Focal adhesion Kinase (FAK) is a nonreceptor protein tyrosine kinase that is overexpressed in tumors and plays a significant role in tumor survival and metastasis. The purpose of the study is to perform correlation of FAK expression with patient prognostic factors using tissue microarrays (TMA) samples.We analyzed FAK expression by immunohistochemical staining in 196 breast primary tumor samples from stage II-IV patients and in 117 metastatic tissues matched to the primary tumors using TMA that were stained with FAK monoclonal antibody.High FAK expression in primary tumors was associated with a younger age of patients (p = 0.033), lymphovascular invasion (p = 0.001) and with the triple-negative phenotype (p = 0.033). FAK expression in 117 metastatic tissues positively correlated with FAK expression in matched primary tumors by Spearman correlation analysis. In addition, a strong positive correlation was observed between high FAK expression and shorter overall survival and progression free survival in patients with metastatic tumors.The data demonstrate a high potential for FAK as a therapeutic target, especially in triple-negative breast cancer patients with high FAK expression. Focal Adhesion Kinase (FAK) is a 125 kDa non receptor kinase which localizes at the focal adhesion sites and is important for tumor survival, metastasis and angiogenesis . FAK wasIn a previous report evaluating FAK levels in 629 breast tumor samples from patients with mostly Stage I or II breast cancer, 25% had high FAK expression, while approximately 75% were classified as having not high FAK levels (11). Those with high FAK expression were noted to have significant associations with poor prognostic features such as higher mitotic index, higher grade, estrogen receptor (ER) and progesterone receptor (PR) negativity, as well as HER-2 overexpression. In the present report, we analyzed FAK expression in 196 stage II-IV breast cancer patients and 117 metastatic samples available matched to the primary tumors using the tissue microarray (TMA) technique which allowed us to analyze hundreds of tissue samples simultaneously using one paraffin block, reducing variability between samples in the case of regular immunohistochemical staining of many samples of large tumor sections . The purBreast cancer tumor samples were collected and all cases of breast cancer diagnosed between November 1994 and January 2008 treated surgically at Roswell Park Cancer Institute (RPCI), Buffalo, NY. Samples were frozen immediately in liquid nitrogen, and other tissue available from blocks fixed in paraffin. Medical records were reviewed to define clinical and pathological characteristics of all cases with tumor samples used for the study. The Roswell Park Cancer Institute Institutional Review Board (IRB) approved this research and approval is consistent with federal, state and local requirements. The clinical and outcome data were de-identified.TMA’s were constructed from formalin-fixed paraffin tissues with tumors grouped based on hormone receptor and HER-2 status. TMA’s containing breast cancer tumors from 196 breast tumor samples were prepared with each tumor in triplicate. Among these samples, 117 samples included tissue from the primary breast tumor and from matched metastatic tumor tissue. Three one-millimeter tissue cores from formalin-fixed paraffin embedded donor blocks were precisely arrayed into a new recipient paraffin block that included tumor specimens and controls, which included multiple cores of normal tissue from 10 different organs. Each patient had three breast tumor tissue cores on a single TMA slide.2O2 for 10 minutes and washed with 1×PBS with 0.5% Tween 20 solution. Slides were loaded on a DAKO autostainer and blocked with serum-free protein block solution (Dako #X0909) which was applied for 5 minutes and then FAK primary antibody (Millipore #05-537) was applied for one hour. The biotinylated goat anti-mouse IgG  was applied for 30 minutes, followed by the Elite ABC Kit  for 30 minutes, and the DAB chromagen  for 5 minutes. The slides were counterstained with hematoxylin, dehydrated, cleared and cover slipped.The immunohistochemical staining was performed with FAK 4.47 antibody (Millipore #05-537). For antigen retrieval, slides were heated in the microwave for 10 minutes in citrate buffer (pH 6.0), followed by a 15 minute cool period. Endogenous peroxidase was quenched with aqueous 3% HThe scoring was performed by a board-certified pathologist (L.Y.), as described . The scoThe associations between FAK scores and categorical factors were assessed using independent sample permutation t-test. Patients with less than two tissue sample cores were excluded from analysis. The median FAK score was used to dichotomize patients into FAK high and FAK low categories. A Cutoff value of 4.0 that was equal to median FAK score in 196 primary tumors was used for tumor classification and patient dichotomizing into two groups. The same cut-off for patient dichotomizing into two groups was used for 117 matched primary and metastatic tissues. Fisher’s Exact and the Wilcoxon Rank sum test were used for categorical and continuous comparisons between dichotomous FAK categories. The Spearman correlation analysis was performed in 117 matched primary and metastatic tissues. Normal and Kernel distribution was performed on these samples for FAK expression. Kaplan Meier curves and Logrank test were used for Overall Survival (OS) and progression free survival (PFS) data. The p-value less than 0.05, was considered statistically significant.TMA’s contained breast tumor samples from 196 patients with a median age of 56 years, range 27 - 91 years . The tumor intensity levels from 0 to 3 are shown in Figure High FAK expression in primary tumors was associated with younger patient age (the median age was 51 versus 57 with low FAK expression) (p = 0.033) Table . The medOf the overall 196 patients with primary tumor samples, there were 117 who also had matched metastatic tumors samples. Although these 117 primary tumor samples had higher percentages of being ER and PR negative, HER2 positive, triple negative and having LVI compared to the overall group of 196, this did not reach statistical significance (not shown). We performed analyses of FAK expression in the 117 metastatic tumors that were available for 117 matched primary tumors Figure A. The meOverexpression of FAK has an important role in tumorigenesis, tumor survival, and metastasis. This paper highlights a method of accurately measuring FAK expression using TMA technique, allowing direct comparison of FAK expression in multiple tissue samples. The prognostic value of FAK and its associated clinicopathologic characteristics is important for development of new and more effective treatment regimens for breast cancer patients, especially as FAK inhibitors enter clinical trials.A recent report that analyzed FAK expression in 98 breast tumor samples using TMA analysis did not show a significant association with FAK and prognostic indicators in breast cancer . That stOur previous report  demonstrAlthough a significant association was not found between FAK and Her-2 in this paper, there is evidence that FAK and Her-2 signaling pathways are linked in breast cancer development. Activated FAK colocalized with Erb-2/3 receptors at cell protrusions and FAK signaling had an essential function in ErbB-induced invasiveness, metastasis and oncogenesis . A recenin vitro (not shown). Thus, these data suggest that targeting FAK in triple-negative breast cancer patients is a promising approach. It is important to note that FAK has many binding partners and integrates multiple oncogenic survival pathways and sequesters tumor-suppressor pathways [This report for the first time demonstrated high levels of FAK in triple-negative breast cancer patients and correlated with shorter overall survival of the triple-negative group (not shown). This association can be important for the development of therapies for triple-negative breast cancer, suggesting an important role of FAK in triple-negative breast cancer survival signaling. This data is consistent with previous data on increased FAK gene amplification by FISH analysis in triple-negative breast cancer tissues . Since tpathways , 22. TheThe data on association of high FAK expression in primary breast cancer tumors with lymphovascular invasion supports the important role of FAK in epithelial and mesenchymal transition , angiogeThe present simultaneous analysis of FAK expression using tissue microarrays allows for a more comprehensive method of analyzing FAK expression in breast tumor samples. This analysis demonstrated the prognostic value of high FAK expression in breast tumors being associated with more aggressive tumor features such as lymphovascular invasion and triple-negative phenotype. In addition, a high positive correlation between high FAK expression in primary tumors and metastatic tissues was shown, with significantly worse overall and progression free survivals found in patients whose metastatic tumors had high FAK expression. These associations are important for understanding the mechanisms of breast tumorigenesis and for the development of novel biomarkers associated with FAK overexpession and new, effective anticancer therapies.


The development of optimized cultivation strategies for mesenchymal stem cells (MSC) is one major field in the area of tissue engineering and cell based therapies. The physiological tissue niche of stem cells is characterized by a complex set of structural, physical and chemical cues, which create a complex three-dimensional dynamic microenvironment. Mimicking these conditions in vitro is a promising strategy for expansion and differentiation of MSC. This also includes the application of physiological oxygen concentrations (e.g. 3-8 % for most tissues).Multiple cell biological effects of varying oxygen supply have been reported . HoweverMSC derived from adipose tissue were cultivated under hypoxic (5% O2 ) and normoxic (21% O2) conditions in αMEM medium supplemented with 10% human serum. Cell culture supernatants were collected after 4 days incubation on a confluent cell layer, corresponding to approximately 700 000 cells/ml. During sample preparation proteins were precipitated with trichloroaceticacid / acetone (v:v1:8) dissolved in IPG-buffer  and protein concentration was determined by Bradford assay. Reproducibility of sample preparation was tested by repeated preparation of 2D gels. Furthermore, we compared two depletion methods  for reduction of high abundant proteins to find a well reproducible strategy that allows analysis of the lower abundant secreted proteins. The proteomics approach was applied to characterize the MSC secretome after removal of high abundance proteins by affinity enrichment and depletion.The first step was the evaluation of the method for protein precipitation and 2D gel electrophoreses, which revealed highly reproducible sample preparation. However, the visualization and detection of low-abundance proteins remained difficult. Therefore, high-abundance serum proteins had to be significantly removed without losing valuable secreted proteins. An effective depletion of serum proteins and thus reduction of high-abundance proteins was observed resulting in a reduced number of protein bands in 1D and a lower number of spots in 2D PAGE was achieved after utilizing Top12 depletion spin columns. The possibility of co-depletion of low abundant proteins was considered to be very likely. Therefore the combinatorial peptide library was also tested, but showed a lower efficiency for serum protein removal (Figure Serum containing cell culture supernatants could be reproducibly plotted on 2D gels but removal of high abundant proteins was required for the intended secretome analysis. Both tested methods efficiently reduced high abundance serum proteins giving access to the interesting secretome of MSC. Future work will focus on DIGE analysis to point out proteome differences between the normoxic and hypoxic secretome of MSC and differently expressed proteins will be identified by mass spectrometry.


Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis.Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn  In addition, alcohol-fed rats had a 5-fold decrease in capacity to clear inoculated Klebsiella pneumonia from their lungs and this, as well as increased oxidative stress in the lung, could be prevented by dietary zinc supplementation in vitro improved their phagocytic function One such factor is zinc (Zn), the second most abundant metal in the body and important for many physiological and pathological processes, especially in the immune system. Increased prevalence of obstructive lung disorders Little is known about Zn in macrophage efferocytosis of apoptotic cells and its effects in chronic lung diseases including COPD. It is also not known how macrophages regulate their levels of Zn including their responses to Zn deficiency. Critical to the normal cellular and tissue homeostasis of Zn is the role of specific membrane Zn transporter proteins. Based on their sequence homology and structural properties, Zn transporters have been assigned to two families: SLC39A  and SLC30A  If efferocytosis is Zn-dependent, this might explain our earlier observations of a marked accumulation of apoptotic epithelial cells in the lungs of mice with combined airway inflammation and nutritional Zn deprivation as well as reduced efferocytosis in the airway and lungs of patients with COPD and smoke exposed mice Human ethics approval was granted by the institutional review board for human studies at the Royal Adelaide Hospital (RAH), which approved the protocols; all procedures were undertaken after written fully informed consent from the subjects. BAL was collected from controls  and COPD subjects who were further sub-classified according to smoking history Zn sulphate (cell culture grade), ovalbumin, TPEN, PMA and DAPI and ß-mercaptoethanol were from Sigma-Aldrich . Other reagents were obtained as described below or in the text.BAL were centrifuged and supernatants frozen at –80°C until assayed for Zn. BAL Zn was assayed by a Zinquin-based fluorometric technique 6) were differentiated into macrophages in 6-well plates in 2 mL of RPMI 1640 medium containing 0.1 µM of PMA over 48 hr at 37°C. Differentiation was assessed by adherence to plastic. and determination of plasma membrane CD11B expression using flow cytometry as described A differentiated THP-1 human monocytic cell line  was applied as these cells behave very much like native monocyte-derived macrophages and have been used extensively to study mechanisms involved in human macrophage differentiation phycoerythrin (PE) for 30 min on ice in the dark. Fluorescence was recorded using a FACS Canto II (BD). Cells were gated relative to an unstained control and the percentage of cells that stained positive was determined. The following controls were used to determine compensation between FITC and PE channels: unstained cells to determine appropriate FSC and SSC PMT settings, and cells loaded with FluoZin-3 AM ester alone and cells stained with CD11b PE alone to determine compensation between both channels. BAL Zn levels were not considered an issue as macrophages were washed twice before the assay and cells further washed with FACS buffer before staining with CD11b PE.AM, THP-1 cells or PMA treated THP-1 cells (scraped from culture dish) were loaded with FluoZin-3 AM ester  in culture medium at 37°C for 30 min, washed with 1 mL of FACS buffer  and resuspended in 100 µL of FACS buffer. Cells were loaded with FluoZin-3 and incubated with anti-CD11b conjugated with Cells were immediately placed in RLT lysis buffer . Total RNA was isolated as per kit instructions . cDNA was prepared from 2 µg of total RNA using a high capacity cDNA reverse transcription kit . Quantitative PCR was performed using 33 ng of cDNA as template in 10 µL reactions using pre-made TaqMan primer/probes (Applied Biosystems) with TaqMan gene expression master mix and run on an Applied Biosystems ViiA7 Real Time PCR system. Hypoxanthine guanine phosphoribosyl transferase (HPRT) and 18S rRNA were used as endogenous controls. Data was analysed by the delta-delta Ct method, and expressed as fold change .Immunofluorescence of Zn transporters in THP-1 cells or formalin-fixed lung biopsies was performed as described for inflammasome proteins Efferocytosis of apoptotic epithelial cells by macrophages was performed as we have previously described using apoptotic human or mouse lung epithelial cells at a ratio of 1:10 macrophages:apoptotic cells as phagocytic targets −/− mice were provided by Jim Geiser and Glen Andrews  −/− null mice and ZIP1+/+ wild-type controls. Mice were maintained in the the Institute of Medical and Veterinary Science  animal care SPF facility (22°C with a 12 h light-dark cycle). Access to water and a non-purified diet  were provided ad libitum.The Animal Care and Ethics Committees of the IMVS and the University of Adelaide approved animal procedures (Permits M-37-08 and M-67-09) and procedures conformed to ARRIVE guidelines, NIH guidelines  and to National Health and Medical Research Guidelines to promote the wellbeing of animals used for scientific purposes: The assessment and alleviation of pain and distress in research animals (2008). In our experiments, all efforts were made to minimize suffering of the mice. Prior to obtaining peritoneal macrophages, mice were anaesthetized with isofluorane and humanely killed by cervical dislocation. ZIP1ip with 1 mL of 4% Brewer’s Thioglycollate broth . After 2 days, mice were sacrificed (see above) prior to obtaining peritoneal macrophages. Cells were centrifuged at 800 RPM and seeded (3×105 cells) into wells of 24-well plates in RPMI+GlutaMax™ with 10% foetal bovine serum and 1% Penicillin:Streptomycin. Plates were incubated for 2 hrs to allow macrophages to adhere to the plate. Non-adherent cells were removed and adherent macrophages resuspended in RPMI+Glutamax™ medium. Phagocytosis of apoptotic murine epithelial cells by mouse peritoneal macrophages was performed as previously described For good yield of macrophages we obtained peritoneal macrophages. Female mice (average size 22 g), randomly selected from null or wild-type controls, were injected Data was analysed using SPSS software and Mann-Whitney or 1-way ANOVA with post hoc for nonparametric analyses. Correlations were performed using Spearman’s rank test. Analyses were performed using SPSS software. P values<0.05 were considered significant.Zn levels were determined in BAL from 20 controls, 17 healthy smokers, 20 current and 19 ex-smoker COPD subjects. Mean BAL Zn concentration was significantly decreased in COPD patients who were current smokers, compared to controls p<0.05  and THP-1 macrophages (2.9-fold (p<0.005). TPEN significantly decreased mRNA levels of ZIP4 in both monocytes  and macrophages . There were similar decreases in ZIP7 but these only reached significance in macrophages ; in monocytes, p value was 0.065. ZIP11 was significantly decreased in monocytes  but not in macrophages. There were no effects of Zn depletion by TPEN on expression of the other nine ZIP transporters . ZIP12 was not expressed in either cell type.ZnT1  and ZnT6  in both monocytes and macrophages. ZnT5 mRNA was also significantly decreased by TPEN treatment, 0.6-fold (p<0.001) and 0.3-fold (p<0.05), respectively, in the two cell types. ZnT3 was significantly decreased by TPEN in macrophages  but not in monocytes while ZnT7 was significantly decreased in monocytes  but not in macrophages. TPEN treatment had no significant effect on ZnT2,4, 8, 9 or 10 mRNA levels  in macrophages of ZIP1−/− mice. Fluorescence was inhibited by pre-treatment of macrophages for 4 h with 16 µM Zn chelator TPEN (not shown), confirming the Zn dependence of the assay.+/+ macrophages, the efferocytic index was 20.9% . When the isolated macrophages were depleted of Zn by treatment with 16 µM TPEN for 4 h, the efferocytic index was decreased by 25% in the macrophages from the ZIP1+/+ mice but there was no further decrease in efferocytosis in the macrophages from the ZIP1−/− mice (not shown).Next we assayed efferocytosis of apoptotic epithelial cells by the peritoneal macrophages as per our published method for AM Recent studies have highlighted the importance of maintaining sufficient lung Zn levels Having shown a Zn deficiency in BAL from COPD patients, we then focussed on the potential role of the zinc deficiency in the defective AM efferocytosis ability that we have previously reported in COPD. Both BAL Zn and AM Zn were positively correlated with the capacity of AM to efferocytose apoptotic bronchial epithelial cells. The link between macrophage efferocytosis ability and Zn was further shown in a murine model: when isolated macrophages were depleted of Zn by treatment with TPEN, the macrophage efferocytic index was decreased by 25%. Importantly this effect was abolished by knocking out of ZIP1, furthermore confirming that Zn was essentially involved. The exact mechanisms by which Zn deficiency impairs efferocytosis are unclear, but current data suggest they are complex. Thus, a pathway of CD44-mediated activation of CR3 which is implicated in phagocytosis and efferocytosis has been shown to be dependent on divalent cations including Zn but not Ca What causes the low lung Zn levels in smokers with COPD is not precisely known although it is possible that cigarette smoke-induced inflammation and lung injury plays a role. In animal models of airway inflammation or acute lung injury, there were abnormalities in airway epithelial or AM Zn transporter expression, including ZIP1, without systemic Zn deficiency A gap in the literature is the paucity of information on Zn homeostasis in the macrophage and the mechanisms by which Zn influences critical functions of the macrophage such as efferocytosis. We have therefore begun to characterize those Zn transporters important for monocyte/macrophage Zn homeostasis. Cellular Zn homeostasis involves, in part, the coordinate action of two families of Zn transporters with opposing actions. ZIP transporters increase cytosolic Zn by importing extracellular Zn or by releasing organelle stores of Zn into the cytosol. ZnT transporters export Zn or compartmentalize it into organelles. In particular, there is a need to identify the plasma membrane Zn importers responsible for bringing Zn into the monocytes and macrophages.in vitro, by exposing to the membrane-permeable Zn chelator TPEN, there was no change in ZIP1 expression indicating that this Zn transporter is unlikely to be involved in maintaining cytosolic Zn levels during Zn deficiency. However, mRNA for ZIP-2 gene increased significantly 28-fold and 3-fold, respectively. This suggests that ZIP-2 is an important Zn transporter for these cells under Zn-deprived conditions. It is interesting that the increase was almost 10-fold greater for monocytes than macrophages. This may indicate that macrophages have more substantial reserves of zinc. Of the three ZIP transporters that decreased at the mRNA level with Zn depletion, ZIP4 is a plasma membrane Zn importer while ZIP5 and ZIP7 are transporters involved in efflux of Zn into the cytosol from ER, Golgi and other organelles. ZIP5 and ZIP7 proteins may, therefore, have a role in maintaining cytosolic Zn levels during Zn deficiency.One strategy is to look for up-regulation of Zn transporters in response to induced Zn deficiency. Of interest, when the THP-1 monocytes or macrophages were rendered Zn-deficient None of the ZnT type Zn transporters increased in gene expression in Zn-depleted monocytes or macrophages, while many decreased in expression. Notably, there were large decreases in ZnT1, a transporter that is primarily involved in efflux of Zn out of the cell. Decrease in ZnT1 expression is likely involved in maintaining intracellular Zn when extracellular Zn is limited. There were also decreases in ZnT5-7. Since these are involved in sequestration of Zn into organelles, their decrease might also help to conserve cytosolic Zn.in vivo lung fluids. The latter is difficult to measure since the process of obtaining lung fluid lavages introduces a dilution factor.Joshi and colleagues have previously identified ZIP1 as being important for lung Zn homeostasis and AM phagocytosis of bacteria −/− mice. It was necessary to use peritoneal macrophages for this, as there were limitations on the number of alveolar macrophages that we could isolate. Pregnant ZIP1−/− mice on a low Zn diet were reported to have increased abnormalities in their embryos compared to those of ZIP1+/+ mice on the same diet, but no difference was observed when the null and wildtype mice were on Zn replete diets In this manuscript we also show that both intracellular Zn and efferocytic capacities of macrophages were decreased in ZIP1The studies described here begin to address the basic science questions underlying the role of Zn in AM, including the first characterization of the subcellular distribution of Zn and Zn transporters in these cells as well as the role that altered Zn metabolism plays in efferocytosis. We have identified candidate ZIP and ZnT type Zn transporters which may function to maintain cytosolic Zn in Zn deficiency. We have also identified two Zn transporters ZIP1 and ZIP2 which are candidate plasma membrane influx Zn transporters for lung macrophages. ZIP1 was unresponsive to a zinc deficiency signal but was important for normal efferocytosis function. ZIP2 may play a special role in maintaining macrophage Zn in the presence of low extracellular Zn concentrations and this might be important in lung inflammation and injury. Its role, if any, in efferocytosis needs further investigation. Future studies demonstrating that addition of Zn to either monocytes or macrophages increases efferocytosis, and assessing Zn transporter mRNA in human AM will be of importance for better understanding the role of Zn in macrophage dysfunction in COPD. Elucidation of the mechanisms leading to defective efferocytosis in lung injury and disease will provide a foundation for our long term goals of better clinical management of COPD.Checklist S1ARRIVE Guidelines Checklist.(PDF)Click here for additional data file.


Investigators from Johns Hopkins University reported a cohort of 27 patients with incidentally-noted rolandic spikes (RS) on EEG. Investigators from Johns Hopkins University reported a cohort of 27 patients with incidentally-noted rolandic spikes (RS) on EEG. The cohort included children aged 3-9, mostly male, with 19% comorbid ADHD and 19% familial epilepsy. Of 27 patients, 7 developed seizures, including 3 with benign rolandic epilepsy (BRE) and 1 with febrile seizures and learning difficulties. The patient with febrile seizures and 6 additional patients were offered levetiracetam. Of 7 patients given levetiracetam, five reportedly ‘improved’; the remaining 2 were not affected and lost to follow-up. [COMMENTARY. While BRE is defined by characteristic seizures and an EEG with RS, the significance of RS found incidentally is more enigmatic. To begin, the true incidence of this finding may depend on both phenotype and type of EEG. Though the incidence of asymptomatic RS is reported as 2–3% of children aged 5–12 [As a diagnosis of BRE confers an additional risk of language and memory deficits, it is tempting to speculate that a child with a similarly abnormal EEG and comparable learning impairments without seizures would benefit from anticonvulsant therapy. As the authors previously described improved speech following treatment of BRE with levetiracetam , the samThis study hints at a larger question, which is whether RS are truly ‘incidental,’ or rather a biomarker for mild cerebral dysfunction. Indeed, it is debated whether characteristics of RS rather than seizure burden itself have a clearer prognostic role in terms of cognitive impairment . A recenMcNally and Kossoff  suggest


Well-characterized cell lines represent useful scientific tools to study the pathophysiology of human disease. Chronic rhinosinusitis (CRS) is a very common condition, though the number of CRS cell lines is limited, as are data showing how closely they resemble primary cells.Searches for available human cell lines were performed using the American Type Culture Collection (ATCC) and European Collection of Cell Cultures (ECACC). Identified cells were cultured and characterized with tinctorial and immunohistochemical staining and ELISA to assess their response to common, disease-relevant inflammatory stimuli. Carefully phenotyped CRS patients were recruited with informed consent. Primary nasal epithelial cell (PNEC) brushings were harvested, cultured, and compared to the available cell lines.Searches identified 1 relevant CRS sino-nasal cell line, RPMI 2650. Cultured PNECs showed strong expression of epithelial markers while being negative for mesenchymal markers. However, RPMI 2650 cells show an atypical mixed epithelial/mesenchymal phenotype. When stimulated by pro-inflammatory ligands, PNECs responded in a dose-dependent manner, whereas RPMI 2650 cells showed limited response.The number and availability of cell lines to study the pathophysiology of CRS greatly underrepresent the disease burden. Additionally, the sole commercially available cell line appears to have a different phenotype and behavior to primary patient-derived cells. The development of further reproducible cell lines would be beneficial in our understanding of CRS. Despite this lack of efficacy, medical treatment has demonstrated little change during an era that has seen significant innovation in surgical management. However effective surgery may now be in the short term, a relatively high disease recurrence rate persists.5 The lack of medical treatment options is however not surprising given our poor understanding of the pathogenesis of CRS. A concerted international effort utilizing a variety of methodological approaches, including clinical, in vivo, and in vitro cellular studies, is being pursued to improve our knowledge of chronic rhinosinusits. Within our CRS group we are currently focused on the association between the sinonasal epithelium and the sub-epithelial fibroblast layer and their roles in the persistent CRS inflammatory environment. To investigate this further, we have employed both patient-derived primary cultures of human sinonasal cells isolated from patients undergoing sinus operations and also sought to use immortalized cell lines for their inherent reproducibility. Cell lines are widely used to interrogate disease mechanisms throughout the body and act as rapid, effective laboratory models for hypothesis testing without the cellular heterogeneity or interindividual variability of patient-derived samples. To our surprise, the number of commercially available cell lines to study CRS is very limited and may not be representative of the parent tissue from within the sinonasal cavity.Chronic rhinosinusitis (CRS) is one of the most common human conditions, with an estimated prevalence of 12.5%,nasal, sinus, and human. Searches yielded 1 relevant cell line, RPMI 2650, which was purchased and grown in standard laboratory cell culture conditions. A vial of 2 × 106 cells was cultured as per the suppliers instructions (ATCC) in Sigma EMEM  with 1% non-essential amino acids (NEAA) , plus 100 iu/ml penicillin/streptomycin , 50 ml fetal calf serum (FCS) , and 2 mM L-Glutamine . Cells were supplied at P26 and were amplified in T75 tissue culture flasks to generate sufficient cell numbers for our experiments. Cells were grown as submerged monolayer cultures in tissue culture flasks for stimulation experiments and on 13 mm coverslips for imaging.Searches for commercially available cell lines were performed via the online catalogs of the American Type Culture Collection (ATCC) and European Collection of Cell Cultures (ECACC) using the search terms 6 were invited to participate in the study, with appropriate ethical and research governance approvals . Primary nasal epithelial cells (PNECs) were harvested from the middle meatus by gentle passage with a cytology brush. Isolated PNECs were cultured in Lonza BEGM cell culture media  plus 100 iu/ml penicillin/streptomycin with media changed every 2 days until cells reached confluence.Participants undergoing elective operations for chronic rhinosinusitis according to the EPOS 2012 international consensus documentoC overnight , followed by fluorophore-conjugated secondary antibody (goat anti-rabbit TRITC conjugated Sigma T6778) at 1:100 dilution in 5% bovine serum albumin PBS-0.2% Tween 20 incubated for 90 minutes in the dark. Negative controls were performed with secondary only antibodies and matched IgG isotype negative controls to identify if there were nonspecific binding or background autofluorescence. Coverslips were mounted on slides with DAPI Vectashield , and images were captured with a Leica LSM 510 confocal microscope.Cells grown on 13 mm coverslips were fixed in 4% paraformaldehyde for 10 minutes and then washed with 1× phosphate buffered saline (PBS). Glycine (100 mM) was added to quench any remaining fixative. Cells were then permeabilized in 0.1% Triton-X100  in 1× PBS for 30 minutes and washed twice in PBS-0.2% Tween 20  and once further in 1× PBS. Cells were then either stained with H&E to assess cellular morphology or using immunocytochemical techniques for epithelial and mesenchymal markers. Primary antibodies were incubated at 4Monolayers of PNECs and RPMI 2650 cells approaching confluence were stimulated with CRS disease-relevant pro-inflammatory ligands: TNF-α 1 ng/ml, 5 ng/ml, 10 ng/ml ; LPS 0.1 µg/ml, 1 µg/ml, 10 µg/ml ; Poly I:C 50 µg/ml, a synthetic viral analogue ; and TGF-β 5ng/ml  for 3 and 24 hours to determine if the cells were able to mount appropriate inflammatory responses. Untreated control cells without inflammatory ligands were cultured in parallel. Standard curves were performed as internal controls to ensure reproducibility between experiments. All cells were treated with identical conditions in triplicate repeats from the same batch of inflammatory ligands. Following stimulation, the conditioned media were harvested and the inflammatory response measured by the amount of IL-8 released into the culture media. Quantification was by ELISA for IL-8 protein as per manufacturer instructions .Primary nasal epithelial cells were successfully grown as monolayers from participants undergoing surgery for CRS. H&E staining of these cells demonstrated a typical confluent cell culture monolayer . ConfocaRPMI 2650 cells were additionally shown to produce very little inflammatory response as measured by IL-8 release to a range of ligands at 2 different time points. When compared to control untreated cells, tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS), poly I:C, and transforming growth factor-β (TGF-β) stimulated cells did not produce the expected inflammatory IL-8 response at either 3 or 24 hours  and 3D. 7 yet antibiotics are not always effective. The position internationally of increasing antibiotic resistance and the slow progress in developing new antimicrobial agents8 illustrate further the unmet need for better CRS treatments. More modern, mechanistically focused anti-inflammatory and biological medications have been used in other chronic inflammatory conditions since the end of the twentieth century such as anti TNF-α therapy in rheumatoid arthritis9 and inflammatory bowel diseases.10 The prolonged use of corticosteroid medications is also not without risk. Although the majority of CRS steroid preparations are given topically rather than systemically, systemic absorption and suppression of the hypothalamo-pituitary axis does occur.11 The development of novel anti-inflammatory treatments for rheumatoid arthritis and inflammatory bowel diseases has followed from a greater understanding of the disease pathophysiology.12 Although such conditions are by no means completely characterized, advances in their detailed knowledge has translated into new therapies for patients. In many ways, CRS appears to show some similar pro-inflammatory and fibrotic disease characteristics,13 though these remain very early findings. Hopefully a more detailed understanding of the CRS disease mechanisms will lead to similar treatment advances for CRS patients.Chronic rhinosinusitis is a very common condition that consumes significant health resources worldwide. There are vast direct health expenditures from the medical and surgical treatments for this condition. The mainstay of medical treatment has not changed from corticosteroids and antibiotics over many decades, at a time that has seen significant advances in the medical management of similar inflammatory conditions. Sinusitis remains the fifth most common indication for an antibiotic to be prescribed,14 A search of the online catalog for ECACC, for example, offers researchers investigating pathology for similar inflammatory conditions in the lower airways with 97 possible cell lines available.15 Here we have identified only 1 commercially available cell line to study CRS, RPMI 2650. Unlike many other human conditions, there has been a paucity of well-validated reliable cellular models to study CRS. The only readily available commercial sinonasal cell line RPMI 2650 is often used in sinonasal studies,1619 yet there is little published data about its relationship to sinonasal cells and its validity as a cellular model.Cell lines represent very useful laboratory tools to study the pathophysiology of human disease. Their use is widespread throughout all organ systems, which is reflected in the number and variety of available cell lines. The ECACC alone at present holds in excess of 40 000 cell lines available for study since its establishment in 1984.21 A major function of cells of the sinonasal cavity is in mucociliary clearance, which due to the nature of submerged culture could not be assessed here.There are of course some shortcomings from the experimental approach that we have utilized to compare patient-derived PNECs with the sinonasal cell line RPMI 2650. First, we have grown both cell types in submerged culture rather than at an air-liquid interface to force differentiation. We chose this approach as in earlier air-liquid culture experiments we were not able to create a differentiated ciliated epithelium with RPMI 2650 cells  in agreement with previous work investigating the use of the cell line in nasal drug delivery studies.2225 While this is initially disappointing, it is perhaps not surprising when we investigate the origin of the cell line. It was derived from an anaplastic squamous cell carcinoma of the nasal septum in a 52-year-old male.26 Tumor cells were isolated from his metastatic pleural effusion and shown to grow as adherent nasal epithelial cells. The cell line has been proven to have a similar diploid karyotype to normal nasal epithelial cells.27 It has also been shown to have some similarity in terms of the expressed surface cytokeratins,28 and it produces a mucoid material that is visible on the cells apical surface.26 To date, the cell line has only been validated as a model to study the regulation of TGF-β biology in house dust mite–related allergic rhinitis.29 The neoplastic source of these cells perhaps explains the mixed epithelial and mesenchymal phenotype and growth pattern in cell culture. As a result, presently we would not recommend the use of RPMI 2650 as an in vitro cellular model for the sinonasal epithelium in CRS studies. In preference, we would advocate the use of primary, patient-derived nasal epithelial cells until such time that better validated sinonasal cell lines are established. The unmet disease burden from CRS would no doubt benefit from better CRS cell lines to aid our progress in understanding and treating this condition.From our investigations we have shown that the sinonasal cell line RPMI 2650 is significantly different from patient-derived PNECs in terms of its cellular morphology, surface marker expression, and biological response to CRS disease-relevant inflammatory ligands such as TNF-α.


The degradation and recycling of cellular components is essential for cell growth and survival. Here we show how selective and non-selective lysosomal protein degradation pathways cooperate to ensure cell survival upon nutrient limitation. A quantitative analysis of starvation-induced proteome remodeling in yeast reveals comprehensive changes already in the first three hours. In this period, many different integral plasma membrane proteins undergo endocytosis and degradation in vacuoles via the multivesicular body (MVB) pathway. Their degradation becomes essential to maintain critical amino acids levels that uphold protein synthesis early during starvation. This promotes cellular adaptation, including the de novo synthesis of vacuolar hydrolases to boost the vacuolar catabolic activity. This order of events primes vacuoles for the efficient degradation of bulk cytoplasm via autophagy. Hence, a catabolic cascade including the coordinated action of the MVB pathway and autophagy is essential to enter quiescence to survive extended periods of nutrient limitation.DOI:http://dx.doi.org/10.7554/eLife.07736.001 Yeast and other organisms have evolved to survive extended periods of starvation by digesting their own proteins and other cell materials and thereby recycle them into new proteins and structures. One way in which these cell materials can be destroyed is by a process called autophagy. A membrane forms around the cell material to isolate it from the rest of the cell. In yeast, the resulting structure fuses with a cell compartment called the vacuole, which contains enzymes that break down the cargo into smaller molecules that can be re-used by the cell.When cells experience starvation, autophagy is not very selective in what it destroys and so it is tightly controlled to avoid damaging important structures in healthy cells. Alongside autophagy, specific proteins in the membrane surrounding a yeast cell can be targeted for destruction by another process called the MVB pathway. Certain membrane proteins are tagged with a small protein called ubiquitin, which leads them to being selectively incorporated into cell compartments called MVBs that then go on to fuse with the vacuole. However, it is not clear how the MVB pathway and autophagy may cooperate to enable the cell to survive periods of starvation.Here, Müller et al. monitored the changes in the proteins present in yeast cells during a period of starvation. The experiments show that many different membrane proteins in the yeast cells were destroyed via the MVB pathway within three hours of the removal of their food source. This was essential to allow the cells to carry on producing new proteins at this early stage in starvation. These new proteins included the enzymes found in vacuoles, which increased the ability of the cells to break down the proteins and other cell materials that were transported there via autophagy.These findings show how the MVB pathway and autophagy are co-ordinated to allow cells to survive periods of starvation. The next challenge is to work out how the MVB pathway is regulated at the molecular level in response to fluctuations in nutrient availability.DOI:http://dx.doi.org/10.7554/eLife.07736.002 Mis-folded proteins in the endoplasmic reticulum (ER) are also ubiquitinated, extracted by the ER-associated protein degradation system (ERAD) and degraded at 26S proteasomes in the cytoplasm  non-selectively transports bulk cytoplasm into lysosomes. Therefore the induction of autophagy is tightly controlled: under normal growth conditions autophagy operates on a basal level because it is suppressed by signaling from the target of rapamycin complex 1 (TORC1) . In respendosomal sorting complex required for transport)-dependent manner via the MVB pathway . This comprehensive and selective remodeling of the plasma membrane appeared to be completed within 3–4 hr of starvation. Autophagy was also immediately activated upon starvation and remained active throughout starvation. Surprisingly, early during starvation the selective degradation of membrane proteins via the MVB pathway was mainly responsible to maintain critical levels of free intracellular amino acids that were sufficient to uphold protein synthesis and promote the corresponding adaptation of the proteome. Most notably this included the de novo synthesis of vacuolar hydrolases, which boosted the proteolytic activity of vacuoles to support the efficient degradation of autophagic cargo. The continuous delivery and degradation of autophagic cargo further enhanced intracellular amino acid recycling and was ultimately essential to restore intracellular amino acid pools of cells during extended starvation. These findings reveal an unexpected role for the MVB pathway in maintaining intracellular amino acid homeostasis and thereby promoting the up-regulation of vacuolar hydrolases early during starvation, which is tightly coordinated with autophagy. This catabolic cascade is ultimately required to allow starving cells to complete their cell division cycle and enter a quiescent state for survival.To understand how the MVB pathway, autophagy and proteasomal degradation cooperate during nutrient limitation, we first analyzed the starvation-induced degradation of model proteins in yeast.atg8∆) mutant (vps4∆) mutant, the MVB pathway was blocked and Mup1-GFP was not delivered into the vacuole but instead accumulated on the class E compartment and at the plasma membrane  . Under r) mutant . In an Emembrane .10.7554/cytoplasm to vacuole targeting (cvt)-vesicles. Therefore Atg8 is degraded together with autophagic cargo inside vacuoles. To be able to compare the degradation of GFP-Atg8 to Mup1-GFP, 10 times more lysate of cells expressing GFP-Atg8 was subjected to western blot analysis  and non-selective (Fba1) autophagic cargoes. Growing yeast cells contain about 200,000 ribosomes that occupy up to 30–40% of the cytoplasmic volume . Upon stanalysis . Small aanalysis  as obseranalysis . Previouanalysis . Hence, utophagy . Pho8∆60arvation . These rarvation .Additionally, we investigated how cytoplasmic proteins were degraded at proteasomes upon starvation. Therefore, we employed a ubiquitin-GFP (Ub-GFP) fusion protein, which is an established reporter for proteasomal activity . It is dOverall, these findings indicate that starvation triggered protein degradation by different selective and non-selective degradation pathways: the constitutive protein degradation via the proteasome was active from the onset of starvation and was previously suggested to play a key role upon acute nutrient restriction . Our resWhile protein degradation at 26S proteasomes provides an immediate amino acid pool for protein synthesis already within minutes of acute starvation  and autovps4∆) or autophagy (atg8∆) mutants as well as double mutants  by liquid chromatography (atg8∆)  , but slie levels .10.7554/1 hr after starvation in synthetic medium without amino acids and ammonium salts, the total free amino acid pool decreased to similar levels in WT cells and all mutant strains . In WT cvps4∆ mutants, autophagy mutants (atg8∆) and WT cells. However, after 2 hr the overall amino acid levels were lower in vps4∆ mutants compared to WT cells and autophagy mutants. The levels of 14 individual amino acids were lower in vps4∆ mutants when compared to WT cells or autophagy mutants. Moreover the amino acid levels failed to recover during extended starvation   .vps4∆ and atg8∆ single mutants  were lower after 2 hr of starvation when compared to WT cells or autophagy mutants (atg8∆), as observed in the auxotrophic strains , protein synthesis declined faster when compared to WT cells or autophagy mutants, which seems consistent with the more rapid decline of intracellular amino acids  the MVB pathway is essential to maintain a critical pool of free amino acids for protein synthesis early during starvation. (ii) In the absence of the MVB pathway autophagy can only partially uphold amino acids levels and protein synthesis (iii) The MVB pathway and autophagy cooperate to maintain intracellular amino acids during starvation, potentially in a consecutive manner.vps4∆ and atg8∆ mutants , TORC1 signaling appeared to be reduced under rich growth conditions, but not completely switched off  or vacuolar acidification (vma4∆) pHluorin-Atg8 was not quenched  and pHluorin-Atg8 positive vesicular structures were detected inside their vacuoles, suggesting that autophagic bodies were not efficiently lysed , the fluorescence of pHluorin-Atg8 was quenched in the vacuoles similar to WT cells, but occasionally few perivacuolar pHluorin-Atg8 positive structures were observed . Overall, it seemed that autophagic bodies were efficiently lysed in acidified vacuoles of vps4∆ mutants  cells changed within the first 3 hr of starvation using stable isotope labeling with amino acids in cell culture (SILAC)  analysis (B) (Next we determined in detail how the MVB pathway would contribute to the starvation program of yeast. Therefore we measured how the proteome of WT ( (SILAC) . WT cellanalysis . In thisysis (B) . 101 proTo identify cellular components and biological processes that were regulated early during starvation, we determined the enrichment of specific Gene Ontology (GO) terms  Figure . Amino a+-ATPase, Pma1, were not dramatically altered. The majority of Pma1 remained at the cell surface and only a small portion was delivered into the vacuole , nucleobases (Fur4), amino acids  and ammonium (Ato3) but also the G-protein coupled receptor Ste2 were down-regulated  and degr vacuole . Moreove vacuole .vps4∆)  than in the vps4∆ mutants (green curve)  . To compnditions . Correlaresponse . Howeverarvation . To addr13C615N2-lysine) and vps4∆ mutants growing under rich conditions . GO analysis of these 135 proteins identified three processes that were primarily differentially regulated between vps4∆ mutants and WT cells upon starvation , Pho8 was not matured (vps4∆ mutants the Pep4-dependent maturation to mPho8 was not impaired under rich growth conditions or starvation (atg8∆ and atg5∆ mutants), sPho8 activity still increased 3–5 fold during the first 3 hr of starvation similar to WT cells, but failed to increase further upon extended starvation  or ESCRT-II (vps36∆) mutants were also severely impaired in increasing the catabolic activity of their vacuoles during starvation  recycles less efficiently between endosomes and the golgi . Hence, pep mutants  in proteins . As expeng Vps10 . Yet, Vpng Vps10  and thusnditions . Despitenditions  and protnditions  in vps4∆vps4∆ mutants but also vps4∆ mutants overexpressing Vps10 could not efficiently up-regulate the de novo synthesis of vacuolar hydrolases in time. Therefore the catabolic activity of vacuoles remained low during starvation in vps4∆ mutants overexpressing Vps10  grew slower when compared to WT cells upon starvation and failed to complete cell division and thus could not enter G1/G0 arrest , ESCRT-II (vps36∆), ESCRT-III (snf7∆) and vps4∆ mutants failed to complete their final round of cell division and hence could not enter a G1/G0 arrest    also fai0 arrest . In this0 arrest . Hence tvps4∆ and atg8∆ mutants gradually lost viability over time, and the vps4∆, atg8∆ double mutants showed severe synthetic survival defects  for Rsp5, the major ubiquitin ligase required for endocytosis in yeast. Thereby TORC1 signaling orchestrates the selective remodeling of the plasma membrane proteome under different growth conditions and additionally could increase protein turnover via the MVB pathway . Our resOur results further demonstrate that the ESCRT-dependent degradation of these membrane proteins in vacuoles is essential to maintain a critical pool of free amino acids for protein synthesis. This enables the de novo synthesis of vacuolar hydrolases that are required to boost the catabolic activity of vacuoles . Thereby1/G0 quiescent state and thereby ensures cell survival 2SO4. For growth on agar plates, yeast cells were diluted to OD600nm = 0.05 and spotted in 10× dilutions on YPD or YNB plates. Protein synthesis was inhibited by treatment with cycloheximide  and proteasomal activity was blocked by MG132 .All experiments were performed with SEY6210 yeast strains, except for Pho8∆60-expressing strains, strains used in Genetic modifications were done by PCR and/or homologous recombination using standard techniques. Where applicable, tags were introduced at the C-terminus to preserve the endogenous promoter sequences. Plasmid-expressed genes including their endogenous promoters were amplified from yeast genomic DNA into centromeric vectors (pRS series). All constructs were analyzed by DNA-sequencing and transformed into yeast cells using standard techniques. Yeast strains and plasmids used in this study are listed in 13C615N2-L-Lysine or unlabeled 12C614N2-L-Lysine. For starvation experiments, logarithmically growing cells from unlabeled medium were washed twice with starvation medium and incubated for 3 hr in starvation medium. Cells were harvested by centrifugation. Labeled and unlabeled cells were mixed in a 1:1 ratio according to their OD600nm. Cell were mechanically disrupted with glass beads at 4°C in PBS containing protease inhibitors . Cell lysates were cleared from intact cells by centrifugation . Cleared cell lysates were TCA-precipitated and washed twice with acetone. Proteins were dissolved in water, lyophilized (in order to remove traces of organic solvents) and solubilized in 100 mM NH4HCO3 (pH 8.0). Solubilization was attained by sonication for 3 × 40 s. Resolubilized proteins were reduced with dithiothreitol, alkylated with iodoacetamide, and in-solution digested with LysC (1:75 wt/wt) in 100 mM NH4HCO3 (pH 8.0). The resulting peptides were fractionated by reverse phase chromatography using an EC 250/4.6 Nucleosil 120-3 µm C18 column  and resulting fractions were analyzed by capillary electrophoresis-mass spectrometry (g) and proteins precipitated in 10% trichloroacetic acid (TCA). Precipitates were washed twice with acetone and resolubilized in SDS/urea sample buffer. SDS-PAGE in Tris-HCl gradient gels  was used to reduce sample complexity. The SDS gel was stained with SimplyBlue Safe Stain , cut into 21 slices per lane and proteins in each slice were in-gel digested by Trypsin  or LysC . Resulting peptides were analyzed by liquid chromatography-mass spectrometry (LC -MS) using an UltiMate 3000 nano-HPLC system (Dionex/Thermo Scientific) coupled to an LTQ Orbitrap XL (Thermo Scientific). MS settings were as described  and kept at mid-log phase for 24 hr. Subsequently, cells were washed with their corresponding labeling medium and then used to inoculate 500 ml of labeling medium. Cells were kept in log phase for 10 generations with either, heavy trometry . Peptidetrometry  to an LTescribed .vps4∆ mutants under rich conditions, we only used proteins with at least three heavy and light peptide counts. Each protein had at least one unique peptide and MS scans in at least two biological replicates. No additional filtering criteria were applied for the WT and vps4∆ data under starvation conditions. Differential regulation was estimated using the significance B (Perseus v1.0.2.13).MS data were analyzed using MaxQuant (Version 1.2.2.5) . The yeahttp://www.geneontology.org/). The enriched GO term at the highest level in the GO hierarchy was selected and its child terms were excluded. In cases when the parent process has a higher p-value, the child term was chosen. A hypergeometric test was used to estimate if the mapped GO term is significantly enriched with the selected proteins. The null hypothesis is that the selected proteins are randomly sampled from all yeast proteins. The resulting p-values were corrected with the Benjamini–Hochberg method. All adjusted p-values below 0.05 were reported. We also calculate the ratio of the observed (dataset frequency) vs the expected number of proteins (genome frequency) associated with the GO term, referred to as enrichment over genome . The significance level was 0.05. The two-sided test resulted with 135 significant differences between WT and vps4∆ protein ratios.The density plots show the computed density estimates of the protein ratios quantified in all the three experiments, using Gaussian kernel (R software environment). The protein ratio distributions were compared pairwise using the Kolmogorov–Smirnov test, under the null hypothesis that the two tested groups are samples from the same distribution, that is, have the same median, variability and distribution shape. The significant protein changes were excluded. Additionally, paired Student's t test was used to test for mean difference in the log2-transformed protein ratios. All p-values were adjusted using the Benjamini–Hochberg test. To individually compare the ratios of each protein in WT and ESCRT mutant cells during starvation, we analyzed the ratio of ratios. For each protein quantified under the two starvation conditions, we took the ratio (fold change) of WT and To explore and visualize the data we used the R language for statistical computing and graphics. To calculate the linear correlation between protein ratios we used Pearson correlation. All p-values below or equal to 0.05 were reported.A Zeiss Axio Imager M1 equipped with standard fluorescent filters and a SPOT Xplorer CCD camera was used. VisiView software was used for image-acquisition. Brightness and contrast were linearly adjusted. For vacuole staining  growing To prepare whole cell lysates, yeast cells were pelleted, resuspended in ice-cold water with 10% trichloroacetic acid (TCA), incubated on ice for at least 30 min and washed twice with acetone. The precipitate was resolubilized in boiling buffer , solubilized with glass beads and boiled at 95°C. Urea sample buffer  was added and the cleared cell lysate was separated by SDS-PAGE. Alternatively, proteins were extracted by alkaline extraction . For anaWhole cell protein extracts were prepared by TCA extraction or alkaline lysis, separated by SDS-PAGE and transferred to PVDF membranes. Antibodies used in this study include: α-Flag , α-GFP , α-Pgk , α-ALP , α-HA , α-Pep12 , α-CPY/Prc1 , α-Ape1 and α-Atg8 . The α-AThe Pho8∆60 assay was performed as described . Soluble35S]-Met/Cys labeling mix  for 5 min at 30°C and stopped with excess L-Met (5 mM) and 75 µg/ml cycloheximide. Cell extracts were analyzed by autoradiography of SDS-PAGE  or quantified by liquid scintillation counting (Beckmann Coulter LS6500).Mid-log cells grown in rich YNB medium (not containing methionine and cysteine) or starved cells were labeled with 15–30 µCi eLife. Your full submission has been evaluated by Vivek Malhotra (Senior editor), a Reviewing editor, and one of the original reviewers.Thank you for sending your revised work entitled “The sequential action of the MVB pathway and autophagy ensures cell survival during starvation” for consideration at The authors have responded to reviewers concerns by including new data, but major concerns still remain that preclude the publication of the paper at this time. The concerns of the reviewers follow.1) The data presented on GFP-Atg8n in new 2) The authors show increased amino acid levels in wild-type cells during starvation. This is puzzling and inconsistent with a previous report , where the amino acid concentration was dramatically reduced even in wild-type cells and more profoundly in autophagy mutant cells. The authors do not discuss this apparent discrepancy. This should be addressed experimentally.3) The data in  1) The data showing the sequential activation of the MVB pathway and autophagy during starvation is not strong. Autophagy can be immediately initiated after starvation. It is recommended to use GFP-Atg8 as another autophagy marker in addition to Rpl25-GFP in, which may show faster kinetics. Indeed, free GFP is clearly detected at 2h after starvation inWe never intended to argue that autophagy is only activated after membrane proteins were degraded via the MVB pathway. Therefore we have also slightly modified our model in A) The delivery of cytoplasm by the autophagic machinery gradually increases during extended starvation. This is a well-accepted concept and supported using a Pho8 60 assay, a sensitive method to measure autophagy early during starvation  Noda, T. This isB) Our new results demonstrate that at the onset of starvation, GFP-Atg8 is approximately 10–20 times less abundant and almost not detectable when directly compared to Mup1-GFP . Both coC) These findings are also consistent with the idea that higher Atg8 protein levels allow to grow larger phagophores that would engulf increasing amounts of bulk cytoplasm .D) This induction of the autophagic machinery is also reflected by our quantitative proteomic data. The protein levels of Atg8 (3.7 fold increase) and other autophagy core components such as Atg1 (3.3 fold increase), Atg3 (1.5 fold increase), Atg7 (1.4 fold increase) and Atg15 (2.5 fold induction) increase during starvation (E) The protein levels of the selective autophagic cargo Ape1 strongly increase early during starvation . SimilarF) All of this is consistent with our results presented in G) Finally our analysis of amino acids levels during starvation demonstrates that the MVB pathway provides amino acids prior to autophagy  bridge the gap in amino acid recycling before an efficient autophagic response develops.2) It is also essential to show that the amino acid pool is indeed reduced in vps4∆ cells during early starvation. It was previously shown that the amino acid levels are reduced in autophagy mutants compared to wild-type cells at as early as 3h after starvation . A similar experiment should be performed using MVB, autophagy, and double mutant cells, which will provide important information on relative contributions of these two pathways, in terms of both time and quantity, to the amino acid pool during starvation.vps4), autophagy mutants (atg8) and the double mutant   . Under r4, atg8) .3) Although the phenotype of vps4∆ cells suggests that starvation-induced cargo degradation in the MVBs is important to supply amino acids for synthesis of vacuolar enzymes, it is still possible that, in vps4∆ cells, delivery of hydrolases to the vacuole is defective, which affects the vacuolar function independently of amino acid supply. These two possibilities are not clearly distinguished. In particular, the data shown insuggest that there may be a defect in the vacuolar function even in growing conditions or during a very short starvation period in vps4∆ cells. This needs to be further clarified to support the authors' main conclusion.pep4∆, prb1∆, prc1∆ mutants but not in ESCRT mutants; The graph in While there is certainly a subtle maturation defect of Ape1 in ESCRT mutants, not only pApe1 but also mApe1 protein levels are increased under rich conditions, so that overall Ape1 activity is compensated for . This isvps4) and hence restored the catabolic activity of vacuoles to >80% of WT levels  The data inshould be quantified because, as the authors mention in Discussion, several papers have reported that the ESCRT machinery is important for a late step of autophagy (i.e. autolysosome formation).pep4∆, prb1∆, prc1∆ and vma4∆ mutant cells.Quantification of the localization of pHluorin-Atg8 in >120 cells demonstrates that pHluorin-Atg8 fluorescence was quenched in the vacuoles of most WT cells and ESCRT (>90%) mutants . In cont5) The authors mention that selected membrane proteins are degraded during starvation. The methionine permease Mup1 is certainly degraded very efficiently . Do they also have examples of membrane proteins that are unaffected? Can they discuss how the cell can distinguish which proteins are degraded selectively?The general amino acid permease Gap1 is strongly induced during starvation and transported to the cell surface while other amino acid permeases such as Mup1 or Can1 are endocytosed and degraded  of specific adaptors for the HECT-type ubiquitin ligase RSP5. We have included this possibility in the Discussion. In addition we have new data that suggest that distinct ubiquitination sites on Mup1 are required for starvation- vs. ligand-induced endocytosis, but this would go beyond the scope of this study.6) In the same experiment, do the authors use Met/Cys free medium when they label cells under nutrient rich conditions? Otherwise, relative activity of 35S-Met/Cys would be different between the rich and starved conditions.We have used Met/Cys free medium for all our experiments and described the media in the Material and methods section.7) The ESCRT-mediated MVB formation would be important for degradation of not only membrane proteins but also cytosolic proteins that are engulfed inside intraluminal vesicles. This is a reminiscence of microautophagy. Indeed, it was reported by Cuervo's lab that MBV formation contributes to the degradation of cytosolic proteins . This possibility should also be discussed.“Interestingly, the ESCRT machinery has recently been described to contribute to selective micro-autophagy on late endosomes in mammalian cells  We thoroughly analyzed the onset of autophagy using GFP-Atg8 in relation to other selective and non-selective autophagic cargoes as well as MVB cargo and we discuss their temporal relationships. Our results are consistent with the essential role of autophagy for amino acid recycling later during starvation (see point b).b) We have improved the amino acid analysis. In WT cells, amino acid levels decrease to about 50% during the first 2 hours of starvation and fully recover in an autophagy-dependent manner at around 4 hours of starvation. Most importantly, our analysis also shows that the MVB pathway is essential to maintain free intracellular amino acid pools in the first 2 hours of starvation.vps4∆ mutants that overexpress Vps10. The results are consistent with the idea that selective membrane protein degradation via the MVB pathway, rather than the mere sorting of vacuolar hydrolases, is essential to maintain sufficient free intracellular amino acids to uphold protein synthesis for proteome remodeling early during starvation.c) We have measured the amino acid pool, protein synthesis and autophagic activity in To our mind these results further strengthen our model: early during starvation, the degradation of membrane proteins via the MVB pathway is required to maintain a critical intracellular amino acid pool for the synthesis of new proteins, including vacuolar hydrolases to boost the catabolic activity of vacuoles. This primes vacuoles for the efficient degradation of bulk cellular material via autophagy, which is essential to enter a stable G1/G0-arrested, quiescent state. Thus, a coordinated action of the MVB pathway and autophagy is key to survive extended periods of nutrient limitation.1) The data presented on GFP-Atg8n in newtogether withshows a relative rapid induction in autophagy activity during starvation. Thus, it is inappropriate to suggest “the degradation of cytoplasmic contents via autophagy passed through an initial lag phase and peaked later during starvation, when compared to membrane protein degradation via the MVB pathway ”. The time at which degradation of a protein peaks depends not only on the degradation activity, but also its rate of synthesis and total expression level. The authors have used selective cargos to monitor MVB pathway, it is also important to use selective cargos (e.g. GFP-Atg8) to monitor autophagy and discuss their temporal relationship and relative contributions. The response of the authors to this concern is not acceptable, for example, the expression levels of Atgs are not direct indicators of autophagic activity.’ with ‘coordinated action’ throughout the manuscript and also in the title.We thank the reviewers for critically pointing out that our statement ‘autophagy passed through a lag phase ’ was mis-leading, especially since we never intended to argue that the MVB pathway and autophagy are sequentially activated. Apparently, our intended message, namely that the MVB pathway has an earlier impact on the cellular amino acid levels when compared to autophagy, was not clearly put into words. To avoid this, we have now replaced ‘sequential actionIn addition, we have performed the recommended experiments and used GFP-Atg8 as an additional marker to determine the vacuolar delivery of all selective and non-selective autophagosomes as well as cvt-vesicles . Small aIn summary our results suggest that both autophagy and starvation induced-endocytosis are simultaneously activated early during starvation (probably by inactivation of TORC1). Autophagy continuously delivers ever-increasing amounts of cytoplasm into vacuoles and remains active during extended periods of starvation, whereas starvation-induced degradation of membrane proteins via the MVB pathway appears to be completed relatively early (within the first 4 hours of starvation). Our analysis of intracellular amino acid recycling supports this idea and demonstrates that autophagy is essential for amino acid recycling later during starvation, whereas the MVB pathway appears to be more important to maintain intracellular amino acid levels early during starvation (see reply to concern 2 and 2) The authors show increased amino acid levels in wild-type cells during starvation. This is puzzling and inconsistent with a previous report , where the amino acid concentration was dramatically reduced even in wild-type cells and more profoundly in autophagy mutant cells. The authors do not discuss this apparent discrepancy. This should be addressed experimentally.vps4) or autophagy (atg8) mutants or double mutants  by HPLC. 1 hour after starvation, the total free amino acid pool decreased to similar levels in WT cells and all mutant strains  The data inreveals a clear defect in processing of mPho8 into sPho8 even under growing conditions. This indicates a defect in vacuolar hydrolytic activity. Indeed, the authors describe a partial sorting defects of vacuolar hydrolases in ESCRT mutants and they have tried to rescue them by over expression of Vps10 . However, this was performed only for data shown inand. Vps10 rescue experiments should be included in other experiments that measure the amino acid pool , protein synthesis , and autophagic activity . These are more relevant to the potential function of the MVB pathway as an amino acid generator. It is also unclear why over expression of Vps10 cannot rescue the pho8 activity under starvation conditions. Taken together, it is likely that defects in protein turnover observed in ESCRT mutants are due to a combination of defects in sorting of vacuolar enzymes and generation of amino acids.vps4 mutants rescued the partial sorting defects for vacuolar hydrolases but not membrane protein degradation. Consistently, Vps10 over-expression almost completely restored the catabolic activity of vacuoles under rich conditions (vps4 mutants overexpressing Vps10 still failed to maintain intracellular amino acid levels and protein synthesis during starvation (vps4 mutants over-expressing Vps10, similar to vps4 mutants alone, could not efficiently up-regulate the de novo synthesis of vacuolar hydrolases and thereby boost the catabolic activity of vacuoles (pep mutants, unlike other endo-lysosomal trafficking complexes such as HOPS, CORVET or retromer (Vps10 over-expression in nditions . Our newvacuoles , which ivacuoles . In consvacuoles . In addivacuoles . Similarvacuoles . In the vacuoles . Most liHence the striking cellular defects of ESCRT mutants during starvation can be mostly attributed to their block in membrane protein degradation, rather than the mere and rather partial sorting defects for vacuolar hydrolases.


Streptomyces . Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, α, β and γ-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (Anthostomella sp.), two Streptomyces and one Bacillus bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales , while the majority of actinomycetes belonged to the genus  Insects are one of the most diverse groups of organisms in nature, with the ability to feed on almost any terrestrial food source. Among insects, Coleopterans, commonly called beetles, are the most diverse order. Of the approximate 300,000 reported beetle species, about 10% can be found in Costa Rica, where they inhabit an array of ecosystems with various conditions of temperature and precipitation across the whole country 9–1011 cells/ml The insect gut represents a confined environment comprising a diverse and complex microbiota including resident and transient bacteria, actinomycetes, archaea and fungi, and it has a microbial density estimated between 10Beetles acquire their microbiota mainly from the materials on which they feed; many of these microorganisms could have adapted to the gut environment and over time, developed mutually beneficial relationships with their host From a biotechnology perspective, studying the cultivable microbiota colonizing beetle guts in tropical ecosystems constitutes an important, yet unexplored tool for the discovery of novel enzymes for processes including industrial production of enzymes through fermentations, lignocellulose hydrolysis, acetogenesis or methane and nitrogen fixation, all of them readily scaled in bioreactors In order to identify microorganisms adaptable to conditions employed in biotechnological applications we explored the cultivable microbiota of beetles from Costa Rican protected ecosystems. After a survey of gut microbial diversity from several beetle genera that thrive on decomposing wood as food source, we focused on microbial isolates from Passalid beetles for in depth analyses of their plant cell wall degrading capabilities. We chose these beetles as a model because their sub-social behavior suggests that beneficial microbial transmission between adults and larvae may play an important role in their ability to degrade the recalcitrant polymers that constitute their main food source. This is supported by decades old research showing that Passalid larvae cannot survive without their parents We collected insect specimens during five field trips to the following National Parks in Costa Rica: Carara, Puntarenas Province ; Caño Island Biological Reserve, 20 km from the Costa Rican Pacific coast, Puntarenas Province ; Arenal Volcano in Guanacaste province  and two in Braulio Carrillo, Quebrada González sector, Heredia Province . Additionally, we collected samples at the private reserve of the Costa Rican National Biodiversity Institute (INBio) in Limon province . Sampling was performed under the collection permits of the Technical Office of CONAGEBIO (Ministry of Environment and Telecommunications), Costa Rica. We sampled the interior of decomposing logs and collected larvae and adults from six different beetle families: Passalidae, Staphylinidae, Scarabaeidae, Cerambycidae, Tenebrionidae and Elateridae. All insects were transported to the laboratory at room temperature inside vented polyethylene containers and surrounded by the woody material in which they were collected . Beetle taxonomic identification was carried out by experts at INBio.Beetles were dissected in sterile conditions, using scissors and forceps to extract the digestive tract. Gut tissues were placed in 1.5 ml tubes containing phosphate buffered saline solution, mixed in a vortex for 10 seconds and sonicated for 30 seconds. Then the homogenized gut tissues were diluted 10,000 fold with PBS solution and plated to Potato Dextrose Agar (PDA) Genomic DNA was obtained from pure microbial cultures. DNA isolations from fungi were performed by employing Nucleospin Plant kit II , placing approximately 200 mg of macerated mycelium in 1.5 ml microtubes containing 400 µl of lysis buffer PL1. The tissue was disrupted mechanically in a vortex after adding 30 µl of proteinase K. Tubes were incubated at 60°C for one hour and DNA was extracted according to manufacturer's specifications. Actinomycetes were treated similarly except that DNA isolation was performed by employing 50 mg of each strain and the Qiagen DNeasy Blood & Tissue Kit, according to manufacturer's instructions. Isolation of genomic DNA from eubacteria was performed using the same approach described for actinomycetes but only for strains whose 16S rRNA gene could not be amplified by direct colony PCR.PCR was performed to amplify the Internal Transcribed Spacer (ITS) region of the 18S rRNA gene from isolated fungal strains using genomic DNA as template. We used ITS-1 as forward primer and ITS-4 as reverse primer http://www.dnastar.com/t-products-seqman-ngen.aspx) and MEGA 5 All sequences were analyzed and manually edited using SeqMan Pro-DNA STAR  agar MEA-Remazol Brilliant Blue R (MEA-RBBR) A standard medium for microbial culturing was employed in these assays including, PDA for fungi , LBA for bacteria  and ISP2 for actinomycetes . Incubations were performed at 25–30°C until growth was evident (two weeks maximum in case of slow growing actinomycetes). To determine lignin oxidation activity, a colloidal preparation of 1 mM 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid  was poured over the standard media plates and incubated at 25°C for 2 days. Green coloration of the substrate demonstrated microbial laccase activity Results for the three assays were scored from “0” to “2”, with “0” meaning no activity, “1” meaning low activity  and “2” representing high activity (complete media discoloration or strong coloration of ABTS). Isolates with positive scores were selected for further semi-qualitative evaluation of their enzymatic activities related to plant cell wall degradation, including glucanase, xylanase and cellobiase activity.Selected isolates were inoculated again on the previously mentioned standard media and grown under the described conditions until healthy colonies were visible. Substrate solutions of Glucanase , Xylanase  and Cellobiase  0,6% w/v dissolved in 50 mM NaOAc; pH 5 were sprayed on each colony until they were completely covered. The plates were incubated at room temperature for 8 hours and monitored every 15 minutes. Substrate hydrolysis and the consequent liberation of 4-nitrophenyl phosphate (4NP) was shown by yellow coloration; therefore, isolates were scored qualitatively as “0” if no coloration occurred and “3”, “2” or “1” if substrate coloration took place in 1, 2 or more hours, respectively.http://www.chibi.ubc.ca/matrix2png/bin/matrix2png.cgihttp://www.chibi.ubc.ca/matrix2png/bin/matrix2png.cgi), which was then used to generate a hydrolytic profile of the isolates. Quantitative determination of enzyme activities was not performed.These results were incorporated into a matrix to build a heat map . Comparisons between groups were performed employing the MANOVA method with a significance criteria of <0.05.Fungi and bacteria were isolated from guts of 52 adults and 71 larvae spanning six families of Costa Rican beetles, including Passalidae , Scarabaeidae , Cerambycidae , Elateridae , Tenebrionidae  and Staphylinidae . Streptomyces with 86% of the isolates. Other detected genera were Microbacterium (3%), Mycobacterium (3%), Nocardia (2%) and Micrococcus (2%). Phylogenetic analyses of isolated actinomycetes showed two major clades, one for Streptomycetaeae and the other for Nocardiaceae , followed by Enterococcus (11%). Most Gram negative isolates clade in two other major groups including, β-proteobacteria and γ-proteobacteria, with only one Rhizobium isolate belonging to the α-proteobacteria. The most commonly found β-proteobacteria family was Burkholderiaceae (13% of the isolates), within this group the most commonly isolated genera were Burkholderia (14%). Finally, among γ-proteobacteria, the Enterobacteriaceae family is the most represented with 25% of all isolates, followed by Pseudomonadaceae with 6.2%. Bacteria in the genera Citrobacter, Klebsiella and Pseudomonas were the most frequently isolated . The most commonly identified genus among the actinomycetes was rdiaceae . Althougrdiaceae . Phylogerdiaceae  revealedisolated .Trichoderma  and Nectria (7%). These isolates make up the largest cluster on the fungal phylogenetic tree , including 88 fungi, 74 eubacteria and 44 actinomycetes in hand , we nextAnthostomella sp.), 4506K (Hypocrea sp.) and 4504W . There was also positive activity detected in the eubacteria 4504F (Bacillus sp.) and 4505D (Pseudomonas sp.) and the actinomycetes 4506AD, 4512 L and 4516K . Cellobiase activity was observed only in 4505L (Anthostomella sp.) and in the eubacteria 4504F (Bacillus sp.) and 4505D (Pseudomonas sp.). Hypomyces sp. (4506K), Hypocrea sp. (4505I) and the Russulales fungal isolate 4509H also showed lacasse activity.The group of 23 isolates that exhibited positive activity on two or more assays on the screening phase was then subjected to further analysis for cellobiase, xylanase and glucanase activities . Eight iAnthostomella) and the actinomycete isolate 4506AD (Streptomyces sp.) had the highest scores both on the initial and the follow up assays, and thus showed the highest potential for the degradation of plant cell wall polymers (Streptomyces isolate (4512L), a Hypocrea isolate (4506K), and a Bacillus isolate (4504F). In general, Streptomyces was the taxonomic group that displayed higher cellulase, lignin peroxidase and xylanase activities. Isolates belonging to the bacterial genera Pseudomonas and Burkholderia, known for their high metabolic versatility, also showed important cellulolytic activity. The heat map in The fungal isolate 4505L . Furthermore, no significant differences were observed in the number and diversity of microorganisms obtained from larvae and adults, instead, both life stages seem to share the most frequently isolated groups. These results may strengthen the hypothesis that some of the microorganisms that are consistently isolated may have a close relationship with their insect hosts Streptomyces (86% of isolates), but despite the lack of genetic diversity shown in their 16S rDNA sequences, they did show higher phenotypic diversity reflected in several different colony morphologies. Streptomyces are well known for their capacity to degrade cellulose and lignin, both resistant to degradation by most microorganisms Even though we isolated 241 actinomycetes in this study, we cannot conclude whether they are dominant members of the gut cultivable microbiota due to the selective media employed for their isolation. However, we can certainly affirm that this type of microorganism can be recovered consistently from the wood-feeding beetle gut. The dominant genus isolated in this study was Cordyceps and Metarhizium may be an example of these phenomena, because their presence could be a result of ingestion within the diet rather than an active infection inside the beetle gut. However, the existence of an ecological relationship between the host and any of these fungi or other microbial symbionts cannot be discarded, specially because several of these microbes are known for their ability to secrete carbohydrate-degrading enzymes, and thus they might confer an adaptative advantage to cope with a continuous flux of complex polymeric materials, allowing the host to gain biomass in a niche filled with recalcitrant materials Trichoderma, which together with Nectria, also detected in the beetle gut, have been characterized as a bioenergy relevant microbial genome Trichoderma is also well known as a biocontrol agent, reducing fungal diseases in crops Trichoderma and the genera Metarhizium, Metacordyceps and Paecilomyces, among others; several isolates from these genera have been involved in relevant biotechnological applications in agriculture, industry, environmental remediation and medicine Fungi are also well known members of insect gut microbial communities, including beetles Anthostomella, family Xylariaceae, had the overal highest score in these tests. Some species of Anthostomella have been described as plant pathogens but little information is available about possible ecological relationships with beetles and other insects. Besides Anthostomella, other members of the Hypocreales group also showed significant scores in the experiment. In addition, microorganisms of the genera Streptomyces, Bacillus and Pseudomonas presented positive activities on these assays. These results strengthen the hypothesis that Passalid beetles depend on their gut microbiota to break down cellulose and obtain energy, liberating nutrients in the intestinal environment that can benefit both the microbiota and the beetle itself.To accomplish this goal, we decided to focus on isolates from Passalid beetles and test their ability to degrade plant cell walls by endogenous enzymes. We chose these insects because our preliminary results indicated a rich and diverse microbiota, and their unusual cooperative and care-giving behavior among Coleopterans of sharing a decomposing log with their brood from birth through adulthood The nutrient-acquisition and biomass-degrading capabilities of the microbiota associated with insects such as termites Figure S1Distribution of isolates from the gut of Costa Rican wood-feeding beetles. A: Distribution of the total number of isolates from all beetles collected according to their family. B: Percentage of isolates obtained by number of specimens sampled in each family .(TIF)Click here for additional data file.Table S1Distribution of bacterial and fungal isolates according to their taxonomy.(DOCX)Click here for additional data file.Table S2Morphological description and Blast analysis of isolates.(DOCX)Click here for additional data file.