.SJL-PtprcaPep3b/BoyJ expressing the CD45.1 allotype were purchased from the mouse breeding core facility at the University of Rochester School of Medicine and Dentistry.Mice were maintained within the Vivarium facility at the University of Rochester School of Medicine and Dentistry in accordance with protocols approved by the University’s Institutional Animal Care and Use Committee, the University Committee on Animal Resources. Wild type C57BL/6J, and B6.129P2-5 molecular weight dextran to precipitate RBCs and the WBC containing supernatant was collected.Bone marrow cells were collected by flushing the long bones of the hind limbs of mice using a 25-gauge needle and FACS buffer . Peripheral blood cells were collected by submandibular bleeds into EDTA coated tubes. CBC counts were performed on a HESKA Hematrue. Peripheral blood white blood cells (WBCs) were collected by incubating blood in a 2% solution of 5 × 107 cells were collected as described above and suspended in 100 μL PBS supplemented with 2% heat inactivated fetal calf serum (FACS buffer) and stained with the appropriate antibodies , resuspended in PBS with 50 μM 127IdU cell identification reagent (DVS-Fluidigm cat# 201127) and incubated for 30 minutes at 37 °C. Cells were washed twice with MaxPar staining buffer, resuspended in 100 μl MaxPar staining buffer containing the appropriate extracellular antibodies .For CyTOF analysis 10 × 10−/− or C57bl/6 (CD45.2+) control mice were mixed with 5 × 105 competitor whole bone marrow cells from B6.SJL-PtprcaPep3b/BoyJ (CD45.1) mice in a total of 0.1 mL of FACS buffer. Recipient 6–8 week old B6.SJL-Ptprca Pep3b/BoyJ mice received a split dose of radiation of 5 Gy each separated by 24 hours. The second dose of radiation occurred 1–2 hours prior to injection of transplanted cells via tail vein.One hundred sorted LSK, Flt3- bone marrow cells from CCL3Dissected hindlimbs were scanned on a Viva CT40  using a 55-kVp, 145-μA current and a 300-ms integration time at a resolution of 12.5 μm. Analysis of trabecular bone was performed on a 1.25 mm region 50 μm below the growth plate of the femur and tibia. Cortical analysis was performed 4375 mm from the growth plate of the femur and tibia and encompassed a region of 375 μm.7. Immunohistochemical staining for F4/8029 and Osteocalcin30 was performed as previously described.Dissected hindlimbs were fixed, decalcified, processed and embedded in paraffin as previously describedSupplementary information


After this article  was publcyr1-, sxa2-) in cyr1-, sxa2-, cpc2-) in In the 8h, 16h, and 32h panels for JY546 panels shown in In the JY1578 appears to have different contrast levels between the cells and surrounding background.In sxa2-, pmp1-) in sxa2-,oe-cpc2+).The image for strain JY1716 in sxa2-,oe-cpc2+).The image for strain JY710, 16h panel is provided in The authors are unable to clarify the concerns raised about background irregularities in cyr1-, sxa2-, pmp1-) cells in sxa2-, pmp1-) panel, and in sxa2-, pyp2-) and JY1717 panels. In the updated sxa2-, pmp1-) panel is included in The authors apologize for the PLOS ONE Editors issue this Expression of Concern.Original data are no longer available for the other figure panels discussed above and other results reported in the article. Due to the nature and extent of image issues and lack of underlying image data for the figures in question, the PLOS ONE Editors apologize for our delay in resolving this matter and thank the authors for replying promptly to journal queries.Some of the above concerns were raised in 2014. The S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Spcdc25) in tobacco results in small cell size, premature flowering and increased shoot morphogenetic capacity in culture. When Arath;WEE1 is over-expressed in Arabidopsis, root apical meristem cell size increases, and morphogenetic capacity of cultured hypocotyls is reduced. However expression of Arath;WEE1 in tobacco plants resulted in precocious flowering and increased shoot morphogenesis of stem explants, and in BY2 cultures cell size was reduced. This phenotype is similar to expression of Spcdc25 and is consistent with a dominant negative effect on WEE1 action. Consistent with this putative mechanism, WEE1 protein levels fell and CDKB levels rose prematurely, coinciding with early mitosis. The phenotype is not due to sense-mediated silencing of WEE1, as overall levels of WEE1 transcript were not reduced in BY2 lines expressing Arath;WEE1. However the pattern of native WEE1 transcript accumulation through the cell cycle was altered by Arath;WEE1 expression, suggesting feedback inhibition of native WEE1 transcription.WEE1 regulates the cell cycle by inactivating cyclin dependent protein kinases (CDKs) via phosphorylation. In yeast and animal cells, CDC25 phosphatase dephosphorylates the CDK releasing cells into mitosis, but in plants, its role is less clear. Expression of fission yeast CDC25 ( However, unlike animal cell cycles, in plants two CDKs, A and B regulate the transition from G2 to mitosis3. CDKA is closely related to the ancestral cdc2 of fission yeast and is able to complement temperature sensitive cdc2- mutants. In tobacco BY2 cells, CDKA transcript4 and protein5 levels are constant throughout the cell cycle, whereas activity peaks at S/G25. The CDKB family  is unique with the highly conserved PSTAIRE domain of cdc2 (and CDKA) altered to PPTLARE/PPTLRE3. Also, Arabidopsis CDKB genes are unable to complement cdc2-/cdc286. CDKB transcripts5, protein and activity all peak at G2/M4.The eukaryotic cell cycle is a conserved phosphorylation cascade in which key substrates require phosphorylation or dephosphorylation prior to the next step in the cell cycle. At the G1/S and G2/M transitions, major phosphoregulation occurs, catalysed by cyclin-dependent protein kinases (CDKs) that are largely conserved in unrelated species8. When Wee1 is over-expressed in fission yeast (Schizosaccharomyces pombe) cells arrest in G2 resulting in highly elongated cells7. Conversely, the phosphatase, Cdc25, dephosphorylates the same tyrosine residue activating the CDK9. The role of CDC25 in the plant cell cycle is less clear. A truncated version of the yeast CDC25 gene containing only the catalytic domain is present in the Arabidopsis genome10 and can induce a short cell length when expressed in fission yeast11. However, its role in the plant cell cycle seems to be limited to the DNA damage replication checkpoint12 as the plants grow and develop normally. Thus perturbation of Arath;CDC25 expression in Arabidopsis resulted in hypersensitivity to hydroxyurea while over-expression resulted in tolerance compared to wild type. Moreover, although Arath;CDC25 has phosphatase activity10 it also has arsenate reductase activity13 suggesting that in plants CDC25 may have additional roles outside of the cell cycle.In fission yeast, Wee1 and Mik1 kinases phosphorylate Tyr15 of the CDK to inactivate it and prevent entry into mitosisSpcdc25) was expressed in plant cells to study the effects of CDK de-phosphorylation14. Expression of the fission yeast CDC25 gene in both tobacco15 and Arabidopsis16, resulted in phenotypes that are consistent with its action in dephosphorylating and activating CDK. Expression of Spcdc25 in tobacco BY2 cells resulted in a reduced mitotic cell size and a reduction in the length of the G2 phase17. Moreover, in these cells, cytokinin levels were greatly reduced and the cells were insensitive to the cytokinin biosynthetic inhibitor, lovastatin indicating a link between CDK de-phosphorylation and cytokinin signalling. In addition, Spcdc25 expression in tobacco cell suspension cultures altered carbohydrate status resulting in an increase of starch and soluble sugars and a higher sucrose:hexose ratio. These changes are inducible in WT by cytokinin treatment, thus, Spcdc25 expression in tobacco had a cytokinin-like effect18. In whole plants, this cytokinin-independent phenotype was supported by an ability of Spcdc25 expressing stem explants to produce shoots in the absence of exogenous cytokinin19. Consistent results were obtained in Arabidopsis plants expressing Spcdc2516, which showed a reduction in primary root length and increased production of lateral roots. Another effect of Spcdc25 expression in tobacco was precocious flowering with a dramatic reduction in both the time to flowering, and the number of leaves and nodes formed prior to flowering20. Moreover, study of flowering of tobacco nodal stem segments in vitro revealed that the typical acropetal flowering gradient in WT plants did not occur in the Spcdc25 transgenic plants21. However when Spcdc25 was expressed in Arabidopsis, flowering time was not affected (Rogers and Francis lab. unpublished data).Given the uncertainties around plant CDC25, fission yeast CDC25  resulted in increased overall WEE1 protein, reduction in CDKA histone H1 kinase activity and an increase in phosphorylated CDKA25. This was accompanied by an increase in cell size and a delay in the G2/M transition in synchronised cells. However, surprisingly, when Arath;WEE1 was expressed in tobacco BY2 cells, there was a shortening of G223. This was reversed by co-expression of the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1), which interacted with WEE1, presumably removing it through the 26 S proteasome.Cultured hypocotyls of Arath;WEE1 expression in tobacco cells are mirrored by effects on the development of whole plants, and is consistent with a perturbation of the native tobacco WEE1, creating a dominant-negative-like effect.Data are presented here showing that the anomalous effects of Arath;WEE1 expression in tobacco  flowered significantly earlier, after about 100 days  were detected compared with the empty vector line (EV) or with WT  and peaked earlier (4–5 h) compared with the minus DEX control in which expression of Arath;WEE1 was not induced , the WEE1 protein level decreased . Hence, changes in WEE1 protein through the cell cycle followed the altered timing of mitosis in the induced cells.To establish the mechanism of the cell cycle changes, effects on the timing of changes in WEE1 protein during the cell cycle were investigated. In synchronised BY2 cell lines transformed with an inducible sed Figs ; S6. WheArath;WEE1 by addition of DEX, WEE1 kinase activity was maximal in early S phase and decreased by 31% in late G2 reaching a minimum during mitosis, consistent with the observed decrease in WEE1 protein level. In induced cultures, the WEE1 kinase activity was again maximal in early S phase and decreased by 29% in G2 and by a further 28% when the mitotic index peaked. Thus, WEE1 kinase activity also followed WEE1 protein levels and the altered timing of the mitotic peak.A WEE1 kinase inhibition assay was used to investigate whether the WEE1 protein levels correlated with changes in the timing of WEE1 kinase activity. WEE1 activity was measured as the inhibitory action of immunoprecipitated WEE1 protein on CDK activity, using histone H1 as substrate Fig. . SamplinArath;WEE1 line 1 with and without DEX induction. CDKA activity was relatively constant regardless of the addition of DEX  and Nicta;CDKB;1 (referred to here as CDKB) in the inducible DEX Figs ; S8. How/G2 Figs ; S8. ThuNicta;WEE1 in exponential phase BY2 cell cultures carrying the Arath;WEE1 inducible construct following DEX induction, was compared with Nicta;WEE1 expression in exponential phase WT BY2 cell cultures  when Arath;WEE1 expression was induced by DEX in BY2 cells carrying the inducible construct compared to exponential phase WT BY2 cell cultures  and number of leaves produced before flowering (a 2.8 fold reduction) was almost identical. However in contrast to tobacco plants expressing Spcdc25, expression of Arath;WEE1 in tobacco plants did not result in additional flowering from lateral branches. Based on grafting experiments20 it was hypothesised that the anticipation of flowering in the Spcdc25 expressing plants may be result from an earlier competence of the shoot apical meristem to respond to the floral stimulus15. A similar mechanism may be operating in the tobacco plants expressing Arath;WEE1. It is also possible that Arath;WEE1 tobacco plants have similar perturbations in cytokinin signalling and carbohydrate status that were noted in Spcdc25 expressing tobacco plants19, although this would require further verification.The flowering phenotype seen in the Arath;WEE1 contrasts with the effect of Spcdc25 in increasing lateral root production noted by26. However, it is consistent with later reports of a restriction in root growth elicited by Spcdc25 expression in tobacco and attributed to a replacement of cytokinin effects in the roots15. Shorter primary roots were also found when Arath;WEE1 was over-expressed in Arabidopsis24 and is consistent with a negative effect of increased WEE1 on root meristematic cell division.The reduction in primary root length and lateral root production in tobacco plants expressing Spcdc25 and Arath;WEE1. However it contrasts with the phenotype seen in Arabidopsis plants over-expressing Arath;WEE1 where cultured hypocotyls from the Arath;WEE1 over-expressors produced fewer shoots than WT24. In fact the phenotype of the tobacco plants expressing Arath;WEE1 in this respect is more similar to the Arath;WEE1 knockout mutant lines, which produced more shoots from cultured hypocotyls than WT24.The spontaneous formation of shoots in the absence of added cytokinins was also seen both in tobacco expressing Spcdc25 and Arath;WEE1 in tobacco. This is surprising given the opposing functions of the enzymes encoded. The difference between the expression of Arath;WEE1 in tobacco and Arabidopsis confirms that Arath;WEE1 does indeed induce the expected phenotype when expressed in its native environment. However the effects of its expression in tobacco are more consistent with a dominant negative effect, somehow repressing the action of the native Nicta;WEE1.Thus at a plant and organ level there are strong similarities between the effects of expressing Arath;WEE1 also had a positive effect on cell division, very similar to that seen with the expression of Spcdc2517. This effect was independent of the insertion location or the construct since multiple tobacco BY2 lines of both constitutively expressed and inducible Arath;WEE1 created multiple times in the lab all had the same phenotype. In most of the transgenic lines, the reduction in mitotic cell area in Arath;WEE1 expressing tobacco BY2 cells was not quite as severe as that seen when Spcdc25 was expressed, and indeed the Arath;WEE1 expression did not induce the formation of double files of cells as was seen in the Spcdc25 expressing cell lines17. However in one line, c-WEE1 line 10 where mitotic cell area was as low as seen in Spcdc25 expressing lines, double files of cells were also visible. This indicates a threshold effect for the production of double cell files. As previously suggested15 the double cell files are reminiscent of the initial divisions in the pericycle that lead to the production of lateral root primordium. It is possible that the increase in lateral roots seen in some Spcdc25 expressing tobacco plants may be related to the severity of the effect on meristematic cell size. The reduction in root mass and in lateral roots in Spcdc25 and Arath;WEE1 expressing plants may therefore be consistent with a less severe cellular phenotype when the transgene is expressed constitutively as was the case here and in Bell et al.14 as opposed to an inducible vector26.At a cellular level expression of Arath;WEE1 or Spcdc25 with both showing a dramatic reduction in the length of the G2 phase and a lengthening of G1 + M phase17. In Spcdc25-expressing cells the anticipated mitotic peak was matched by an earlier increase in CDKB activity. Consistent with previous reports5, CDKB activity was also high at G2/M in uninduced cells. However, it peaked much earlier, in S phase in the cultures expressing Arath;WEE1. The anticipation of the mitotic peak when Arath;WEE1 expression was induced, was also accompanied by a premature fall in WEE1 protein and kinase activity, consistent with the changes in WEE1 seen in WT cells23. Thus at a cellular level the induced Arath;WEE1 expressing cell cultures are consistent with an early induction of mitosis after a short G2 resulting in a smaller mitotic cell size.Effects on cell cycle progression again were strikingly similar between BY2 cells expressing Arath;WEE1 in tobacco BY2 cells resulted in the opposite phenotype to that found with Solly;WEE1 expression in BY2 cells25 and indeed over-expression of Nicta;WEE1 in the tobacco BY2 cells essentially had no effect. The results here also contrast with the effects on cell size seen when Arath;WEE1 was over-expressed in Arabidopsis plants24 where root meristematic cells were larger than in WT.Expression of Arath;WEE1 in the BY2 cells was causing an overall reduction of WEE1 protein perhaps due to a reduction of the native Nicta;WEE1 transcript. However overall WEE1 protein was higher, and neither Nicta;WEE1 or overall WEE1 transcript  changed dramatically on induction of Arath;WEE1 expression in exponentially growing BY2 cells. This indicates that the phenotypic effect is not due to a cell cycle-independent activation of the RNAi degradation pathway, which can be activated even with sense expression of transgenes27. A sense silencing mechanism is also less plausible given that in all three vector systems  used to express Arath;WEE1 in BY2 cells the orientation of the constructs is such that read through of antisense transcript from the selectable marker construct is not possible. This was shown to be a key factor in sense-mediated silencing27.One hypothesis to explain these unexpected results was that the expression of Nicta;WEE1 may form the underlying mechanism for the activation of a premature mitosis with the resulting phenotypic effects seen at a cellular, organ and whole plant level. In both WT and uninduced BY2 cells, Nicta;WEE1 transcripts are most abundant during S phase. This is consistent with the slightly later accumulation of WEE1 protein during S + G2 phase. However, when Arath;WEE1 is expressed, the peak of Nicta;WEE1 transcripts in S phase seems to be replaced by a later expression peaking in M/G1. Arath;WEE1 expression in these induced cultures is expressed more evenly through the cell cycle with a slight peak in S phase. This pattern is broadly consistent with reports on the expression of the 35S promoter during the cell cycle which show either a peak in S phase28 or constant expression throughout all phases29. One possible mechanism is that Arath;WEE1 transcript production and translation into protein during S + G2 results in a feedback to Nicta;WEE1 transcription, delaying the accumulation of native WEE1 transcripts. This could be mediated through the large number of transcription factors that are thought to regulate WEE1 expression that include AtTCP1530, SOG131 and many others. An alternative mechanism may act at the protein level. The accumulation of Arath;WEE1 protein in S/G2 may activate the proteasome machinery prematurely due to differences in its sequence  and P36SS (5′GCACACTAGTCGACTCAACCTCGAATCCTAT-3′) and cloned into the BIN HYG TX vector32 under an attenuated form of the 35S promoter (as described in33) for constitutive expression, or into the inducible vector pTA700234 using Xho I/Spe I. Individual clones were sequenced and a clone for each construct in which the amino acid sequence was intact was chosen for further work. For expression in whole tobacco plants, Arath;WEE1 was cloned into pkanII-SPYCE(M)35 as described in Lentz Grønlund et al.36. Nicta;WEE1 was cloned into the pTA7002 vector as described in Cook et al.23.For expression of Nicotiana tabacum) BY2 cells was achieved using a modified version of the method described by37 with the addition of 20 µM acetosyringon (Sigma-Aldrich) during co-cultivation of the Agrobacterium (LBA4404) with the BY2 cells. Transformants were selected on solidified BY2 medium (0.8% agar) supplemented with 250 µg/ml Timentin and 80 µg/ml hygromycin. Calli were cultured in 50 ml BY2 medium, 250 µg/ml Timentin and 80 µg/ml hygromycin until stationary phase (1–3 weeks). Cultures were subjected to at least four rounds of sub culturing before being used in synchrony experiments.Stable transformation of tobacco  to a final concentration of between 1 µM and 100 µM. Induction of Nicotiana  tabacum var Samsun plants grown in soil were surface sterilised in 5% hypochlorite solution containing 100 ul/l Triton X-100 for 5 min with gentle agitation. Leaves were rinsed three times in sterile distilled water and cut into 1 cm2 squares using a razor blade. Leaf squares were co-cultivated for 20–30 min in 100 ml of Rhizobium radiobacter (Agrobacterium tumefaciens) LBA4404 cell suspension (containing the WEE1 construct) at OD600 of 0.5 in 1 × MS medium in 140 mm diameter Petri dishes. Leaf squares were then transferred to shooting medium . Following 48 h at 22 °C in the light, Leaf squares were then transferred to shooting medium including 50 ug/ml hygromycin and 200 ug/ml carbenicillin and incubation was continued for 4–6 weeks with a weekly subculture until calli and shoots were visible. Shoots were then excised and further cultured in rooting medium  to induce rooting. Plantlets were transferred to soil and grown to maturity. Expression of the transgene was analysed by PCR using primers AtWEE1fw (AGCTTGTCAGCTTTGCCT) and AtWEE1rv (TCAACCTCGAATCCTATCA). Two lines expressing the transgene (lines #2 and #8) were selected for further experiments.Young leaves from −2), and a relative humidity 50–75% as described in38. The leaves were numbered from the base (1 oldest) and when the first flower bud emerged, the length of leaves without the petiole was measured and leaves above 10 cm in length were counted. The age of the plants is given as days of growth after sowing.Wild type and transgenic tobacco plants were grown from seed in a growth chamber at 22/18 °C day/night thermoperiod with 16 hrs illumination  as described in38. The length of the main root was measured and lateral roots counted semi-automatically with Smart Root software. For visualisation of root primordia the clearing method was used. The roots were fixed in acetone overnight and then fixed in phosphate buffer and mounted in 65% aqueous glycerol. They were observed with an Olympus BX51 microscope equipped with anvApogee U4000 digital camera.Sterilized tobacco seeds were sown on a square Petri dish containing MS medium  containing 3% sucrose, 2 cm apart. After 21 days of cultivation at 25 °C with 16 h illumination with PFD (photon flux density) approximately 100 μmol m38. After 21 days of cultivation, the number of shoots and protruded shoot primordia were counted.Tobacco stem segments, 1 cm long, were placed onto MS medium  containing 3% sucrose, or SIM (shoot inducing medium) consisting of MS medium with 3% sucrose, 0.1 mg/l NAA , and 2 mg/l BAP (benzylaminopurine) as described in39. The mitotic index was measured at hourly intervals after removal of aphidicolin by scoring ≥300 Hoechst-stained cells per slide in random transects using fluorescence microscopy . Mitotic cell area was measured for approximately 300 cells per experiment.BY2 cells were subcultured every 7 d and division was synchronized as previously described33. Total RNA was extracted from BY2 cells using TRI reagent  and residual genomic DNA was removed by DNase treatment . RNA (5 µg) was reacted with Superscript II reverse transcriptase . To study expression of Arath;WEE1, primers were designed which do not amplify the endogenous tobacco Wee1 gene : Arath;WEE1fw, and Arath;WEE1R: GTGCATCTCCTTCTTCTACT. Thermocycle conditions were: 35 cycles of 95 °C (1 min), 55 °C (1 min), 72 °C (1 min). Two sets of specific primers for Nicta;WEE1:  were used to analyse the expression of the endogenous tobacco WEE1 gene, Nicta;WEE1 (Tm = 60 °C and 55 °C respectively). The first set was used for the quantification of Nicta;WEE1 expression in synchronised cells while the second set were used to quantify expression in exponential phase cultures. For detection of Nicta;WEE1 transgene expression only, a primer was designed to bind to the vector sequence: (35STRS 5′-ACGCTGAAGCTAGTCGACTC) and used in conjunction with NtWEE15R 5′-TTATCCCCATCGGCAGCATCAG. Histone H4 primers (H4F: 5′-GGCACAGGAAGGTTCTGAGGG ATAACA and H4R: 5′-TAACCGCCGAAACCGTAGAGAGTCC) were used to verify cell cycle stage, and primers to 18S rRNA: PUV2 5/-TTCCATGCTTAATGTATTCAGA and PUV4, 5/-ATGGTGGTGACGGGTGAC were used as a control17 (Tm = 60 °C). Thermocycler conditions were as above.RT-PCR was performed as described in40 .For all semi quantitative RT-PCR experiments, cycle number was reduced and optimised rigorously as described previously40 Fig.  so that 42. The WEE1 antibody and Western blotting were described in35. The antibody was used at a dilution of 1:1000 followed by α-rabbit IgG at 1:2500 . ECL reagents  were used to visualise the proteins.Proteins were extracted from Arabidopsis or tobacco leaves essentially as described inet al.42. Immunoprecipitations were carried out using antisera raised to Nicta;CDKA;1 and Nicta;CDKB1 as described in Sorrell et al.4. H1 protein kinase assays were as previously described42 using 2 µl of antiserum. Incorporation was assayed by quantitation of autoradiographs using the GeneGenius .For histone kinase assays proteins were extracted from 5 ml of synchronised cultures and assayed essentially as described in Cockcroft Supplementary Figures


There are errors in the placement of references 17 and 18. The sixth sentence of the fifth paragraph in the Introduction section should have no citations. The seventh sentence should have cited references 17 and 18, in addition to references 19 and 20.The correct sentences should read: The same drawback is also observed with smaller affinity tags. This problem has been solved through the use of proteolytic enzymes whose cleavage sites enable the formation of native Aβ  [17], [18], [19], [20].


Alzheimer’s disease (AD) is the most common form of dementia characterized by the deposition of extracellular amyloid-β (Aβ)-containing plaques, the formation of intraneuronal neurofibrillary tangles as well as neuroinflammatory changes. As the key player in the brain innate immune system, microglia has now taken a center stage in AD research. A large number of AD risk loci identified by genome-wide association studies are located in or near the genes highly expressed in microglia. Among them, the triggering receptor expressed on myeloid cells 2 (TREM2) has drawn much attention. A rare variant in TREM2 increases AD risk with an odds ratio comparable to the strongest genetic risk factor apolipoprotein ε4 allele. In the past 6 years, extensive studies have dissected the mechanisms by which TREM2 and its variants modulate microglial functions impacting amyloid and tau pathologies in both animal models and human studies. In addition to the full-length TREM2, research on the soluble form of TREM2 (sTREM2) has facilitated the translation of preclinical findings on TREM2. In this review, we summarize our current understanding of the biology and pathobiology of sTREM2 including its origin, its emergence as a disease biomarker, and its potential neuroprotective functions. These aspects are important for understanding the involvement of sTREM2 in AD pathogenesis and may provide novel insights into applying sTREM2 for AD diagnosis and therapy. Alzheimer’s disease (AD) is the most common cause of dementia in the elderly, typically presenting with a slow but progressive behavioral and cognitive impairment. Key histopathological hallmarks of AD include the deposition of extracellular neurotoxic plaques primarily composed of Aβ, and intracellular neurofibrillary tangles resulting from the aggregation of hyperphosphorylated tau  since the rare R47H variant of TREM2 increases AD risk almost three-fold  has facilitated the translation of preclinical findings on TREM2. sTREM2 was produced from either the proteolytic cleavage of the membrane-anchored TREM2 receptor or the alternative splicing of TREM2 lacking the transmembrane domain . However, the predicted molecular weight of this transcript is ~27 kDa, which is larger than the 20 kDa deglycosylated sTREM2 protein detected in the CSF. Therefore, alternative splicing may not be the major source of sTREM2 protein.The existence of sTREM2 at the protein level was first reported in 2008 by immunoblotting of the precipitates from either human CSF or cultured dendritic cells supernatant fluid  gene cluster as a key modulator of sTREM2 concentration in the CSF. Variants in the MS4A gene region have been previously linked to the risk of developing LOAD  is associated with reduced CSF sTREM2 levels, increased AD risk and accelerated age at onset. The study further provided functional evidence that CSF sTREM2 levels can be modified via modulating MS4A4A expression or targeting MS4A4A with a specific antibody. However, the study only focused on the common variants with a minor allele frequency larger than 0.02. Therefore, additional studies in a larger sample size will be necessary to fully identify the genetic modifiers of CSF sTREM2 levels. Moreover, future studies are necessary to uncover the molecular mechanisms by which the MS4A gene cluster modulates sTREM2 level. The MS4A transmembrane proteins are thought to play a role in intracellular protein trafficking  and other CNS inflammatory diseases  are less potent in both suppressing apoptosis and triggering inflammatory responses in microglia. Thus, our results may constitute previously unknown molecular mechanisms linking TREM2 variants with increased risk of AD.Initially, sTREM2 was postulated to block the function of full-length TREM2 by competing with the ligand binding, in a way similar to the soluble version of another TREM family member, TREM1. Nevertheless, accumulating evidence suggests that sTREM2 possesses important biological and pathobiological functions rather than acting as a decoy receptor opposing full-length TREM2 signaling. One study presented the first evidence that sTREM2 efficiently prevents macrophage apoptosis when cultured under low concentrations of colony stimulating factor 1 (Wu et al., 181 levels in the CSF predicted slower rate of clinical progression. These findings suggest that higher CSF sTREM2 levels are associated with attenuated cognitive and clinical decline in AD.Since the CSF levels of sTREM2 are intimately associated with AD progression, it has been proposed that sTREM2 might modulate AD pathology. Accumulating evidence from the association analysis between CSF sTREM2 levels and regional brain volumes in AD cohorts suggests that sTREM2 functions are likely neuroprotective. One study reported that higher CSF sTREM2 levels were correlated with increased gray matter volume in MCI patients, when controlled for age, sex, and p-tau-associated brain atrophy (Gispert et al., Trem2 deficient 5xFAD mice, while no such findings were observed in mice expressing the R47H variant (Song et al., It was reported that sTREM2 bound to plaques and neurons when the common variant of human TREM2 was expressed on a bacterial artificial chromosome in The majority of AD therapies currently under investigation target Aβ accumulation by modulating the activity of key secretases involved in APP processing. Alternatively, antibodies were developed to prevent Aβ aggregation and/or to promote Aβ plaque clearance (Cao et al., X-FC and LZ reviewed the literature and drafted the 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.


Rf4) and hence cannot be utilized directly in the hybrid rice breeding. To convert the partial restorer to complete restorer, a cross was made between Swarna and a stable restorer KMR3R possessing Rf3 and Rf4 genes and developed BC1F5 and BC2F4 populations by marker-assisted back cross breeding (MABB). The SSR marker DRRM-RF3-10 linked to Rf3 gene located on chromosome 1, clearly distinguished restorers from partial restorers. All the improved lines of Swarna possessing Rf3 and Rf4 genes showed complete fertility restoration in test crosses with higher grain yield heterosis. Few rice hybrids developed by using converted restorers were evaluated in multi location testing under the All India Co-ordinated Rice Improvement Project (AICRIP). The results indicated that new rice hybrids expressed higher heterosis with matching grain quality attributes like Swarna. This study provides significantly novel and relevant restorers to enhance and economize future hybrid rice breeding programs.The major constraints in hybrid rice breeding are availability of limited number of parental lines with specific desirable traits and lower frequency of restorers among elite breeding lines. The popular, high-yielding mega-rice variety Swarna, has been identified to be a partial restorer (as it has only one of major fertility restorer genes,  O. sativa L.) is the most important staple food crop for more than half of the world’s population and it is cultivated in an area of 44.5 million hectare in India with the production of 106.5 million tonnes during the year 20161. After the advent of high yielding semi-dwarf rice varieties, hybrid rice technology has been touted as a major strategy for enhancing the genetic yield potential of rice. The success of hybrid rice technology has been very well demonstrated in China, which produces 146.5 million tonnes of rice from 30.32 million hectares1. This significant increase in production in China is mainly due to cultivation of hybrid rice (with >50% area and production under rice hybrids). Several technical challenges, market and policy constraints has limited the development and diffusion of hybrid rice outside China2. In India, hybrid rice is cultivated in an area of ~3 million hectares3, which is about 6.7% of total area of rice cultivation. Hybrid rice accounts for less than 10% of the area under rice cultivation in Bangladesh, Indonesia, and the Philippines and just 10% in Vietnam.Rice (5. The most widely used CMS system in rice is based on wild abortive (WA) cytoplasm derived from Oryza sativa f. Spontanea7. The WA-CMS system is highly stable with complete pollen sterility8. Hybrid rice seed production using CGMS/three line system involves a CMS (A) line, a maintainer (B) line and a restorer (R) line carrying the fertility restorer genes.Hybrid rice technology aims to increase the yield potential of rice by exploiting the phenomenon of hybrid vigour or heterosis. Cytoplasmic male sterility coupled with fertility restoration controlled by nuclear genes is a very useful tool in exploiting heterosis in self pollinated crops. In rice, three CMS systems viz. Wild Abortive (WA), Boro II (BT) and Honglian (HL) are deployed for commercial hybrid rice seed production7. Rice fertility restoration is governed by two independent dominant genes and one of the genes appeared to be stronger in action9 than the other. Bharaj et al.10 reported that fertility restoration is governed by two dominant genes, the restorers with dominant alleles of the two genes in homozygous or heterozygous condition will be fully fertile and the genotypes having dominant alleles of one of the two genes in homozygous or heterozygous condition but homozygous recessive alleles of the other gene will behave partially sterile or partially fertile and vice-versa.The restorers with strong restoration ability have two major genes along with modifier genes and a restorer with semi-restoring ability have either one of the two major genesRf3 and Rf4 and have been mapped on chromosome 1 and 10 using Zhenshan 97A near-isogenic lines (NILs) mapping population12. The chromosomal location of Rf genes has been resolved using RFLP (Restriction Fragment Length Polymorphism) markers and it showed that effect of the locus on chromosome 10 is larger than chromosome 113. Mapping of two Rf genes of WA-CMS system on the long and short arm of chromosome 10 using SSLP (Simple Sequence Length Polymorphism) markers was done by Jing et al.14. The SSR primer RM258 located on chromosome 10 was found linked to the restorer gene at a distance of 9.5 cM15. Fertility restorer gene linked to RM6100 was mapped at a distance of 6–7 cM on chromosome 10 in the restorer lines viz., PRR 78 R, IR 40750 and MTU 999216. The candidate gene based marker, DRRM-RF3-10 associated with Rf3 locus showed maximum selection accuracy in identifying restores in comparison with other reported markers viz., RM10305, RM10318, DRRM-RF3-5 and DRRM-RF3-617.The major loci restoring the fertility has designated as 1 for their pollen and spikelet fertility. According to the male fertility of F1 plants, the test lines can be classified as maintainers, restorers, partial restorers and partial maintainers9. Molecular markers linked to the fertility restoration trait are useful for evaluation of large number of germplasm/breeding lines to identify restorers in rice without progeny testing18. Based on molecular screening with markers linked to Rf4 and Rf3 genes19 reports that lines possessing both the Rf genes showed higher fertility than the genotypes containing Rf3 or Rf4 individually. In hybrid rice breeding, elite lines and released varieties from varietal improvement programme are being tested for their fertility restoration ability by crossing with the CMS lines to identify restorers and maintainers. The restorer frequency among indica lines is only 40%20. The majority of the popular, mega varieties released in India like Swarna, Samba Mahsuri, and MTU1010 were found to be partial or incomplete restorers and hence cannot be utilized as such to produce experimental hybrids.The process of screening for the trait of fertility restoration involves test crossing with a set of CMS lines and evaluation of hybrids/F21, occupying more than 30% of total rice area in eastern India22. Swarna possesses good grain and cooking quality with desirable grain type. However, as mentioned earlier, it cannot be utilized in hybrid breeding as it is a partial restorer. Therefore in the present investigation, an attempt was made to convert the partial restorer Swarna to a complete restorer by transferring major fertility restorer gene(s) through marker-assisted back cross breeding (MABB), and demonstrated the complete fertility restoration ability of the improved lines and developed new rice hybrids with attributes like Swarna.The major issues in hybrid rice breeding are lack of parental lines with desirable specific traits and lower frequency of restorers and maintainers among elite breeding lines. One of the major constraints, which limit the spread of hybrid rice area in shallow low lands and coastal areas, is non availability of long duration hybrids, which can mature in 145–150 days or more. The popular mega rice variety, Swarna has been widely adopted by farmers for its high yielding ability and adaption under low input conditions, shallow low lands. It is cultivated in an approximate area of 4.5 M ha1 hybrids were produced by crossing Swarna and KMR3R with four CMS lines viz., APMS 6A, IR79156A, IR68897A and IR58025A. The F1 hybrids were evaluated for their pollen and spikelet fertility during both kharif (wet) and rabi (dry) seasons  and seven plants were selected based on genome recovery. The chromosome 4, 7, 8 and 11 were shown complete recurrent parent type polymorphism, whereas the remaining allele ranged from 20% (Chromosome 5) to 55% (Chromosome 3). In BC2F1, out of 22 plants, three plants were selected based on foreground and background selection, which had shown complete genome recovery on chromosome 2, 3, 5 and 6 with 91% of recovery of Swarna genome.A set of 728 hyper-variable SSR markers were utilized for surveying polymorphism between Swarna and KMR3R across the 12 linkage groups of rice. Of these, 89 were polymorphic between the parents (12.22% polymorphism). In this study, chromosome 8 and 6 recorded the highest and lowest polymorphism percentage of 19.60% and 5.88%, respectively  along with better agro morphological traits (i.e. similar to Swarna) was backcrossed with Swarna to produce BC2F1 seeds. Simultaneously, the selected BC1F1 plant with maximum recurrent parent genome was selfed to produce BC1F2 seeds. By foreground selection in BC2F1 and BC1F2 generation plants carrying Rf3 gene in homozygous and heterozygous conditions were identified  possessing superior phenotypes were subjected to background selection analysis. BC2F1 and BC1F2 plants with maximum recurrent parent genome (RPG) were selfed to produce BC2F2 and BC1F3 seeds. The BC2F2 and BC1F3 plants were raised and subjected to foreground selection for the presence of Rf3 gene. Marker-assisted screening resulted in identification of 35 plants to carry Rf3 in homozygous condition in BC2F2 and 40 plants in BC1F3 generation. The identified homozygous plants were selfed to raise BC2F3 and BC1F4 generations. Phenotypically superior plants were identified based on morphological traits at BC2F3 and BC1F4 generations and were selfed to produce the subsequent generation seeds. BC2F4 and BC1F5 lines were evaluated in three rows and plants were subjected to stringent phenotypic selection and agro morphological evaluation.One superior BCied Fig. . A total2F4 and BC1F5 generations derived from the cross Swarna x KMR3R, were evaluated in alpha lattice design along with parents and checks in 6 sq. m plot during kharif  2015 at Indian Institute of Rice Research, Rajendranagar, Hyderabad. The observations were recorded on five plants in each replication and mean data is presented in  and 23.9 cm (KMR3R). The panicle length of converted restorer lines were significantly longer (>7 cm) than Swarna. With respect to mean grain yield per plant of selected backcross derived lines, ranged from 19.0 to 25.0 gm, which is significantly higher than the recurrent parent, Swarna (15.28 gm). The test weight of Swarna (1000 grain weight) was 15.9 gm with short bold grains, whereas KMR3R possessed 26.5 gm of test weight with long bold grain type. The converted restorer lines test weight ranged from 15.1 to 21.9 gm with the grain type of short slender to short bold. Days to 50% flowering (DFF) of selected backcross derived lines ranged from 91 to 121 days  and possessing plant height of more than 100 cm were crossed with above mentioned CMS lines to produce F1 hybrids. F1 hybrids were evaluated for pollen and spikelet fertility percentage and grain yield heterosis along with different duration checks. Based on days to 50% flowering three hybrid groups viz., early (<100 days), medium (101–110 days) and late (>110 days) were constituted for evaluation.  in different zones in India. The hybrid entries with a yield superiority of more than 5% and 10% over the best hybrid and varietal checks either on over all mean basis or on zonal mean basis are promoted from IHRT to next stages of testing in Advance Varietal Trial 1 (AVT 1) and AVT 223.Based on the performance of converted improved restorer lines in the research station trial, two high yielding hybrids namely IIRRH-111 and IIRRH-112 were nominated for multi-location evaluation in AICRIP (All India Co-ordinated Rice improvement project)-IHRT trial during kharif 2016 multi-location testing. With respect to quality data collected from the AICRIP progress report (2016), this hybrid possessed short bold grains as that of Swarna and preferred intermediate amylose content (26.22%) with 60% high head rice recovery. In AICRIP testing rice hybrids were evaluated for not only yield and also for their grain quality characters. The grain quality characters of hybrids developed by crossing CMS lines with improved restorers are presented in Table The rice hybrid IIRRH-112 showed a superior performance over the checks  and registered yield superiority over checks during 24. In India, as a result of concerted efforts by both public and private sectors, a total 102 hybrids have been released for commercial cultivation in the country. In spite of having great potential to enhance rice production and productivity, area expansion under hybrid rice has not increased significantly in the last few years due to major constraints like non availability of long duration hybrids suitable for shallow lowlands and coastal areas, poor grain and cooking quality, marginal heterosis, higher seed cost, non-involvement of public sectors in hybrid rice seed production25. The popular, high yielding, mega rice variety Swarna, with good grain and cooking quality, cannot be utilized as such in hybrid breeding due to their partial restoration of fertility of CMS lines (pollen and spikelet fertility is less than 80%)  and Improved Samba Mahsuri, Kavya and MTU 1010  particularly Rf3 gene located on chromosome 1. Backcross breeding is the most commonly used method for incorporating any essential genes into a rice cultivar. Backcross breeding along with marker-assisted foreground and background selection contributes immensely to accelerate recurrent parent genome (RPG) recovery27.An earlier study28 considered to be an efficient strategy for transferring targeted specific genes to elite lines. MABB is a most effective breeding strategy in rice for improving simple and complex traits. Some of the popular rice varieties released for cultivation in India through MAS/MABB for biotic and abiotic stress tolerance are Pusa Basmati 1 with bacterial blight (BB) resistance genes xa13 and Xa21 involving single back cross generation followed by selfing and pedigree based selection29 and Improved Samba Mahsuri with three BLB resistance genes viz., xa5, xa13 and Xa21 involving four back crossing30. Swarna Sub1 with Sub1 QTL for the submergence tolerance31 and IR64 Drt 1 with two yield QTLs under drought situations namely qDTY2.2 and qDTY4.1 (IR64 NILs) for drought tolerance32 are the best examples of abiotic stress tolerance varieties derived through MAS.Marker-assisted backcross breeding which includes MAS for the target gene(s) known as foreground selection and MAS for the recovery of recurrent parent genome known as background selectionRf3 gene. We could recover Swarna genome in the backcross derived lines of converted restorers of BC1F5 and BC2F4 generations by 80% and 92.3%, respectively. The agro morphological evaluation of converted restorers (RP5934-66 to RP 5934-100) in the background of Swarna exhibited desirable traits for an ideal restorer lines viz., improved plant height, longer panicles, more number of productive tillers with heavy pollen load along with traits specific for Swarna, like brown glume or golden husk colour with stay green type etc  in order to develop improved parental lines in the background of an elite Indian mega- variety of rice and thereby confirming the major and minor role of Rf4 and Rf3. The grain quality characters of the converted (i.e. improved) restorer lines in the genetic background of Swarna were similar to that of the original parent. In a panel test for grain quality traits conducted at ICAR-IIRR, Hyderabad, it was observed that, the Rf3 gene introgressed lines were indistinguishable from Swarna in terms of color of paddy, grain size and shape. Further, replicated field trials that were carried out at twelve different locations across India under the all India coordinated rice improvement project (AICRIP), showed that yield levels of the improved lines were significantly higher than Swarna and the check lines indicating that there is no apparent yield penalty associated with presence of the Rf3 gene3. Considering these points, improved version of restorers developed in the genetic background of Swarna would be of great use in hybrid rice breeding for developing late duration hybrids. Large scale seed production of potential hybrids involving long duration improved restorers with different WA-CMS lines are under pipeline for multi location testing. Some of the improved lines were also demonstrated their potentiality as higher heterotic hybrids in the early and medium duration category , whereas Swarna is a very popular rice variety and occupying major area under cultivation.Introgression of fertility restorer genes without recovery of other characters of recurrent parent would have been a futile exercise, if the developed lines do not meet expected grain and cooking quality with higher yields. We feel that this was made possible because of the extensive phenotypic selection and also because of the use of molecular markers for background selection. The selected BCles Fig. . This waRf genes through marker-assisted backcross breeding. Thus, in the present study, we have successfully developed restorers with characteristic features of popular mega Indian rice variety Swarna for utilization in hybrid rice breeding, specially for development of late maturity hybrids.To the best of our knowledge, this is the first attempt to convert partial restorer to complete restorer by transferring Rf gene(s) was KMR3R (Jaya/IR29723-143) a restorer parent of popular rice hybrid KRH-2. KMR3R has been identified to carry both fertility restorer genes Rf4 and Rf3 genes18. To determine the fertility restoration status of recurrent and donor parent, crosses were made with four CMS lines namely APMS 6A, IR 79156A, IR 68897A and IR 58025A to produce F1 hybrids during rabi 2011. These F1 hybrids were evaluated for their pollen and spikelet fertility and grain yield heterosis during two rice crop seasons viz., kharif 2012 (i.e. wet season 2012) and rabi 2013 (i.e. dry season 2013) to confirm their fertility restoration status.The experimental materials consist of Swarna (MTU 7029) a popular mega rice variety, which occupies larger area under cultivation in India, derived from the cross Vasistha x Mahsuri and is a partial restorer and it was utilized as the recurrent parent for improvement of its fertility restoration ability. The donor parent for the fertility restoration trait/2-KI) solution43, on a glass slide and three randomly selected microscopic fields were counted. Stained, well filled and round pollen grains were counted as fertile, while unstained, shrivelled and empty pollen grains were considered as sterile. Pollen fertility was calculated and expressed in percentage as given belowPollen fertility was measured using anthers collected from the spikelets at one or two days before anthesis. The anthers from each spikelet were smeared in a drop of 1% Iodine-potassium iodide , partially fertile (51–80%), partially sterile (21–50%) and completely sterile (0–20%)The panicles that emerged from the primary tiller were bagged before anthesis to avoid out crossing and the number of filled grains and chaffs in the panicle were counted at the time of maturity. The ratio of filled grains to the total number of spikelets was expressed as spikelet fertility as given below:Plants were classified into four classes based on spikelet fertility percentage, namely, fertile (more than 85% spikelet fertility), partially fertile (50–85%), partially sterile (21–50%) and completely sterile (0–20%)44. PCR analysis was carried out using 30–50 ng DNA as template, 5 pmoles of each primer, 0.05 mM dNTPs, 10X PCR buffer (TaKaRa Taq™ DNA Polymerase) and 1 U Taq DNA polymerase in a total volume of 10 µl. Template DNA was initially denatured at 94 °C for 5 min followed by 35 cycles of PCR amplification with the following parameters: a 30 s denaturation at 94 °C, a 30 s annealing at 55 °C and 1 min of primer extension at 72 °C. A final extension was done at 72 °C for 7 min. The amplified products were electrophoretically resolved on a 3% agarose gel containing 0.5 µg/ml of ethidium bromide in 0.5X TBE buffer and visualized under UV and results were documented. The recurrent parent Swarna, donor parent KMR 3 R along with few popular varieties  were screened with 20 reported SSR markers linked to Rf4 & Rf3 genes to identify polymorphic marker between restorer and partial restorers for foreground selection . The backcross breeding procedure followed is presented in the Fig. 1 plants of Swarna x KMR3R were screened using DRRM-RF3-10 identified polymorphic SSR markers between donor and recurrent parent. The true F1s were then backcrossed with Swarna to generate BC1F1s, which were confirmed for the presence of fertility restorer gene(s) Rf3 and Rf4 with the help of DRRM-RF3-10 and PPR3 markers (i.e. foreground selection). The plants which were positive for the restorer genes Rf4 and Rf3 were subjected to background selection with a set of 89 identified polymorphic SSR markers to identify a single BC1F1 plant, possessing maximum recovery of the recurrent parent genome (RPG). This plant was selfed to generate BC1F2s and also backcrossed with Swarna to generate BC2F1s. The MABB process involving foreground and background selection as explained above was repeated among the BC2F1 plants and the best BC2F1 plant was selfed to generate BC2F2 seeds. The BC1F2 and BC2F2 were analyzed with markers specific for Rf3 and Rf4 to identity homozygous plants. Homozygous BC1F2 and BC2F2 plants were then advanced through pedigree method of selection for further phenotypic evaluation based on duration of flowering (days), plant height (cm), number of tillers, panicle length (cm) and single plant yield (g). Phenotypically superior plants were advanced for further evaluation.A cross was made between Swarna x KMR3R to improve the fertility restoration trait of the recurrent parent Swarna, during 1F5 and BC2F4 generations along with the parents were transplanted in the main field with the spacing of 20 × 20 cm and fertilizer dosage of 120:80:60 (N:P:K) kg/ha during kharif 2014. The experimental plots were arranged in alpha lattice design in four blocks with two replications. Standard agronomic practices were followed while raising the rice crop. Data was recorded on randomly selected five plants in each replication for the agronomic traits, viz. flowering duration, plant height (cm), number of productive tillers, panicle length (cm), grain yield per plant, and 1000-grain weight. The plants were visually observed for the following traits viz., heavy pollen load during anthesis, strong culm, non-lodging type and Swarna’s specific traits of stay greenness and golden hull colour for selecting superior phenotypic plants as restorers. The data was tabulated and statistically analyzed using standard Microsoft office excel and SPSS package.Thirty days old seedlings at BC1F5 and BC2F4 generations possessing Rf4 and Rf3 genes were crossed with two CMS lines viz., APMS 6A and CRMS 32A to produce F1 hybrids during kharif 2014 (i.e. wet season 2014). Thirty days old seedlings of experimental hybrids along with checks were transplanted to the field with the spacing of 20 × 20 cm in two replications. Data on days to flowering, plant height, productive tillers, panicle length, pollen and spikelet fertility percentage, grain yield and grain yield heterosis were estimated. Seeds of Swarna and selected experimental hybrids were stored for three months after harvesting and grain quality tests were carried out by standard grain quality evaluation protocols (as explained in30) with the rice grains having 12 to 14% moisture content. Superior rice hybrids were identified to produce hybrid seed in larger plots during rabi 2016 (i.e. dry season 2016) and nominated to multi-location evaluation of AICRIP trials during kharif 2016 (i.e. wet season 2016).The phenotypically superior lines from BC


SLC26A4 gene cause hearing loss with enlargement of the vestibular aqueduct (EVA). Some patients also have a thyroid iodination defect that can lead to multinodular goiter as part of Pendred syndrome. A haplotype of variants upstream of SLC26A4, called CEVA, acts as a pathogenic recessive allele in trans to mutations affecting the coding regions or splice sites of SLC26A4. Our first hypothesis is that CEVA, acting as a pathogenic recessive allele, is correlated with a less severe phenotype than mutations affecting the coding regions and splice sites of SLC26A4. Our second hypothesis is that CEVA acts as a modifier of the phenotype in patients with EVA caused by mutations affecting the coding regions or splice sites of both alleles of SLC26A4 or EVA caused by other factors.Recessive mutations of coding regions and splice sites of the . To test our first hypothesis, we compared the thyroid and auditory phenotypes of subjects with mutations affecting coding regions of both alleles of SLC26A4 with those of subjects carrying CEVA in trans to mutations affecting the coding regions. To test our second hypothesis, we compared the phenotypes associated with the presence versus absence of CEVA among subjects with no coding region mutations, as well as among subjects with mutations affecting coding regions of both alleles.This was a prospective cohort study of 114 individuals and 202 ears with EVAtrans to a mutation of SLC26A4 have a normal thyroid phenotype and less severe hearing loss in comparison to individuals with mutations affecting coding regions of both alleles of SLC26A4. In subjects with no mutant alleles of SLC26A4, hearing loss was more severe in subjects who carry the CEVA haplotype in comparison to non-carriers. There was no correlation of CEVA with the phenotype of subjects with mutations affecting coding regions of both alleles.Subjects carrying CEVA in SLC26A4. CEVA may act as a genetic modifier in patients with EVA caused by other factors.CEVA, acting as a likely pathogenic recessive allele, is associated with a less severe phenotype than alleles with a mutation affecting the coding regions or splice sites of  SLC26A4 gene on chromosome 7q (OMIM 274600) [SLC26A4 can cause bilateral EVA and a thyroid iodination defect that can lead to multinodular goiter as part of Pendred syndrome (PS) (OMIM 274600) [SLC26A4 can also be associated with nonsyndromic EVA (NSEVA), also referred to as nonsyndromic recessive hearing loss DFNB4 (OMIM 600791) [Enlarged vestibular aqueduct (EVA) is the most common temporal bone malformation detected in ears with sensorineural hearing loss . Some in 274600) . The iod 600791) .SLC26A4 with the presence or absence of the PS thyroid phenotype. Variants originally reported to be specifically associated with NSEVA/DFNB4 [Until recently, there was no known correlation of specific mutations or variants of VA/DFNB4 . HoweverVA/DFNB4 , 9.SLC26A4 with the thyroid phenotype [SLC26A4 (M2) [SLC26A4 [We have reported correlations of the number of mutant alleles of henotype , 11, bi−henotype , 13, andhenotype . Pendred[SLC26A4 , 13.SLC26A4-linked haplotype, called Caucasian EVA (CEVA), comprised of 12 variants (10 single-nucleotide substitutions and two single-nucleotide deletions) upstream of SLC26A4 [SLC26A4. Some of the variants are intergenic and some are found within introns of the other genes which include BCAP29, DUS4L, COG5, HBP1, and PRKAR2B. None of these five genes are known to be associated with EVA or any phenotype consistent with EVA. We showed that a chromosome 7 with CEVA and no mutations of coding regions or splice sites of SLC26A4 acts as a mutant allele with incomplete penetrance in trans to the allele with a mutation affecting the coding regions or splice sites in M1 patients [We recently reported an  SLC26A4 . The fre SLC26A4 . CEVA wa SLC26A4 . The 12 patients . The prepatients .SLC26A4 allele with the CEVA haplotype is correlated with a less severe phenotype than mutations affecting the coding regions or splice sites of SLC26A4. The aim of our current study was to test this hypothesis by comparison of the phenotypes of M2 subjects without CEVA with those of M1 subjects carrying CEVA in trans to a mutation affecting the coding regions or splice sites. A second aim of our study was to test the hypothesis that CEVA acts as a genetic modifier of the phenotype caused by mutations affecting coding regions or splice sites of both alleles of SLC26A4 (M2) or by factors other than SLC26A4 mutations (M0).Based on these findings, we hypothesized that the SLC26A4 genotypes and CEVA haplotypes have been previously reported [SLC26A4 (M0).This study was approved by the Combined Neurosciences Institutional Review Board (IRB) of the National Institutes of Health . Written informed consent was obtained from all adult subjects and parents of minor subjects. Race and ethnicity were classified according to our IRB reporting designations. All Caucasians with EVA on at least one side were included in this study. We originally defined a vestibular aqueduct as enlarged if the diameter > 1.5 mm , but subreported , 18, 19.reported . Two prereported  , Pendred syndrome (PS), or indeterminate (I). Ultrasonography was used to evaluate thyroid size and texture , 11. SubSeverity of hearing loss was classified using a four-frequency (0.5/1/2/4-kHz) pure-tone air-conduction threshold average (PTA) calculated from the most recent complete audiogram for each ear with EVA , 13. WheSLC26A4 genotype-haplotype combination. The following comparisons were made: (1) M2 vs. M1/CEVA (2) M2 vs. M2/CEVA (3) M0 vs. M0/CEVA. The Mann-Whitney test was used to compare pure-tone threshold average (PTA) for ears with different genotype-haplotype combinations. These analyses were performed using GraphPad Prism 7 for Mac OS X . Stata  was used to perform multivariate linear regression analysis to investigate associations of hearing loss in M2 R/R and M1 C/R EVA patients as a function of genotype status (M1 or M2), CEVA (reference or CEVA) haplotype status, age, sex, and EVA laterality. We performed the same analysis in M0 EVA patients but without genotype status as a variable since all patients were M0.Fisher’s exact test was used to investigate associations of sex, EVA laterality , and thyroid phenotype with SLC26A4, we compared the phenotypes of M2 subjects without CEVA to those of M1 subjects with CEVA in trans to an allele with the reference haplotype and a mutation affecting the coding regions or splice sites but no CEVA. Among subjects with a determinate thyroid phenotype (Pendred syndrome or nonsyndromic), 10 of 11 M2 subjects without the CEVA haplotype had a Pendred syndrome thyroid phenotype and six of six M1 subjects with CEVA in trans had a nonsyndromic phenotype . Twenty-five (93%) of 27 M2 subjects without CEVA and nine (82%) of 11 M1 subjects with CEVA in trans had bilateral EVA. This difference was not significant (p = 0.564). The median pure-tone average  in the M2 without CEVA group was significantly different  from the median pure-tone average  in the M1 with CEVA group . These results indicate that, as a recessive Mendelian allele in trans to an allele with a mutation of the coding regions or splice sites of SLC26A4, an allele with CEVA but no coding region or splice site mutations is associated with a normal thyroid phenotype and less severe hearing loss in comparison to alleles with a mutation of the coding regions or splice sites and the reference haplotype.To test the hypothesis that CEVA, acting as a recessive mutant allele, is correlated with a less severe phenotype than mutations affecting the coding regions or splice sites of SLC26A4 mutations, we compared the phenotypes of M0 subjects without CEVA with the phenotypes of M0 subjects heterozygous or homozygous for the CEVA haplotype . The median pure-tone average  in the M0 without CEVA group was significantly different  than the median pure-tone average  in the M0 with homozygous CEVA group  than M0 subjects heterozygous for CEVA  . These results indicate that CEVA modifies the severity of hearing loss in M0 EVA ears.To test the hypothesis that CEVA acts as a modifier of the phenotype of patients with EVA caused by factors other than subjects ). Among SLC26A4 alleles with the CEVA haplotype within the cohort of M2 subjects (p = 0.3346). These results do not support the hypothesis that CEVA modifies the severity of hearing loss caused by mutations affecting the coding regions or splice sites of both alleles of SLC26A4.We did not observe any significant differences between hearing loss severity and number of SLC26A4, termed CEVA, that acts as a pathogenic recessive allele in trans to an allele with a mutation affecting the coding regions or splice sites of SLC26A4 in M1 EVA patients [We recently reported a haplotype of 12 variants upstream of patients . When Ampatients , resultipatients .SLC26A4, individuals carrying an SLC26A4 allele with the CEVA haplotype in trans to a mutation of SLC26A4 have a normal thyroid phenotype (i.e. nonsyndromic EVA or DFNB4) and tend to have less severe hearing loss. Although our previous comparisons of phenotypes of M2 subjects with those of M1 subjects revealed that the M1 phenotypes were less severe than the M2 phenotypes, some of the M1 subjects had the reference haplotype in trans to the mutant allele, and some of the M2 subjects had CEVA in cis with one or both mutant alleles. Our current result and conclusion are novel because we performed a comparison that was not confounded by inclusion of M1/reference or M2/CEVA subjects.In contrast to individuals with mutations affecting coding regions or splice sites of both alleles of SLC26A4. Alternatively, our observation may result from ascertainment bias or other unknown factors among the M0 subjects. In contrast, we did not observe any correlations between hearing loss severity and number of SLC26A4 alleles with the CEVA haplotype within the cohort of M2 subjects. However, the power of these analyses was limited by the number of M2 subjects with CEVA (four). We did not perform this analysis in M1 subjects since CEVA is etiologic in those subjects and cannot also be considered a modifier. Furthermore, we cannot be certain of the etiology of EVA in M1 subjects without CEVA. We could therefore not test the hypothesis that CEVA acts as a genetic modifier in M1 subjects.Our analysis of M0 subjects suggests that CEVA, acting as a genetic modifier, increases the severity of hearing loss associated with EVA caused by factors other than mutant alleles of SLC26A4 that cause hearing loss and nonsyndromic EVA or modify the severity of hearing loss in ears with EVA. However, the co-segregation of EVA with SLC26A4-linked markers in M1 families, and the near-zero probability of EVA in the siblings of M0 EVA probands, indicate that such variants would be extremely rare Mendelian causes of nonsyndromic EVA [It is possible there are pathogenic variants in regions or genes unlinked to omic EVA . Our resSLC26A4 exons and splice sites for individuals with hearing loss and EVA because the detection of CEVA provides a definitive diagnosis for individuals with a mutation affecting the trans allele of SLC26A4 [SLC26A4 genotype-haplotype result are unlikely to develop the thyroid abnormality and increased severity of hearing loss associated with Pendred syndrome and mutations affecting the splice sites or coding regions of both alleles of SLC26A4. Finally, this study indicates that CEVA may act as a genetic modifier to increase the severity of hearing loss in ears with EVA caused by factors other than mutations affecting the coding regions or splice sites of SLC26A4.We previously recommended inclusion of testing for CEVA along with analysis of  SLC26A4 . Our curSLC26A4, is associated with less severe auditory and thyroid phenotypes than alleles with a mutation affecting the coding regions or splice sites of SLC26A4. CEVA may act as a genetic modifier in patients with EVA caused by other factors.CEVA, acting as a likely pathogenic recessive allele of


Mecp2 promoter methylation in adolescent and adult brain tissue of offspring exposed to prenatal immune activation on gestation day 9 and offspring of saline exposed mice. PCR based methylation assays using Sequenom EpiTYPER was used to quantify DNA methylation at promoter CpG methylation of Long Interspersed Elements-1 (LINE1 or L1) and Mecp2. The dataset also includes global DNA methylation and Mecp2 promoter methylation profile at 6 and 12 weeks following early dietary intervention with omega-3 (n-3) PUFA.Prenatal exposure to infection and inflammation increases the risk of neurodevelopmental disorders such as schizophrenia and autism. The etiology could be partly through transgenerational and modifiable DNA methylation changes in the adult offspring's brain. This data descriptor presents a dataset of global DNA methylation (using LINE1 assay) and  Lists are sorted on ID column with summary information . Other columns are body weight in grams at 12 week or 6 week, MIA group or saline control group, assigned diet (n-3 or n-6), sex, mean LINE1 promoter methylation, mean Mecp2 promoter methylation. Missing data marked as ‘na’ are either not measured or samples with <70% data.This manuscript describes methylation datasets from offspring exposed to prenatal infection and subsequent inflammation in the striatum and prefrontal cortex  and matched controls . Half of the animals in each group received dietary intervention with n-3 poly unsaturated fatty acids (PUFA) from weaning. 2C57BL/6 N mice were bred and mated in the Laboratory Animal Unit (LAU), The University of Hong Kong. The animals were maintained under ad libitum food and water, kept in 12:12 h normal light-dark cycle (lights off at 19:00) and temperature and humidity-controlled  animal vivarium. Pregnant females were not disturbed, except for weekly cage cleaning. All experiments were performed in accordance with relevant institutional and national guidelines and regulations approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) at The University of Hong Kong and every effort was made to minimize the number of animals used and their suffering.PolyI:C administered C57BL6N MIA mouse model was generated as described elsewhere 2.1The pups were weaned, weighed, and littermates of the same sex were caged three to four per cage on PND 21. Both saline control group and polyI:C group were split into two halves with diet enriched with n-3 was matched to a standard rodent American Institute of Nutrition 93 (AIN93) diet or diet enriched with n-6. Diets differ only in the ratio of n-3/n-6 fatty acids used and provides 16% energy from fat diets supplied by Harlan Laboratories, Madison, and water. Please see study design Approximately 2 gm n-3/day was administered through diet as an early dietary intervention. The calorific value and total fat content of both n-3 and n-6 diet was balanced. A detailed list of nutrient contents of the rodent AIN93 and modified diet is shown in 2.2At 6-week and 12-week of age, the mice were sacrificed by cervical dislocation and brains removed quickly and transferred to chilled PBS solution. Striatum and prefrontal cortex were collected in 1.5ml tubes using microdissection on a cold platform referring to the Allen Mouse Brain Atlas 2.3EZ DNA methylation kit from Zymo Research, CA, USA was used to treat five hundred nanograms of genomic DNA with sodium bisulfite in duplicate following the manufacturer's standard protocol. The kit exploits the three-step chemical modification that converts unmethylated cytosine to uracil and the methylated cytosine will be protected from sodium bisulfite 2.4Mecp2 promoter were amplified using previously reported primers in LINE1 elements and 2.5Mecp2 is provided in the dataset as values from 0 to 1 (0 represents not methylated and 1 represent fully methylated).MALDI-TOF MS readings were interpreted by EpiTYPER software and generates quantitative information about individual CpGs in each analyzed amplicon. Blank, fully methylated and fully unmethylated controls were confirmed for their corresponding epigram and methylation levels. CpGs with missing data (>20%) and samples with less than 70% data recorded across the CPGs were deemed unfit for subsequent analysis. All flagged data from EpiTYPER such as low mass, high mass  were discarded. Mean CpG methylation for LINE1 and


Streptococcus pneumoniae. We identified only 17 cases by culture, but molecular methods identified S. pneumoniae in 68% (92/135) of culture-negative samples. The most frequent serotypes were 3, 1, and 19A, together accounting for 62% (68/109) of cases. Nineteen cases attributable to 13-valent pneumococcal conjugate vaccine (PCV13) serotypes (mostly serotype 3) were detected among 22 children age-appropriately vaccinated with PCV13. The dominance of the additional serotypes included in PCV13 among PCPP cases in Portugal continues, even with PCV13 available on the private market (without reimbursement) since 2010 and with average annual coverage of 61% among age-eligible children. Our data suggest reduced effectiveness of PCV13 against serotype 3 PCPP.Despite use of 7-valent pneumococcal conjugate vaccine, incidence of pleural effusion and empyema  is reportedly increasing globally. We cultured and performed PCR on 152 pleural fluid samples recovered from pediatric patients in Portugal during 2010–2015 to identify and serotype  Streptococcus pneumoniae (pneumococcus) is the leading cause of bacterial pneumonia in children and is the most common pathogen isolated in pleural effusions and empyemas .We asked 61 hospital laboratories and pediatric departments located throughout Portugal to report all cases of possible PCPP (in patients <18 years of age) for which pleural fluid was available for analysis and to submit these samples for characterization. We included in our study only samples recovered during January 2010–December 2015, but we performed no audit that would ensure reporting compliance. Because our network includes all secondary and tertiary care hospitals in which PCPP is likely to be treated and a pleural fluid sample obtained, we assume our catchment population is the entire population of Portugal <18 years of age. During 2010–2015, this population steadily decreased, from 1,929,331 in 2010 to 1,802,196 in 2015  . For this study, we classified serotypes as vaccine serotypes  and nonvaccine serotypes.We identified all bacteria as S. pneumoniae by using conventional PCR to target the lytA and wzg genes (2010–2011) and later (2012 and later) by using real-time PCR (rPCR). We serotyped positive samples by conventional PCR, rPCR, or a combination of both. We confirmed negative results by conventional PCR by using rPCR on samples stored at −80°C.When identification of disease etiology by conventional microbiologic methods failed, we sent pleural fluid to the central laboratory, where total DNA was extracted from 200 μL of the patient sample by using the High Pure PCR Template Preparation Kit  or DNeasy Blood and Tissue Kit  according to the manufacturers’ instructions. We used a conventional PCR amplification of 2 human genes (encoding human β-actin and RNaseP) as control for the quality of the purified DNA. We initially tested the presence of S. pneumoniae genes (lytA and wzg) (We used a multiplex PCR for the amplification of the genes encoding human β-actin and RNaseP (lytA and wzg genes) (2 (50 nmol/L), and water, for a final volume of 25 μL. Cycling conditions were as follows: 95°C for 10 min, followed by 50 cycles at 95°C for 15 s and 60°C for 1 min. We performed detection of lytA and wzg in singleplex PCR. We defined negative results as those with cycle threshold (Ct) >40 and positive results as those with Ct<35  by using the Platinum quantitative PCR SuperMix-UDG . PCR reactions contained 5 μL of DNA, 5 pmol/L of each primer .S. pneumoniae in the pleural fluid by molecular methods, but we confirmed the remaining 92 cases (68%) to be PCPP by molecular methods. The total numbers of requests for molecular testing and samples positive for S. pneumoniae were approximately constant over the years  PCPP cases occurred in nonvaccinated children (median age 2.6 [IQR 1.4–8.5] years), and the vaccination status was unknown for 28 (26%) patients. The remaining 47 (43%) PCPP cases occurred in children who received >1 vaccine dose (median age 3.2 [IQR 3–5] years). All vaccinated children received PCV7, PCV13, or both; PCV10 had not been administered to any child in the study.Among the 109 PCPP case-patients (17 identified by culture and 92 by molecular methods), 56 were male and 52 female; the sex of 1 patient was not available. Patient age ranged from 4 months to 17 years (median age 4 [IQR 2.3–6] years); 27 cases were in children >0.198 by Fisher exact test). Whenever possible, conventional PCR serotyping enabled the identification of serotypes 7F, 6B, and 9V, whereas rPCR detected only the groups 9V/9A, 7F/7A, and 6 without further discrimination. On the 17 culture-positive samples, only 5 serotypes were found: 1 and 3 (n = 7 each) and 14, 10A, and 19A (n = 1 each). Apart from serotype 10A, which was detected exclusively in 1 isolate, all other serotypes were also detected by PCR from culture-negative samples. Most of the PCPP case-patients for whom a serotype was unambiguously identified were infected with the addPCV13 serotypes (n = 47 [43%]), followed by the addPCV10 serotypes (n = 25 [23%]), whereas PCV7 serotypes were responsible for a small fraction of PCPP cases (n = 7 [6%]). Among the remaining cases, 6 were caused by nonvaccine serotypes, and in another 6 cases, we were unable to classify capsular types as vaccine or nonvaccine type. These cases included samples for which rPCR tested positive for serogroup 6 (n = 1), 7F/7A (n = 4), or 9V/9A (n = 1).Except for 18 (16%) samples, we were able identify the serotypes of the pneumococci responsible for illness . OverallAmong the 47 vaccinated children, 35 were age-appropriately vaccinated . Of thesS. pneumoniae among children age-appropriately vaccinated with PCV13 (n = 22/27) and among the other case-patients for whom vaccination status was known (n = 59/71) (p = 1 by Fisher exact test).We compared the number of addPCV10 and addPCV13 PCPP cases  among children age-appropriately vaccinated with PCV13 and among the other case-patients for whom vaccination status was known and serotype could be unambiguously determined. Serotype 3 was overrepresented among PCPP cases in children age-appropriately vaccinated with PCV13 (p<0.001 by Fisher exact test). We detected a similar number of positive samples for S. pneumoniae in pleural fluid samples was greatly improved by the use of molecular methods, as previously reported , which would increase the likelihood that any PCPP cases in this group would be caused by serotype 3.>3 years of age (n = 12/17), but the distribution was similar to that of serotype 3 case-patients not vaccinated with PCV13 (p = 0.353 by Fisher exact test), so it does not seem likely that this was attributable to faster waning of the immune response to this serotype. In fact, cases of serotype 3 PCPP occurred in younger children than did cases caused by serotype 1, a serotype also included in PCV13 and for which only 1 vaccine failure was detected. Because the synthesis of the serotype 3 capsular polysaccharide proceeds through a synthase mechanism, the polysaccharide is not covalently linked to the peptidoglycan and can be released during growth, thereby potentially reducing opsonophagocytosis (The effectiveness of PCV13 against serotype 3 has been questioned in several studies. In a large surveillance study performed in the United States, no reduction in IPD caused by serotype 3 was observed despite reductions in IPD incidence and evident decreases in IPD caused by PCV13 serotypes 19A and 7F (The proportion of children asymptomatically colonized with serotype 3 increased in the period preceding PCV13 introduction in Portugal (In agreement with our observations, other reports document serotype 3 vaccine failures. A study in Greece found 5 cases of complicated pneumonia caused by serotype 3 pneumococci among vaccinated children, although most vaccine failures occurred in children who received a single dose of PCV13, which could offer only limited protection (S. pneumoniae in pleural fluid samples and emphasizes the potential role of molecular techniques when evaluating disease incidence. Another limitation of our study is lack of detailed information about the immune status or other underlying conditions in age-appropriately vaccinated children with PCPP by vaccine serotype. However, given the high prevalence of serotype 3 in this group, it is unlikely that all cases could be explained by host characteristics, indicating that specific properties of serotype 3 must be responsible for this behavior.Although our study was prospective and involved both pediatric and microbiology departments, it was not designed to estimate the incidence of PCPP because it did not identify cases in which there were clinical or radiographic criteria for complicated pneumonia and for which pneumococci were identified in either blood or respiratory samples. Although this certainly resulted in an underestimation of PCPP, the use of conventional PCR and rPCR techniques greatly enhanced the ascertainment of PCPP cases by improving the detection of In summary, we describe data that are compatible with a lower individual effectiveness of PCV13 against PCPP, a presentation of IPD for which PCV13 is specifically recommended. The public health consequences of such possible lower protection might be mitigated by a reduction in circulating serotype 3 by vaccination with very high coverage, such as those achieved through inclusion in NIPs. Carriage studies and continued surveillance are necessary to determine the group effect of the introduction of PCV13 in the NIP to clarify the effectiveness of PCV13 in the prevention of infections caused by serotype 3.


We hypothesized that MRI, when used in an optimal This prospective case-control study was approved by the institutional review board. 3D MRI acquisitions including saturation-recovery T1 mapping and DIXON imaging was performed at 4.0 T on 9 human LA samples collected from patients who underwent cardiac surgery. Histological quantification of fibrosis and fat was obtained. MRI T1 maps were clustered based on a Gaussian Mixture Model allowing quantification of total, interstitial and fatty fibrosis components. Fat maps were computed from DIXON images and fat fractions were calculated. MRI measurements were performed on the same location as the histological analysis (plane) and on the entire sample volume (3D).High correlations and levels of agreement were observed between MRI and histology for total (r = 0.93), interstitial (r = 0.93) and fatty fibrosis (r = 0.98) and fat (r = 0.96). Native T1 correlated with the amount of fibrosis from MRI and histology. The 3D MRI total, interstitial and fatty fibrosis ranges were between 6% and 23%, 4% and 17.3%; and 1.4% and 19.7% respectively.ex vivo MRI was able to quantify different LA myocardial components with high agreement in 2D with histology and moreover to provide 3D quantification of such components whereas in vivo application remains a challenge.High Field  Most. Most2].o by MRI . Subsequin vivo quantitative myocardial tissue characterization non-invasively 8], p[8], pin , p[8], pvasively 10]11].[11].in v.[11].in  effects . Few att effects . However effects 15]..in vivo ex vivo setting  to study the ability of MRI to characterize complex fibrotic and fat components of the LA wall. The DIXON method has been proposed to generate fat maps of the myocardium . The. Thek-spin vivo observations at close field strength 25],[25],in v,[25],in strength 9]10][2[10][2in [2[10][2istrength 27]..in vivo in vivo studies analyzing left ventricular myocardial biopsies in subjects undergoing aortic valve replacement (r = 0.65) 30].[30].in v.[30].in ex vivo. This issue has been raised by Kellman et al. ..versus hIn vivo assessment of myocardial fat has been proposed by spectroscopy or imaging [in vivo when applied to the thin (2 mm) left atrial wall [ex vivo results to patients in the clinical setting and uniquely provide compartmental quantitative tissue characterization of the LA wall in vivo. Such data at the tissue level would fill an important gap in currently available information in vivo as LA biopsies are not performed and could increase our understanding of LA cardiomyopathy and pave the way for novel management strategies. imaging  but the ial wall . Improveex vivo T1 mapping sequence used here in vivo in a moving heart with a sufficient spatial resolution to reliably study the thin left atrial wall. However, our primary goal was here to demonstrate the ability of MRI to characterize the complex LA myocardial compartments and define the determinants of native T1 in “ideal” conditions. This technique may be immediately useful in analyzing myocardial biopsies to reduce sampling errors or in animal validation studies in the whole heart before technical improvements make them ready for translation to in vivo human studies. Further studies should be performed to overcome the challenging technical issues which will allow translation to a moving heart.This study has several limitations. First, there is a relatively small sample size. This reflects the high difficulty to obtain tissue samples in patients. Second, it is not currently possible to apply the ex vivo on human LA myocardium provided highly accurate quantification of myocardial components including fibrosis, fat and fatty fibrosis, as revealed by highly significant correlations with histology. Increased native T1 was strongly related to increased interstitial fibrosis and the extent of adipose tissue tended to decrease T1 values. Furthermore, MRI was able to propose a 3D distribution of these myocardial tissue components over the sample.T1 mapping combined with DIXON MRI sequences acquired S1 Fig(DOCX)Click here for additional data file.