Occupational exposures to chromium (Cr), which can have adverse effects on immune function, have not yet been extensively investigated. Hexavalent chromium (Cr (VI)), used in mineral tanning processes, poses a threat to the health of workers in the leather tanning industry.The aim of the present study was to evaluate the effects of long-term Cr exposure on the physical health and immunological parameters of male tannery workers in Bangladesh compared with a control group.A health examination was performed with tannery workers (N=195) and control subjects (N=125) by physicians, demographic data were recorded in questionnaires and peripheral blood samples were collected. Serum Cr levels were analyzed by an atomic absorption spectrophotometer (AAS), immunoglobulin (Ig)G, IgA, and complement components C3 and C4 were determined by nephelometry, IgE was measured by sandwich enzyme-linked immunosorbent assay and complement function was assayed by bactericidal activity.2), was not significantly different from the controls (22.7±3.2 kg/m2). The mean serum level of Cr in 30 long-term exposed tannery workers (26.97±21.11 μg/dL) was significantly higher than that of 30 randomly selected control subjects (7.38±6.81 μg/dL). The tannery workers had rough skin, rashes, itchy and decolorized skin, allergic diseases and respiratory illness, and had significantly lower levels of serum IgG, IgA, C3 and C4, but significantly higher levels of IgE than the controls. IgG, IgA and C3 levels were all inversely associated with Cr, while IgG, IgE and bactericidal activity showed an inverse correlation with duration of exposure.The mean duration of work exposure for the tanners was 9.4±7.1 years. Their body mass index (21.8±3.0 kg/mThe results of the present study suggest that chronic exposure to Cr is associated with impaired immune function in male tannery workers.ObtainedThe present study was approved by the Ethical Review Committee of the faculty of Biological Sciences, University of Dhaka, Bangladesh.The authors declare no competing financial interests. Chromium (Cr) is an essential nutrient required for normal energy metabolism. Insufficient dietary Cr has been linked to maturity-onset diabetes and cardiovascular diseases. However, Cr exposure can be hazardous to human health. Among its many valence states, hexavalent chromium (Cr (VI)) is a major health concern. Epidemiological studies have reported a high incidence of lung cancer among workers occupationally exposed to Cr (VI) by inhalation.7Tanning processes involve several chemicals and some of them are known to be potentially carcinogenic with adverse health effects. The tanning process involves basic trivalent chromium (Cr(III)) sulfate binding to collagen fibers which stabilizes the leather. Tannery workers are under constant risk of adverse health effects due to excessive exposure to nitrosamines, chromate pigments, soft sodium sulphide, lime, ammonium sulphate, benzidine-based direct dyestuffs, formaldehyde, leather dust and aromatic organic solvents.Several studies have investigated the possible effects of Cr on lymphocytes. A study in Guiyu, China, found DNA (deoxyribonucleic acid) damage in lymphocytes in the umbilical cord blood of neonates.13BMIBody mass indexCComplement componentcfuColony forming unitsELISAEnzyme-linked immunosorbent assayIgImmunoglobulinILInterleukinPBSPhosphate buffered salineA study investigating metal-induced modulation of nitric oxide production by murine macrophages in vitro found that Cr, copper and lead moderately suppressed inducible nitric oxide synthase while other heavy metals like cadmium, mercury, or cobalt did not produce any significant effect. These observations suggest that environmental toxicant metals may directly modify the nitric oxide synthase enzyme or cofactor activity and have suppressive effects on phagocytic function of macrophages to exercise defense mechanisms.17Immunoglobulin (Ig)G is the principal immunoglobulin of adaptive immune response that gives protection against invading pathogens and their toxins, while IgE functions against intestinal helminths and IgA gives mucosal immunity. Complement components C3 and C4 play important roles in bacterial killing, inflammation and cellular activation. These immunological parameters may be affected by chronic exposures to chemicals used in mineral tanning processes. Previous studies evaluated serum IgE levels and investigated their relationship with a wide range of allergic diseases, including allergic contact dermatitis, allergic rhinitis, allergic urticaria and non-specific allergy in tannery workers.Tannery workers were enrolled in the present study in Hazaribagh, the largest industrial tanning area in Bangladesh, located in the southwestern part of the capital city of Dhaka. This cross-sectional study was conducted with 320 subjects: 195 tannery workers exposed to Cr and 125 unexposed control subjects. Inclusion criteria included at least two years of work in tanneries in sections with exposures to Cr. As there were very few female workers who met the inclusion criteria of two years of tannery work with Cr exposure, and in order to make equal comparisons, all of the control subjects were male.The control subjects generally worked in the same region, in offices, shops, banks and student dormitories. Exclusion criteria included those suffering from impaired renal and liver functions and any other chronic conditions. Simple random and availability sampling was applied to collect samples.The present study was approved by the Ethical Review Committee of the faculty of Biological Sciences, University of Dhaka, Bangladesh. Each individual was informed about the objectives and significance of the study. Only full consenting volunteers were enrolled in the study. The guidelines of the Ethical Review Committee were followed during physical health examination, participant interviews and peripheral blood sample collection.Supplemental Material). The health of participants was assessed by a physician.Demographic information, including age, body weight, height, blood pressure, working hours per day, duration of work exposure (years) and type of work etc., was recorded by questionnaire , rashes, and headaches caused by ingestion of certain foods. Scaly, superficial infections on skin and nails by fungus and bacteria were also noted. The health information of the study subjects was carefully recorded on questionnaires which were subsequently analyzed for demographic data.Samples were analyzed for the measurement of Cr by an atomic absorption spectrophotometer (AAS) (Varian 240) that used acetylene flame to vaporize the acid-treated samples. The AAS utilized a hollow cathode lamp specific for Cr detection in gaseous state and detected the total Cr content in the samples. Only 30 serum samples from the workers involved in activities in various sections of the tanneries (at least 3 from each section) and samples of 30 randomly picked control subjects were investigated for this parameter due to a scarcity of serum, as one milliliter of each sample was necessary for this analysis.https://www.healthcare.siemens.com).All reagents used for the determination of immunoglobulins IgG and IgA, and complement components C3 and C4 were purchased from Siemens Healthcare Diagnostics, GmbH, Germany  technique. The ELISA plate and the reagents were obtained from DRG Diagnostics, Germany. The sandwich ELISA technique utilized one anti-IgE antibody in the solid phase and another one in the antibody-enzyme conjugate solution. The test sample was added to the anti-IgE coated microtiter plates and the content of IgE in the serum samples was determined by standard procedure.Escherichia coli DH5α cells, according to the procedure described elsewhere.E. coli cells, and the remaining viable cells were serially diluted with PBS to 1:10 000 and then 20 μL of each was spread on 3 agar plates, incubated for 16–18 hours, and the number of colonies formed was counted. For the negative control, 20 μL of PBS was added to the bacterial cell suspension, incubated and then serially diluted with PBS to 1:50 000 and spread on agar plates. The number of colonies formed was recorded.Serum complement function was assessed by bactericidal activity on Data were analyzed using the Statistical Package for Social Sciences . Student's t-test and nonparametric tests, including chi-squared and Mann-Whitney U tests (as appropriate) were used for comparison of different parameters between two groups (tannery workers and unexposed group). Spearman's rho bivariate tests were used for correlation analyses between different parameters of the study populations. GraphPad Prism 7.05  was used to analyze bactericidal activity data. The results were considered significant when p was ≤ 0.05.2 and their working hours ranged from 7 to 16 hours per day, including overtime. The age of the control subjects varied from 17 to 62 years and their BMI ranged from 16.2 to 31.5 kg/m2. About 15% of the tannery workers were underweight (BMI <18.5 kg/m2) compared to only 3% of the control group .Demographic data on the study subjects were collected in questionnaires and the findings were analyzed and presented in Figure 1). All of these health complaints were significantly higher in the tanners than the controls subjects (χ2 test). A small percentage of the workers had other health problems like body pain, general weakness, diabetes, hypertension, arthritis, goiter, bronchospasm, conjunctivitis and frequent fever. While some of the workers had more than one health problem, a number of tannery workers reported no symptoms (28%). There were very few cases of hypertension, diabetes and allergic diseases in the control group.The physical health examination data of the study subjects are also shown in The tannery workers enrolled in this study came from the wet blue section (working moist Cr (III) oxide-tanned blue leather, 21%), drum section , technical section , and chemical section . Some workers were involved with removing hair from hides, cutting, washing, drying and finishing (20%). Additionally, some workers involved in unloading, storing and weighing chemicals (8%), and packing (4%) were included in this study because of their close contacts with possible exposure.Thirty serum samples of the tannery workers involved in different types of work .The mean levels of serum IgG and IgA in the tannery workers, 11.67 ± 3.58 g/L and 1.50 ± 0.62 g/L, respectively, were significantly lower than the corresponding values in the control subjects (Table 3).In the present study, the serum IgE level of the control subjects varied from 3–750 IU/mL, and the level varied from 4–950 IU/mL in the tannery workers. Statistical analysis showed that the IgE level in the tannery workers was significantly higher (p< 0.01) than in the control subjects (Table 3).About 40% of the tannery workers had a serum complement C3 level within the normal range , whereas 60% were below the normal level. The mean levels of serum complements C3 and C4 in the tannery workers were significantly lower than those in the control subjects (6 colony forming units (cfu)/mL with a mean value of 1437 × 106 cfu/mL. Upon treatment with serum complements, the number of colonies formed varied from 26 - 242 × 106 cfu/mL, with a mean value of 110 × 106 cfu/mL for the control subjects, while the number of colonies varied from 60 - 324 × 106 cfu/mL with a mean value of 133 × 106 cfu/mL for the tannery workers. Compared to PBS, complements of both the tannery workers and control subjects exhibited significant bactericidal activities (p<0.001 for both). The bactericidal activity of serum complements was calculated in terms of percentage of killing. It was found that complements from the control subjects killed 88.3 to 97.1% of bacterial cells with a mean value of 93.2 ± 2.1%. On the other hand, complements from the tannery workers killed 81.2 to 94.4% of bacterial cells with a mean value of 89.7 ± 3.2%, which was significantly lower (p< 0.001) than for the control subjects .A total of 35 control subjects and 35 tannery workers were investigated for the bactericidal activity of serum complements. Since complements are heat-labile and readily activated to generate split products, these samples were thawed only once. Thus, samples used in other assays could not be tested for this function. The number of colonies grown in PBS (negative controls) varied from 600 - 2420 × 10Correlation analyses of the immunological, nutritional and Cr data were carried out to investigate the relationships between different parameters in the study subjects. These findings are shown in There was a significant linear correlation between the levels of serum IgE and blood eosinophils in the control subjects  which was not found in the tannery workers. Eosinophils were counted from blood films stained with Giemsa stain using an Olympus microscope.The present study examined serum immunoglobulins, complement proteins and immune function in long-term Cr-exposed tannery workers and the findings were compared with unexposed control subjects. During leather manufacture, the widely used Cr(III) is oxidized to form Cr(VI), which is more soluble, highly toxic and capable of penetrating skin with its ability to form free radicals, and thus can damage the skin of tannery workers.24Table 1). Both complaints were significantly higher than the unexposed controls, which may have been caused by direct contact and inhalation of various chemicals, including Cr. It was observed that many tannery workers in Hazaribagh were not using gloves, masks and protective boots while handling various chemicals, despite policies requiring protective equipment. This warrants further study.Tannery effluents have also been found to suppress immune function in tilapia.Table 1). The purposes of the chemicals used in the tanning processes are to remove hair from hides, harden, decolorize, soften, lime and de-lime hides, among other functions. As the tanners sometimes perform this work with bare hands, these chemicals may produce similar results on their own skin, i.e., decolorized skin, caused hardening, roughness and skin rash. Chromium and other chemicals are well known to cause inflammation. Chromium exposure through inhalation leads to respiratory complications like breathing and chest problems, wheezing, cough etc., which could be an influencing factor for elevated levels of serum immunoglobulin E. Immunoglobulin E plays an important role in the onset of allergic responses, although IgE has a beneficial role against parasitic worms. In Western countries, the normal serum concentration of IgE is 0–200 IU/mL, whereas in tropical countries this level may vary up to 2000 IU/mL.18We found decolorized skin on the upper and lower limbs of 16.4% of the tannery workers exposed to chemicals  and Cr (VI) on phytohaemagglutinin-induced blastogenesis in human lymphocytes.30The present study found significantly elevated levels of serum Cr in chronically exposed tannery workers (26.97 μg/dL) compared to the control subjects who also had high levels of Cr in serum, 7.38 μg/dL .Table 4). Various factors such as Cr(V) and Cr(VI)-induced apoptosis of lymphocytes32In the present study, serum IgG and IgA levels of the tannery workers were found to be inversely associated with the level of Cr in serum .34Complement C3 is the major protein in the complement system that can kill bacteria directly by making pores on the target cell surface. Complement C3 becomes activated via activation pathways during infection. In the present study, both complements C3 and C4 levels were found to be significantly lower in the tannery workers, suggesting impaired function of the classical complement pathway. The bactericidal activity of the workers was significantly lower, which could be due to lower levels of C3 and C4. Correlation analyses of the data showed bactericidal activity had no relation with Cr, which could be due to the small number of samples analyzed for this function. However, bactericidal activity was inversely correlated with the duration of work exposure. The lower levels of IgG and IgA presented in this study are in agreement with the observation of Qian et al., suggesting that adverse changes in the immunological status in tannery workers are possibly caused by occupational exposure to chromate and other chemicals.Table 4), suggesting elevated IgE levels were not due to intestinal helminths, rather it could be a direct effect of inhalation of Cr-containing leather dusts and fumes of chemicals used in tanneries. In the present study, 12.3% of tannery workers were found to be suffering from respiratory illness. A similar study reported that respiratory diseases (16.7%) were mainly responsible for a higher morbidity among exposed tannery workers in Kanpur, India.18The present study found serum IgE levels to be significantly elevated in the tannery workers. It should be noted that IgE is only produced in response to a select group of antigens . We found a significant linear correlation of IgE with eosinophil count in the control subjects which was not found in the tanners (Table 4), suggesting that underweight/malnourished workers were more affected by the toxic effects of Cr that induced allergic responses leading to respiratory diseases; elevated IgE levels could be due to the toxic effects of Cr. Immunoglobulin G and IgE both had an inverse correlation with duration of work exposure, although unlike IgG, the effect on IgE was not significant. Immunoglobulin G and IgA are important components of humoral immunity and thus decreases in levels generally indicates a decline in the immune system, although elevated IgE signals a deteriorating health status. The present study found that 15% of tannery workers were underweight (BMI <18.5), which was significantly higher (p<0.001) than the control population. The inverse correlation of IgE with BMI suggests that the poor nutritional status of the workers may be associated with Cr-induced allergic responses leading to respiratory diseases and elevated IgE in the tannery workers.The present study showed a significant inverse correlation of IgE with BMI among tannery workers (Workers in the leather tanning industry in Hazaribagh face a serious threat of Cr toxicity. Tannery workers directly exposed to Cr salts had considerably higher levels of Cr in serum than those working in the finishing sections. The present study found significantly lower levels of serum IgG, IgA, C3 and C4, but significantly higher levels of IgE in workers with, skin problems, infections, allergies and respiratory illness. Tannery workers also had lower bactericidal activity, suggesting impaired immune function. Levels of IgG, IgA and C3 were all inversely correlated with serum Cr. Direct exposures to Cr and prolonged work duration were correlated with adverse health effects among these workers. Elevated levels of serum IgE could be a toxic effect of Cr on the tannery workers.Tannery workers in Hazaribagh were observed working with bare hands and feet. The use of gloves, rubber boots and gas masks could greatly reduce Cr exposure in these workers. Monitoring occupational conditions, assessment of leather dust, Cr particles, fumes of organic solvents, and fungal and bacterial spores in the tannery air could reduce health risk factors for these workers. Additionally, regularly scheduled physical health examinations would benefit the health and longevity of the tannery workers.The present study was subject to certain limitations. The sample size was inadequate and might not reflect the full picture of this population. It must be noted that only 30 samples of chronically exposed tannery workers and 30 samples of control subjects were investigated for Cr contents due to a shortage of serum samples. Another limitation of the study was exclusion of female tannery workers. This was because very few female tanners worked in sections using Cr for 2 years or more. Additionally, bactericidal activity was assessed on 35 random samples from each group as there was a scarcity of unthawed serum samples with which to study complement function. Therefore, the results might not reflect the whole population; furthermore, correlations of some parameters might have been missed. Finally, the study could not take into account other toxic chemicals used in tanneries, which might have affected the results.Click here for additional data file.


Ethylene, the plant ripening hormone of climacteric fruit, is perceived by ethylene receptors which is the first step in the complex ethylene signal transduction pathway. Much progress has been made in elucidating the mechanism of this pathway, but there is still a lot to be done in the proteomic quantification of the main proteins involved, particularly during fruit ripening. This work focuses on the mass spectrometry based identification and quantification of the ethylene receptors (ETRs) and the downstream components of the pathway, CTR-like proteins (CTRs) and ETHYLENE INSENSITIVE 2 (EIN2). We used tomato as a model fruit to study changes in protein abundance involved in the ethylene signal transduction during fruit ripening. In order to detect and quantify these low abundant proteins located in the membrane of the endoplasmic reticulum, we developed a workflow comprising sample fractionation and MS analysis using parallel reaction monitoring. This work shows the feasibility of the identification and absolute quantification of all seven ethylene receptors, three out of four CTRs and EIN2 in four ripening stages of tomato. In parallel, gene expression was analyzed through real-time qPCR. Correlation between transcriptomic and proteomic profiles during ripening was only observed for three of the studied proteins, suggesting that the other signaling proteins are likely post-transcriptionally regulated. Based on our quantification results we were able to show that the protein levels of SlETR3 and SlETR4 increased during ripening, probably to control ethylene sensitivity. The other receptors and CTRs showed either stable levels that could sustain, or decreasing levels that could promote fruit ripening. Worldwide, tomato is the second most important vegetable crop in terms of production . It is wArabidopsis, which is also a negative regulator of the ethylene response  were grown in a greenhouse at the Research Station for Vegetable Production of Sint-Katelijne-Waver (Belgium). Plants were hydroponically cultivated on rockwool under natural light. Twelve biological replicates from each maturity stage  were harvested (April 2016) after visual inspection. Mature green corresponded to fully developed tomatoes that had not started the ripening process yet; breaker, to tomatoes in which ripening was initiated and the first degreening was visible; orange, to the ones in which no green color was visible anymore and red tomatoes, to the ones which matched the final red-ripe stage. Pericarp tissue of 24 tomatoes  were directly homogenized and processed for protein extraction. The pericarp tissues of the other 24 samples  were flash frozen in liquid nitrogen, crushed with a grindomixer  and stored at -80°C for gene expression analysis.Tomato plants , and centrifuged at 100,000 g for 1 h at 4°C. The pellets were re-suspended in homogenization buffer containing 10% SDS, 10 mM Tris pH 7.5, and the samples were boiled at 95°C for 5 min. Protein concentrations of solubilized pellets were determined with a DC protein assay kit  using bovine serum albumin as standard.The protein extraction method was adapted from Hundred μg of protein per sample were denaturated and reduced by addition of Laemmli buffer for 5 min at 95°C and then alkylated by addition of 60 mM iodoactetamide for 30 min at RT in the dark. The samples were loaded on an SDS-PAGE gel (4% stacking and 12% resolving) and were migrated until the smallest protein band of the pre-stained protein standard  reached the end of the gel. Proteins were stained overnight with colloidal blue Coomassie staining. For each gel lane, one band fraction containing the proteins ranging from 163 to 52 kDa was excised from the gel and cut into small pieces. Gel pieces were de-stained in 25 mM ammonium bicarbonate/50% acetonitrile (ACN) at 37°C, then incubated in ACN for 15 min. Gel pieces were dried in a speed-vac until the ACN was completely evaporated. Gel pieces were incubated overnight in 500 ng of trypsin in 50 mM ammonium bicarbonate at 37°C. Next, 200 μL 10% formic acid (FA) and 200 μL 100% ACN were added to the gel pieces and incubated during 15 min at 37°C. The supernatant was retained, and gel pieces were re-incubated with 200 μL 100% ACN and 200 μL 10% FA. Supernatants were pooled and dried in a speed-vac. Finally, the pellets were re-suspended in 2% ACN and 0.1% FA and the peptide concentration determined with a Pierce Quantitative Colorimetric Peptide Assay .In silico tryptic digestions of protein sequences obtained from UniProt  for assay development were purchased from JPT Innovative Peptide Solutions . Samples were run using a 60 min gradient from 5% up to 35% solvent B. Analytes were transferred to the gaseous phase with positive ion electrospray ionization at 2.0 kV. Precursors were targeted with a 2 m/z selection window around the m/z of interest. Precursors were fragmented in high-energy collisional dissociation mode with normalized collision energy of 28. A single MS1 scan was performed at a mass resolution of 17,500, an automatic gain control (AGC) target of 106 ions and a maximum C-trap fill time of 200 ms. Subsequently, 10 PRM scans were performed at a resolution of 70,000, an AGC target of 105 ions and a maximum injection time of 200 ms. Initial screening for targets transitions was unscheduled but retention-time scheduling of PRM (sPRM) was adopted for peptide quantification, allowing analysis of 42 peptides in a single acquisition.Samples (1 μg) were analyzed in PRM acquisition mode on a Q Exactive Plus mass-spectrometer , using a 75 μm × 2 cm, C18, 3 μm, 100 Ȧ trapping column  and an Easy nLC 1000 system (Thermo Scientific). Peptides were separated with a 50 μm × 15 cm, nanoViper, C18, 2 μm, 100 Ȧ column (Acclaim PepMap) retrofitted to a NanoSpray Flex source with a flow rate of 300 nL/min  of the unlabeled peptides and the endogenous peptides of the tomato sample. Peptide identification was based on retention time, the presence of the main transition ions and a low mass error (less than 5 ppm). Labeled synthetic versions were ordered for candidate peptides with the most consistently detectable transitions.The labeled synthetic peptides were spiked into endogenous peptides digests  from tomato samples at the following concentrations: 0, 1, 5, 10, 100, and 200 fmol. The aliquots were measured by LC–MS in PRM mode using retention time scheduling. Based on a comparison of the XIC of the labeled and endogenous peptides, final concentrations of labeled peptides were chosen for absolute quantitation experiments such that signal intensity was similar to that of endogenous peptides.This experiment was also used to evaluate the linearity of the dilution curves for the individual peptides. The ratio of sum of the area-under-the-curve (AUC) of the transitions Table  of the hAfter spiking the samples with labeled peptides, two sets of precursor ions were detected upon PRM analysis: heavy-isotope labeled (mass difference + 8 if containing a lysine or + 10 if containing an arginine) and non-labeled (from digested endogenous protein). The XIC from each individual peptide was manually checked in Skyline to ensure the correct identification of the peptide across biological replicates. Furthermore, the mProph algorithm was used to calculate the FDR  of the targeted peptide identifications trained with the second best peak option. The information extracted from this analysis is provided in Supplementary Table g for 10 min at room temperature. The supernatant was transferred to a gDNA eliminator spin column  and centrifuged at 8,000 g for 2 min at room temperature. Half a volume of ethanol was added to the effluent, then the mixture was loaded and washed through the RNeasy mini column (Plant RNeasy Extraction Kit) and finally the RNA was eluted with RNAse free water. The amount of total RNA extracted was measured by spectrophotometry using the NanoDrop 2000  and its purity determined by the 260/280 or 260/230 nm ratio. RNA integrity was checked on an ethidium bromide stained 1% agarose gel. One microgram of purified RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) in a total volume of 20 μL following the manufacturer’s protocol.Total RNA was extracted from tomato fruit pericarp samples. Ground tissue samples (500 mg) were homogenized in 800 μL of extraction buffer containing cetyltrimethylammonium bromide, as described previously . The mix® Green PCR Master Mix  on a Rotor Gene Q . The selected primers, designed with the Primer3 web tool1, are listed in Supplementary Table Gene expression studies were performed following Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines . Real-tip < 0.05) was used to compare between ripening stages. Statistical differences in gene expression between ripening stages were analyzed with the one-way ANOVA procedure and Tukey’s HSD test (p < 0.05). All analyses were performed using JMP 12 statistical software .Given an individual protein was represented by up to three different peptides, protein data were analyzed using the mixed models procedure. In this approach ‘ripening stage’ was considered a fixed categorical factor while ‘peptide’ was treated as a categorical random factor introducing a repeated structure ‘sample’ to account for the fact that the various peptides were covariates measured on the same fruit samples. In the case of a single peptide per protein the classical one-way ANOVA was applied. In both cases, Tukey’s honestly significant difference (HSD) test  a list of unique peptides was established. Those unique peptides that also followed the criteria for being identifiable in MS1, were combined with a selection of unique peptides identified during the previous shotgun approach  and the output of the mProphet analysis can be found in Supplementary Table Arabidopsis the amino acid residue where the proteolytic cleavage of the C-terminal domain of EIN2 is produced after ethylene binds to the receptor-CTR complex. We performed a Clustal alignment with UniProt between the EIN2 protein of Arabidopsis and tomato and both proteins only have 48% sequence similarity  and SlCTR1 the increase in the expression levels is not reflected at the protein level. However, a significant correlation between both kind of data was found during ripening, indicating that the protein abundance was directly controlled by gene expression. We hypothesize that in this specific case, the high transcription was counteracted by a fast rate of protein degradation of the newly formed protein after the binding of ethylene to the receptor-CTR complex. This could be the reason why no peak in protein levels was observed. Given SlCTR1 is the most abundant CTR and because of its specific behavior, it might be the strongest regulator of the tomato CTRs. Likewise the transcript levels of SlCTR4 behaved, but its low abundancy did not allow its identification in spite of using the highly sensitivity targeted acquisition proteomics method PRM.In the case of It is remarkable that SlETR1, SlETR2 and SlETR5 and SlCTR2 protein levels rapidly declined as soon as ripening started in the breaker stage. However, no comparative decline of their transcript levels could be observed during ripening, neither correlation between protein and mRNA. This suggests that protein abundance of these signaling components is likely controlled by post-translational modifications, like degradation, and not by a transcriptional regulation. Although SlETR1, SlETR2, and SlETR5 are the three least abundant ethylene receptors, it is possible that their higher protein levels in the mature green stage influence ethylene sensitivity by restraining ethylene signaling in this maturity stage due to their negative action. Their subsequent decrease in abundance during ripening could release this inhibitory action of ethylene sensitivity and perhaps eventually trigger fruit ripening. In this scenario, these receptors together with SlCTR2, could influence the initiation of tomato fruit ripening.SlEIN2 expression is silenced, show a delayed fruit ripening phenotype, confirming that SlEIN2 is a positive regulator of ethylene signaling in tomato , CTRs and EIN2 in tomato pericarp. The strategy is composed of (i) microsomal membrane extraction, (ii) fractionation of the protein sample through 1-D gel, (iii) in-gel tryptic digestion and (iv) identification and absolute quantification through the monitoring of several unique peptides of the target proteins by PRM. The combined quantification of protein and mRNA levels of the ethylene signaling components during ripening has revealed different patterns between gene expression and protein abundance which might collectively modulate and control ethylene sensitivity and thus the timing and rate of fruit ripening. Our hypothesis is that some receptors would largely control the ethylene sensitivity and, therefore, the ripening process, like SlETR3 and SlETR4 with the help of SlCTR1, some of the most abundant proteins, and possibly SlCTR4. Other signaling components such as SlETR6, SlETR7, and SlCTR3 show an unaltered protein abundance during the onset of ripening and might therefore be important to sustain the ripening process. Finally, proteins such as SlETR1, SlETR2, SlETR5, and SlCTR2 show a rapid decline in protein abundance, which might suggest that they could control the initiation of ripening. SlEIN2, being a positive regulator of ethylene signaling, also show a declining abundance profile, and could therefore also control ethylene sensitivity during climacteric fruit ripening of tomato. In conclusion, it seems that ethylene sensitivity is differently controlled by a balanced turnover of the different components of the ethylene signaling pathway, combining positive and negative feedback regulations.Future mass spectrometry analyses are needed to reveal the specific proteolytic cleavage site of SlEIN2 and to study the phosphorylation dynamics of both the receptors and SlEIN2 during ripening. Finally, a broad quantitative proteomics study including additional downstream signaling transcription factors such as the EILs and ERFs could help us to better understand ethylene sensitivity and signaling during climacteric fruit ripening of tomato.CM, BF, HP, MH, and KL designed the experiments. CM carried out the experiments, data analysis, prepared the figures, and wrote the manuscript. CM, BF, HP, MH, GVR, GB, BVdP, KL, and BN helped to improve the manuscript and participated in discussions. All authors provided feedback on the manuscript and gave their final approval for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Results of this work identified phytotoxic effects of chicken manure and milorganite, but not dairy manure, to collard greens. Potentially phytotoxic chemicals such as copper, zinc, nickel, and salts were also higher in chicken manure and milorganite compared to dairy manure. In particular, nickel in chicken manure and milorganite aqueous extracts was 28-fold and 21-fold, respectively, higher than previously reported toxic levels to wheat seedlings. The results demonstrate the need for more research on phytotoxicity of commercial organic soil amendments to ensure their safe use in vegetable and fruit crop production systems.Small-scale vegetable and fruit crop producers in the USA use locally available commercial organic fertilizers and soil amendments recycled from municipal and agricultural wastes. Organic soil amendments provide crops with their nutrient needs and maintain soil health by modifying its physical, chemical, and biological properties. However, organic soil amendments might add unwanted elements such as toxic heavy metals or salts, which might inhibit crop growth and reduce yield. Therefore, the objective of this study was to evaluate phytotoxicity of three commercial organic amendments, chicken manure, milorganite, and dairy manure, to collard greens using the seed germination bioassay and chemical analysis of the organic amendments. The seed germination bioassay was conducted by incubating collard greens seeds to germinate in 1:10 ( Small-scale vegetable and fruit crop growers are transitioning from conventional farming to environmentally friendly and more profitable semi-organic or organic farming  compost aqueous extracts  seeds in aqueous extracts of chicken manure, milorganite, and dairy manure. Furthermore, we analyzed the chemical properties and composition of the aqueous extracts. Our studies identified phytotoxicity of chicken manure and milorganite to collard greens possibly due to higher levels or synergetic effects of micronutrients, salts, and heavy metals.A widely used germination test protocol for identifying phytotoxicity is incubation of seeds to germinate in 1:10 , granulated and dehydrated chicken manure (Medina), and composted dairy manure (Black Kow). Milorganite (Milwaukee Organic Nitrogen) is prepared from heat-dried microbes that digested municipal and industrial wastes from Milwaukee Metropolitan Sewerage District (MMSD) Archer . Milorgaw/v) on a rotary shaker for 1 h. The solutions were centrifuged at 5000g for 15 min, and then filtered through 0.8-μm membrane filter. A range of dilutions of the aqueous extracts using deionized water as diluent was prepared. Commercial collard greens seed variety Tiger (F1) was obtained from a seed company (Johnny’s Selected Seeds). Ten collard greens seeds were set to germinate on Whatman filter paper wetted with 4 ml aqueous extracts or deionized water (control) in 100-mm-diameter and 25-mm-height Petri dishes (Fisher Scientific) incubated at 25 °C. After 3 days, the number of germinated seeds and the radicle length of each germinated seed were documented. A seed with a radicle length of at least 2 mm was considered as germinated. A second round of aqueous extracts (2nd extract) were prepared by adding 10 ml deionized water to the residues from the first aqueous extract preparations (1st extract), incubated for 1 h on a rotary shaker, centrifuged at 5000g for 15 min, and then filtered through 0.8-μm membrane filter. Germination tests were conducted by incubating ten collard greens seeds with a range of dilutions of the second aqueous extracts. After 3 days, the number of germinated seeds with radicle length at least 2 mm and the radicle length of each germinated seed were documented. The whole experiment including preparation of fresh aqueous extracts and germination test was repeated four times, and the results were analyzed by determining the relative seed germination (RSG), relative radicle growth (RRG), and germination index (GI) as shown below:To conduct seed germination bioassay, fresh aqueous extracts (1st extract) were prepared by shaking 1 g of milorganite, chicken manure, or dairy manure with 10 ml of deionized water , electrical conductivity (EC) meter (Milwaukee MW 802), and an ICP-OES. Heavy metals and plant macro- and micronutrients analyzed include arsenic (As), chromium (Cr), cadmium (Cd), mercury (Hg), copper (Cu), nickel (Ni), lead (Pb), selenium (Se), zinc (Zn), boron (B), iron (Fe), sodium (Na), and phosphorus (P). Calibration standards for the ICP-OES analysis were prepared from single elements  (Sigma) and pre-mixed elements  (Agilent) diluted with 1% HNO+ stock solution was prepared by dissolving 2.54 g NaCl in 50 ml deionized water. The amount of Na+ in the stock solution was confirmed using ICP-OES. A range of NaCl dilutions were prepared from the stock solution containing from 50 to 10,000 ppm Na+ in deionized water to a final volume of 4 ml and used in germination test in Petri dishes using the same method as described for the aqueous extracts of chicken manure, milorganite, and dairy manure.The effect of sodium salts on the germination and radicle growth of collard greens seeds was investigated using sodium chloride (NaCl) solution. A 20,000 ppm NaN, where SD is standard deviation of the means, and N is the number of biological replicates. The physical properties and chemical analysis results reported in this paper were also analyzed similarly.The number of germinated seeds and the average radicle length in each of the four biological replicates were determined. The RSG, RRG, and GI of each biological replicate were calculated using the equations shown in the “The seed germination bioassay identified potential phytotoxic effects of chicken manure and milorganite, but not dairy manure, to collard greens. As shown in Fig. In addition to GI, we analyzed the relative seed germination (RSG) and relative radicle growth (RRG) to see the effect of chicken manure, milorganite, and dairy manure aqueous extracts specifically on seed germination and root growth of collard greens. Both RSG and RRG were improved in 100% chicken manure and milorganite second aqueous extracts compared to the level in the first aqueous extracts, with more improvement in RSG than RRG Fig. . The RSGTo identify phytotoxic factors in chicken manure and milorganite, we analyzed the chemical properties and composition of the aqueous extracts used in the seed germination bioassay. As shown in Table Electrical conductivity measurements are used to determine the level of salts in soil  content of the first aqueous extracts of chicken manure, milorganite, and dairy manure was 442.2 parts per million (ppm), 63 ppm, and 36.3 ppm, respectively , zinc (Zn), and iron (Fe) were relatively higher in chicken manure than in milorganite and dairy manure aqueous extracts Fig. . Fe was +  could result in the inhibition of seed germination and radicle growth  is commonly used to determine phytotoxicity of compost  growth than either Ni or NaCl on its own (Yusuf et al. Ni is a micronutrient essential for plant growth at very low concentration, but could be toxic to germinating seeds when present at higher level (Seregin and Kozhevnikova Commercial scale conversion of municipal and agricultural wastes into organic soil amendments provides solutions to the growing demand of semi-organic or organic farmers for organic soil amendments and appropriate management of pollutions. Commercial organic fertilizers and soil amendments might pose little concern for human health and the environment. However, using widely accepted method for determining phytotoxicity of compost to crops, we identified strong phytotoxicity of commercial chicken manure and milorganite to collard greens. We also identified a higher level of potentially phytotoxic chemicals such as Ni and sodic salts in the aqueous extracts of chicken manure and milorganite. It is possible that there may be additional phytotoxic chemicals that this study did not address. In addition, there is variation in the degree of tolerance of crops species to phytotoxic levels of micronutrients, salts, and heavy metals. Therefore, further studies of phytotoxicity of commercial organic fertilizers and soil amendments are needed for developing strategies for their safe use in semi-organic or organic vegetable and fruit crop production systems.


After the 30-day treatment period, biochemical parameters were analyzed in the pancreas, liver, and serum. Histopathological changes in pancreatic tissue were examined by Hematoxyline & Eosin staining and the insulin content in the islet measured by immunohistochemistry with anti-insulin antibody. The glycogen content in the hepatocytes was quantified by Periodic Schiff Acid staining. The QV formulation reduced the glycemia, preserved the pancreatic architecture, increased insulin levels, furthermore ameliorated lipid profile and to promote higher survival rate of animals. Together, our data suggest that the QV formulation treatment was able to normalize metabolic homeostasis in type 1 diabetic rats.This study aimed to investigate the potential of an oral formulation (QV formulation) containing Quercetin and a Dipeptidyl Peptidase-4 Inhibitor (DPP-4 inhibitor), Vildagliptin, in improving metabolic homeostasis in type 1 diabetes model. Female albino Fischer rats were divided into four groups: untreated control animals (C), untreated diabetic animals (D), diabetic animals treated with QV formulation (DQV), and diabetic animals treated with insulin (DI). Diabetes was induced by injection of alloxan (135 mg kg body mass) The individuals affected may find it difficult to perform glycemic control, resulting in less adherence to treatment. This is mainly due to invasive treatment, which consists of the subcutaneous administration of insulin at regular intervals. Conventional treatment generates pain, anxiety, and loss of quality of life, which impacts the life of individuals and their families. In addition, the high financial cost results in medical care and decreased productivity, making this disease burdensome2. Different strategies have been developed to provide greater comfort for patients and to prevent frequent episodes of severe hypoglycemia, such as glucose sensors, oral and dermal insulins3, and oral spray. However, access to these innovations is unfeasible for a large part of the population due to the high cost, considering that 75% of patients with T1DM live in low-income countries. Recently our group demonstrated that drugs indicated for treatment of T2DM have been studied in T1DM and show a variable degree of benefits4. DPP-4 inhibitors participate in numerous biological processes, via glycemic control, weight reduction, and improvement in beta cell function and cardiovascular risk markers5, resulting in additional beneficial effects in metabolic homeostasis. In addition, in T1DM, DPP-4 inhibitors as add-on to insulin therapy can reduce the daily insulin requirements6. Animal studies indicate that Vildagliptin, an oral DPP-4 inhibitor, has a promising effect on T1DM, stimulating the neogenesis of pancreatic beta cells7. Other authors have also reported effects of Vildagliptin on the stimulation of insulin secretion8 and on the maintenance and repair of beta cells9 in animal model. However, no studies to date have demonstrated the effect of Vildagliptin on the regulation of metabolism in the T1DM model. In addition, the use of Quercetin, a flavonoid present in several foods of plant origin, has also been demonstrated to have antidiabetic potential, improving the hyperglycemic state, reducing the incidence of peripheral complications10 and increasing the secretion of insulin and protection of pancreatic cells11. Studies have shown that both Quercetin and Vildagliptin, when administrated alone, have antidiabetic potential, but their combined effect on metabolic homeostasis in the T1DM model is controversial. Considering different mechanisms of action, either by direct effects of Vildagliptin on the inhibition of DPP-4 enzyme, or by the antioxidant activity of Quercetin, we hypothesized that the combination of these two compounds are capable of to generate benefits in biochemical and histological parameters, promoting an improvement in metabolic homeostasis in type 1 diabetic rats.Type 1 diabetes (T1DM), prevalent in 5 to 10% of the diabetic population, is a result of the immune-mediated destruction of pancreatic beta cells. It manifests earlier than type 2 diabetes (T2DM), and the incidence among children has been increasing in many countries, particularly in individuals under 15 years oldAfter 30 days of treatment, the group receiving insulin alone and the group that received the QV formulation had a reduction in the hyperglycemic condition Fig. . The perHistological sections of pancreatic islets from the experimental groups reveled positive labeling with anti-insulin antibody Fig. . The QV No inflammatory signs, degenerative or fibrotic areas were found in any of the experimental group animals, either in the acinar components or in the pancreatic islets Fig. . The numNone of the treatments showed an effect on the GLUT2 (glucose transporter 2) level present in the pancreas Fig. . The GLPThe activity of hexokinase in hepatocytes of animals that received QV formulation was reestablished Fig. . Both trGLUT2 concentration, as well as GLP-1R concentration present in the liver did not reveal differences between the experimental groups who received the treatments Fig. .Figure 6Lipid profile dosages showed that treatment with QV formulation reduced the levels of the non-HDL fraction (particle non high density lipoprotein-cholesterol - atherogenic particles) and increased HDL fraction in relation to the untreated diabetic group. No differences were found between groups in relation to cholesterol total, however the QV formulation exhibited lower triacylglycerol levels in relation to insulin administration. The QV formulation did not exhibit alterations in ALT , AST (aspartate aminotransferase) and albumin concentrations. Assessment of urea levels showed that treatment with QV formulation was able to promote an improvement in this parameter in comparison to the untreated diabetic group. In contrast, the serum creatinine levels showed no change in any of the groups evaluated Table .Table 1E13, however clinical trials and other research in T1DM indicate that administration of each of these agents separately is not consistent in altering glycemic parameters14, that is, results are conflicting and the final outcomes are inconclusive. The development of the QV formulation was mainly based on previous studies that showed that treatment with Vildagliptin and Quercetin alone did not increase serum insulin levels sufficiently to the extent of reducing glycemia , while daily administration of insulin was responsible for increasing triacylglycerol levels when compared to treatment with the QV formulation. Although we did not observe improvement in triacylglycerol levels, some authors report that increased incretin plasma concentration is related to the reduction of intestinal absorption of triacylglycerol in animal studies34, besides Vildagliptin be able to lowers triacylglycerol-rich lipoprotein postprandial levels in T2DM, suggesting that DPP-4 inhibitors mobilize and burn fat during meals, and thus the fat that is associated with cardiovascular risk is not accumulated35. Our research group has previously suggested that treatment with Vildagliptin for 30 days improved the lipid profile in chronic T1DM model. Treatment with Vildagliptin alone reduced total cholesterol levels, and this reduction was due to decrease in the concentration of atherogenic fractions, since there was no change in the HDL fraction. Additionally, there was reduction of triacylglycerol levels7. Nath et al. also showed that Vildagliptin ameliorated lipid profile in diabetic mice33. In contrast, Maeda et al. did not observe alterations in total cholesterol, HDL fraction, and triacylglycerol levels in T1DM rats after treatment with Vildagliptin36. The effect of Quercetin has also been reported in the prevention and treatment of chronic non-infectious diseases, such as diabetes, obesity, and hyperlipidemia, mainly for its antioxidant effects. Studies have shown that Quercetin was able to improve the regulation of lipid metabolism37 and suppress the formation of atheromatous plaques in an atherosclerosis mouse model31. Therefore, supported by literature, our results reveal that 30-day treatment with QV formulation was effective in improving the lipid profile of animals.The QV formulation also had a positive effect on the lipid profile of diabetic rats, reducing non-HDL fraction and increasing HDL fraction, which is of particular significance because lipidic metabolism is directly related to dyslipidemia as well as microvascular and cardiovascular complications are typical of diabetes38, occasioning possible damage to cells which express this carrier. Treatment with the QV formulation also did not normalize the serum levels of albumin, a protein produced in the liver. Diabetes is responsible for reducing blood levels of albumin, which is also indicative of liver failure. Vildagliptin add-on insulin therapy in T2DM also did not alter the level of liver enzymes32, whereas in a hepatic fibrosis model the administration of Quercetin resulted in maintenance of ALT/AST levels and increased serum albumin levels, suggesting a possible hepatoprotective effect39. Diabetes is characterized by redox imbalance in the kidneys that can lead to nephropathy40. In addition, alloxan toxicity may also affect this organ due to the presence of the GLUT2 transporter in renal tubular cells38. Based on this, in relation to renal function, urea levels increased in diabetic animals, which may be justified by the higher protein catabolism and higher excretion of this metabolite. The QV formulation treatment, unlike the insulin treatment, promoted an improvement in this parameter to values similar to the control group, characterizing a possible renoprotective effect. Some studies indicate possible beneficial effects of DPP-4 inhibitors on renal complications caused by diabetes. In addition to providing protective effects against tubular damage and slowing the progression of chronic kidney disease, it has been suggested that the renoprotective effects of DPP-4 inhibitors may be due to increased levels of GLP-141. Although our study showed no difference in creatinine levels, research using an experimental model of diabetic nephropathy showed that Quercetin treatment reduced serum creatinine, restored clearance, and reduced glomerular hyperplasia and oxidative stress40.Diabetes is responsible for causing damage to various organs including the liver and kidney. Assessment of liver function parameters revealed that QV formulation treatment did not reduce serum levels of ALT and AST enzymes. In contrast, treatment with insulin increased AST levels and since this enzyme is found in other compartments such as the heart, muscles, kidneys, and the brain and is released into the blood after some type of damage, it suggests the presence of some extrahepatic injury. Differently, the ALT enzyme is found largely in the liver, being released into the circulation as a result of liver damage. Alloxan hepatic metabolism may have been responsible for the increase in ALT/AST levels in diabetic animals, since the structural similarity with the glucose molecule allows its absorption by the cell through the GLUT2 transporter42. In this study, both the QV formulation and insulin reduced the MMP-2 activity, which was increased in untreated diabetic animals. Besides, we also confirmed that there is a strongly positive correlation between glycemic values and MMP-2 activity  was commercially acquired in the pharmacy . Quercetin and Alloxan  was purchased from Sigma-Aldrich .ad libitum. The animals were randomly divided into four experimental groups: untreated control animals (C) received 1 mL of the formulation containing the vehicle (n = 6); untreated diabetic animals (D) received 1 mL of the formulation containing the vehicle (n = 11); diabetic animals treated with QV formulation (DQV) received 1 mL of the formulation containing Vildagliptin49 (10 mg kg body mass)−1 and Quercetin (50 mg kg body mass)−1 (n = 14); and the diabetic animals treated with insulin (DI) received 2UI of insulin intraperitoneal injection* (n = 12). The animals of C, D, DQV and DI groups were submitted to orogastric treatment  daily, and in this order, for 30 consecutive days , approximately 120 days of age and weighing 200 g, were used for this study. During the experimental period, the rats were maintained in well-ventilated cages with controlled temperature, ventilation, and humidity. They had access to water and commercial rat chow ays Fig. . Animals*Daily and for 30 consecutive days, at 9:00 AM, 2UI of exogenous insulin (NPH recombinant human insulin −10 mL suspension/100UI/mL concentration) were administered intraperitoneally and with the aid of a syringe. The insulin flask was kept under refrigeration (2–8 °C).−1 dissolved in 0.2 mL sterile saline22. Control animals received only 0.2 mL of sterile saline solution. Three days after alloxan administration, animals were considered to be diabetic when after-fasting tail-vein blood glucose concentrations of ≥16 mmol/L, measured by an AccuChek Glucometer .‘T1DM was induced by a single intraperitoneal injection of 135 mg alloxan (kg body mass)For biochemical analysis, the blood samples collected at the end of the experiment were used to measure glucose, triacylglycerol, total cholesterol, and high-density lipoprotein-cholesterol (HDL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea and albumin levels all by Labtest Kits . The serum insulin and glucagon levels were determined using a commercial Rat/Mouse Insulin enzyme-linked immunosorbent assay (ELISA) kit Merck Millipore  and by competitive inhibition enzyme immunoassay technique for quantitative measurement, using the enzyme-linked immunosorbent assay kit from Cloud-Clone Corp. , respectively.The pancreas and liver were also collected at the end of the experiment. For determination of Hexokinase activity in pancreas and liver, 10 mg of tissues were rapidly homogenize with 200 µl ice cold HK Assay Buffer* for 10 minutes on ice. The homogenates were centrifuged at 12.000 rpm for 5 minutes e the supernatant was collect and store at −80 °C for further analysis. .*HK Assay Buffer: Hexokinase Colorimetric Assay Kit .−1, pH 7.0–7.2) to remove excess blood thoroughly and weighed before homogenization. Minced the tissues to small pieces (20 mg) and homogenized them in 1 mL lysis buffer* with a homogenizer on ice. The resulting suspension was sonicated with an ultrasonic cell disrupter to further break the cell membranes. After that, the homogenate was centrifuged for 5 minutes at 5.000 × g, for GLP-1R, and centrifuged for 5 minutes at 10.000 × g, for GLUT2. The respective supernatants were collected in aliquot and store at −80 °C for further analysis. Evaluated by sandwich enzyme immunoassay, using the Enzyme-linked Immunosorbent Assay Kit from Cloud-Clone Corp. .For determination of GLUT2 (Glucose transporter 2) and GLP-1R (glucagon-like peptide 1 receptor) concentrations in pancreas and liver, tissues were rinsed in ice-cold PBS  to differentiate the architecture of pancreatic islets, and stained with anti-insulin antibody (1:5000 dilution)  for immunohistochemical staining of insulin in pancreatic tissue. . To quantify glycogen, the liver slides were stained with Periodic Schiff Acid (PAS). Photomicrographs were obtained using Leica Application Suite analysis software, magnification x440, and quantification was performed on Leica QWin Plus software version 3.0 .et al.50.MMP-2 activity was detected using gelatin zymography, as previously described by Sung The normality of the data was tested by the Kolmogorov-Smirnov test and followed normal distribution (parametric data). The results were expressed as mean ± standard error and statistical analysis was performed using One-Way Analysis of Variance (ANOVA), followed by Bonferroni post-test for multiple comparisons. Differences were considered statistically significant when p < 0.05. All analyses were performed using GraphPad Prism software version 6.01 for Windows .All procedures followed were in accordance with the international standards of animal protection and the ethical principles of the National Council of Animal Experimentation and was approved by the Ethics Committee on Animal Use of the University Federal of Ouro Preto (#2014/17).Supplementary Information


Lactose and mannitol are some of the most commonly used powders in the pharmaceutical industry. The limited research published so far highlights the effects of process and formulation parameters on the properties of the granules and the tablets produced using these two types of powders separately. However, the comparison of the performance of these two types of powders during twin screw wet granulation has received no attention. The present research is focused on understanding the granulation mechanism of different grades of two pharmaceutical powders with varying properties . Three grades each of lactose and mannitol were granulated at varying liquid to solid ratios (L/S) and screw speed. It was noticed that primary powder morphology plays an important role in determining the granule size and structure, and tablet tensile strength. It was indicated that the processed powders such as spray-dried and granulated lactose and mannitol can be used in formulation for wet granulation where flowability of active pharmaceutical ingredient (API) is poor. In the pharmaceutical industry, granulation of powders to form structured products  is one of the most important operations in solid oral dosage form manufacturing to improve flow, compaction or homogeneity of powders. It can be carried out using either dry or wet approaches. In wet granulation, liquid binder is added onto a bed of primary powder particles that is being agitated by an impeller (in a high-shear granulator (HSG), screws (in twin screw granulator (TSG), or air (in a fluidized bed granulator (FBG)) to form wet granules.Amongst various excipient (filler/diluent) powders available for the wet/dry granulation and tableting in the pharmaceutical industry, lactose and mannitol are some of the most common ones. Both lactose and mannitol are commercially available in different grades  presenting different particle characteristics  ,2.Lactose occurs in α and β forms possessing different melting points, solubility, and hardness . It is kMannitol is used as a filler/diluent in tablet making with both wet/dry granulation and direct compaction methods in the pharmaceutical industry . MannitoThe particle characteristics of lactose and mannitol powders can play important roles in granulation. For instance, primary powder particle size plays a critical role in determining the size of nuclei during the nucleation step in wet granulation , which fIn batch high-shear granulation, the effect of primary powder morphology on granulation behavior and properties of granules and tablets have been studied previously ,4,5,6. Kw/w) and lower using a batch high-shear granulator. The effects on granule size, flowability, and other product attributes were investigated. It was noticed that granule size increases with an increase in the particle size primary powder. It was also found that spray-dried lactose powder produce granules with better flowability, narrow granule, size distribution, and tablets with good hardness and low friability. It can also reduce the degree segregation/agglomeration of granules throughout the manufacturing process.Huang, et al.  comparedCompared to batch high-shear wet granulation, the effect of primary powder  morphology on granulation behavior and properties of granules and tablets has received limited attention in continuous twin screw wet granulation ,19. MoreIt is only recently that Vanhoorne, et al.  investigThe objective of this study was to compare the performance of different grades of lactose and mannitol powders during twin screw wet granulation. Different types of lactose and mannitol powders having different powder morphology were granulated separately at different L/S and screw speeds and their impact on the properties of the granules  and tablet (tensile strength) was investigated.Different grades of lactose and mannitol powders were used in this study for comparing the effects of their physical properties on the granulation behavior and granule properties.Three types of lactose powders were used in the study: α-lactose monohydrate (Pharmatose 200M), spray-dried lactose (SuperTab11SD), and anhydrous lactose SuperTab21AN). All powders were supplied by DMV-Fonterra Excipient GmbH and co., Goch, Germany. α-lactose monohydrate powder is manufactured by slow crystallization of a supersaturated lactose solution below 93.5 °C accompanied by roller drying resulting in single crystals of α-lactose monohydrate. The crystals are further milled to produce Pharmatose 200M grade powder  (β-mannitol ), spray-A co–rotating twin screw granulator (TSG) (Euro lab 16 TSG (L/D-25/1), Prism, Thermo Scientific (Thermo Electron GmbH), Karlsruhe, Germany) was used for the granulation experiments. A gravimetric, loss-in-weight twin screw powder feeder  having a pair of co-rotating screws was used to feed powder into the granulator. A peristaltic pump  was used to inject granulation liquid (distilled water) into the granulator.Powder particle size was measured using Camsizer-XT (X-jet module) . The powder shape and surface were examined using scanning electron microscopy (SEM) . The lactose and mannitol particles were nonconductive, hence, were coated (for 40 s) with a thin layer (~25 nm) of gold using a coating machine .K for all six powders was determined from the relationship between the uniaxial stress σ and the resulting powder bed apparent density ρ, as shown in Equation (1) [K was determined from the slope of a logarithmic plot of the apparent density as a function of stress.σ1 = major principal stressσ2 = minor principal stressρ1 = powder bulk density at σ1ρ2 = powder bulk density at σ2The compressibility of factor tion (1) . It is ktion (1) . This waLactose and mannitol powders were granulated separately using distilled water in the TSG. The granules (60 g in each experiment) were collected after 1 min, when the system reached the equilibrium (based on the stabilization time (~30 s at powder feed rate of 2 kg/h), which was determined by monitoring feed factor variability over time) for a gravimetric, loss-in-weight twin screw powder feeder. The granulation was carried out using the full length of the granulator keeping the screw configuration unchanged .The experimental design is shown in 50) of the granules was measured using the particle size analyzer: Camsizer  . Three repetitions were performed for the size analysis.The granules were air dried at room temperature for 48 h. The median size (dThe X-ray tomography (XRT)  of granules was carried out to gain more information on the change in the internal structure of the granules. The granules collected from various processing conditions had varying shapes. For this reason, only the middle area of the granules was scanned to determine the porosity. The stack of 240 images (slices) was threshold to differentiate the void and solid particle using ImageJ Software  . The blaThe granules produced at two extremes of L/S  at 4 screw speeds were sieved into different size classes  and compressed in a 12 mm die at 10 kN compression force  to produce tablets of about 405 mg using an Instron testing machine. This was done to study the impact of granule size range on the tablet tensile strength [σ) was determined by inputting maximum force (F), tablet diameter (D), and thickness (T) in Equation (2) [The tablets so produced were analyzed for their dimensions  and tensile strength. The thickness and diameter were measured using a digital caliper. The tensile strength of tablets was measured by diametric compression method using Zwick/Roell Z 0.5  instead of an Instron testing machine. This was done because Zwick/Roell provides more suitable force range compared to the Instron testing machine. The tablets were compressed diametrically  until they fractured. The force-displacement data was recorded. Ten tablets were used for each experimental condition to produce reproducible data. The strength of tablets (tion (2) ,28.(2)σ50) amongst lactose and mannitol powders, respectively.The morphology of powder  is discussed in this section. The primary particle size of six different powders is shown in The scanning electron microscopy (SEM) images for lactose and mannitol powders are shown in K) for different powders. The compressibility factor results for lactose and mannitol powders were anomalous. Based on the particle size, the expected trend for K value was Pharmatose 200M < SuperTab11SD < SuperTab21AN for lactose and Pearlitol 50C < Pearlitol 200SD < Pearlitol 300DC for mannitol; however, it was not the case. The K value was higher for crystalline Pharmatose 200M and Pearlitol 50C having smaller particle size, while it was lower for spray-dried large sized (meaning higher compressibility) SuperTab11SD and Pearlitol 200SD, respectively. It was also expected that the pre-processed SuperTab21AN and Pearlitol 300DC (which are suitable for direct compression in tableting) to have lower K than SuperTab11SD and Pearlitol 200SD, respectively, but it was not the case. This can be attributed to the structure of the spray-dried lactose and mannitol particles. Scanned electron microscopy images of SuperTab11SD particles  compared to Pearlitol 300DC, which is a granular compact.articles c,d show articles c,d clearIn the case of mannitol a–d, incrX-ray tomographic images of granules produced using different grades of mannitol are presented in Granules produced at L/S of 0.048 and 0.113 were sieved in size range of 212–1400 μm and into further subdivisions of 212–600 μm, 600–1000 μm, and 1000–1400 μm. This was done to study the effect of different granule size range on the tableting and tablet tensile strength.50) amongst the three lactose powders, but it produced granules comparable to SuperTab21AN, which had larger primary particle size. This indicates that the smaller particles of Pharmatose 200M which promoted the dissolution of particle surface (more than anhydrous lactose [50-172.3 µm), and hence, limited dissolution of particle surface [From  lactose ), potent lactose ,33. As sAt L/S of 0.07 and screw speeds of 200 rpm and 450 rpm, SuperTab11SD produced relatively larger granules amongst the three powders. At screw speeds of 700 rpm and 950 rpm, SuperTab21AN had relatively larger median granule size amongst the three powders. This indicated that L/S of 0.07 and a screw speed of 700 rpm may be the parameter settings where SuperTab21AN started exhibiting granule growth. The presence of slightly more granulation liquid (compared to L/S of 0.048) with additional shear/compression force within TSG at higher screw speed resulted in the formation of stronger liquid bridges between SuperTab21AN particles, thus the increase in median granule size.With further increase in the L/S to 0.1 and 0.113, the solubility of lactose powders increased further resulting in the formation of stronger and higher numbers of liquid bridges between primary particles, meaning formation of bigger and stronger granules. However, apart from the solubility, there are other factors such as compressibility which can also play a key role during wet granulation ,34. PharIn the case of SuperTab11SD, at lower L/S of 0.048 and 0.07 , no significant difference was observed due to the porous structure of SuperTab11SD, where liquid was absorbed into the powder particles reducing the availability of liquid on the surface . The absIn the case of SuperTab21AN, increasing screw speed at lower L/S of 0.048 did not show a significant effect on the granule growth, because liquid was not sufficient to form an adequate number of liquid bridges between the particles. When increasing screw speed from 200 rpm to 450 rpm at an L/S of 0.07, there was no significant change in granule size, but as the screw speed was increased further to 700 rpm and 950 rpm, the granule size increased noticeably. This is because at lower screw speeds 200 rpm and 450 rpm, the barrel fill level was high and residence time was longer ,38. ThisFrom 50) amongst all mannitol powders, generally produced relatively larger granules in almost all cases, while Pearlitol 200SD produced small size granules. This was not the case with similar sized lactose powder ) where granule size was either similar or smaller than the other two lactose powders at various conditions. Pearlitol 300DC, which has the largest primary particles amongst the three mannitol powders, produced granules comparable to Pearlitol 50C. This indicates that there are more dominant variable(s)/properties that controlled the granule size than the primary particle size of the powder. As discussed in the Comparing the three powders, Pearlitol 50C, which has the smallest primary particle size . It behaves like Pearlitol 50C at higher L/S. Pearlitol 300DC had large particles, but they are not porous, as in case of Pearlitol 200SD. This means that the liquid was not absorbed in the structure and was available for the inter-particle liquid bridge formation and thereby for granule growth. Additionally, there was less reduction in the volume due to relatively lower compressibility. Hence, it can be concluded that when there is significant difference in the structure and the compressibility, the granule growth is not just controlled by the primary particle size of powder in twin screw wet granulation. The effect on granule size can be a combination of one or more properties of a powder.X-ray tomography images of granules produced using different lactose powders at varying L/S  are shown in Pearlitol 50C a–d and PIt was found that the tensile strength of tablets was similar at both L/S of 0.048 and 0.113  a–d. The The tensile strength of tablets of SuperTab11SD granules was similar at L/S of 0.048 and 0.113 (at varying screw speed) for different size classes a–d.From Subdividing the granules into narrower size classes showed a screw speed dependent effect on the tablet strength to some extent b. The taFrom In Pearlitol 200SD, varying screw speed at lower L/S of 0.048 showed no significant change in tablet tensile strength for broader (212–1400 µm) and subdivided granule size ranges  a,b. The It is clear that the tensile strength of tablets was low for all three mannitol powders at both lower and higher L/S at all screw speeds. It is likely that the differences in the granule strength or compressibility may have been concealed by the compression force used in the study .Powders differ in their size and structure due to the way they are manufactured for their intended application (suitability for dry or wet granulation or direct compaction). In this study, the effects of the properties of powder on twin screw wet granulation behavior and properties of so-produced granules and tablets were studied. It was found that the granulation behavior of powder is controlled by size, structure, and compressibility of primary powder, which agrees with findings in previous research works ,18,19,46


Pretreatment based on lignin oxidation have demonstrated to reach the highest delignification yield, also in terms of monosaccharides recovery. In fact, all the delignified samples were then saccharified with enzymes (cellulase and beta-glucosidase) and hydrolysis efficiency was evaluated in terms of final sugars recovery before fermentation. Analysis of Fourier transform infrared spectra (FTIR) has been carried out on treated samples, in order to better understand the structural effects of delignification on lignocellulose. Active chlorine oxidations, hypochlorous acid in particular, were the best effective for lignin removal obtaining in the meanwhile the most promising cellulose-to-glucose conversion.Valuable biomass conversion processes are highly dependent on the use of effective pretreatments for lignocellulose degradation and enzymes for saccharification. Among the nowadays available treatments, chemical delignification represents a promising alternative to physical-mechanical treatments. Banana is one of the most important fruit crops around the world. After harvesting, it generates large amounts of rachis, a lignocellulosic residue, that could be used for second generation ethanol production, via saccharification and fermentation. In the present study, eight chemical pretreatments for lignin degradation ( Among tnd so on . Therefond so on . Lignoceoduction  and for  concept .Various technological developments based on saccharification and fermentation have improved for the conversion of these substrates into bioethanol . AgriculDifferent pretreatment technologies have been already extensively described in terms of the mechanisms involved, advantages and disadvantages, and economic assessment . They inorganosolv) [Even though a biological approach would be in perspective the ideal solution, because it is less harmful to the environment and needs milder conditions, it still necessitates improvements in process duration and cost reduction, factors that strongly limit their actual efficient industrial application ,16. To danosolv) , and oxianosolv)  or activanosolv) . Recent anosolv) . Saccharomices cerevisiae or other microorganisms via alcoholic fermentation [After delignification, residual biomass must be hydrolyzed to produce glucose which is then converted to bioethanol by entation . Even thentation , hydrolyentation . There aentation .In the present study, eight chemical pretreatments and some combinations thereof for lignin degradation have been tested on lignocellulosic feedstock as banana rachis and the effects evaluated. organosolv) which remove lignin and hemicellulose by solubilization and extraction [Treatments with different mechanism have been chosen: with organic solvents  represent a waste and in producing countries as Ecuador or India the majority of the producers prefer to leave these residues to decompose outdoors, causing environmental problems such as the spread of diseases or polluting groundwater . Some atMusa paradisiaca var. barraganete) cultivated and collected in the region of Guayas, Ecuador. 1000 g of samples were locally cut in small pieces and sent to our laboratory for subsequent treatments. The material was ground up, sieved using a <0.5 mm mesh size to obtain a homogeneous powder and then dried at 60 °C overnight (2 at room temperature.The lignocellulosic residue was rachis from banana plants (vernight . Sample v/v) and pH adjusted to 2.7 with HCl 1M. Treatment was carried out on lignocellulosic substrate (20 g) by adding 250 mL of the acid-organosolv mixture for 30 min at boiling temperature under reflux. After treatment, the residual biomass was filtered on paper filter, extensively washed with water and buffered to neutral pH with NaOH 1M, and dried in a hot air oven at 60 °C overnight. The dried treated was weighed and stored in a desiccator with CaCl2. A mixture of glacial acetic acid (>99.85%), acetone (>99.5%)  and water has been prepared at a ratio 10:50:40 (2.Rachis powder (20 g) was treated with 250 mL of 96% ethanol (>96.5%) (Sigma Aldrich) for 30 min at boiling temperature. The residual biomass was filtered on paper filter, extensively washed with water and dried in a hot air oven at 60 °C overnight. The dried treated was weighed and stored in a desiccator with CaCl2.A solution of NaClO 5% (250 mL) was added to rachis powder (20 g) at room temperature for 30 min. The residual biomass was filtered on paper filter and extensively washed with water and buffered to neutral pH with HCl 1M, and dried in a hot air oven at 60 °C overnight. The dried treated was weighed and stored in a desiccator with CaCl2.Rachis powder (20 g) was treated with Electro Chemical Activated solution based on Hypochlorous Acid (ECA)at pH 6 (250 mL) for 10 min at room temperature. According to Tamburini et al. , ECA sol2O2 2% (v/v) (900 mL) was added . In sample hydrogen peroxide with alkali (HPA) NaOH solution 5% (w/w) (100 mL) was added alongside. The final volume of HP and HPA was completed up to 1000 mL with water. Both treatments were kept under stirring for 90 min at room temperature. The the residual biomass was vacuum filtered in glass microfiber, washed with water until the hydrogen peroxide was completely removed, buffered to neutral pH with HCl 1M and dried in a hot air oven at 60 °C overnight. The dried treated was weighed and stored in a desiccator with CaCl2.To rachis powder (20 g) H2.To rachis powder (40 g) 96% ethanol (500 mL) was added and refluxed for 30 min. The solid suspension was separated by filtration and dried at 60 °C overnight. The dried powder (20 g) was thereafter mixed up with ECA solution (250 mL) for 10 min at room temperature. After filtration the powder was dried at 60 °C up to constant weight, weighed and stored in a desiccator with CaCl2.To rachis powder (40 g) a mixture of glacial acetic acid:acetone:water (10:50:40) (500 mL) was added and refluxed for 30 min. The solid suspension was separated by filtration and dried at 60 °C overnight. The treated powder (20 g) was thereafter put in contact with ECA solution (250 mL) for 10 min at room temperature. After filtration the powder was dried at 60 °C up to constant weight, weighed and stored in a desiccator with CaClTrichoderma reesei , supplemented with 50 μL of beta-glucosidase from Aspergillus niger  were used for saccharifying the cellulosic material. Enzymatic hydrolysis has been carried out on 1 g of sample in 50 mM citrate phosphate buffer (pH 5.0). The substrate with buffer was pre-incubated at 50 °C on a rotatory shaker  at 150 rpm for 2 h. 0.3 g of Tween 80  was also added to the reaction mixture and the reaction continued up to 72 h [Delignificated rachis samples have been submitted to enzymatic hydrolysis. 50 μL of cellulase from + (8%), 300 × 7.8 mm  was used at 80 °C. Isocratic elution was carried out with H2SO4 0.01M water at 0.6 mL/min. Samples were analyze in triplicate. Infrared spectroscopic analysis of residual lignin has been carried out by means of Fourier transform infrared (FT-IR) spectrophotometer (Perkin Elmer Spectrum 1000), using a KBr disc containing 1% finely ground samples. Through the Spectrum 10® software, supplied with the instrument, the corresponding absorbance spectra were collected within the range of 4400 cm−1 to 500 cm−1, and processed for baseline correction, noise correction (smooth) and identification of the wave numbers of the main peaks. The chemical composition  of untreated rachis sample and of all the residual solid fraction post pretreatments were determined following standard Technical Association of Pulp and Paper Industry (TAPPI) protocols . The mony-intercept and slope of the regression line. The curves were validated by means of the analysis of variance (ANOVA).All pretreatments trials have been carried out in triplicate and the results reported as average. Linearity was evaluated by linear regression analysis, which was used to calculate the correlation coefficient, Banana rachis pretreated materials was the substrate used in this study. As previously reported in the Materials and Methods section, the raw material was analyzed to determine the chemical composition. In the best of the Authors’ knowledge, in literature compositional analysis of banana rachis has been reported only from Guerrero et al. , otherwiTo facilitate a general view of the different conditions applied during chemical treatments, a synoptic table is proposed .The different combinations of times and temperatures of treatments resulted from a series of trial-and-error experiments in our laboratory with the aim to maximize delignification yield for each treatment. Notably, taking the amount of raw material as starting invariant, conditions were very different, and unavoidably had different impacts, in terms of costs and in terms of environmental burden. Even though a detailed description of both of those aspects are beyond the aim of this study, a balance between efficiency and energy/water consumption should be done when defining the best treatment. organosolv are the most commonly used methods to pretreat lignocellulosic biomass, they have proved not to be so successful for banana rachis. Reporting data in terms of percentage of lignin loss, i.e., delignification capacity of various treatments, compared with 0% of not-treated sample (NT), it is even more evident that treatments based on the oxidant effect of chlorine show high and comparable efficiency.Effects of different chemical pretreatments on rachis powder, compared to not-treated sample, are shown in organosolv does not produce a significant advantage over the absolute yield of the dignified biomass treated only with the oxidizing ECA solution. The oxidizing treatment with the ECA solution simultaneously removes a similar amount of soluble substances and lignin, without adding solvents. The results indicate that the pretreatment of biomass with oxidizing ECA solution combined with both organosolv. Anyway, in a perspective of practical use, it is worthwhile pointing out that sodium hypochlorite is a chemical reagent that needs to be removed from the solution at the end of the reaction, hypochlorous acid naturally decades to chloride after 48–72 h , reducing the overall environmental impact of the treatment.Even SH pretreatment has permitted to reach good lignin losses, comparable to ECA solution, alone or in combination with A mass balance for each treatment has been carried out based on the chemical characterization of samples before hydrolysis . Measuring mass of the samples before and after the delignification treatments allowed for calculation of the weight loss caused by the delignification. As expected, all treatments lead to material losses in the range 40–50% . Others The results of enzymatic hydrolysis and quantification of monosaccharides recovery are shown in The presence of small amount of free monosaccharides in not-treated rachis sample could derive from natural phenomena, and they have been quantified as a benchmark. The overall amount of free pentoses (xylose and arabinose) were low, and their concentration in some cases decrease after treatments, compared with NT sample, probably due to degradation occurring in the severe conditions maintained during chemical delignification. The enzymatic saccharification of all pretreated samples showed a significant conversion of cellulose to glucose because of lignin and/or hemicellulose removal during pretreatment had permitted to cellulase and beta-glucosidase to act on their elective substrate . It is worthwhile noting that, in general, glucose recovery followed the same trend of delignification, namely higher is the lignin loss higher is the enzymes efficiency of hydrolyze cellulose. From one side it is due to the barrier disruption which “unlocks” cellulose to enzymes, from the other side the limited enzymatic saccharification in the presence of higher lignin content can be due to the high affinity of cellulase towards lignin, which resulted in unavailability of enzyme to cellulose moieties and led to poor saccharification yields. Similar observation have been reported by other Authors ,47.organosolv treatments. This observed reverse correspondence between percentage of delignification and glucose recovery in the case of organosolv (AA and ET) and hydrogen peroxide (HP and HPA) treatments can be right explained by different actions of various chemical agents on lignin structure [Among the different samples evaluated here, those treated with the active chlorine were observed to be more vulnerable to enzymatic hydrolysis and resulted in maximum glucose recovery. The higher enzymatic saccharification in SH, ECA, ECA + AA, and ECA + ET pretreated samples can be attributed to the presence of higher free cellulose content with a minimum amount of residual lignin (3.7–4.2%). On the other hand, the hydrogen peroxide treatments with minimum lignin removal showed higher glucose recovery than both tructure . It has tructure .2 > 0.97; p = 0.05) for all treatments, except for organosolv-treated samples that resulted as outliers , and hydrogen peroxide (HP), as examples of the paradigm of chemical pretreatments here applied.Non-destructive elucidation and analysis of the lignin and hemicellulosic samples are nowadays carried out primarily by spectroscopic methods, which constitute important tools in connection with the characterization of the polymers . In this−1 corresponding to OH-stretching and at 2900–2800 cm−1 corresponding to CHn stretching, together with signals at 1170–1100 and 1060 cm−1, corresponding to C–O–C stretching of pyranose ring skeletal and C–OH alcoholic bonds of sugars, are found in FTIR spectra of pure cellulose and hemicellulose [−1). This group of complex and superimposed peaks could indicate rachis lignin rich of metoxyl–O–CH3, C–O–C aryl-alkyl ether linkages containing compounds and C=C bonds from aromatic rings .In the spectrum of not-treated samples a, typicaellulose . Lignin −1 of C=O stretching of carbonyl bonds and comparing weak signals at 1500 and 1250 cm−1, probably due to degradation fragments. Moreover, two new peaks are evidenced at 2351 and 2137 cm−1, probably due to the production of nitrogen-based degradation products. During the oxidative delignification process, oxidation reagents release a large number of free radicals, resulting in remarkable oxidative fragmentation and removal of lignin from lignocellulosic matrix. Oxidant treatment with ECA solution b seems torganosolv AA  and oxidant effects with active chlorine in form of hypochlorite/hypochlorous acid and hydrogen peroxide, with and without alkali. Hypochlorous acid was the highest performing in terms of delignification yield, and its combinations with organosolv did not reach a significant improvement. In general, active chlorine oxidations were the most effective for lignin removal, meanwhile obtaining the most promising cellulose-to-glucose conversion. Further research will be focused on optimizing the ECA pretreatment up to be applied as a valuable industrial delignification process. Delignification and saccharification of banana rachis for bioethanol production were performed and evaluated. Pretreatments have been chosen to compare solvolitic effects (


TM as an MAS second layer with the same thickness. A cost analysis indicated that armor vests with fique fabric composites as an MAS second layer would be 13 times less expensive than a similar creation made with Kevlar™.A relatively unknown natural fiber extracted from the leaves of the fique plant, native of the South American Andes, has recently shown potential as reinforcement of polymer composites for engineering applications. Preliminary investigations indicated a promising substitute for synthetic fibers, competing with other well-known natural fibers. The fabric made from fique fibers have not yet been investigated as possible composite reinforcement. Therefore, in the present work a more thorough characterization of fique fabric as a reinforcement of composites with a polyester matrix was performed. Thermal mechanical properties of fique fabric composites were determined by dynamic mechanical analysis (DMA). The ballistic performance of plain woven fique fabric-reinforced polyester matrix composites was investigated as a second layer in a multilayered armor system (MAS). The results revealed a sensible improvement in thermal dynamic mechanical behavior. Both viscoelastic stiffness and glass transition temperature were increased with the amount of incorporated fique fabric. In terms of ballistic results, the fique fabric composites present a performance similar to that of the much stronger Kevlar TM. In spite of numerous publications dedicated to this subject, the growing interest for engineering applications is continuously demanding research works on less common promising natural fibers and fabrics. An example is the relatively unknown fiber extracted from the leaves of an Andean plant (Furcraea andina). This fique fiber has been brought to attention for its potential as a composite reinforcement [The beginning of this new century witnessed a surge in research works dedicated to the use of fibers extracted from plants in engineering applications. Several specialized and review articles ,13,14,15orcement ,37,38,39orcement  and therorcement  as well orcement  and tensorcement  dedicate3 for the fique fiber, 2. An isophtalic polyester resin hardened with 1% methyl-ethyl-ketone, fabricated by Dow Chemical and supplied by Resinpoxy, Rio de Janeiro, Brazil, was used as the composite matrix. Composites were prepared by accommodating the previously dried fabric (60 °C for 24 h) in a steel mold, and then pouring the still fluid resin-hardener mixture between fabric layers. In this way, 120 × 150 × 50 mm3 plates of laminate composites with 10, 20, and 30 vol % fique fabric associated with 4, 8, and 12 layers were respectively produced. These plates were kept under 3 MPa pressure for 24 h, at room temperature (25 °C), until complete solid cure. Test samples were cut from the laminate composite plates and standard flexural specimens with 10, 20, and 30 vol % fabric, as well as neat polyester for control, were prepared.Fique fabric commercially available in Colombia was supplied by Compañia de Empaques, Itagüí, Antioquia, Colombia. A piece of as-received fabric is shown in DMA tests were performed in fique fabric composites, using flexural specimens according to standard. All analyses were conducted in triplicate to evaluate the degree of dispersion in corresponding curves. Less than 10% difference was found between curves for identical conditions, basically confirming the same results. Therefore, only one typical curve is used to illustrate each condition.Each DMA test was carried out in the interval from −20 to 160 °C in a model Q/800 TA Instruments  in three-point flexural mode, 1 Hz frequency, and a heating rate of 3 K/min (3 °C/min) under nitrogen atmosphere. The storage modulus (E′), loss modulus (E″), and tangent delta curves were provided by the equipment. Prismatic standard specimens with dimensions of 50 × 13 × 3 mm were used for the tests.2 NATO military ammunition with a 9.7 g projectile propelled from a gun barrel. The tests were conducted at the Brazilian Army shooting facility, CAEX, in the Marambaia Peninsula, Rio de Janeiro, with three samples of 10 and 20 vol % of fique fabric for each type of multilayered armor system (MAS) [In addition to DMA, preliminary ballistic tests were carried out according to the NIJ 0101.06 standard using 7.62 × 51 mmem (MAS) ,42. FiguThe ruptured surface of each MAS component after the ballistic test was analyzed by scanning electron microscopy (SEM) in a model QUANTA FEG250 Fei  microscope operating with secondary electrons at 20 kV.Tg) of the polymeric matrix. Therefore, the results in Tg [Tg with the increasing amount of fique fabric in the polyester matrix. Consequently, the results in The variation of the tan δ with the temperature for the neat polyester resin and the composites incorporated with fique fabric is shown in Tg . MoreoveAfter ballistic tests, the fracture surface of MAS components were observed by SEM. TM), and glass fiber [A cost model associated with the use of fique fabric composites in comparison to conventional engineering composites is presented in ss fiber , as wellss fiber  and for ss fiber .TM (aramid fiber laminate) is the most expensive composite, more than 13 times the price of any common natural fiber composite, including the presently investigated fique composites. Even a less expensive glass fiber composite is more than five times as costly as any natural fiber composite. Therefore, the incorporation of any common natural fiber-based material, including fique fabric, reduces the price of the composite.In this table, is it shown that KevlarTg), indicating less mobility in the polyester resin chains of the matrix by interaction with the fique fabric. The maximum in tan δ curves, associated with end of Tg, suffers not only a reduction in its amplitude but also a shift towards higher temperatures with the introduction of fique fabric. Hence, a high attenuation of internal vibration and increase in Tg occur with an increasing amount of fique fabric in the polyester matrix.The introduction of fique fabric raises the viscoelastic stiffness level and tends to shift the curves of the storage modulus (E′) to higher temperatures. This leads to a delay in the onset of the thermal softening of the composite. The peak α of the loss modulus (E″) is also shifted to higher glass transition temperatures (A multilayered armor system (MAS), in which conventional Kevlar™ was replaced by a polyester matrix composite reinforced with 10 or 20 vol % of fique fabric as second layers, attended the NIJ trauma limit after ballistic tests with 7.62 mm ammunition. The depth of penetration into 20 vol % fique fabric composite, 15 mm, demonstrated this composite to be more efficient than conventional Kevlar™ with 23 mm as a second MAS layer.More than ballistic performance, the significantly lower cost in association with the environmental and societal benefits of using a natural material favor the substitution of fique fabric composite as an MAS second layer. As an economical advantage, armor vests with fique fabric composites would be 13 times cheaper than similar ones made with Kevlar™.