Pneumonia is the leading cause of death in children worldwide and kills an estimated 1.2 million children under the age of five every year; more than AIDS, malaria and tuberculosis combined . In the In 2010, the disease previously dubbed the “forgotten killer of children” by the United Nations Children’s Fund (UNICEF) and the World Health Organization (WHO), was made a global health priority and a resolution was passed by the World Health Assembly recognising pneumonia as the world’s leading infectious killer of children [Haemophilus influenzae type b and pneumococcal infections that cause bacterial pneumonia, and secondly, by preventing infections such as measles and whooping cough that can lead to pneumonia as a result of complications [Increasing attention has been placed on childhood pneumonia, primarily since the launch of the first Global Action Plan for Prevention and Control of Pneumonia (GAPP) in 2009. The GAPP has as its core aim, to increase the awareness of pneumonia as a major cause of child death and to spur action to deal more effectively with the problem, by implementing proven interventions for the prevention and treatment of the disease . The Gloications .As the mean age of the population increases however, it is expected that the burden of pneumococcal infection is also likely to increase . FactorsEarlier this year, a new Integrated Global Action Plan for the Prevention and Control of Pneumonia and Diarrhoea was released by the WHO and UNICEF, stating its potential to save up to 2 million extra children every year from deaths caused by pneumonia and diarrhoea . The Actpneumonia, has been an initiative in development for some years. The original concept of the journal was born at the International Symposium on Pneumococci and Pneumococcal Diseases-5 (ISPPD5) held in Alice Springs, Australia, in 2006 and a subsequent Tri-nation  meeting that was held in Sydney, Australia, in 2009 [pneumonia was launched and is an online, open access journal that provides a platform for bringing together knowledge related to the pathogenesis, treatment and prevention of the disease.The online journal  in 2009 . All parpneumonia’s Editorial Board are passionate and dedicated to advancing the fight against childhood mortality due to pneumonia. The journal’s Editorial Board are well represented across the globe from countries including Argentina, Australia, Belgium, Israel, the Netherlands, Papua New Guinea, Spain, South Africa, the United Kingdom and the United States — all combining efforts to raise the global profile of pneumonia. The members of the Board have extensive academic qualifications and are internationally renowned researchers that have published over 7000 papers combined over the course of their careers. As an open access journal, published articles are freely available from the journal’s website which therefore supports a greater global exchange of knowledge. This international collaboration and ease of access is particularly important for researchers and authors in developing countries, as it provides a forum for publishing their research findings without the associated costs of manuscript submission, publication and online access.pneumonia, papers were published from Malawi, examining oxygen supply and demand in a large teaching hospital setting [In volume 1 of  setting ; Austral setting ; and Fra setting .In volume 2, papers have been published from Australia, on the threats facing the success of the pneumococcal conjugate vaccine, specifically serotype replacement ; the propneumonia are responsible for all aspects of the peer-review and publication process. All activities are performed in-house including manuscript receipt and check-in, reviewer and author correspondence, copy and layout editing and electronic publication; systematically tracking every stage of the process along the way.The staff of the Editorial Office of The staff have also been developing policy and procedure manuals and been highly involved in the long-range planning of the journal, which has meant addressing the effects of the electronic world on publication. Author submission guidelines have been methodically written for ease of manuscript submission and to assist the editorial staff to promptly progress papers through the peer-review process in a timely manner.pneumonia accepts original research articles, case studies, reviews, critical commentaries, correspondence, highlights and news on all aspects of pneumonia and is able to process and publish these free of charge. Although the journal is in its infancy, the site is making impressive progress with worldwide interest. This highlights the significance of the deadly disease and the importance of having an international forum such as pneumonia, devoted to raising the global profile of pneumonia. Programs such as the Bill and Melinda Gates Foundation and their partnership with the GAVI Alliance, the WHO and UNICEF Global Action Plan for Pneumonia and Diarrhoea, activities led by the Global Coalition Against Child Pneumonia such as World Pneumonia Day and the introduction of the scientific journal pneumonia, are all helping to ensure that pneumonia receives the funding and attention that it deserves and is no longer labelled as the forgotten disease.


Increased liver stiffness exerts a detrimental role in driving hepatocellular carcinoma (HCC) malignancy and progression, and indicates a high risk of unfavorable outcomes. However, it remains largely unknown how liver matrix stiffness as an independent cue triggers epithelial-mesenchymal transition (EMT) and facilitates HCC metastasis.Buffalo rat HCC models with different liver stiffness backgrounds and an in vitro Col I-coated cell culture system with tunable stiffness were used in the study to explore the effects of matrix stiffness on EMT occurrence and its underlying molecular mechanism. Clinical significance of liver stiffness and key molecules required for stiffness-induced EMT were validated in HCC cohorts with different liver stiffness.HCC xenografts grown in higher stiffness liver exhibited worse malignant phenotypes and higher lung metastasis rate, suggesting that higher liver stiffness promotes HCC invasion and metastasis. Cell tests in vitro showed that higher matrix stiffness was able to strikingly strengthen malignant phenotypes and independently induce EMT occurrence in HCC cells, and three signaling pathways converging on Snail expression participated in stiffness-mediated effect on EMT including integrin-mediated S100A11 membrane translocation, eIF4E phosphorylation, and TGF β1 autocrine. Additionally, the key molecules required for stiffness-induced EMT were highly expressed in tumor tissues of HCC patients with higher liver stiffness and correlated with poor tumor differentiation and higher recurrence.Higher matrix stiffness as an initiator triggers epithelial-mesenchymal transition (EMT) in HCC cells independently, and three signaling pathways converging on Snail expression contribute to this pathological process. This work highlights a significant role of biomechanical signal in triggering EMT and facilitating HCC invasion and metastasis. Extracellular matrix proteins, as the most abundant noncellular solid-state component in tumor microenvironment, not only maintain three-dimensional morphological architecture of tumor cell/tissue, but also produce biochemical or biophysical signals to influence biological functions of tumor cells. Currently, the biochemical effects of matrix proteins on invasion and metastasis of tumor cells have been well documented –3, but tHigher matrix stiffness promotes proliferation and chemotherapeutic resistance , upregulEMT frequently occurs in the initiation of tumor invasion and metastasis. Soluble factors, ECM components, and hypoxia are able to induce a transition of tumor cells from epithelial state to mesenchymal state . Except In the study, using buffalo rat HCC models with different liver stiffness backgrounds and an in vitro Col I-coated cell culture system with tunable stiffness, we clarified the underlying molecular mechanisms by which matrix stiffness alone as an initiator induced EMT occurrence in HCC. This work highlights a significant role of biomechanical signal in triggering EMT and facilitating HCC invasion and metastasis, and opens a promising therapeutics approach targeting on matrix stiffness for prevention of HCC metastasis.4 (3 ml/kg) followed by 50% CCl4 olive solution (2 ml/kg) once a week for 12 weeks, the rats in group H with 100% CCl4 (3 ml/kg) followed by 50% CCl4 olive solution (2 ml/kg) twice a week for 12 weeks, and the rats in group N with saline for 12 weeks. Live stiffness of the rats in 3 groups (6 rats each group) were measured using a TA.Xtplus Texture Analyzer . Subsequently, a subcutaneous tumor tissue mass (2 × 2 × 2 mm3) derived from McA-RH7777 cells was orthotopically transplanted into livers of other 18 rats in 3 groups (6 rats each group). Twenty-five days later, HCC tumor-bearing rats with different liver stiffness backgrounds were formed. All animal care and experiments were in accordance with the guideline for the Care and Use of Laboratory Animals published by the National Academy of Science, and the related experiment design met with approval from the Animal Care Ethical Committee of Fudan University.Thirty-six buffalo rats  were randomly divided into 3 groups. The rats in group M were intraperitoneally injected with 100% CCl7 MHCC97H cells or 1.5 × 107 Hep3B cells were suspended in C solution (100 μl serum-free medium), Matr solution (6 mg/ml Matrigel in 100 μl serum-free medium), Matr+L solution (6 mg/ml Matrigel and 3.5 mg/ml COL1 in 100μlserum-free medium), and Matr+H solution (6 mg/ml Matrigel and 70 mg/ml COL1 in 100 μl serum-free medium), respectively. The suspended HCC cells were subcutaneously injected into the upper left flank region of nude mice, and the growth of subcutaneous tumors was observed.Twenty-four 4-week-old male BALB/c nude mice were obtained from Shanghai SLAC Laboratory Animal Co., Shanghai, China. Appropriate 1 × 10An in vitro system of mechanically tunable COL1-coated polyacrylamide gels was done as our previous study . For detWe retrospectively collected the clinical data of 74 HCC patients who received preoperative assessments for liver stiffness by two-dimensional shear wave elastography and underwent curative resection in the Zhongshan Hospital of Fudan University  during July 2015 to August 2017. Patients did not receive any preoperative anticancer therapy. We classified these patients into three groups according to the values of liver stiffness: group I (≤ 8 kPa), group II (> 8 and < 12 kPa), and group III (≥ 12 kPa). Thirty HCC tissue specimens of 74 patients (10 tissue samples each group) were obtained from tissue bank. Tumor differentiation was assessed according to the Edmondson grading system. Histological grading of inflammation (G0–G4) and staging of fibrosis (S0–S4) for non-tumoral liver were evaluated according to the Metavir scoring criteria. All the patients after surgical resection were followed up to October 2018, with a median follow-up of 30.2 months (range 3.6–38.5 months). The study was approved by the Zhongshan Hospital Research Ethics Committee, and written informed consent obtained from each patient.n). The chi-squared test and rank sum test were used to compare qualitative variables, and quantitative variables were analyzed by the analysis of variance (ANOVA) test among three groups and Student's t test between two groups. A P < 0.05 (two-tailed) was considered statistically significant.Statistical analyses were performed using SPSS 23.0 . Continuous variables were expressed as the mean ± standard deviation (SD), and categorical data were displayed as absolute number  is an enzyme located at cell membrane and produces reactive oxygen species (ROS) which is associated with EMT , 28. TheThe eIF4E overexpression enhanced higher stiffness-induced EMT in HCC cells, while its knockdown attenuated the expression of EMT markers Fig. a, b. AddIn spite of higher stiffness-triggered EMT in HCC cells independent of exogenous TGF β1 Fig. e, we stiP = 0.026) and higher HCC recurrence (P = 0.036)  and the high-stiffness group . Compared with that in the low-stiffness group, integrin β1 or Snail is highly expressed in the high-stiffness group  Table , demonst Table , Liver stiffening as a remarkable pathological phenotype frequently emerges in the late stage of chronical liver diseases and HCC. Now, increased liver stiffness has become an important clinicopathological parameter of HCC to indicate its pathological grade and the risk of unfavorable outcome , 32. Alt, HCC cells exhibited a fibroblast-like morphology, greater motility ability, and higher expression of metastasis-associated genes under higher stiffness stimulation. These alterations clearly stated that matrix stiffness signals strengthen the invasion and metastasis of HCC and modulate its malignant properties. More importantly, in the absence of exogenous TGF β1, HCC cells also acquired an obvious mesenchymal attribute grown on higher stiffness substrate. Meanwhile, the suppression of integrin β1 or α5 reversed the expressions of EMT markers. All these findings strongly support that matrix stiffness alone is sufficient to drive EMT in HCC cells, in agreement with results in breast cancer cells [We first established buffalo rat HCC models with different liver stiffness backgrounds to clarify the relationship between liver stiffness and HCC progression. The data suggested that higher liver stiffness facilitated HCC growth and enhanced its metastasis. Subcutaneous tumor analysis also showed an obvious acceleration in HCC growth and invasion/metastasis gene expression in a group of higher matrix stiffness. In vitroer cells . As expeer cells –40 and rer cells  and is rer cells . Here, her cells , while Rer cells . Our resIn addition, we found that another pseudopod protein eIF4E was also involved in stiffness-induced EMT in HCC. eIF4E highly expresses in a variety of human malignancies and is relevant to cancer development and progression , 44. elFConsidering that there was a significant increase in Smad2/3 phosphorylation level under higher stiffness stimulation, we continued to analyze whether TGFβ1 autocrine existed in HCC cells during this pathological process. A loss or gain of miRNAs always occurs in EMT-increased tumor invasion and metastasis . Matrix Subsequently, a clinical significance of higher liver stiffness in pathological grade and HCC prognosis was validated in HCC samples and TCGA analysis, and the potential values of the required proteins for higher stiffness-induced EMT were clarified in indicating poor tumor differentiation and HCC recurrence.Although the three signaling pathways described above participate in higher stiffness-induced EMT in HCC metastasis, we are still unable to exclude the existence of other signal pathways in this pathological process. With the development of high-throughput signal pathway screening technology and the establishment of ideal animal model, other molecular mechanisms related to stiffness-induced EMT will be better elucidated.In summary, the three signaling pathways converging on Snail expression contribute to higher stiffness-induced EMT in HCC metastasis, including S100A11 membrane translocation, eIF4E phosphorylation, and TGFβ1 autocrine Fig. . To the Additional file 1:. Supporting Information.Additional file 2: Figure S1. Higher liver stiffness promotes the growth of HCC and facilitates HCC invasion and metastasis in vivo. (A) The mRNA expression of integrin β1, LOX and AFP in orthotopic HCC tumors in groups N, M and H. (B) Liver function analysis of buffalo rat HCC models with different liver stiffness backgrounds. B, Healthy buffalo rats, C, HCC buffalo rats with normal liver stiffness, M, HCC buffalo rats with medium liver stiffness, S, HCC buffalo rats with high liver stiffness. (C) Gross appearance and wet weight of subcutaneous tumors derived from Hep3B cells mixed in Matrigel and varied concentration of COL1. (D) Histochemistry analysis of subcutaneous tumors and their Ki-67 expression. (E) The expressions of integrin β1 and FAK in subcutaneous tumors. (F) The expressions of MMP2, MMP9, SPP1, CD44 in subcutaneous tumors. C, Hep3B cells; Matr., Hep3B cells in Matrigel; Matr+L., Hep3B cells in Matrigel and low concentration COL1; Matr+H., Hep3B cells in Matrigel and high concentration COL1.Additional file 3: Figure S2. An in vitro system of COL1-coated polyacrylamide gels with tunable stiffness. (A) Preparation of an in vitro system of COL1-coated polyacrylamide gels with tunable stiffness (B) Stiffness values of different stiffness substrates (C) Morphology alteration of HCC cells grown on FN/LN- coated gels with tunable stiffness and their Snail expression. (D) Expressions of integrin α5 or integrin β1 in HCC cells transfected with LV-shRNA-ITG α5 and LV-shRNA-ITG β1.Additional file 4: Figure S3. S100A11 participates in stiffness-induced EMT in HCC cells. (A) The expression of EMT markers in HCC cells co-transfected with pFU-GW-shRNA-S100A11 and LV-shRNA-ITGβ1 or pEGFP-OE-S100A11 and LV-shRNA-ITGβ1 under higher stiffness stimulation. (B) The expression of EMT markers in HCC cells co-transfected with pFU-GW-shRNA-S100A11 and LV-shRNA-ITGα5 or pEGFP-OE-S100A11 and LV-shRNA-ITGα5 under higher stiffness stimulation.Additional file 5: Figure S4. eIF4E participates in stiffness-induced EMT in HCC cells. (A) The expression of EMT markers in HCC cells co-transfected with pFU-GW-shRNA- eIF4E and LV-shRNA-ITGα5 or pEGFP-OE-eIF4E overexpression and LV-shRNA-ITGα5 under higher stiffness stimulation (B) The expression of EMT markers in HCC cells co-transfected with pFU-GW-shRNA- eIF4E and LV-shRNA-ITGα5 or pEGFP-OE-eIF4E and LV-shRNA-ITGα5 under higher stiffness stimulation.Additional file 6: Figure S5. The levels of TGF-β1 in culture supernatants of HCC cells grown on different stiffness substrates.Additional file 7: Table S1. Primer pairs used for qRT-PCRAdditional file 8: Table S2. Different phosphorylation signal proteins in expression pattern of “a”. Table S3. Different phosphorylation signal proteins in expression pattern of“b”. Table S4. Different phosphorylation signal proteins in expression pattern of “c”. Table S5. Different phosphorylation signal proteins in expression pattern of “d”.


L. major infection by initiating a signaling cascade that results in production of pro-inflammatory cytokines and recruitment of both innate and adaptive immune cells. Upon infection with L. major, CXCL1 is produced locally and plays an important role in the recruitment of neutrophils to the site of infection. Herein, we report that L. major specifically targets murine CXCL1 for degradation. The degradation of CXCL1 is not dependent on host factors as L. major can directly degrade recombinant CXCL1 in a cell-free system. Using mass spectrometry, we discovered that the L. major protease cleaves at the C-terminal end of murine CXCL1. Finally, our data suggest that L. major metalloproteases are involved in the direct cleavage and degradation of CXCL1, and a synthetic peptide spanning the CXCL1 cleavage site can be used to inhibit L. major metalloprotease activity. In conclusion, our study has identified an immune evasion strategy employed by L. major to evade innate immune responses in mice, likely reservoirs in the endemic areas, and further highlights that targeting these L. major metalloproteases may be important in controlling infection within the reservoir population and transmittance of the disease.Leishmaniasis is a global health problem with an estimated report of 2 million new cases every year and more than 1 billion people at risk of contracting this disease in endemic areas. The innate immune system plays a central role in controlling  L. major metalloprotease in cleaving and degrading murine CXCL1. Indeed, L. major metalloprotease did not cleave murine CXCL2 or human CXCL1, CXCL2 and CXCL8. CXCL1 is a critical chemokine required for neutrophil recruitment to the site of infection; thus, we propose that this metalloprotease may have evolved to evade immune responses specifically in the murine host. We have further identified that the C-terminal end on CXCL1 is targeted for cleavage by the L. major metalloprotease. Finally, this cleavage site information was used to design peptides that are able to inhibit CXCL1 degradation by L. major. Our study highlights an immune evasion strategy utilized by L. major to establish infection within a murine host.Our study discovered a highly specific role for  Leishmania spp. are unicellular eukaryotic protozoan parasites that are transmitted to mammalian hosts by sandfly (Phlebotomine and Lutzomyia spp.) bites [L. major promastigotes , the promastigotes are quickly taken up by neutrophils, macrophages and keratinocytes [Leishmania spp. promastigotes hijack the phagocytic vacuole and transform into amastigotes (round body lacking an anterior flagellin) [Leishmania spp. amastigotes then proliferate within the vacuole and establish infection within the host [Leishmania spp. transmission, several studies have reported a vertical transmission of these parasites in mammalian hosts, from a pregnant female to its offspring [Leishmania spp. infection has been found to be endemic in foxhound dog populations in the United States, where the vectors are not present [Leishmania spp. can infect several hosts, including rodents and dogs (in addition to humans) [.) bites . Upon trinocytes –6. Withiagellin) . The Leithe host , 9. Whilffspring –13. Spec present –17. Give humans) , these sLeishmania spp. have evolved to evade the host immune response by using its armada of virulence factors to avoid host killing [Leishmania spp. include leishmanolysin metalloprotease glycoprotein 63 (GP63) and lipophosphoglycan (LPG), and both have been extensively studied for their roles in immune evasion [Leishmania spp. can hijack the host immune responses to establish infections as shown by Leishmania chagasi (L. chagasi)-mediated activation of TGF-β and Leishmania major (L. major)-induced activation of NLRP3 inflammasome, events that promote L. major survival and pathology [L. major.Our immune system is extremely efficient in killing pathogens. Professional phagocytes such as macrophages and neutrophils phagocytose and kill the invading pathogen in the intracellular phagosome-lysosome compartment. In addition, these phagocytes also respond to the foreign pathogens by secreting pro-inflammatory cytokines and chemokines to recruit neutrophils to the site of infection –21. Leis killing . The two evasion , 24. GP6 evasion , 26, inh evasion , 28, and evasion , 30 for athology –33. HereL. major is the most common Leishmania spp. and a major cause of cutaneous leishmaniasis that affects 600,000–1,000,000 people globally [L. major promastigotes first come in contact with keratinocytes, which then secrete pro-inflammatory cytokines to recruit innate immune cells [L. major as less than 1–2% of human keratinocyte have been shown to internalize L. major in vitro; which are often non-productive infections [L. major promastigotes are taken up by the resident macrophages, which then secrete essential cytokines and chemokines to elicit an innate immune response [L. major include CXCL1 [Leishmania spp. infection. Several studies have shown that neutrophils infiltrate the site of Leishmania spp. infection as early as 1-hour post-infection and are important for optimal resolution of the infection [L. major targets CXCL1 to evade early host innate immune responses.globally . Once dene cells , 35. Kerresponse . One of de CXCL1 . CXCL1 ide CXCL1 –42. Kerade CXCL1  and macrde CXCL1 , 45 havenfection , 46. GivL. major metalloprotease and a possible immune evasion strategy employed by this parasite to establish a successful infection within the murine host. We further report that L. major promotes proteolytic cleavage of murine CXCL1 at the C-terminal end to initiate its degradation. Finally, we have designed a synthetic peptide spanning the CXCL1 cleavage site that inhibits L. major protease activity. In conclusion, our study has uncovered a specific mechanism employed by L. major to degrade murine CXCL1 which may help the parasite to establish infection within the murine host.In this current study, we have identified murine CXCL1 as a highly specific substrate for L. major impacts innate immune responses elicited by macrophages, we stimulated bone marrow-derived macrophages (BMDM) with lipopolysaccharide (LPS) in the presence or absence of L. major (WHOM/IR/-173) infection following established protocol [Fig 1A), BMDM were stimulated with 20 ng/ml LPS in the presence or absence of 20 MOI L. major promastigotes for 48 hours. LPS stimulation of BMDM induced robust production of IL-6, TNF and CXCL1. Simultaneous LPS stimulation and L. major infection did not impact the production of IL-6 and TNF by BMDM whereas CXCL1 levels were significantly blunted . The reduction of CXCL1 was directly proportional to the concentration of L. major as demonstrated by L. major concentration dependent reduction in the levels of CXCL1 from LPS-stimulated BMDM . These data suggest that L. major specifically targets LPS-induced CXCL1 production by BMDM.To investigate how protocol . As detaL. major targets signaling pathways to evade immune responses and establish infection [[L. major specifically targeted CXCL1 expression and production, we designed an experiment whereby BMDM were infected with 20 MOI L. major for 6 hours, extracellular L. major were washed, then stimulated with 20 ng/ml LPS for the next 42 hours . Interestingly, L. major-infected BMDM produced equal levels of CXCL1 when compared to controls . These results show that inhibition of CXCL1 by L. major may not occur through modulation of intracellular signaling pathway that promotes CXCL1 expression and/or production by BMDM. L. major promastigotes once inside the macrophages transform into amastigotes. To this end, we tested the ability of lesional amastigotes to directly modulate rm-CXCL1 detection. Our results show that lesional L. major amastigotes prepared from L. major-infected footpads were able to inhibit rm-CXCL1 detection . Altogether, these results suggest that both extracellular (promastigote) and intracellular (amastigote) forms of L. major are able to modulate CXCL1 levels.Previous studies have shown that fection . To exaL. major pre-infection of BMDM prior to LPS stimulation did not affect CXCL1 production, we posited that L. major regulates secreted CXCL1 in the extracellular milieu. To this end, we used supernatants derived from LPS-stimulated BMDM as a source of CXCL1 and cultured 500 μl of these cell-free supernatants with 20 × 106L. major promastigotes for 24 hours . While levels of IL-6 and TNF detected in the supernatants remained unchanged, levels of CXCL1 were significantly reduced after addition of L. major to the supernatants. The reduced levels of CXCL1 were not due to its short half-life in the culture, as the levels of CXCL1 in the cell free supernatants were stable up to 48 hours . Thus, L. major directly dampens CXCL1 detection.Given that L. major on CXCL1 detection was dependent on live parasites. To this end, we cultured supernatants from LPS-stimulated BMDM with L. major lysates (Lm lysate: generated by 3× freeze-thaw cycles of L. major) or supernatants from L. major culture . Similar to the addition of live L. major, addition of Lm lysate or Lm sup also reduced CXCL1 in the supernatants while IL-6 and TNF remained unaffected . These results demonstrate that: first, L. major need not be alive to dampen CXCL1 detection; second, L. major lysate can dampen CXCL1 detection; and finally, L. major secreted factors dampen CXCL1 detection. Boiling Lm lysate or Lm sup for 20 minutes at 100ºC rescued CXCL1 detected in the supernatants, suggesting that the responsible L. major components are susceptible to heat treatment . Given the sensitivity to heat treatment, we propose that the CXCL1 regulating L. major components are proteinaceous.We next examined whether the observed effect of L. major components on CXCL1 [S3 Fig). Importantly, Lm sup addition to rm-CXCL1 dampened its detection by ELISA in a time-dependent manner, and boiling Lm sup rescued rm-CXCL1 detection .Macrophages secrete several hundreds of different proteins that may indirectly alter our observed effect of on CXCL1 . To thisL. major in reducing rm-CXCL1 levels, we examined rm-CXCL2, rh-CXCL1, rh-CXCL2 and rh-CXCL8 sequence homology by CLUSTAL-W alignment and found significant homology between these recombinant proteins . Despite the significant homology, L. major failed to reduce levels of rm-CXCL2, rh-CXCL1, rh-CXCL2 or rh-CXCL8, demonstrating specific regulation of rm-CXCL1 . As expected, L. major did not inhibit rm-TNF levels . While the C-terminal cleavage site of murine CXCL1 has high degree of sequence homology to murine CXCL2 and human CXCL1, CXCL2 and CXCL8; L. major fails to cleave murine CXCL2 or human CXCL1, CXCL2 and CXCL8. Several possibilities that may explain these discrepancies could be: 1) a requirement of specific sequence for Lm protease that is only present in murine CXCL1, and 2) the three-dimensional structure of murine CXCL1, specifically at the c-terminal end, is different from that of other CXCL homologs and orthologs. Given the specific nature of L. major (WHOM/IR/-170) in dampening rm-CXCL1 levels, we next determined whether this activity was specific to the WHOM/IR/-170 isolate. However, supernatant from L. major . Furthermore, exosomes from the L. major supernatants from both isolates were able to reduce rm-CXCL1 levels, suggesting that the active components are also present in the L. major-derived vesicles .To further investigate the specificity of  in Iraq ) was alsL. major reduced CXCL1 levels as demonstrated by ELISA . Possible reasons why CXCL1 levels were reduced when incubated with L. major include: 1) L. major proteins bind to CXCL1, limiting the ability of anti-CXCL1 antibody in ELISA to interact with CXCL1 or 2) L. major proteases cleaves and degrades CXCL1. Considering both outcomes, it is possible that CXCL1, either masked or cleaved by L. major components, could still be biologically active. To this end, we performed a functional assay where rm-CXCL1 or rm-CXCL1+Lm lysate was used to stimulate BMDM . As demonstrated previously, when rm-CXCL1 is incubated with L. major, rm-CXCL1 levels are significantly reduced . Lm lysate alone has very little stimulatory activity as demonstrated by mRNA induction of Cxcl1, Tnf and Il6 . As expected, rm-CXCL1 induced modest increase of Cxcl1, Tnf and Il6 but rm-CXCL1+Lm lysate failed to induce Cxcl1, Tnf and Il6 mRNA . These results altogether demonstrate that L. major inactivates biological activity of rm-CXCL1.Our data show that L. major, we incubated rm-CXCL1 with Lm lysate for various time periods and examined rm-CXCL1 by silver staining . Rm-CXCL1 was detected at approximately 10KDa by silver stain. Interestingly, when incubated with Lm lysate rm-CXCL1 showed two bands as early as 30 minutes after incubation, suggesting Lm lysate-mediated cleavage of rm-CXCL1 . By 2 hours, only the cleaved rm-CXCL1 was observed and this cleaved band intensity decreased over time, suggesting further degradation . L. major-mediated cleavage of rm-CXCL1 was specific because similar experiments done with rm-CXCL2, rh-CXCL1 and rh-CXCL2 did not result in cleavage or degradation of these recombinant proteins .To determine whether CXCL1 is degraded by Fig 5A) and a smaller fragment . To identify the cleavage product and site, we processed the full-length rm-CXCL1 and Lm lysate-cleaved rm-CXCL1 bands by trypsin digestion and performed mass spectrometry to determine the sequence of the bands . The tryptic peptide sequence readouts of full-length rm-CXCL1 covered the whole rm-CXCL1 peptide sequence , while the peptide coverage of cleaved rm-CXCL1 covered all of the rm-CXCL1 peptide sequence except the last 7 amino acids (MLKGVPK) at the C-terminal end . Thus, L. major cleaves rm-CXCL1 after lysine 65 (K65) residue that results in a large N-terminal rm-CXCL1 fragment lacking 7 amino acids at the C-terminal end .Based on the cleavage pattern of rm-CXCL1 (less than 1KDa shift), cleavage either at the N-terminal or C-terminal end would result in a large fragment  . Roche cOmplete protease inhibitor is a broad inhibitor of proteases including serine, cysteine and metalloproteases, and Sigma P8340 inhibitor is reported to inhibit serine, cysteine, acid proteases and aminopeptidases. However, the presence of protease inhibitors from Roche (cOmplete) or Sigma (P8340) did not inhibit Lm sup-mediated degradation of rm-CXCL1 as demonstrated by ELISA . Marimastat, a broad inhibitor of matrix metalloproteases [Lm sup . EDTA treatment, which chelates metal ions such as Ca2+ and Fe3+ , did not rescue rm-CXCL1 cleavage by Lm sup . Interestingly, 1,10-Phenanthroline, a Zn2+ metalloprotease inhibitor [Lm sup . While the addition of 1,10-Phenanthroline resulted in the upward shift of rm-CXCL1 bands in SDS-PAGE gels; importantly, no cleavage or degradation of rm-CXCL1 was observed .nfection . MetalloL. major . To examroteases , did notnhibitor , rescued2+ metalloprotease present on all Leishmania spp and is often used in biochemical assays to inhibit non-specific proteolytic activity of GP63 [Lm lysate using anti-GP63 antibody . Immunoblotting the GP63+ve and GP63-ve IP fractions for GP63 showed that GP63 was exclusively present in the IP+ve fraction and not present in the IP-ve fraction demonstrating successful immunoprecipitation of GP63 . More importantly, when these fractions were cultured with rm-CXCL1, the IP-ve (Lm lysate lacking GP63) but not the IP+ve (i.e. GP63) fraction degraded rm-CXCL1 suggesting a role for GP63-independent metalloprotease in the specific degradation of rm-CXCL1 .Several studies have shown that 1,10-Phenanthroline inhibits GP63, a Zn of GP63 . To deteL. major Zn2+ metalloprotease specifically cleaves rm-CXCL1 after the K65 residue leaving a 7-mer amino acid sequence (MLKGVPK). To further examine the specificity of this L. major metalloprotease, we designed a blocking peptide that covered this cleavage site . The addition of blocking peptide was able to rescue Lm lysate-mediated degradation of rm-CXCL1 in a dose-dependent manner . The peptide sequence from the signal peptide region of rm-CXCL1 . These data demonstrate that a synthetic peptide spanning the murine CXCL1 cleavage site can competitively inhibit L. major-mediated rm-CXCL1 degradation.Our data suggest that Leishmania spp. infection in the skin [Leishmania spp. infection of mice in vivo has been understudied. Charmoy et al showed that mice deficient in CXCL1 have a slight increase in lesion size and reduced numbers of neutrophil infiltrates in chronic lesions, but the overall pathology remained similar between WT and Cxcl1-/- mice [L. major Seidman strain (LmSd) that causes a non-healing infection in C57BL/6 mice [Tlr2-/- mice produce significantly lower levels of CXCL1 following L. major infections and develop smaller lesions suggesting a pathogenic role for CXCL1 [Tlr2-/- during L. major infection resulted in increased parasite numbers and disease pathology [L. major infection in mice and humans.CXCL1 is a potent neutrophil chemoattractant and is rapidly upregulated following the skin ; however-/- mice . HoweverL/6 mice . A recenor CXCL1 . Furtherathology . Thus, mL. major infections, other studies have shown no role or even amelioration of disease pathology [L. major strain, route of infection and timing of CXCL1 release may all contribute to the fate of the infection and overall pathology. In line with this thought, two independent L. major strains were tested in our study, and both strains degraded CXCL1 in our experimental systems. Whether this degradation of murine CXCL1 is a specific feature of L. major or a more general feature of all Leishmania spp. will need further investigation.Importantly, even depletion of neutrophils using anti-Gr1 neutralizing antibody does not always lead to increased parasitic burden, lesion or pathology . While sathology , 53–61. L. major in culture with macrophages effectively inhibited LPS-induced CXCL1 production. Further examination showed that intracellular L. major within the macrophages did not inhibit LPS-induced CXCL1 production. These experiments suggest that L. major does not interfere with signaling pathways that promote CXCL1 production but directly regulates secreted CXCL1 in the extracellular milieu. L. major are present as metacyclic promastigotes in sandflies [L. major infect macrophages and resides within the vacuole where they transform into amastigotes, multiply and establish infection [L. major-infected macrophages which contain amastigotes did not inhibit CXCL1 production, it could be posited that only the promastigote form of L. major degrades CXCL1. However, genome microarray analysis shows that the majority of genes are constitutively expressed in both L. major promastigotes and amastigotes (>90%) and therefore the major genes and virulent factors expressed by these different stages of L. major may not be different [L. major promastigotes and amastigotes may similarly cleave and degrade murine CXCL1 in an in vitro assay. In addition, infected macrophages can secrete exosomes containing L. major proteins [L. major secreted microvesicles (i.e. exosomes) contain these metalloproteases that can degrade murine CXCL1. Physiologically, L. major promastigotes released during a sandfly bite may act locally to limit acute CXCL1 produced in response to the infection. Once L. major are phagocytosed and transformed into amastigotes, they are physically separated from the CXCL1 due to their location (amastigotes are in the vacuole while CXCL1 are secreted by the macrophages and are extracellular) and may only impact CXCL1 production through the L. major component laden exosomes.Our initial observation demonstrated that andflies . When innfection . Becauseifferent . Thus, bproteins , 66, whiL. major cleaves murine CXCL1. Given that 1,10-Phenanthroline (Zn2+ chelator) but not EDTA  or Marimastat  rescued L. major-mediated CXCL1 degradation, it is possible that the unknown metalloprotease is a Zn2+ metalloprotease. Our studies also demonstrate that the L. major metalloprotease is highly specific for murine CXCL1 in that its closest murine homolog, CXCL2, or human homologs, CXCL1, CXCL2 or CXCL8, were not degraded. We hypothesize that this particular L. major metalloprotease may have evolved to specifically evade host immune response in rodents. Given that human CXCL1 homologs are not susceptible to this metalloprotease, one can argue that our results may not have any importance from a public health standpoint, but we reason otherwise. Our results are highly relevant to public health because rodents are ubiquitous, serve as a reservoir for Leishmania spp. and we highlight a rodent-specific L. major evolution in targeting murine CXCL1 [Our studies suggest that a yet-unknown metalloprotease from ne CXCL1 .L. major and by 4 hours the cleavage is complete in that only the cleaved bands are observed. Interestingly, we did not observe any accumulation of the cleaved CXCL1 band, suggesting continuous degradation of the cleaved form. While our data clearly demonstrate that the unknown L. major metalloprotease mediates cleavage of murine CXCL1, how the cleaved CXCL1 is further degraded will be the subject of future investigation. The cleaved murine CXCL1  may be highly unstable and undergo spontaneous degradation overtime. Alternatively, the cleaved murine CXCL1 may be susceptible to additional L. major proteases which promote its subsequent degradation.Our studies demonstrate that the murine CXCL1 is first cleaved at the C-terminal end after K65 releasing a 7aa residue. CXCL1 cleavage occurs as early as 30 minutes of incubation with L. major that is highly specific to murine CXCL1 . Specifically, L. major-associated metalloprotease cleaved murine CXCL1 at K65 residue and released a C-terminal 7 amino acid fragment to promote its degradation. Finally, we have designed a peptide spanning the cleavage site of CXCL1 that inhibited murine CXCL1 cleavage by L. major. Our study altogether uncovered an immune evasion strategy employed by L. major that may have evolved in rodents and highlights how parasites may utilize diverse immune evasion strategy to establish infection within its diverse mammalian hosts.In conclusion, we have identified an immune evasion strategy utilized by Experimental procedures that utilized mice were all approved by the University of Iowa Animal Care and Use Committee  and performed in accordance to the Office of Laboratory Animal Welfare guidelines and the PHS Policy on Humane Care and Use of Laboratory Animals.6 cells in 12-well cell culture plates in IMDM media containing 10% FBS, 1% nonessential amino acids, and 1% penicillin-streptomycin. BMDMs were primed with LPS (20 ng/ml) and infected with 1, 2, 5, 10 or 20 MOI L. major promastigotes for 24 and 48 hours (L. major supernatant (Lm sup) and L. major lysate (Lm lysate). BMDMs were also treated with rm-CXCL1 (20 ng/ml) or rm-CXCL1+Lm sup preparations in some experiments.BMDMs were prepared as described previously (32). Briefly, bone marrow cells were harvested from the hind limbs of BALB/c mice  and cultured in L cell-conditioned IMDM medium supplemented with 10% FBS, 1% nonessential amino acid, and 1% penicillin-streptomycin for 5–7 d to differentiate into macrophages. BMDMs were counted and seeded at 1 × 1048 hours . For somL. major strains WHOM/IR/-173 [L. major promastigotes for 48 hours. Conditioned L. major supernatant (Lm sup) was prepared after L. major reached the stable growth phase at approximately 20 × 106 / ml. Following centrifugation (3000×g) to pellet L. major, supernatants were collected and filtered using a 0.2μm vacuum filter to harvest Lm sup. Lm lysate was prepared by collecting the L. major pellet, washing 3 times with PBS followed by 3 freeze-thaw cycles of 7 × 109L. major /ml in PBS. For the preparation of L. major exosomes, promastigotes were grown to stable phase and changed to serum-free media and incubated at room temperature overnight. Supernatant was obtained as above and then separated using a 100kDa molecular weight Amicon ultra-15 centrifugal filter . The concentrated vesicles were washed twice with PBS by ultracentrifugation at 110,000xg at 4°C for 1 hour . The final pellet (exosomes) was resuspended in PBS and used in in vitro biochemistry assays./IR/-173  and IA0 /IR/-173  were gro6L. major promastigotes / footpad. 4 weeks post infection when footpad lesions were visible, mice were euthanized and footpad harvested and homogenized in PBS. L. major burden in the footpad were determined by limiting dilution assay as described previously [L. major amastigotes. These footpad lysates with L. major amastigotes at a concentration of 2.5 x 108 and 1.25 x 108 (determined by limiting dilution assay) were incubated with 2ng rm-CXCL1.BALB/c mice were injected with 2 x 10eviously . FootpadL. chagasi promastigote GP63 at a concentration 1:1500 incubated overnight at 4°C. The membranes were then probed with HRP-tagged secondary antibodies at room temperature for 1 hour and developed using Immobilon Forte Western HRP Substrate (MilliporeSigma) and imaged with an Odyssey Fc Infrared Imaging System . Immunoprecipitation was performed using Protein G Dynabeads and Leishmania GP63 monoclonal antibody clone 96–126 (Thermofisher). As instructed by the manufacturer, 50μL beads were washed in PBS with 0.02% Tween 20 and bound to 5μg antibody for at least 10 minutes. Following a wash, the antibody bound beads were incubated with Lm lysate for another 10 minutes and finally either antigen-antibody binding was negated by acidic (pH 2.8) 50mM Glycine treatment or bead bound antibody-antigen complexes were used directly for biochemistry.For in vitro reactions, 125ng of total recombinant protein was separated on 4–20% or 10–20% Novex WedgeWell Tris-Glycine precast gels . For silver stain, the Pierce Mass Spec compatible silver stain kit was used per manufacturer’s instructions (Thermofisher). Synthetic peptides were generated and purchased from Pierce Biotechnology (Thermofisher) and are composed of the following sequences: peptide#1: CAALLLLATSRLA; peptide#2: RLATGAPIAN; blocking peptide: LVQKIVQKMLKGVPK. For Western blot analysis a semi-dry transfer was completed using trans-blot turbo system  with Immobilon PVDF membrane. The membranes were blocked in 5% non-fat milk for 1 hour at room temperature. Membranes were probed with sheep polyclonal antibody Sp180 raised against 3 pieces, and washed in 100mM ammonium bicarbonate:acetonitrile  and 25mM ammonium bicarbonate /acetonitrile , respectively, to achieve complete de-staining. The gel pieces were further treated with acetonitrile (ACN), to effectively “dry” the gel segments and then reduced in 50μl of 10mM DTT at 56°C for 60 min. Gel-trapped proteins were alkylated with 55mM chloroacetamide (CAM) for 30 min at room temperature. The gel pieces were washed with 25mM ammonium bicarbonate: acetonitrile  twice to remove excess DTT and CAM. 50μl of cold trypsin solution at 10ng/μl in 25mM ammonium bicarbonate was then added to the gel pieces and they were allowed to swell on ice for 60 min. Digestion was conducted at 37°C for 16 h. Peptide extraction was performed three times, adding 100μL of 50% acetonitrile/0.1% formic acid for 0.5 h, combining the supernatants. The combined extracts were concentrated in a lyophilizer and rehydrated with 15μl of mobile phase A.In-gel trypsin digestion: The gel was stained using a Pierce mass spec compatible silver stain kit (Thermofisher) per manufacturer directions. A procedure slightly modified than that described by Yu et al  was usedLC-MS/MS: Mass spectrometry data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermofisher) coupled to an Easy-nLC-120 System (Proxeon P/N LC1400). The autosampler is set to aspirate 3μl (estimated 0.3ug) of reconstituted digest and load the solution on a 2.5cm C18 trap  coupled to waste, HV or analytical column through a microcross assembly . Peptides are desalted on the trap using 16μl mobile phase A for 4 min. The waste valve is then blocked and a gradient begins flowing at 0.4μl/min through a self-packed analytical column, 10cm in length × 75μm id. The fused silica column was tapered from 100μm ID (Polymicro) to ~8μm at the tip using a Sutter P-2000 laser puller then packed with 2.7micron Halo C18 particles using a He-pressurized SS cylinder. Peptides were separated in-line with the mass spectrometer using a 70 min gradient composed of linear and static segments wherein buffer A is 0.1% formic acid and B is 95%ACN, 0.1% formic acid. The gradient begins first hold at 4% for 3 min then makes the following transitions : , , , , .Tandem mass spectrometry on the Thermo Q-Exactive hf: Data acquisitions begin with a survey scan (m/z 380–1800) acquired on a Q-Exactive Orbitrap mass spectrometer (Thermofisher) at a resolution of 120,000 in the off-axis Orbitrap segment (MS1) with automatic gain control (AGC) set to 3E06 and a maximum injection time of 50 ms. MS1 scans were acquired every 3 sec during the 70-min gradient described above. The most abundant precursors were selected among 2–6 charge state ions at a 1E05 AGC and 70 ms maximum injection time. Ions were isolated with a 1.6 Th window using the multi-segment quadrupole and subjected to dynamic exclusion for 30 sec if they were targeted twice in the prior 30 sec. Selected ions were then subjected to high energy collision-induced dissociation (HCD) in the ion routing multipole (IRM). Targeted precursors were fragmented by (HCD) at 30% collision energy in the IRM. HCD fragment ions were analyzed using the Orbitrap . Both MS2 data were recorded as centroid and the MS1 survey scans were recorded in profile mode.Proteomic searches: Initial spectral searches were performed with both Mascot version 2.6.2 (MatrixScience) and Byonic search engines (Protein Metrics ver. 2.8.2). Search databases were composed of the Uniprot KB for species 10090 (mouse) downloaded February 6, 2016 containing 58436 sequences. In either search, an equal number of decoy entries were created and searched simultaneously by reversing the original entries in the target database. Precursor mass tolerance was set to 5 ppm and fragments were searched at 10 ppm. A fixed 57 Da modification was assumed for cysteine residues while variable oxidation was allowed at methionine. A variable GG modification at lysine was set to monitor ubiquitylation and potential phosphorylation was accessed at Ser and Thr residues. The false discovery rate was maintained at 1% by tracking matches to the decoy database. Both Mascot and Byonic search results were combined and validated using Scaffold ver. 4.8.5 (Proteome Software). Protein assignments required a minimum of two peptides established at 70% probability  and an overall 95% protein probability (assigned by Protein Prophet). Approximately 300 protein families (including common contaminants) were assigned at a total FDR to 1.2%. Proteins were annotated with GO terms from goa_uniprot_all.gaf downloaded on May 3, 2017.Cytokine ELISAs and multiplex immunoassays were performed according to manufacturer instructions. Mouse and human CXCL1 and CXCL2 as well as human IL-8 (CXCL8) were obtained from R&D Systems  and multiplex CXCL1, IL-6, and TNF were completed using ProcartaPlex assays (Thermofisher) which were run on the BioRad Bio-Plex . We obtained our recombinant proteins from Tonbo biosciences which included mouse CXCL1, CXCL2, and TNF as well as human CXCL1, CXCL2, and CXCL8.mCXCL1: 5’-CAATGAGCTGCGCTGTCAGTG-3’, 5’-CTTGGGGACACCTTTTAGCATC-3’; mTnf: 5′-CATCTTCTCAAAATTCGAGTGACAA-3′, 5′-TGGGAGTAGACAAGGTACAACCC-3′; mIL-6: 5’-CAAGAAAGACAAAGCCAGAGTC-3’, 5’-GAAATTGGGGTAGGAAGGAC-3’. Threshold cycle Ct values were normalized to GAPDH, and gene fold change was determined by the relative comparison method, relative to the 0 h time point.RNA was extracted from BMDM cultures in TRIzol reagent (Thermofisher) followed by chloroform extraction and isopropanol precipitation. The extracted RNA was reverse-transcribed into cDNA by using qScript Supermix . Real-time quantitative PCR was performed on Eppendorf realplex EPgradient S Mastercycler  using PerfeCTa SYBR Green SuperMix ROX (Quanta Biosciences) and the appropriate primers. The sequences of the quantitative RT-PCR primers are as follows: t tests (two-tailed and Mann-Whitney) for two groups, and one-way ANOVA (with Dunnett’s or Tukey’s multiple comparisons tests) for three or more groups. For the purpose of alignment presentation, sequences were obtained from the protein manufacturer or the NCBI protein database and figures were prepared using Jalview 2 [GraphPad Prism 8.0 software was used for data analysis and figure presentation. Data are shown as mean ± SEM. Statistical significance was determined by alview 2 .S1 FigL. major  for 24h and cell culture supernatants were analyzed for the indicated cytokines. (B) BALB/c mice were infected with 2 x 106L. major promastigotes / footpad, and 4 weeks later footpad harvested and homogenized. L. major burden in the footpad were determined by limiting dilution assay. Footpad lysates with L. major amastigote equivalent of 2.5e8 and 1.25e8 (based on limiting dilution assay) were cultured with rm-CXCL1 for 24 hours and the levels of rm-CXCL1 determined by ELISA. (C) Conditioned supernatants from 24h LPS (20 ng/ml) stimulated BMDMs were collected and further cultured for 0, 24, or 48h and stability of CXCL1, IL-6 and TNF in the culture were determined by ELISA. Data are representative of at least three independent experiments. Results are represented as mean ± SEM. ***P<0.001, ****P<0.0001.(A) BMDMs were treated with LPS (20 ng/ml) in the presence or absence of (TIF)Click here for additional data file.S2 FigLm lysate or Lm sup. Levels of CXCL1, IL-6 and TNF were determined by ELISA. Results are represented as mean ± SEM. ****P<0.0001.(A) Experimental Design. (B) Conditioned supernatants from 24h LPS (20 ng/ml) stimulated BMDMs were collected and subjected to 24h treatment with control or boiled (20 min at 100°C) (TIF)Click here for additional data file.S3 FigLm sup (control or boiled) for up to 48 hours in culture. The quantity of rm-CXCL1 in the culture at 0, 24 and 48 h were determined by ELISA. Data are representative of at least three independent experiments. Results are represented as mean ± SEM.Rm-CXCL1 were left alone or treated with (TIF)Click here for additional data file.S4 FigLm lysate for acute time points as indicated to determine the precise time course of rm-CXCL1 cleavage. Silver staining was used to visualize rm-CXCL1. Data are representative of at least three independent experiments.Rm-CXCL1 were left alone or treated with (TIF)Click here for additional data file.S5 Fig(A) A silver-stained SDS-PAGE showing cleavage of full length (BAND 1) and cleaved rm-CXCL1 (BAND 2). (B) Mass spectrometry analysis of BAND 1 (full length CXCL1) and tryptic peptide coverage analysis confirm a full length rm-CXCL1. (C) Mass spectrometry analysis of BAND 2 (cleaved CXCL1) and tryptic peptide coverage analysis confirm a C-terminal end cleavage after K65 residue.(TIF)Click here for additional data file.S6 FigLm sup in the presence or absence of Roche cOmplete protease inhibitor. Levels of CXCL1, IL-6 and TNF were determined by ELISA. (B) Rm-CXCL1 was treated with Lm sup in the presence or absence of Marimastat (10μM) for indicated time-points and ELISA was performed to evaluate the levels of CXCL1. Data are representative of at least three independent experiments. Results are represented as mean ± SEM. ***P<0.001, ****P<0.0001.(A) Conditioned supernatants from 24h LPS (20 ng/ml)-stimulated BMDMs were collected and treated with (TIF)Click here for additional data file.


Policymakers, practitioners, and researchers need a balanced, thoughtful, and analytical resource to meet the challenge of global aging at a rate that’s historically unprecedented. The Journal of Aging & Social Policy (JASP), which was founded in 1989, serves this role by drawing contributions from an international panel of policy analysts and scholars who assume an interdisciplinary perspective in examining and analyzing critical phenomena that affect aging and the development and implementation of programs for elders from a global perspective. Study settings extend beyond the United States to include Europe, the Middle East, Australia, Latin America, Asia, and the Asia-Pacific rim. This presentation will document the scope, content, and focus of JASP, including the rise of international submissions, which now account for approximately half of articles published. Opportunities for publishing in JASP will be discussed; so too will strategies for navigating the peer-review process successfully.


The mission of applied gerontology is to bridge science and practice to benefit the health and well-being of older persons, their families, their communities, and other contexts. This presentation will provide insights from the Journal of Applied Gerontology and its attempts to publish and disseminate scholarship that has international application. Following an overview of the growing internationalization of peer-reviewed submissions to the Journal of Applied Gerontology on a variety of topics and from a range of perspectives, the presentation will highlight key achievements as well as ongoing concerns and opportunities to better achieve the goals of applying gerontological scholarship to aging contexts worldwide. Concluding comments will examine how outlets for dissemination and authors themselves can better position their work to enhance their influence on aging in an international context.


We present a case of a 72-year-old male patient who was referred to us with a 1.2 x 0.6-cm left alar rim notching and retraction defect . The alaWhere in the ear can chondrocutaneous graft be harvested for nasal reconstruction?What is the upper limit for a chondrocutaneous graft before the graft viability is compromised and what can be done to overcome composite graft failure?What is the postoperative appearance sequelae of chondrocutaneous graft take?What is the typical postoperative care?,2Nasal defects in the alar rim are challenging to reconstruct. Thin skin coverage, cartilage support, and thin lining are needed to replace this cosmetically prominent site. Several reconstructive choices are available for this region, but the best cosmetic result is achieved with a composite full-thickness graft from the ear. The auricle is an excellent donor source because it provides a graft with thin skin attached to a delicate segment of contoured cartilage. The cartilage provides structural support and the skin that closely resembles the adjacent nasal skin of the nostril margin.1,3-1,3Auricular composite chondrocutaneous grafts have high metabolic demands. Composite cartilage grafts only interface with the recipient bed around the graft's perimeter. The traditional recommendation is to limit the size of composite grafts to 1 cm or less due to limited blood flow, which otherwise will lead to tissue necrosis of the graft.1b) due to lack of sufficient vasculature; the graft obtains its nourishment through plasma imbibition. However, as it progresses toward inosculation around 1 to 2 days postoperatively, it will acquire a congested blue color (c), denoted as deep cyanosis, and even crustiness.7,Fig 4d).Composite chondrocutaneous grafts follow a predictable healing pattern. Successful grafts transition in color during the first week: white, then blue, and then progressively pink/red. Immediately following introduction to local anesthetic and harvesting from the ear, the composite graft will appear pale b due to ue color c, denotePatient education and postoperative care are extremely important. Upon discharge, patients are given the following instructions: 10 minutes on/10 minutes off ice-saline compress application for the first 1 to 2 days; minimize all activities; avoid nose blowing; sneeze with an open mouth; and wipe gently with tissue. Bacitracin should be applied with a Q-tip regularly and gently around the incision inside and outside the nose. Teeth should only be brushed with a soft toothbrush. Avoid manipulation of the upper lip and excessive smiling for 1 to 2 weeks. Only wear clothing that fastens in the front or the back, and avoid slipover sweaters, tight T-shirts, and turtle necks. The only dietary restriction related to the alar rim composite graft reconstruction is avoiding foods that require prolonged chewing. Washing the face gently is permitted, as long as avoiding touching the nose. Finally, avoid sun exposure and a hot environment for 6 weeks postoperatively.


The molecular structural parameters of six normal rice starches with different amylose contents were investigated through their iodine absorption spectra and gel permeation chromatography of fully branched and debranched starches. The thermal and digestion properties of starches were also determined and their relationships with molecular structural parameters were analyzed. Results showed that the molecular structural parameters of maximum absorption wavelength, blue value (BV), optical density 620 nm/550 nm (OD 620/550), amylose, intermediate component, and amylopectin, including its short branch-chains, long branch-chains, and branching degree, had high correlation in different determining methods. The intermediate component of starch was significantly positively related to amylose and negatively related to amylopectin, and the amylopectin branching degree was significantly positively related to amylopectin content and negatively related to amylose content. The gelatinization temperatures and enthalpy of native starch were significantly positively related to BV, OD 620/550, and amylose content and negatively related to amylopectin short branch-chains. The gelatinization temperatures and enthalpy of retrograded starch were significantly negatively related to amylopectin branching degree. The digestions of gelatinized and retrograded starches were significantly negatively related to the BV, OD 620/550, amylose, and intermediate component and positively related to amylopectin and its short branch-chains and branching degree. Starch contains two main glucan polymers in its molecular structure: highly branched amylopectin and mainly linear amylose . The molmax), blue value , optical density 620 nm/550 nm ratio (OD 620/550), and apparent amylose content (AAC) [The molecular structure of starch includes the content and molecular weight distribution of amylose and amylopectin and is normally analyzed using the corresponding iodine absorption spectra and gel permeation chromatography (GPC) of fully branched and debranched starch ,2,3. Amynt (AAC) ,4,5. Amynt (AAC) . For debThe thermal properties of starch are important indicators of starch quality. The morphology, size, molecular structure, and crystalline structure of starch all have significant effects on its thermal properties ,10,11. SIn this study, we determined and compared many molecular structural parameters of six normal rice starches using their iodine absorption spectra and GPC. The thermal and digestion properties of starches were also investigated and their relationships with molecular structural parameters were analyzed. Our objective is to evaluate the molecular structural parameters of starch and their relationships with functional properties and provide important information for starch application through determining the molecular structural parameters.max is related to the polymerization degree and average chain length of amylose and amylopectin. BV reflects the iodine affinity of starch [max, BV, OD 620/550 nm, and AAC of starches are presented in The absorption spectra of starch–iodine complexes were clearly different among the six rice starches . λmax isf starch ,4. OD 62f starch . AAC reff starch . The λmaThe molecular weight distributions of fully branched starches are shown in The GPC chromatograms of debranched starches are shown in max, BV, OD 620/550, and AAC derived from starch–iodine absorbance spectrum had highly positive correlation (p < 0.01), and were all positively related to amylose (ACB and ACD) (p < 0.05) and negatively related to amylopectin  and branching degree (AP-S/L) (p < 0.05). For molecular weight distribution of fully branched starch determined by sepharose CL-2B GPC, amylopectin content had significantly negative correlation with the contents of intermediate component and amylose (p < 0.01), and intermediate component showed high correlation with ACB (p < 0.01). Similar correlation was also reported in previous studies [p < 0.05) and were all negatively related to amylose (ACD) (p < 0.05). The results are in agreement with previous reports in rice [B derived from Sepharose CL-2B GPC were positively related to amylose (ACD) derived from debranched starch (p < 0.05) but negatively related to the short branch-chain, long branch-chain, and branching degree of amylopectin (p < 0.05). Similar relationship is also reported in maize starch [The correlations among different molecular structural parameters are shown in  studies ,20. For  in rice ,22. Bothe starch . The aboThe DSC thermograms of native and retrograded starches are presented in p < 0.05), but negatively related to amylopectin short branch-chains and branching degree (p < 0.05). These data partly contradicted the previous studies. Park et al. found that gelatinization temperature correlated positively with AC and amylopectin long branch-chain, but no significant correlation was observed between gelatinization properties and amylopectin short branch-chains [max, BV, OD 620/550, and AC (p < 0.05) but negatively related to intermediate component and amylopectin, including short and long branch-chains (p < 0.05). However, the enthalpy and retro% of retrograded starch were only significantly positively related to amylose (ACD) and negatively related to amylopectin short branch-chains and branching degree (p < 0.05)  . Shi andAspergillus niger (AAG) are shown in In vitro digestion properties of gelatinized and retrograded starches by both α-amylase from porcine pancreatic (PPA) and amyloglucosidase from p < 0.05) but positively related to amylopectin and its short branch-chains and branching degree (p < 0.01). The RS of gelatinized starches was significantly positively related to BV, OD 620/550, AC, and intermediate component (p < 0.05) but negatively related to amylopectin, including its short branch-chains, long branch-chains, and branching degree (p < 0.01). However, the RS of retrograded starches was only positively related to OD 680 and AC (p < 0.05) and negatively related to amylopectin short branch-chains and branching degree (p < 0.05). Several studies also showed that the digestibility of starch is significantly negatively related to AC but positively related to amylopectin short branch-chains [The correlation between molecular structural parameters and in vitro digestion properties of rice starches are presented in h-chains ,13,14,21h-chains . For reth-chains . TherefoJaponica rice and the others are Indica rice. They were grown in the experiment field of Yangzhou University  in May 2015 and harvested in September 2015.Six normal rice varieties of 9311, Changjiang II, Guihuahuang, Huanghuazhan, Teqing, and Wuyunjing 8 were selected as plant materials in this study. The varieties of Changjiang II, Guihuahuang, and Wuyunjing 8 are Starch was isolated from mature brown seeds as previously described .v/v) methanol and dissolved in urea dimethyl sulfoxide solution as previously described [The protein was removed from starch granules by using protease and sodium bisulfite. Starch was defatted with 85% (escribed . The iodescribed .g). The supernatant (1 mL) was mixed with 4 mL of absolute ethanol to precipitate the starch, and the residual supernatant was used for subsequent debranched starch in g) and the supernatant was sieved through a 5 μm filter. Two milliliters of supernatant was injected into a GPC column (1.6 cm × 50 cm) packed with Sepharose CL-2B . The column was eluted at 0.5 mL/min with 25 mM NaCl and 1 mM NaOH solution, and 1.5 mL fractions were collected. The quantity of carbohydrate was determined using anthrone-H2SO4 method.The fully branched starch was analyzed as previously described  with somThe starch in the residual supernatant in The thermal properties of native starch were measured by a differential scanning calorimeter  as previously described . Five mi3, 2.5 mM CaCl2, 4 U PPA, 4 U AAG) was added in the centrifuge tube. The sample was incubated at 37 °C for 20 or 120 min using ThermoMixer at 1000 rpm, and then 240 μL of 0.1 M HCl and 2 mL of 50% ethanol were added immediately to terminate the hydrolysis. The sample was centrifuged , and the glucose quantity in the supernatant was determined using the glucose assay kit . The digested starches within 20 min and between 20 to 120 min were RDS and SDS, respectively, and the undigested starch at 120 min was RS.The in vitro digestion of starch was determined using both PPA  and AAG  as previously described . Brieflyp < 0.05, and Pearson’s bivariate correlations were performed using SPSS 19.0 software .Analysis of variance of the means, comparisons of multiple means using Tukey’s multiple-range tests at The molecular structural parameters of starch measured by different methods could reflect different structural information and had significant correlation. The intermediate component of starch was significantly positively related to amylose and negatively related to amylopectin including its short branch-chains, long branch-chains, and branching degree. The gelatinization temperatures of native starches and peak temperature of retrograded starches were significantly positively related to BV, OD 620/550, and AC and negatively related to amylopectin, including its short branch-chains and branching degree. The gelatinization enthalpy and retro% of retrograded starch were significantly negatively related to amylopectin short branch-chains and branching degree and positively related to AC of debranched starches. The resistance of starch to in vitro digestion could be increased by the BV, OD 620/550, AC, and intermediate component and decreased by amylopectin, including its short branch-chains and branching degree.


Accurate segregation of chromosomes to daughter cells is a critical aspect of cell division. It requires the kinetochores on duplicated chromosomes to biorient, attaching to microtubules from opposite poles of the cell. Bioriented attachments come under tension, while incorrect attachments lack tension and must be released to allow proper attachments to form. A well-studied error correction pathway is mediated by the Aurora B kinase, which destabilizes low tension-bearing attachments. We recently discovered that in vitro, kinetochores display an additional intrinsic tension-sensing pathway that utilizes Stu2. The contribution of kinetochore-associated Stu2 to error correction in cells, however, was unknown. Here, we identify a Stu2 mutant that abolishes its kinetochore function and show that it causes biorientation defects in vivo. We also show that this Stu2-mediated pathway functions together with the Aurora B-mediated pathway. Altogether, our work indicates that cells employ multiple pathways to ensure biorientation and the accuracy of chromosome segregation. The precise regulation of cell division is critical to processes such as self-renewal, proliferation and development. A key event in the cell cycle is the partitioning of every pair of duplicated chromosomes to daughter cells. Defects in chromosome partitioning lead to aneuploidy, a condition that is a common hallmark of cancer cells and the cause of some birth defects. Chromosomes segregate using their kinetochores, the specialized protein structures that are assembled on centromeric DNA sequences and attach to spindle microtubules. Here, we report that a protein that associates with kinetochores called Stu2 ensures that each kinetochore attaches to the proper microtubules. We identified a Stu2 mutant that does not associate with kinetochores and found that it generates aneuploidy. Together, our work identifies a previously unknown mechanism where cells ensure that chromosomes are accurately inherited during cell division. Faithful partitioning of the genetic material is a fundamental aspect of cell division. Chromosome segregation errors are the most prevalent genetic alteration in tumor cells and are proposed to be a major factor in the evolution of cancer (reviewed in ). SegregThe canonical “error correction” pathway is mediated by the conserved protein kinase Aurora B. The activity of Aurora B destabilizes kinetochore-microtubule interactions that are not under tension by phosphorylating multiple outer kinetochore components –8. HowevHere, we address whether the kinetochore-associated function of Stu2 promotes biorientation in cells. Stu2 has multiple functions and regulates spindle microtubules (reviewed in ), so we Stu2 has important roles in both the formation of the mitotic spindle and the regulation of kinetochore function ,12–14. Wstu2-AID allele at the endogenous locus [stu2 mutations was recently examined in cells [To determine the element(s) within Stu2 required for kinetochore-association, we individually deleted each of these domains and expressed the mutant alleles from an ectopic locus in a strain harboring a conditional us locus . Each ofin cells , but onlin cells . While mTo further confirm the requirement of these domains, we performed in vitro binding assays. We purified and immobilized kinetochores lacking endogenous Stu2 and examined the ability of each Stu2 variant (purified independently) to bind the immobilized kinetochores. Consistent with our co-purification results, deletions of the coiled-coil, C-terminal, and TOG1 domains disrupted the ability of purified Stu2 to ‘re-bind’ the kinetochores . These iAs a complementary approach to define regions of Stu2 required for its kinetochore interaction, we conducted crosslinking mass spectrometry with recombinant Stu2-eGFP and its outer kinetochore receptor, the Ndc80 complex (Ndc80c) . There wInterestingly, even though Stu2’s coiled-coil domain is required for its kinetochore association, we did not detect any crosslinks between this domain and Ndc80c. This observation suggests that Stu2’s coiled-coil domain may not contain sequence-specific elements that directly mediate Ndc80c binding; instead, the protein may simply need to form a homo-dimer. Consistent with this, replacement of the coiled-coil domain with the homo-dimerization of the Gcn4 bZIP transcriptional activator  restoredstu2ccΔ) exhibited impaired growth and benomyl sensitivity when the endogenous Stu2-AID protein was depleted  and measuring the distance between spindle poles (labeled by Spc42-CFP). We performed this experiment in cells carrying the stu2-AID allele with an ectopic copy of the STU2 variant to be examined. As expected, stu2-AID cells that were treated with auxin, and that did not express a covering STU2 allele, arrested with a short bipolar spindle . Thus, while mutants that alter Stu2’s dimerization exhibit polymerization defects in vitro [stu2ccΔ mutant is defective in kinetochore binding yet supports a normal mitotic spindle, it provides a tool to specifically examine Stu2’s kinetochore function.We sought a Stu2 mutant that lacked kinetochore association but maintained spindle and microtubule functions –14. Whil42 foci; ,13,14), in vitro ,18, thisΔcc mutant disrupted this function. As before, we used an optical trapping-based ‘‘force-ramp” technique in which native Ndc80c was linked to beads and then attached to growing microtubule tips using a laser trap  residual kinetochore/Ndc80c binding of this mutant  and kinetochore distribution was assayed using Mtw1-3GFP. As expected, the vast majority of stu2-AID cells treated with auxin that expressed a wild-type allele of STU2 exhibited normal spindle length and a normal bi-lobed kinetochore distribution (89% ± 1%) , the clusters appeared closer together than STU2WT and some cells also displayed unattached kinetochores  . Consistlignment . Althoug; STU2WT . The celstu2ccΔ expressing cells cause a cell cycle delay. Cells were arrested in G1 and released into auxin-containing media to degrade the endogenous Stu2-AID protein, and ectopically expressed either STU2WT or stu2ccΔ. Anaphase was delayed in cells expressing stu2ccΔ . Thus,  stu2ccΔ , suggestanaphase , consiststu2ccΔ mutants when the spindle checkpoint was abolished using a fluorescently marked centromere on chromosome VIII. stu2-AID cells ectopically expressing either STU2WT or stu2ccΔ in the presence or absence of MAD3 were released from a G1 arrest into auxin-containing media. The mis-segregation of chromosome VIII was notably increased in stu2ccΔmad3Δ cells . We preustering  is consiome VIII  and arreome VIII . In metanct foci . As expestu2ccΔ allele specifically disrupting the function of kinetochore-associated Stu2, it was recently reported that a similar Stu2 mutant protein that lacks the coiled-coil as well as its C-terminal domain displays reduced microtubule polymerase activity in vitro [stu2 mutant that partially restored microtubule polymerase activity in vitro. This mutant adds positively charged residues to the basic linker domain to restore the dimer-equivalent number of basic residues [stu2ccΔ mutant, displaying equivalent defects in both kinetochore association as well as biorientation  to establish biorientation , likely due to mitotic spindle collapse . These results suggest that Stu2’s kinetochore-associated function is only required for the establishment of correctly bioriented attachments and is dispensable thereafter.Aurora B function is dispensable once bioriented attachments have been made , so we tentation . We thencollapse . In contstu2ccΔ mutant and the ipl1-321 temperature sensitive allele of Aurora B [ipl1-321 cells grow nearly as well as wild-type cells. We found that under conditions where both pathways were perturbed (i.e. in the presence of auxin and at the semi-permissive temperature of 30°C), the cells expressing the stu2ccΔ allele and harboring ipl1-321 displayed a significant additive growth defect over either of the single mutations  and at the semi-permissive temperature of 30°C and examined chromosome mis-segregation in anaphase (i.e. the percentage of cells that failed to segregate a copy of chromosome III to each of the resulting daughter nuclei). As expected, control cells expressing wild-type STU2 and IPL1, as well as ipl1-321 cells with the Aurora B pathway partially disrupted alone, displayed low levels of chromosome mis-segregation , however, the stu2ccΔipl1-321 double mutant cells displayed a significant increase in the rate of chromosome mis-segregation compared to either single mutant alone , Our results strongly indicate that Stu2 has an important role in biorientation; how Stu2’s kinetochore-associated function is regulated, however, remains unanswered. An interesting observation that may shed light on this question is related to the SK-rich region-Ndc80c interaction. We observed that Stu2’s SK-rich region is dispensable for kinetochore binding, yet the majority of crosslinks we observed by XL-MS are between this domain and a region near Ndc80’s conserved hairpin region. Recently, the SK-rich region was found to be involved in an intra-molecular regulation of Stu2, in which tubulin occupancy of the TOG domains regulates the ability of the SK-rich domain to bind to the microtubule lattice . An intrIt has been proposed that Stu2's function at the kinetochore is solely facilitated by regulation of the microtubule tip . ConsistErroneous microtubule attachments must be selectively destabilized by the cell’s error correction machinery until all attachments become correctly bioriented (reviewed in ). Error Saccharomyces cerevisiae strains used in this study are described in pCUP1-GFP-LacI and ipl1-321 are described in [CEN3::lacO:TRP1 and CEN8::lacO:TRP1 are described in [MTW1-3GFP, TUB1-CFP and mad3Δ are described in [DSN1-6His-3Flag is described in [stu2-3V5-IAA7, stu2-3HA-IAA7, and SPC24-6His-3Flag were constructed by PCR-based methods [cdc20-IAA17 from Eris Duro and Adèle Marston; SPC42-CFP from Sue Jaspersen; pMET-CDC20 from Frank Uhlmann). pADH1-TIR1-9Myc is described in [pGPD1-TIR1 integration plasmids  as well as a 3V5-IAA7 tagging plasmid (pSB2065) were provided by Leon Chan. Construction of a 3HA-IAA7 tagging plasmid (pSB2229) was described previously in [LEU2 integrating plasmid containing wild-type pSTU2-STU2-3V5 (pSB2232) is also described in [STU2 variants were constructed by mutagenizing pSB2232 as described in [pSTU2-stu2(Δ2–281)-3V5, i.e. stu2TOG1Δ), pSB2255 (pSTU2-stu2(Δ2–550)-3V5, i.e. stu2TOG1-2Δ), pSB2257 (pSTU2-stu2(Δ282–550::GDGAGLlinker)-3V5, i.e. stu2TOG2Δ), pSB2259 (pSTU2-stu2(Δ551–657::GDGAGLlinker)-3V5, i.e. stu2SK-richΔ), pSB2261 (pSTU2-stu2(Δ658–761::GDGAGLlinker)-3V5, i.e. stu2ccΔ), pSB2306 (pSTU2-stu2(R200A)-3V5, i.e. stu2R200A), pSB2307 (pSTU2-stu2(R519A)-3V5, i.e. stu2R519A), pSB2308 (pSTU2-stu2(R200A R519A)-3V5, i.e. stu2R200A R519A), pSB2309 (pSTU2-stu2(Δ658–761::GDGAGLlinker-GCN4(249–281)bZIP-GDGAGLlinker)-3V5, i.e. stu2ccΔ::bZIP), pSB2357 (pSTU2-stu2(Δ762–888)-3V5, i.e. stu2C-termΔ(762–888)), pSB2358 (pSTU2-stu2(Δ855–888)-3V5, i.e. stu2C-termΔ(855–888)), pSB3103 (pSTU2-stu2(2x basic_Δ658–761::GDGAGLlinker)-3V5, i.e. stu22x basic_ccΔ) . Primerauxin inducible degron (AID) system was used essentially as described [The escribed . Briefly600 ~1.0 from which a serial 1:5 dilution series was made and spotted on YPD+DMSO or YPD+100–500 μM IAA (indole-3-acetic acid dissolved in DMSO) plates. Plates were incubated at 23–30°C for 2–3 days unless otherwise noted.For the spotting assay, the desired strains were grown overnight in yeast extract peptone plus 2% glucose (YPD) medium. The following day, cells were diluted to ODExponentially growing cultures were treated with 500 μM auxin for 2h, then fixed (see below) and analyzed for Mtw1-3GFP localization and spindle morphology (Spc42-CFP or Tub1-CFP). An aliquot of cells was fixed with 3.7% formaldehyde in 100mM phosphate buffer (pH 6.4) for 5 min. Cells were washed once with 100mM phosphate (pH 6.4), resuspended in 100mM phosphate, 1.2M sorbitol buffer (pH 7.5) and permeabilized with 1% Triton X-100 stained with 1 μg/ml DAPI . Cells were imaged using a Nikon E600 microscope with a 60X objective (NA = 1.40), equipped with a Photometrics Cascade 512B digital camera. Five Z-stacks (0.3 micron apart) were acquired and all frames with nuclear signal in focus were maximally projected. NIS Elements software (Nikon) was used for image acquisition and processing.cdc20-AID containing strains) or media lacking methionine (for pMET-CDC20 containing strains in cdc20-AID containing strains in pMET-CDC20 containing strains in maintenance of biorientation was determined in pMET-CDC20 containing strains by the addition of 500μM IAA (to degrade endogenous Stu2-AID protein) for 2h and monitoring biorientation over time.In metaphase, sister kinetochores become bioriented and are transiently stretched apart by opposing microtubule pulling forces –25, whicMATa cells also carrying a tandem array of lacO sequences integrated either proximal to CEN3 or CEN8 [Cells were grown in YPD medium. Exponentially growing  or CEN8  and a La or CEN8 ,39 were S. cerevisiae cells as described below (unless otherwise noted in the text). For kinetochore particles, an α-Flag immunoprecipitation of Dsn1-6His-3Flag was performed  containing protease inhibitors  and phosphatase inhibitors  followed by ultracentrifugation at 98,500 g for 90 min at 4°C. Lysates prepared using the beadbeater were instead centrifuged at 16,100 g for 30 min at 4°C. Dynabeads conjugated with α-Flag or α-V5 antibodies were incubated with extract for 3 h with constant rotation, followed by three washes with BH containing protease inhibitors, phosphatase inhibitors, 2 mM dithiothreitol (DTT) and either 150 mM KCl (kinetochores) or 1 M KCl (Ndc80c and Stu2). Beads were further washed twice with BH containing 150 mM KCl and protease inhibitors. Associated proteins were eluted from the beads by gentle agitation of beads in elution buffer (0.5 mg ml−1 3Flag peptide or 0.5 mg ml−1 3V5 peptide in BH with 150 mM KCl and protease inhibitors) for 30 min at room temperature. To eliminate the co-purification of the Spc105 complex with the native Ndc80c, we immunoprecipitated Spc24-6His-3Flag from cells carrying an spc105-AID allele that were treated with 500 μM auxin for 30 min.Native kinetochore particles, Ndc80c and Stu2 were purified from asynchronously growing ribed in ). To purribed in  or mechaused were a sheep anti-mouse antibody conjugated to horseradish peroxidase (HRP) (GE Biosciences) at a 1:10,000 dilution or a donkey anti-rabbit antibody conjugated to HRP (GE Biosciences) at a 1:10,000 dilution. Antibodies were detected using the SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific).For immunoblot analysis, cell lysates were prepared as described above (Protein biochemistry section) or by pulverizing cells with glass beads in sodium dodecyl sulfate (SDS) buffer using a bead-beater (Biospec Products). Standard procedures for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were followed as described in ,56. A ni−1 3Flag peptide in BH with 150 mM KCl and protease inhibitors) for 30 min at room temperature.To examine the binding of Stu2 to purified kinetochores, Stu2-3V5 and native kinetochores were purified from exponentially growing yeast cells as described above (Protein biochemistry section). Prior to eluting purified kinetochores (with approximately 75–100 ng of Dsn1-Flag bound) from α-Flag dynabeads, beads were incubated with 15 μl (30–90 ng) of purified Stu2-3V5 for 30 min at room temperature with gentile agitation. Beads were then washed twice with BH containing 150 mM KCl and protease inhibitors. Associated proteins were eluted from the beads by gentle agitation of beads in elution buffer . Expression was induced in E. coli using Arctic Express Cells and purified as in [The escribed ,57,58. Sed as in .2, 10 μM taxol) was incubated for 15 minutes at room temperature. Cross-linking was initiated by adding 3.75 μL of 145 mM EDC plus 1.88 μL of 145 mM sulfo-NHS dissolved in BRB80. Cross-linking was allowed to proceed for 30 minutes at room temperature before quenching by addition of 5 μL 1M ammonium bicarbonate plus 0.5 μL 2-mercaptoethanol. Samples were reduced with 10 mM dithiothreitol (DTT) for 30 mins at 37°C followed by alkylation with 15 mM iodoacetamide (IAA) for 30 mins at room temperature. Tryptic digestion was performed at 37°C for 4 hours with shaking at a substrate to trypsin ratio of 18:1. After digestion, samples were acidified with 5 M HCl and stored at -80°C until analysis. Mass spectrometry was performed on a Q-Exactive HF (Thermo Fisher Scientific) in data dependent mode by loading 0.4 μg of sample onto a fused-silica capillary tip column (75-μm i.d.) packed with 30 cm of Reprosil-Pur C18-AQ . Peptides were eluted from the column at 0.25 μl/min using a 120 min acetonitrile gradient. Spectra were converted into mzML using msconvert from ProteoWizard [http://www.kojak-ms.org). Percolator version 2.08 [https://proxl.yeastrc.org/proxl/viewProject.do?project_id=52 along with the raw MS spectra and search parameters used.Cross-linking reactions, mass spectrometry and data analysis were carried out as described previously . A proteeoWizard . ProteineoWizard . Cross-leoWizard  availablion 2.08  was usedion 2.08  at: http-1 κ-casein). 10 nM of purified native Ndc80c was incubated with 6 pM beads for 1 h at 4°C, unbound protein was removed by pelleting the beads , washing with ~200 μl of incubation buffer, pelleting beads again  and resuspending in original volume. For the addition of purified Stu2 to Ndc80c, Ndc80c-decorated beads were prepared as above and purified Stu2-3V5 was added to the microtubule growth buffer (see below) to a final concentration of 2 nM as in [Optical trap-based bead motility assays were performed as in ,10,65. S−1 glucose oxidase, 25 mM glucose, 30 μg ml−1 catalase, 1mM DTT, 1.4–1.5 mg ml−1 purified bovine brain tubulin, and blocking protein (1 mg ml−1 κ-casein). Assays were performed at 23°C. Rupture force experiments were performed as in [-1 until bead detachment. Records of bead position over time were collected and analyzed using custom software  and used to determine the rupture force, which was marked as the maximum force sustained by the attachment during each event. All the individual rupture force values and calculated mean rupture strengths are provided in Dynamic microtubule extensions were grown from coverslip-anchored GMPCPP-stabilized microtubule seeds in a microtubule growth buffer consisting of BRB80, 1mM GTP, 250 μg mled as in ,10,47,54S1 Figstu2-AID cultures expressing various STU2-3V5 alleles from an ectopic locus treated with auxin 30 min prior to harvesting . Stu2-3V5 was purified by α-V5 IP, followed by washes in buffer containing 1.0 M KCl (BH 1.0) then V5 peptide elution. Eluate was run on an SDS-PAGE gel and analyzed by silver stain. Background bands were previously determined by mass spectrometry to be the highly homologous heat shock proteins Ssa1, Ssa2 (70 kDa), and Ssb1, Ssb2 (66 kDa), which are common co-purifying proteins in IPs from yeast lysates [STU2-3V5 alleles , SBY14267; stu2C-termΔ(855–888), SBY14273). C) Protein lysates were prepared from exponentially growing cultures containing Dsn1-6His-3Flag Stu2-AID (SBY13772) treated with 500 μM auxin for 30 min prior to harvesting cells. Kinetochore particles were immobilized by α-Flag IP. Immobilized kinetochore-beads were incubated with 30 ng of Stu2-3V5 variants (purified as in A) for 30 min at room temperature, washed and eluted with Flag peptide. Kinetochore-bound proteins were analyzed by immunoblotting with α-Flag, α-V5, α-Ndc80 and α-Ctf19 antibodies. Note: Stu2ΔSK-rich and Dsn1-6His-3Flag co-migrate on an SDS-PAGE gel. For technical reasons (that are not entirely clear), detection of Dsn1-6His-3Flag was affected by first probing for Stu2ΔSK-rich, however similar Dsn1-6His-3Flag levels were observed for all samples when these sample eluates were run on an SDS-PAGE gel and analyzed by silver stain. D) As in (C) using Stu2-3V5 variants (purified as in B). E) Exponentially growing stu2-AID cultures expressing an ectopic copy of Stu2 , SBY14269) or without an ectopic allele  that also contained Dsn1-6His-3Flag were treated with auxin 30 min prior to harvesting. Protein lysates were subsequently prepared and kinetochore particles were purified by α-Flag immunoprecipitation (IP) and analyzed by immunoblotting. F) Protein lysates were prepared from exponentially growing cultures containing Spc24-6His-3Flag Spc105-AID (SBY14022) treated with 500 μM auxin for 30 min prior to harvesting cells. Ndc80c was immobilized by α-Flag IP. Immobilized Ndc80c-beads were incubated with 30 ng of Stu2-3V5 variants (purified as in A) for 30 min at room temperature, washed and eluted with Flag peptide. Ndc80c-bound proteins were analyzed by immunoblotting with α-Flag, α-V5, and α-Ndc80 antibodies. G) Summary table of kinetochore binding and viability data from A)Protein lysates were prepared from exponentially growing (TIF)Click here for additional data file.S2 Figstu2-AID  and stu2-AID cells expressing various STU2-3V5 alleles from an ectopic locus  were serially diluted (5-fold) and spotted on YPD or 5 μg/ml benomyl plates containing either DMSO or 100 μM auxin. C) Wild-type (SBY3), stu2-AID  and stu2-AID cells expressing various STU2-3V5 alleles from an ectopic locus [STU2WT, SBY13901; stu2C-termΔ(762–888), SBY14263; stu2C-termΔ(855–888), SBY14269) were serially diluted (5-fold) and spotted on YPD or 5 μg/ml benomyl plates containing either DMSO or 100 μM auxin.A) Schematic of Stu2’s domain architecture. B) Wild-type (SBY3), (TIF)Click here for additional data file.S3 FigA) Kinetochore distribution (distance between bi-lobed kinetochore clusters) and spindle length (spindle pole-to-pole distance) was measured for cells described in Figs  & 4C. n (TIF)Click here for additional data file.S4 Figstu2-AID  and stu2-AID cells expressing various STU2-3V5 alleles from an ectopic locus  were serially diluted (5-fold) and spotted on plates containing either DMSO or 500 μM auxin. B) Exponentially growing Stu2-AID cultures with an ectopic copy of STU2  that also contained Dsn1-6His-3Flag were treated with auxin 30 min prior to harvesting. Protein lysates were subsequently prepared and kinetochore particles were purified by α-Flag immunoprecipitation (IP) and analyzed by immunoblotting. C) Exponentially growing stu2-AID pMET-CDC20 cells that contained a fluorescently labeled CEN3 and an ectopically expressed STU2 allele  were arrested in metaphase by the addition of methionine for 3 h. Concurrent with methionine addition, auxin was added to degrade the Stu2-AID protein and the percentage of cells that display two distinct GFP foci (i.e. bioriented CEN3) was quantified. Error bars represent SD of three independent experiments; n = 150–200 cells for each time point. Note: We used the Stu2-AID system to examine the effect of depleting all cellular Stu2 on spindle structure after the cells had already formed a mitotic spindle. For these experiments, we arrested cells in metaphase by depleting Cdc20  and found that the subsequent degradation of the Stu2-AID protein led to a significant decrease in both spindle length and collapse of the bi-lobed kinetochore clusters to a mono-lobed focus . A recent study used an “anchor away” system to address this same question [pMET-CDC20 MTW1-3GFP TUB1-CFP and either the wild-type STU2 (SBY17105) or stu2-AID allele (SBY17106) at the endogenous STU2 locus were arrested in metaphase by depleting Cdc20 (by the addition of methionine to the media). Once cells were arrested in metaphase, auxin was added to induce degradation of the Stu2-AID protein, and the cells were subsequently analyzed for kinetochore distribution and spindle morphology . Representative images for each are shown for pre-auxin and 60 min post-auxin addition. Kinetochore distribution was determined to be bi- or mono-lobed. E) Kinetochore distribution (distance between bi-lobed kinetochore clusters) was measured for cells described in (A). n = 46–63 cells; p values were determined using a two-tailed unpaired t test (n.s. = not significant). F) Spindle length (length of Tub1-CFP) was measured for cells described in (A). n = 38–41 cells; p value was determined using a two-tailed unpaired t test.A) Wild-type (SBY3), question . Essentiquestion ,47. Addiquestion , there i(TIF)Click here for additional data file.S5 Figstu2-AID  and stu2-AID cells expressing various STU2-3V5 alleles from an ectopic locus  or also containing an ipl1-321 allele  or ipl1-321 alone (SBY630) were serially diluted (5-fold) and spotted on YPD or 5 μg/ml benomyl plates containing either DMSO or 500 μM auxin and incubated at 23°C (permissive) or 30°C (semi-permissive).Wild-type (SBY3), (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.