This study demonstrated that silicon-enhanced root hydraulic conductance through up-regualtion of aquaporin gene expression resulted in improved root water uptake under osmotic stress in sorghum. Sorghum bicolor L.) growing under polyethylene glycol-simulated osmotic stress in hydroponic culture and water deficit stress in sand culture were investigated. Osmotic stress dramatically decreased dry weight, photosynthetic rate, transpiration rate, stomatal conductance, and leaf water content, but silicon application reduced these stress-induced decreases. Although silicon application had no effect on stem water transport capacity, whole-plant hydraulic conductance (Kplant) and root hydraulic conductance (Lp) were higher in silicon-treated seedlings than in those without silicon treatment under osmotic stress. Furthermore, the extent of changes in transpiration rate was similar to the changes in Kplant and Lp. The contribution of aquaporin to Lp was characterized using the aquaporin inhibitor mercury. Under osmotic stress, the exogenous application of HgCl2 decreased the transpiration rates of seedlings with and without silicon to the same level; after recovery induced by dithiothreitol (DTT), however, the transpiration rate was higher in silicon-treated seedlings than in untreated seedlings. In addition, transcription levels of several root aquaporin genes were increased by silicon application under osmotic stress. These results indicate that the silicon-induced up-regulation of aquaporin, which was thought to increase Lp, was involved in improving root water uptake under osmotic stress. This study also suggests that silicon plays a modulating role in improving plant resistance to osmotic stress in addition to its role as a mere physical barrier.The fact that silicon application alleviates water deficit stress has been widely reported, but the underlying mechanism remains unclear. Here the effects of silicon on water uptake and transport of sorghum seedlings ( Silicon is the second most abundant element in the soil after oxygen, and comprises 31% of the earth’s crust . Althouget al., 2010; +-dependent glyceraldehyde-3-phosphate dehydrogenase . Previous research has indicated that silicon enhances drought resistance through decreasing the plant transpiration rate, which leads to a reduction in water loss in rice and maize , which consists of leaf, stem, and root hydraulic conductance (leaf) is coordinated with leaf water potential (sc), is mainly controlled by vessel characteristics and embolism . Furthermore, in a preliminary experiment, in which sorghum was subjected to short-term (<3 d) osmotic stress, the transpiration rate was also higher in silicon-treated seedlings than in untreated seedlings. Therefore, it was speculated that silicon reduces the decrease in sorghum transpiration rate and that the relative water content (RWC) is involved in the observed changes in Kplant, especially the root hydraulic conductance (Lp), under short-term osmotic stress.Upon long-term (>3 d) exposure to drought stress, roots can respond with marked surface and anatomical alterations, which in turn cause profound changes in their water transport capacity; however, before any changes in root surface and anatomy can be observed, the water permeability is already changed to regulate water uptake capacity in the root . In sorgplant, Lsc, Lp, and the function of aquaporin were measured in sorghum seedlings grown in both hydroponic and sand cultures. In hydroponic culture, it is easy to obtain intact roots to measure Lp and aquaporin function, whereas sand culture promotes stem growth and facilitates the measurement of Lsc.The purpose of this study was to test the hypothesis that silicon improves water uptake by reducing the decrease in plant hydraulic conductance, and thereby enhances sorghum resistance to osmotic stress. To accomplish this objective, KSorghum bicolor (L.) Moench. cv. Gadambalia] were cultivated in a growth chamber under cycles consisting of 14h of light (450 μmol m−2 s−1) at 28 °C and 10h of darkness at 23 °C. The relative humidity was 40–50%.Seedlings of sorghum [2SiO3 was added to the culture solution starting the third day after transplanting. The culture solution was continuously aerated, and the pH was adjusted to 6.0 with 0.1M HCl or 1M KOH every day. Twelve days after transplanting, 10% PEG-6000 (–0.2MPa) was added at 08:00h to induce osmotic stress, and, unless stated otherwise, samples were collected and measurements were made from 10:00h to 13:00h on this day.Sterilized seeds were germinated for 4 d in an incubator at 25 °C. After germination, healthy seedlings were transplanted into a plastic container with 8 litres of one-quarter strength Hoagland culture solution. After 6 d, the culture solution was changed to half strength. After a total of 9 d after transplanting, the culture solution was changed to full strength. For silicon treatment, 0mM or 1.67mM Na2SO4 and 40% HF. The silicon concentration was determined by the colorimetric molybdenum blue method at 811nm. The silicon concentration was expressed as μmol g–1 dry weight.The dry weight of sorghum seedlings was measured after 3 d and 7 d of PEG treatment. The silicon concentration in plant materials was determined according to –2 s–1; the flow rate through the chamber was 500 μmol s–1 and leaf temperature was 28 °C. The leaves were typically 1.7cm wide and the area in the chamber was determined for each leaf. Transpiration rates used to calculate Kplant were also determined gravimetrically in the first 2h from the onset of PEG treatment. Each treatment includes five replications.The photosynthetic rate, stomatal conductance, and transpiration rate were measured with a portable photosynthesis system . The new fully expanded leaf was placed in a chamber at a photon flux density of 500 μmol mThe leaf RWC was measured according to New fully expanded leaves were covered with aluminium foil prior to excision from the plant. The water potential was measured by using a pressure chamber . Each treatment includes five replications.2 pressurized to 2 bar. About 15 μl of root xylem sap was collected and sealed in a microtube. The osmotic potential of the collected sap was determined using a vapour pressure osmometer . Each treatment includes five replications.The osmotic potential of the root xylem sap was measured according to the method of plant was calculated according to the following equation  was –0.07MPa or –0.09MPa under control conditions or with silicon application, respectively. Under PEG treatment, the soil water potential  was –0.2MPa. This experiment includes five replications in each treatment.3 m–2 s–1. Root Lp is calculated from the slopes of Jvr against driving force, and the driving force consisted of Pgas and osmotic gradient. In this study, however, the osmotic driving force was too small to be considered; as a result, Lp was determined from the slopes of Jvr against Pgas only, according to the following equation:The Lp based on the root surface area was measured with a pressure chamber according to the method of After the exuded sap was measured, the root was sampled to determine root surface area using a scanner and analysed by WinRHIZO PRO 2009 software . Root surface area was calculated from projected areas of root that were assumed to be cylindrical in shape. Each treatment includes five replications.2. One group of seedlings was used for measuring the transpiration rate. The other group was treated with 50 μM HgCl2 for 5min and subsequently rinsed with distilled water before being returned to the culture solution (devoid of HgCl2) where the transpiration rate was measured again. The reversibility of the effect of HgCl2 on aquaporin activity was tested by treating roots first with 50 μM HgCl2 and then placing them for 15min in 5mM DTT before measuring the transpiration rate. In addition, to confirm further the participation of aquaporin in silicon-induced water transport, NaN3, another widely used aquaporin inhibitor, was also applied in this study in the same manner as HgCl2.According to the method of SbPIP) aquaporin genes were identified based on data from the NCBI. The genes, as well as the sequences of their specific primers, are presented in SbPIP gene.Root tips 3cm in length were collected after 4h and 24h PEG treatment and frozen in liquid nitrogen to measure the expression of aquaporin genes. Eight sorghum plasma membrane intrinsic protein  according to the manufacturer’s instructions, and treated with recombinant DNase I  to remove the remaining genomic DNA. Reverse transcription was performed using an iScript™ cDNA Synthesis Kit  according to the manufacturer’s instructions. The cDNA was then diluted 50-fold in water, and 2 μl of cDNA was used to carry out the quantitative RT-PCR (qRT-PCR). The iQ™ SYBR® Green Supermix (Bio-Rad) was used for the qRT-PCR on a MiniOpticon™ CFD-3120J1 instrument (Bio-Rad). A melting curve analysis was performed to confirm the absence of multiple products and primer dimers. To confirm further that the single peaks from the melting analysis corresponded to a unique amplification product of the correct size, the PCRs were run on a 1% agarose gel (data not shown). Data acquisition and analysis of qRT-PCR was done using Bio-Rad CFX manager software (version 2.0), and the expression levels of target genes were normalized to that of the internal control gene Actin1 using the 2ΔCt method. Each treatment includes three replications and each replication includes two technical replications.Total RNA was extracted from 100mg of frozen root samples using an RNeasy–1 by watering with Hoagland solution. Starting after 30 d of growth, seedlings were watered with Hoagland solution containing 0mM or 1.67mM Na2SiO3. Starting after 38 d, half of the seedlings (including some silicon-treated and some untreated) were left unwatered until the soil water content had decreased to 0.03g g–1; the pot was weighed continuously, and the soil water content was calculated according to the weight  was measured according to the method of sc of the stem was calculated as the flow rate divided by the pressure gradient along the segment and the leaf area above the node. Each treatment includes five replications and each replication includes two seedlings.The leaf-specific conductivity of the stem  using Statistical Analysis System (SAS version 8.0) software. Differences between the means were compared by means of the Tukey–Kramer test at Under control conditions, the total dry weights of silicon-treated and untreated seedlings were not different. Under osmotic stress, the total dry weights of untreated seedlings decreased by 38% and 42% after 3 d and 7 d of osmotic stress, while those of silicon-treated seedlings decreased by only 12% and 15%, respectively . SiliconSupplementary Fig. S1 available at JXB online). In addition, silicon showed a similar effect on changes in photosynthetic rate, stomatal conductance, and transpiration rate of sorghum seedlings grown under sand culture . These results show that silicon could reduce the decrease of photosynthetic rate, stomatal conductance, and transpiration rate under osmotic/water deficit stress in sorghum seedlings.Under control conditions, the leaf photosynthetic rate was not affected by silicon . When plUnder control conditions, RWC was not affected by silicon in the present study . Under oplant was observed to occur due to the presence or absence of silicon treatment. Under osmotic stress, however, a sharp decrease in Kplant was seen in the absence of silicon treatment, but the Kplant was 45% higher in silicon-treated seedlings than in untreated seedlings .The Lp was not affected by silicon application under control conditions , B, but 2, the transpiration rate decreased sharply, and the difference between osmotic-stressed plants with and without silicon application disappeared .In the presence of HgClappeared . After aSbPIP1;3/1;4, SbPIP1;3/1;4 (2), SbPIP1;6, SbPIP2;2, and SbPIP2;6 were up-regulated by silicon application, especially SbPIP1;3/1;4 (2) and SbPIP2;6, whose expression levels were up-regulated by 1.64- and 4.55-fold, respectively. Only SbPIP2;5 expression was decreased by silicon application under osmotic stress. After 24h PEG treatment, almost all of the SbPIP aquaporin genes were up-regulated, with the exception of SbPIP1;5 and SbPIP2;5, whose expression was not affected by silicon.As shown in sc of the stem was remarkably decreased by water deficit stress, but it was not affected by silicon application under either control or water deficit conditions .The Lnditions . FurtherSupplementary Fig. S2 at JXB online). The silicon concentration was found to be far higher in silicon-treated plants than in untreated plants . In addition, it is worth noting that Lsc was much higher than Kplant, and that osmotic stress decreased Kplant greatly, but Lsc only slightly. This suggests that Lsc is not the limiting factor for water transport in sorghum seedlings under water deficit stress. The results support a previous study’s conclusion that the leaf and root contribute the major portions of whole-plant hydraulic resistance . This indicates that silicon did not affect water uptake by influencing root surface area or root anatomy. It has been suggested that water can move radially toward the xylem along three pathways: the apoplastic, symplastic, and transcellular pathways. The symplastic and transcellular pathways are collectively referred to as the ‘cell-to-cell’ pathway (b). Previous studies have suggested that, under drought stress, the cell-to-cell pathway plays an important role in water transport in the root and is driven by the osmotic gradient between the soil and root xylem sap (Root hydraulic conductance (Lp) is usually the lowest within the liquid component of the soil–plant–air continuum , B. Lp r pathway , b. Prevylem sap . In thisylem sap . These r2 application. Under osmotic stress, most of the transpiration was repressed by HgCl2, and the difference between plants with and without silicon disappeared , stomatal conductance (B), and transpiration rate (C) in sand culture.Figure S3. Effects of silicon application and osmotic stress on vessel diameter (A) and number (B) in root.Figure S4. Effects of an aquaporin inhibitor (NaN3) on transpiration rate with and without silicon application under osmotic stress.Figure S5. Effects of silicon application and water deficit stress on vessel diameter (A) and number (B) in internodes in sand culture.


We also explored the contribution of MHC class II gene expression on transformation. Cellular alterations, such as squamous cell atypia (ASC), atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions were observed in 42% of the pups and in 67% of the adult females. Normal genital epithelium was more common in male than female pups. ASC was five times more likely to occur in older pups. Epithelial alterations were unrelated to infection by the potentially oncogenic otarine type I gammaherpesvirus (OtHV-1), but ASCUS was more common in pups with marked and severe inflammation. Expression of MHC class II DRB loci (Zaca DRB-D) by peripheral antigen-presenting leucocytes showed a slightly ‘protective’ effect for ASC. We propose that transformation of the California sea lion genital epithelium is relatively common in young animals, increases with age and is probably the result of infection by an unidentified pathogen. Expression of a specific MHC class II gene, suggestive of presentation of specific antigenic peptides to immune effectors, appears to lower the risk of transformation. Our study provides the first evidence that epithelial transformation of the California sea lion genital tract is relatively common, even from an early age, and raises questions regarding differences in sea lion cancer-detection and -repair success between geographical regions.An unusually high prevalence of metastatic urogenital carcinoma has been observed in free-ranging California sea lions stranded off the coast of California in the past two decades. No cases have been reported for sea lions in the relatively unpolluted Gulf of California. We investigated occurrence of genital epithelial transformation in 60 sea lions ( CurrentZalophus californianus) stranded along the central California coast and are now diagnosed in 26% of adult animals examined at necropsy [Carcinomas of urogenital origin (UGC) were first reported in the 1980s [necropsy . Histolonecropsy .As happens with most types of cancer, there are a number of intrinsic factors that appear to be associated with occurrence of sea lion UGC. The causal or mechanistic link for such associations remains unknown. However, interesting correlations have been reported. For instance, otarine type I gammaherpesvirus OtHV-1) ,12, and  ,12, andZaca DRB-A, increased the risk of UGC nearly fourfold [Genetic factors have also been reported to influence occurrence of sea lion UGC. For instance, lower levels of heterozygosity are more common in UGC-afflicted sea lions : this fifourfold .In contrast with the marked increase in cases of UGC in sea lions from the species’ northern breeding distribution, this condition has yet to be reported in sea lions from the Gulf of California, even though mortality and pathology surveys are conducted routinely . Taking To date, surveys to determine the presence of potentially oncogenic OtHV-1 in sea lions from the Gulf of California have not been conducted, and neither has the possibility that precancerous transformation of the genital epithelium occurs in these animals been explored. Owing to the species’ migratory behaviour and natural history, we predicted that OtHV-1, which is most likely host-adapted and must harbour a long coevolutionary history with their host, will be present in sea lions from within this geographical area in similar levels to what has been recorded for other areas of the sea lions’ distribution. If OtHV-1 is detected, taking into account the absence of reports of UGC in animals from this area, these pathogens might have a lesser role as causal factors for sea lion UGC than has been proposed. Thus, in order to test our predictions, we conducted thin-layer cell smears of the genital tract to search for cellular phenotypes typically considered as precancerous or transformed cells in humans and mice –31, and 2.2.1n=24 male and 33 female) and three adult females were sampled in 2012 at Granito rookery , in the Midriff region of the Gulf of California during three sampling seasons: July , October  and January . None of the pups sampled at one season were resampled at a later season.Fifty-seven California sea lion pups  and were protected from sunlight until staining. A 7 ml blood sample was collected from the caudal gluteal vein using a heparin-coated vacuum tube . Blood was homogenized and centrifuged immediately on site to separate the buffy coat, which was preserved with RNAlater  and kept in a liquid nitrogen dry shipper until processing. A 3 mm2 skin sample was collected from the third digit of the right hind flipper and preserved in 96% ethanol.Sea lions were captured using hoop nets and were manually restrained. Genital epithelial swabs were collected using sterile cytobrushes. For male pups, the penis was manually exposed by pushing the prepuce back and the penile shaft was gently swiped longitudinally 8 to 10 times. For female pups, the cytobrush was carefully inserted into the cervix and swiped 8 to 10 times using circular movements. The adult females were sampled as indicated above, but a sterile vaginal speculum was used to reach the cervix. Three swabs were collected as described above from each pup; one was stored in a vial containing 96% ethanol while the other swabs were used to make cytology smears. Slides were chemically fixed on site by spraying with Cytofix2.2Genomic DNA was extracted from the ethanol-preserved genital swabs and skin samples using a proteinase K digestion followed by a phenol–chloroform protocol and isopropanol precipitation . DNA wasZaca DRB) expression patterns of circulating antigen-presenting cells (APC). Fifteen micrograms of RNA were reverse-transcribed using Quantitect Reverse Transcription Kit (Qiagen). DNA contamination was avoided by the addition of DNAse I (2.5 U/reaction) during RNA extraction [Total RNA was extracted from the buffy coat samples using Trizol reagent  to determine MHC Class II DRB (hereafter traction .2.3We amplified a 510 bp fragment of the DNA polymerase (Dpol) gene of the type I otarine gamma herpesvirus in DNA extracted from the genital swabs (Dpol 697 5′-GCGGGAACGCAACTATATCCT and Dpol 65 5′-TCTTCGTCCAGTATCATTG) . All 12.http://www.ncbi.nlm.nih.gov/genbank/).Amplified products were electrophoresed on a SYBR gold-stained 1% agarose gel, and visualized on a UV transilluminator. To ensure the amplified products were not the result of non-specific amplification, bands were gel excised, column purified , cloned and bidirectionally sequenced for confirmation. Each sequence was visually inspected and compared with those reported in GenBank , was used as an internal control for amplification. SSP-PCR was performed on an ABI 3100 PCR system (Applied Biosystems) and each reaction was conducted in duplicate under the following cycling conditions: initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 45 s, with a final extension step of 72°C for 10 min. Amplified products were visualized on a 1.5% agarose gel stained with ethidium bromide. Constitutive and expressed Zaca DRB multiplicity was determined as the number of genes detected in the skin DNA and in the buffy coat cDNA, respectively. These analyses were only performed on the 57 pups sampled.Genomic DNA was examined with 10 sequence-specific primer pairs (SSP)  flankinghrough J ,25. Buff2.54) were added to 90 ml of Harris haematoxylin stain, 20 μl of hydrochloric acid (HCl) were added to 60 ml of OG6 stain and 100 μl of acetic acid (C2H4O2) were added to 60 ml of EA50 eosin prior to staining the slides. Incubation, washing and decolourization times were run according to published protocols [2 squares was placed beneath each slide to calculate the total smear surface under the stereoscope microscope. The total number of cells in the genital smears and the differential cell counts were calculated and standardized to cells per mm2. Cellular phenotypes taken into account were cells with a large, well-demarcated, clear perinuclear zone surrounded by a dense peripheral cytoplasmic rim [Epithelial slides were stained using a Papanicolaou protocol (PAP). This protocol allows the staining of nuclear chromatin, differential cytoplasmic counterstaining and cytoplasmic transparency . The PAProtocols . A 3′′×1smic rim  , squamous cell atypia (ASC), atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL), based on the 2001 Bethesda System Terminology . Brieflyalactiae –43.2.6Zaca DRB multiplicity [Zaca DRB diversity, and the presence/absence of specific Zaca DRB expressed genes. For this, we built independent logistic regression models. The effect of explanatory categorical values was examined by the use of contingency tables.For each diagnostic category we investigated the predictive value of iplicity , expressZaca DRB genes and the number of each of the transformed cell types was examined by constructing a series of independent generalized linear models, which included sex and age  as explanatory covariates. Before analyses were conducted, the distribution of each explanatory variable was assessed visually and deviations from normality were assessed by a Shapiro–Wilk test. The distributions of the transformed cell phenotypes  violated the assumptions for normality and showed a negative binomial distribution, and their values were subsequently log transformed. Heteroscedasticity, residual distribution and disproportionate influence of outliers were examined for each model [The relationship between the presence of specific ch model . The goo3.n=2) of the pups and in 33.3% (n=1) of the adult females. The Dpol sequences generated from the OtHV-1 PCR products shared high nucleotide similarity indexes with sequences previously reported .OtHV-1 was detected in the genital epithelial samples of 3.5% (2) followed by binucleated cells  and koilocytes , while adult females had higher numbers of koilocytes (mean=17.57±27.55/100 mm2). Intranuclear inclusion bodies were present only in two of the sampled sea lion pups (mean=0.01±0.05/100 mm2) and in one of the adult females (0.12/100 mm2). None of these individuals were positive for OtHV-1. Severity of inflammation increased with age  were categorized as LSIL. None of the 60 samples showed evidence of HSIL . Thirty-three pups (57.89%) and three females (100%) had at least one of the following transformed cell types: koilocytes, binucleated cells and reactive cells, with varying numbers of each . Of theswith age .Table 1F3,59=7.15, p=3×10−4, figure 1a), while koilocytes increased with age . Binucleated cells increased when pups were older, and decreased again in adult females . The occurrence of moderate and marked inflammation (infiltration of lymphocytes and neutrophils) increased with age .Some of the cellular phenotypes varied with age ; table 1p>0.05 for each cell type); however, the number of normal epithelial cells tended to be higher in male pups . When examining each pup age class separately, males had more normal epithelial cells than females at 6–8 weeks of age and at 18–20 weeks of age , nor was it related to the number of lymphocytes and neutrophils found in the cervical smear. Atypical squamous transformation (ASCUS) was more common in animals whose genital epithelium was marked and severely inflamed , and this pattern held when excluding adult females from the analysis . When examining this relationship in terms of the number of lymphocytes and neutrophils, sea lions with ASCUS tended to have more lymphocytes  but not more neutrophils than normal individuals.Infection by OtHV-1 was not associated with any of the cell types or diagnostic category (Zaca DRB multiplicity ranged from 6 to 10 genes (mean=8.81). Diversity of APC-expressed Zaca DRB genes ranged from 3 to 10 loci (mean=5.77). Neither constitutive Zaca DRB gene multiplicity nor expressed Zaca DRB gene multiplicity explained occurrence of LSIL, ASCUS and ASC .Pups’ constitutive Zaca DRB genes did not influence any of the cellular phenotypes or diagnostic categories . Similarly, individual expression of Zaca DRB genes did not appear to play a significant role in the occurrence of any of the abnormal cytological diagnostic categories, nor did it influence the number of abnormal cell types except for Zaca DRB-D, which showed protective effects. Specifically, pups that expressed Zaca DRB-D had a slightly lower risk of having ASC . The number of infiltrated neutrophils and lymphocytes was not influenced by the expression of any of the 10 Zaca DRB genes .The presence or absence of specific 4.The high prevalence of California sea lion urogenital carcinoma detected in animals stranded along the central California coast has raised concerns about sea lion health and welfare and led to interesting questions about the pathogenesis and risk factors associated with this disease . One of To date, our knowledge on the prevalence of urogenital carcinoma in sea lion populations has depended on having access to post-mortem examinations of stranded individuals ,46. Thisα) expression has strengthened the suggestion that reproductive hormones and cofactors could play a role in malignant transformation and proliferation [To date, urogenital carcinoma has been detected only in sub-adult and adult sea lions, and observed loss of oestrogen receptor  coulIn terms of the pathogens previously associated with urogenital carcinoma, the prevalence of OtHV-1 in the genital tract of the sampled animals was low. Prevalence in pups was similar to that found in the genital tract of pups sampled in US waters (between 1.5 and 12.5% ) and lowCandida, Chlamydia, Trichomonas and Mycoplasma [An explanation of the relatively common ASC found in the genital epithelium of pups could be shifts in the local microbiome during neonatal development, as has been reported for humans  and micecoplasma –43, somecoplasma ,63. ThesZaca DRB-D in circulating leucocytes had a slightly protective effect for developing ASC. Such an effect would be expected under a scenario where circulating lymphocytes that express this gene present antigens from a pathogen potentially associated with these cellular alterations of the genital epithelium, thus eliciting an immune response that decreases the pathological outcome [Zaca DRB-D would not adequately implement an acquired response against this hypothesized pathogen, so the alterations caused to epithelial cells would be higher.It is interesting to note that the expression of  outcome . Thus, aZaca DRB-A for urogenital carcinoma in adult sea lions, where animals that had that gene were four times more likely to develop cancer [Zaca DRB genes on LSIL or ASCUS. There are several explanations for the apparent discrepancy of results between the two studies. First, we examined the presence of transcribed (i.e. expressed) DRB genes, while the abovementioned study assayed the presence of specific genes in genomic DNA, which might not be expressed. Second, we focused on cytological alterations typical of transformation, not on the occurrence of cancerous tumours, which could potentially occur long after initial transformation and at that point be associated with OtHV-1, as has been reported [A previous study reported a harmful effect of p cancer . In our reported ,58. FutuIn conclusion, as recently suggested , the Cal


Chronic hepatitis C is commonly associated with extrahepatic manifestations. Cryoglobulins are observed in 40% to 60% of such patients and their presence seems to modify response to therapy. The new antivirals are greatly improving the sustained virological response (SVR); however, their high cost limits the use, leaving pegylated interferon plus ribavirin (PR) still the standard-of-care therapy worldwide. Since PR therapy is burdened with several side effects, pretreatment predictions of patients who are unlikely to respond to this regimen may avoid ineffective treatment. Variants of the interleukin-28B (IL28B) gene correlate with an SVR to PR, and combined IL28B polymorphisms may improve the prediction of treatment outcome.The potential role of both rs8099917 and rs12979860 IL28B single nucleotide polymorphisms (SNPs) combined with presence of cryoglobulins in predicting SVR to PR in hepatitis C virus (HCV)-chronically infected patients was analyzed in the present study.Single and combined IL28B SNPs (rs12979860 and rs8099917) were analyzed in 64 chronic HCV patients treated with PR showing circulating cryoglobulins and compared to 108 noncryoglobulinemic subjects to verify the predictive value on the SVR.The association of rs12979860CC or rs8099917TT with SVR was confirmed in the noncryoglobulinemic group but not in cryoglobulinemic patients. Moreover, the combined determination of both SNPs improved the prediction of SVR in noncryoglobulinemic patients but not in the cryoglobulinemic subgroup.We report that both single and combined determination of IL28B rs12979860 and rs8099917 SNPs in chronic HCV patients with circulating cryoglobulins treated with PR may have a reduced predictive value of SVR. It has been reported that more than 70% of HCV-positive patients may display an extrahepatic condition with a variable clinical impact.2 HCV infection should be considered a systemic diseases with multifaceted pathogenetic implications and clinical features. The most common and well-defined HCV-associated disease is mixed cryoglobulinemia (MC), an immune complex-related vasculitis. MC can also be considered a benign, low grade lymphoproliferative disorder sustained by B-cell clonal expansions, able to evolve into a B-cell non-Hodgkin's lymphoma (B-NHL). Even if the presence of circulating cryoglobulins may be demonstrated in 40% to 60% of HCV-infected patients, only in 10% to 15% of them a clear cryoglobulinemic vasculitis (CV) may occur.2 In a 10-year prospective study on 343 HCV-positive patients, half of which with detectable circulating cryoglobulins, it was shown that the progression rate to cirrhosis and extrahepatic complications were not different in cryoglobulinemic and noncryoglobulinemic patients.3Hepatitis C virus (HCV) infection represents a major global health problem that requires widespread active interventions for its prevention and control. Even though the exact number of HCV infections worldwide is unknown, a recent report suggests that between 64 and 103 million individuals have chronic HCV infection.4 However, pegylated interferon plus ribavirin (PR) will remain the standard of care for many HCV-infected patients worldwide,5 at least for those with lower grade of fibrosis. Since PR therapy is burdened with several side effects, pretreatment predictions of patients who are unlikely to respond to this regimen may avoid ineffective treatment. Viral load and genotypes as well as the stage of liver disease strongly predict the response to treatment.7 Moreover, host genetic differences such as age and race may also influence the response to HCV therapy.9 A genome-wide association study of patients infected by genotype 1 HCV reported that single nucleotide polymorphisms (SNPs) linked to the cytokine IFNλ3  may strongly predict the response to PR therapy.13 λ-Interferons may inhibit HCV in vitro and may trigger an antiviral cascade through the JAK-STAT pathway.14 Two biallelic SNPs rs12979860 (CT) and rs8099917 (TG), located upstream of IL28B gene, have a strong association with both spontaneous and treatment-induced HCV clearance.15 Both SNPs are in strong linkage disequilibrium, but the allele frequency of rs8099917 differs between populations worldwide so that its predictive power may vary among different cohorts.12 Nevertheless, recent findings demonstrate that, in carriers of rs12979860CT, the determination of additional genotype of rs8099917 SNP significantly improves the prediction of SVR.17In the last years, the advent of new antivirals has been greatly improving the sustained virological response (SVR), defined as undetectable HCV RNA 12 or 24 weeks after therapy conclusion, thus reducing disease progression.18IL28B rs1297860 polymorphism has been evaluated in relation to different HCV infection statuses in a series of patients by using different outcome and including hepatocellular carcinoma, MC, and NHL. The study confirmed the predictive role of IL28B C allele in spontaneous viral clearance but also a weak relation between IL28B T allele and progression to hepatocellular carcinoma, confirming a carcinogenetic model in which IL28B TT genotype could facilitate cancer development by promoting a persistent viral infection. On the other hand, IL28B CC genotype was more frequently observed in patients with MC than in those with hepatocellular carcinoma or NHL, confirming the hypothesis that viral persistence may represent a risk factor for the development of both liver and hematologic malignancies.19 The influence of IL28B rs8099917/rs12979860 on the presence of MC and response to PR treatment was retrospectively examined on 541 patients with chronic hepatitis, 175 of whom with MC. Major genotype TT/CC associated with SVR and, interestingly, was also predictive of MC.20The rs12979860/rs8099917 IL28B polymorphisms have also been analyzed in a large, prospective study of HCV-positive patients with and without MC confirming IL28B genotype as a strong independent predictor of response to interferon-based therapy also in MC patients.21We also contributed to defining the potential role of IL28 in HCV patients affected by CV and treated with PR, demonstrating that TT genotype was more commonly observed in cryoglobulinemic patients and that CC genotype was associated with higher frequency with expanded B cell clonalities in the blood, with kidney involvement and B-NHL. Very interestingly, CC genotype was immunologically characterized by a restricted B cell response and clinically associated with higher risk of renal damage and hematologic malignancies.On these bases, in the present study we aimed to verify the potential role of both rs8099917 and rs12979860 IL28B SNPs combined with presence of cryoglobulins in predicting SVR in HCV-chronically infected patients treated with PR.The study cohort included 172 Caucasian chronic hepatitis C patients attended at the CURE  or at the Institute of Internal Medicine “Baccelli” of the University of Bari. All patients were treated with PR. Treatment duration ranged from 24 to 48 weeks, depending on the individual treatment response and HCV genotype.The characteristics of the study cohort are represented in Table Baseline evaluation included disease history and stage, current signs and symptoms, and previous medications. Physical examination and laboratory values were recorded.©, Arrow Diagnostics, Genova, Italy), according to the manufacturer's instructions. The assay discriminated the different genotypes: wild-type homozygote , heterozygote , replaced homozygote , for rs12979860 and rs8099917, respectively. Fluorescence data were measured at the end of every cycle by the following profile: denaturation time of 3 min at 95°C, 35 cycle at 95°C for 15 s, and 61°C for 45 s. The results were analyzed using Allelic Discrimination Analysis. The samples were tested by duplicate and the IL28B genotypes were assigned by analysis of the reference cycle numbers for each fluorescence curve.All the DNA samples were genotyped for 2 sets of IL28B SNPs, rs12979860 and rs8099917, specifying by C or T and T or G allele, using a real-time polymerase chain reaction by fluorescent probes  medical device, using the M or the XL probe after overnight fasting following standard requirements of the Echosense.23 A stiffness of 14.8 kPa was considered the cut-off for the diagnosis of liver cirrhosis.24Liver fibrosis was assessed by transient elastography, performed with the FibroscanP values <0.05 were considered to be statistically significant. The odds ratio (OR) and the 95% confidence interval (CI) were calculated. Here, ORs >1 imply a higher chance for SVR relative to the reference category. Multivariate binary logistic regression analysis was used to identify the association of the rs12979860 and rs8099917 variations and haplotypes with SVR. In doing so, adjustments were performed regarding age, gender, HCV-RNA levels, cryoglobulinemia, and fibrosis stage. A further multivariate binary logistic regression analysis was performed to identify the association of the rs12979860 and rs8099917 variations with the presence of cryoglobulinemia, using age, gender, HCV-RNA levels, and fibrosis stage as covariates. The selection of covariates in the multivariate analyses was performed by backward selection, using P < 0.1 as a cut-off, with age and sex forced into the model. IL28B SNPs’ comparisons were made using a dominant model, in which patients carrying 1 or 2 copies of minor allele were compared with others. Statistical analysis was performed with the Statistical Package for Social Sciences version 18.0  and the package GraphPad Prism 6.0 for Windows .Data were expressed as count and percentages for qualitative values, and as mean ± standard deviation of the mean (SDM) for quantitative variables. Gaussian distribution of the samples was evaluated by Kolgomorov–Smirnov test. The significance of differences was assessed in contingency tables by Pearson's Chi-squared test and Fisher's exact test. All tests were 2-sided, and P = 3.2 × 10−5; rs8099917: P = 0.025. However, the Hardy–Weinberg equilibrium in genotype distribution was observed for the IL28B rs8099917 SNP in patients with cryoglobulinemia (P = 0.378).The overall distribution of IL28B rs12979860 CC, CT, and TT was 36.0%, 50.6%, and 13.4%, and the distribution of IL28B rs8099917 TT, TG, and GG was 58.7%, 33.7%, and 7.6%, respectively. Significant deviations from Hardy–Weinberg equilibrium in genotype distribution were observed for the SNPs as follows: rs12979860: Frequency of genotype distribution of IL28B SNPs was similar between cryoglobulinemic and noncryoglobulinemic patients  when all HCV genotypes were considered.Our data show that PR therapy is less effective in cryoglobulinemic HCV patients , HCV genotype 1, the presence of autoantibodies, cirrhosis and the presence of cryoglobulinemia  were associated with non-SVR, while no association was found for gender, geographical origin, insulin resistance, or diabetes (Table P < 0.0001) and in cirrhotic , but was not associated with a specific HCV genotype. A multivariate analysis was performed to verify the most important factors associated with the presence of cryoglobulinemia and showed that female gender was the best independent predictor , followed by age .Univariate analysis of factors negatively affecting response to PR in cryoglobulinemic CHC patients revealed that older age ( 1.106–2.22) were The association between IL28B SNPs using a dominant genetic model  and SVR is reported in Table P = 0.013; CC vs TT: P = 0.008) in good agreement with previous reports.12The association of a single IL28B SNP with SVR after PR therapy revealed a rate of 59.7%, 46.8%, and 26.1% for rs12979860 CC, CT, and TT, and 51.5%, 36.2%, and 46.2% for rs8099917 TT, TG, and GG, respectively. When the overall population was considered in the univariate analysis, the rs12979860 CC was confirmed to be significantly associated with SVR ; the presence of serum cryoglobulins was strongly associated with reduced SVR  as well as cirrhosis . In the overall population, the multivariate analysis showed a significant association for rs12979860  but not for rs8099917 .A multivariate regression model was applied to verify the best predictors of SVR .When the same model was limited to cryoglobulinemic patients  with frequencies more than 20%, which are associated with treatment outcome of at least 20% in the subpopulation of noncryoglobulinemic patients.P = 0.945; rs12979860CC/rs8099917TG vs rs12979860CC: OR = 0.901 [0.185–4.378]; P = 0.936). Similarly, in patients with the heterozygous variants of the rs12979860 nonresponder T allele, the pattern of the rs8099917 SNP did not affect the chances of achieving SVR .Considering the total cohort studied, subgroup analysis revealed that in patients with homozygous rs12979860CC genotype, the additional determination of rs8099917 had no significant effect on the prediction of SVR rate  but, once again, no significant association was observed in HCV-positive patients with cryoglobulins .We also verified if the combination of rs12979860 and rs8099917 might improve the prediction of SVR in the HCV cryoglobulinemic patients where single SNP seemed not predictive. When the subgroup analysis was performed in noncryoglobulinemic patients, the additional determination of rs8099917 to the heterozygous variants of the rs12979860 nonresponder T allele, significantly strengthens the chances of achieving SVR .In the present study, we investigated the association between the IL28B polymorphic variants in patients affected by chronic hepatitis C and circulating cryoglobulins, and whether combination of IL28B SNPs rs12979860 and rs8099917 might improve SVR prediction in such patients.13 and accordingly to some authors they may be useful in other genotypes,32 thus we did not limit the analysis to HCV genotype 1 infection. However, it is worth to note that we registered a very low number of patients infected by HCV genotype 4, which represents a limitation of the present study. Our data reveal similar IL28B genotypes distribution in HCV patients with circulating cryoglobulins as compared to chronic HCV patient without, in good agreement with previous report suggesting that genetic variations in IL28B genes proximity do not influence the development of cryoglobulinemia.18 Nevertheless, a different distribution of IL28B genotypes has been reported by a recent study, even though this result may be dependent on the geographical origin of the enrolled patients.20It has been clearly demonstrated that the IL28B SNPs are strongly predictive of SVR to PR therapy in patients infected by HCV genotype 1,33; however, 2 recent studies have reported that cryoglobulinemia is negatively associated with SVR in chronic hepatitis C patients.35 A further study reported that cryoglobulinemia is associated with low SVR rates in symptomatic patients, while asymptomatic MC presents with similar SVR rates than noncryoglobulinemic subjects.20 It is conceivable that the discrepancies might be dependent on ethnic differences.40 Moreover, this study confirms that IL28B SNPs are associated with SVR, irrespective of HCV genotype, in patients with no circulating cryoglobulins but not in those with presence of serum cryoglobulins.SVR rates resulted lower in HCV positive patients with circulating cryoglobulins. The association between cryoglobulinemia and SVR in chronic hepatitis C has been previously investigated, leading to conflicting results. In fact, association between cryoglobulinemia and higher SVR rates has been reported in a Brazilian cohort16 Even though only indicative, the present observation seems to support the utility of the additional genotyping of rs8099917 in patients without circulating cryoglobulins. On the contrary, our data suggest that the combined determination of rs12979860 and rs8099917 does not increase the predictive value of the SVR to PR in chronic hepatitis C patients with circulating cryoglobulins.The combined assessment of all SNPs showed frequencies for the most prevalent genotypes rs12979860CC/rs8099917TT, rs12979860CT/rs8099917TT, and rs12979860CT/rs8099917TG that reached the highest values of frequency (23% to 32%) as compared to the variants rs12979860CC/rs8099917GG and rs12979860CT/rs8099917GG, that had a frequency rates <2%. A previous observation found that in patients with a heterozygous variant for the nonresponder rs12979860 T allele, the additional genotyping of rs8099917 significantly improved SVR prediction, suggesting the combined determination of rs12979860 and rs8099917 genotype as a further diagnostic procedure for improving treatment decisions.20 found the combined assessment of both IL28B SNPs useful in MC; nevertheless, the Authors included a consistent group of Egyptian patients presenting with different distribution and predictive value of IL28B with respect to Caucasians.Boglione et al42In conclusion, we report that both single and combined determination of IL28B rs12979860 and rs8099917 SNPs in chronic HCV patients with circulating cryoglobulins treated with Peg-IFN plus Ribavirin may have a reduced predictive value of response. This could be dependent on several hypotheses, such as the different genetic background as well as other viral reservoirs like mononuclear cells that could explain a longer viral persistence in peripheral blood mononuclear cells with a compartmentalization of viral quasi-species.Considering the adverse effects of PR-based regimen therapy we hope that, almost in cryoglobulinemic patients and in those affecting by any kind of HCV-dependent lymphoproliferative disorder more choice will likely mean greater options both in terms of different regimens to choose as well as perhaps lower costs. This enormous progress is something that we are hopeful we will all be able to take advantage of very soon.


Penicillium expansum, the causal agent of blue mold of stored pome fruits, and several other species of filamentous fungi. This mycotoxin has genotoxic, teratogenic and immunotoxic effects in mammals, and its presence in pome fruits and derived products represents a serious health hazard. Biocontrol agents in the Pucciniomycotina, such as the yeasts Sporobolomyces sp. strain IAM 13481 and Rhodosporidium kratochvilovae strain LS11, are able to resist patulin and degrade it into the less toxic compounds desoxypatulinic acid and ascladiol.Patulin is a mycotoxin produced by Sporobolomyces sp. IAM 13481 and then study the changes of gene expression in Sporobolomyces sp. exposed to patulin. Patulin treatment leads to ROS production and oxidative stress that result in the activation of stress response mechanisms controlled by transcription factors. Upregulated Sporobolomyces genes were those involved in oxidation-reduction and transport processes, suggesting the activation of defense mechanisms to resist patulin toxicity and expel the mycotoxin out of the cells. Other upregulated genes encoded proteins involved in metabolic processes such as those of the glutathione and thioredoxin systems, which are essential to restore the cellular redox homeostasis. Conversely, patulin treatment decreased the expression of genes involved in the processes of protein synthesis and modification, such as transcription, RNA processing, translation, protein phosphorylation and biosynthesis of amino acids. Also, genes encoding proteins involved in transport of ions, cell division and cell cycle were downregulated. This indicates a reduction of metabolic activity, probably due to the high energy requirement by the cells or metabolic arrest while recovering from the insult caused by patulin toxicity.In this investigation we applied a transcriptomic approach based on RNAseq to annotate the genome of Complex mechanisms are activated in a biocontrol yeast in response to patulin. The genes identified in this study can pave the way to develop i) a biodetoxification process of patulin in juices and ii) a biosensor for the rapid and cost-effective detection of this mycotoxin.The online version of this article (doi:10.1186/s12864-016-2550-4) contains supplementary material, which is available to authorized users. Aspergillus and Penicillium. P. expansum, the causative agent of the blue mold disease of stored apples, is the main PAT producer and, as a consequence of its attack, PAT is found in different fruits especially pome fruits and derived products. PAT contamination poses a major risk for children, who consume great quantities of fruit juices.Mycotoxins are food contaminants with harmful impact on human and animal health. Their occurrence in commercialized food and feed is a consequence of fungal attacks on crops in the field and/or on stored products. Patulin (PAT) is a mycotoxin produced by species in the fungal genera The mechanisms of PAT toxicity and its effects on living cells have been explored for decades , 2. The Saccharomyces cerevisiae on cells exposed to PAT revealed increased levels of transcripts of genes involved in proteasome activity, metabolism of sulfur amino acids, and stress responses, which included transporters involved in detoxification and multidrug resistance, oxidative stress scavengers (thioredoxin and glutathione) and DNA repair genes [Schizosaccharomyces pombe confirmed the ROS-induced PAT toxicity through glutathione (GSH) depletion, and the activation of antioxidants and redox systems controlled by the transcription factor Pap1 [S. cerevisiae [Cryptococcus neoformans [S. pombe PAT also causes plasma membrane fluidization [Genome-wide analysis of the model yeast ir genes . Anothertor Pap1 , which irevisiae , 12 and oformans . Moreovedization  and chandization .In mammals, the primary target organs of PAT toxicity are the gastrointestinal tract, kidney, liver and the immune system, with acute symptoms of intestinal hemorrhage, ulceration, nausea, edema, dyspnea, convulsion, and agitation. The carcinogenic risk of PAT is classified in group 3 by the International Agency for Research on Cancer , since tP. expansum, which is still controversial and likely dependent on host factors [In addition to adverse effects on mammals, PAT inhibits microbial growth, having antibacterial, antiviral and antifungal activities, and damages plant cells . As with factors –26.P. expansum infections is crucial to prevent PAT accumulation in stored fruits. Currently, this is achieved through a combination of cold storage and treatment with chemical fungicides. However, because of ethical, technical and health issues, there is an increasing demand for alternative methods to reduce the use of chemicals; the use of biocontrol agents and/or controlled atmosphere are very promising strategies [Postharvest control of rategies .P. expansum attack on stored fruits, strategies to detoxify PAT also could provide safer juices for consumption. A number of microbes have been identified that are naturally resistant to PAT, and some have the ability of degrading it. The influence of biocontrol agents (BCAs) that are effective against P. expansum on PAT accumulation is an emerging and attractive field of study, and major findings have been recently reviewed [Pichia ohmeri and Candida sake, and a bacterium, Pantoea agglomerans, were able to reduce PAT accumulation; however, the observed reductions were not attributed to direct PAT metabolization but to the protection of fruits from infection by PAT-producing P. expansum, or by PAT absorption by cell wall and/or into the cells [. Conversely, the BCA Rhodosporidium kratochvilovae strain LS11, which is a basidiomycete yeast in subphylum Pucciniomycotina, was able to resist and detoxify PAT in vitro through two independent pathways that led to the formation of breakdown products that are much less toxic than the mycotoxin. The isomers (E) and (Z) of ascladiol, intermediate in PAT biosynthesis, were found as transient products, while desoxypatulinic acid (DPA) was identified as the final product of PAT degradation [Sporobolomyces sp. strain IAM 13481, also converts PAT to DPA and ascladiols, but in this case (E) ascladiol was found as a transient metabolite, while DPA and (Z) ascladiol were the two final breakdown products [Rhodosporidium paludigenum and other Pucciniomycotina species were found to be able to degrade PAT to DPA and/or ascladiols . Amongst the fungi there are only two studies that investigated the effect of PAT at the genome-wide level, both on S. cerevisiae, with one analyzing the highly expressed genes, and the second focusing on the differential transcriptional response of a sod1∆ mutant in comparison with the wild type strain and on the scavenging effect of ascorbic acid [Therefore, in the present study we analyzed the effects of PAT treatment on bic acid , 35. TheSporobolomyces sp. is available on the JGI website at the US Department of Energy (http://genome.jgi-psf.org/Sporo1/Sporo1.home.html). Its size is 21.2 Mb and includes 76 scaffolds, four of which contained about half of the genome, and 5536 gene models predicted and with putative functions annotated using the JGI annotation pipeline. Although a key taxon used for comparative genomics with the rust fungi [The genome sequence of the monokaryon strain IAM 13481 of Sporobolomyces sp. annotation.Several approaches were performed for the generation of a reference transcriptome, such as the use of both strand-specific and non-strand specific sequencing, combined or not with the existing gene annotation. According to BLAST similarity analyses of resulting assembled transcripts, the most accurate transcriptome was obtained by merging new genomic traits revealed by strand-specific sequencing to the existing Sporobolomyces sp. 1) yeast cells grown in the presence and in the absence of 5 μg/ml of PAT , 2) yeast cells collected from YPD agar medium after 2 days of incubation, 3) ballistospore cells fired on a YPD agar mirror plate, a spore dispersal system that we previously described [Strand-specific libraries were prepared from RNA isolated from escribed . The tabSporobolomyces genome sequence using Tophat [Sporobolomyces sp. IAM 13481 genome, for a > 95.0 % overall read mapping rate. Tophat outputs were used as inputs for Cufflinks to generate a primary transcriptome for each growth condition; in order to use the existing annotation as a guide for the assembling and for discovering novel transcripts, the RABT (Reference Annotation Based Transcript) assembly was performed. All the Cufflinks outputs were merged in a single final transcriptome through Cuffmerge. The new generated transcriptome consisted of 6595 unique genes and a total of 14462 gene models due to alternative splicing or variable 5′ and 3′ untranslated regions (UTRs), whose sequences are found in the Additional file Gene annotation was performed using the Tuxedo tools. In particular, reads were cleaned and then mapped to the g Tophat . ApproxiSporobolomyces sp. IAM 13481 were evaluated by monitoring the yeast growth in the presence of different concentrations of the mycotoxin. As showed in Fig. Sporobolomyces growth, which was characterized by a lag-phase at the higher concentrations of mycotoxin tested (50 and 75 μg/ml). As we previously reported, the lag-phase is longer when the concentration of PAT increases, and during this stage the mycotoxin is not actively degraded; when the yeast start growing, PAT is degraded and its decrease parallels the onset of the PAT degradation products [Sporobolomyces in the presence of PAT and consequently its degradation. At these tested concentrations, PAT had no morphological effects on Sporobolomyces sp. cells as examined by microscopy.As a first step the effects of PAT on products . Also a Sporobolomyces sp. cultures were incubated in the presence and in the absence of 50 μg/ml of PAT. Cells for poly(A) RNA extraction were collected at two different growth levels, within the lag-phase at optical density 595 nm (OD595) ~0.08, and OD595 ~ 0.25 during the phase of active growth . Although informative, the large amount of data obtained and the lack of biological replicates did not allow us to focus on genes playing key functions in PAT resistance and/or degradation.Non-strand specific sequencing was performed, which we found to be inadequate for an improvement of the existing annotation of oformans . DespiteSporobolomyces sp. growth just started increasing and reached an OD595 value of ~ 0.08 . As expected, species distribution of all BLAST hits reveals that the majority of them belong to the Basidiomycota phylum, with the most represented belonging to species in the Pucciniomycotina , followed by the Agaricomycotina , and the Ustilaginomycotina (Pseudozyma antartica)  Fig. . DEGs weS. cerevisiae gene name is used; where not available, the test_id number generated by Cufflinks is used.Blast2GO analysis of upregulated DEGs assigned GO annotation for 78 genes (~77 %) that were grouped under the following GO terms: oxidation-reduction process (21 DEGs), metabolic process (19 DEGs), transport (12 DEGs), and transcription factor activity (7 DEGs). Further sub-classification of the metabolic process group found 6 DEGs involved in glutathione biosynthesis and turnover and 3 DEGs involved in the thioredoxin system. For the remaining 33 upregulated DEGs either no GO terms were assigned or they belonged to underrepresented GO term groups Fig. . For genOYE2, SRX1, ZTA1, ADH1, GLR1, CCP1) and/or response to DNA replication stress , fatty acid beta oxidation (SPS19) and general stress response .The DEGs in the oxidation-reduction process group included oxidases, medium and short chain dehydrogenases, and oxidoreductases that are involved in oxidative stress resistance (ENV9 (log2FC = 4.78) and GRE2 (log2FC = 2.35). ENV9 encodes a NADP-binding protein proposed to be involved in vacuolar functions, and whose S. cerevisiae mutant shows defects in carboxypeptidase Y (CPY) processing and vacuolar morphology [GRE2 encodes a D-lactaldehyde dehydrogenase that is induced by several stresses , it is regulated by the High Osmolarity Glycerol (HOG) pathway, and is overexpressed in response to DNA replication stress [S. cerevisiae [The GO group of metabolic process included DEGs encoding proteins involved in the metabolism of different cellular compartments and that play various functions in the cell. The two most expressed DEGs of this group were rphology . GRE2 enn stress , 42. Morrevisiae .S. cerevisiae ortholog URE2 (log2FC = 4.15), which encodes a bifunctional protein that is involved in both nitrogen catabolite repression and oxidative stress response. Ure2 binds glutathione with high affinity and it has been shown to exhibit glutathione peroxidase activity in vitro; also, its crystal structure shows that it has structural similarity to glutathione S-transferase [GSH1, GTT2 and GLR1. Furthermore, our data revealed the upregulated expression of the three genes of the cytoplasmic thioredoxin system, the thioredoxins TRX1 and TRX2, and the thioredoxin reductase TRR1.Based on previous reports indicating an important involvement of glutathione , 9 and tnsferase –45, whicFLR1-encoding plasma membrane transporter of the major facilitator superfamily (MFS) (log2FC = 3.22). Flr1 is involved in the efflux of fluconazole, diazaborine, benomyl, methotrexate, and other drugs [S. cerevisiae exposed to PAT [TPO1 (log2FC = 2.29), known to export polyamines  from the cell during oxidative stress [PDR15 (log2FC = 2.61) implicated in general stress response and cellular detoxification, and the yeast cadmium factor YCF1 (log2FC = 2.25), a vacuolar glutathione S-conjugate transporter required for vacuole fusion and that has a role in detoxifying metals, and transports to the vacuole glutathione disulfide (GSSG) that is not immediately reduced [The transport category included several genes encoding transmembrane transporters, the most expressed of which was the er drugs , and it d to PAT . Other te stress , the ATP reduced , 49.CRZ1, YAP1 and SKO1) known to activate transcription of stress response genes.The last group of upregulated DEGs included those encoding predicted transcription factors classified under GO molecular function terms as specific signal transducer activity and nucleic acid binding. They included two putative transcription factors whose fungal orthologs are non-characterized, and others that include three  and DNA repair  had an expression level lower than 1.Besides the DEGs grouped under the most represented GO terms, other upregulated DEGs of interest included those encoding for a stress responsive A/B barrel domain (TCONS_00005994 log2FC = 3.99), for two retrotransposon nucleocapsid proteins  and for a predicted dienelactone hydrolase . Last, DEGs for proteins involved in cell wall assembly and maintenance  only in the presence of PAT. In order to avoid artifacts, genes having a FPKM value greater than 1.5 were selected , with the remaining having no GO sequence description and/or GO classification. Therefore, for a more comprehensive analysis of downregulated Sporobolomyces DEGs were grouped under 7 biological process categories, such as transport (28 DEGs), metabolic and biosynthesis processes  and four other groups including genes that are involved in different steps of the protein synthesis process such as transcription (8 DEGs), RNA processing (20 DEGs), translation (18 DEGs) and protein phosphorylation (7 DEGs). As mentioned above, other genes were grouped according to the model yeast databases as follows: organization of the cytoskeleton (8 DEGs), cell division (4 DEGs), chromatin functions (8 DEGs), cell wall organization and function (4 DEGs) and mitochondrial functions (11 DEGs)  Fig.   proteins that are involved in ions and sugar homeostasis, with calcium being the most represented , and ii) proteins involved in vesicle-mediated transport functions from endoplasmic reticulum (ER) to Golgi, vacuole and other cellular compartments. Within the metabolic and biosynthesis groups, the most represented DEGs were those involved in amino acid biosynthesis and regulation. This result agrees with the downregulation of DEGs involved in different steps of the protein synthesis processes, which also corroborates previous findings in revisiae , 50. MorSporobolomyces sp. incubated in the presence of 50 μg/ml of PAT at two different growth levels , 155 upregulated genes were identified, and 31 of them were in common with DEGs found in samples obtained after treatment with the lower PAT concentration (indicated as PAT5); 208 downregulated DEGs with log2FC ≤ -2 were found, and 19 were in common with PAT5  we found 204 upregulated and 188 downregulated DEGs, of which 37 and 10 were in common with PAT5, respectively  RNA extracted from 595 ~ 0.08) an increase of PAT concentration from 5 to 50 μg/ml led to a higher expression of all the DEGs selected, with the exception of YML131W and URE2. Also, for the majority of the selected genes there was a minimum of a one-unit log2FC increment of expression at 50 μg/ml, indicating that the abundance of transcripts for proteins such as Oye2, Adh1, Flr1, and others, relate to PAT toxicity and thus specifically required to defeat the cellular insult caused by the mycotoxin. Conversely, the expression of other genes  did not considerably differ at the two tested PAT concentrations, suggesting that the encoded proteins might be involved in general response to a toxic molecule , some of the most upregulated DEGs in all the samples were selected and their expression levels compared at different PAT concentrations and at two different growth stages Fig. . As expeule Fig. . FurtherPAT Fig. , suggestOYE2, ENV9, URE2, FLR1, TCONS_00006843 and TCONS_00005994 by PAT treatment were tested. All six genes had high transcript levels in response to different patulin concentrations, confirming the regulation of these genes defined by the RNAseq data (data not shown).Real time reverse transcriptase PCR was used to validate the expression levels of a subset of common genes identified in the three transcriptomic datasets. The relative expression levels of S. cerevisiae, we provide an overview of the global effects of PAT on Sporobolomyces sp. and other eukaryotes  is a major ROS involved in signaling responses and is produced by NAPDH oxidases in conjunction with superoxide dismutase [Sporobolomyces sp. adaptive response to oxidative stress generated by PAT is regulated by the transcription factors identified in our analysis, which in conjunction activate the expression of stress-response proteins, and antioxidant and detoxification systems. Of note, it is likely that an important role is played by Yap1, since this bZIP transcription factor is a key component in H2O2-mediated signaling transduction in S. cerevisiae [S. cerevisiae transcription factors upregulated following PAT and ROS exposure are Skn7, Msn2 and Msn4. However, they were not identified in our study, suggesting that the response of Sporobolomyces and S. cerevisiae to ROS involves different transcription factors, with the exception of Yap1.Based on a number of lines of evidence, hydrogen peroxide  and mitochondrial proteins  that are upregulated following oxidative stress are designed with the GO molecular function term “oxidoreductase activity”. Interestingly, the majority of these stress responsive genes were overexpressed in S. cerevisiae also in response to DNA damaging-agents [Genes of g-agents , implicag-agents , 56.S. cerevisiae [Sporobolomyces genes associated with the antioxidant molecules glutathione and thioredoxin. However, as regard the glutathione cycle, we only found upregulation of URE2, GSH1 and GLR1, while in S. cerevisiae Iwahashi and colleagues found also GRX2 and PRX1 that encode respectively the cytoplasmic glutaredoxin and the mitochondrial thioredoxin. Also, we did not detect the genes encoding for the antioxidant proteins Tsa1, Tsa2, Ahp1, Ahp2 (peroxiredoxins) and Hyr1 (thiol peroxidase). Another important scavenger system activated by Sporobolomyces in response to PAT is the thioredoxin one: on the basis of our transcriptomic data, this system involves mainly the three cytoplasmic proteins Trr1, Trx1 and Trx2. In S. cerevisiae the thioredoxin system is preceded by the peroxiredoxin cycle [Sporobolomyces sp. exposed to PAT, although not statistically significant according to Cuffdiff. These antioxidant systems are well conserved amongst fungi and their function in Sporobolomyces sp. is likely the same as in S. cerevisiae [C. neoformans in which they have been characterized [In concordance with the findings of Iwahashi et al. in revisiae , our anain cycle  whose corevisiae  and in tcterized .S. cerevisiae [Another mechanism mediated by the transcription factors in response to the oxidative stress caused by PAT is the activation of genes encoding transmembrane transporters involved in export of metabolites from the cell. The main transporters identified were the MFS member Flr1 and the ABC transporter Pdr15. We suggest that these proteins are involved in PAT efflux in a similar way as occurs in the multidrug resistance in fungi and bacteria. Also, the MFS transporter Tpo1 likely exports polyamines during PAT-induced oxidative stress, as suggested for revisiae .Sporobolomyces sp. through two independent pathways that lead to the formation of DPA and ascladiols [R. kratochvilovae [R. paludigenum [Rhodotorula mucilaginosa (our unpublished data). Following our transcriptomic analysis we suggest that PAT degradation occurs through different ways and an essential step is the formation of enzymatic adducts of PAT with GSH, as documented by Fliege and Metzler [S. cerevisiae to the oxidative stress-inducing heavy metals cadmium, nickel, arsenic and mercury [S-transferase activity [PAT is degraded in cladiols . The samhvilovae , 29, R. udigenum  and Rhod Metzler . We prop mercury –60. Of nactivity  such as Clonostachys rosea [Sporobolomyces and other sequenced Pucciniomycotina genomes. Therefore, we searched for genes encoding proteins with predicted hydrolase activity on lactone rings and found in our DEGs a predicted dienelactone hydrolase (TCONS_00000158). We previously generated a targeted mutant for this gene as well as for another gene encoding a dienelactone hydrolase, the latter not detected as being differentially expressed in our analysis. Surprisingly, both mutants had the same kinetics of PAT degradation as the Sporobolomyces wild type strain (unpublished data), indicating that these genes are not involved in PAT degradation. Perhaps suitable candidates for DPA generation could be the newly identified gene (TCONS_00005994) that encodes a protein containing a stress responsive A/B barrel domain of unknown function, another gene (TCONS_00013529) that encodes a protein designed as a catalytic LigB subunit of aromatic ring-opening dioxygenase (TCONS_00013529), or one of the upregulated short or medium chain dehydrogenases that are potentially able to degrade xenobiotic compounds.DPA is the most important metabolite of PAT degradation because i) it is characterized by a lower cytotoxicity than PAT, most likely because of its very low reactivity with thiol groups , ii) it ys rosea . Ortholo(E)-ascladiol is oxidized to PAT in one step enzymatic reaction in PAT biosynthesis [(E)-ascladiol from PAT. It has been recently suggested that the P. expansum gene PatE encoding a Glucose-methanol-choline (GMC) oxidoreductase is responsible for the last step in PAT biosynthesis [P. expansum PatE corresponds to the Sporobolomyces GMC oxidoreductase that was found to be upregulated in the present study (TCONS_00004874), and potentially this enzyme may be responsible for the conversion of PAT into ascladiol. Future studies using reverse genetics will allow the elucidation of the role of the upregulated Sporobolomyces sp. DEGs in PAT resistance and degradation.As regards ascladiol, from a chemical viewpoint it results from the hemiacetal opening of PAT and reduction of the derived aldehyde. ynthesis ; thus, iynthesis , even thS. cerevisiae exposed to ROS [Besides the role of ROS as signaling molecules, they are mainly responsible for PAT cytotoxicity. In fact, despite the rapid upregulation and high efficiency of antioxidant defenses, cells can still sustain severe organelle damage and inhibition of biological processes essential for cellular life. The cytotoxic effects of PAT have been studied for over 30 years, as reviewed in 2005 and 2010 [d to ROS . Lipid pd to ROS .P. expansum. Our study when compared with previous findings from S. cerevisiae about PAT cytotoxicity provides compelling evidence for common cellular targets and responses, yet it also discloses new aspects such as novel mechanisms of stress response activation, which may represent a specialized feature of the Pucciniomycotina fungi. Two attractive long term goals are 1) the development of a biosensor, based on upregulated Sporobolomyces and S. cerevisiae genes in response to or even that bind the mycotoxin, for user-friendly and economical procedures for PAT detection and quantification in products ready for commercialization; and 2) the functional characterization of genes involved in conversion of PAT to DPA and/or ascladiol that will create the premises for production of enzymes responsible for PAT detoxification in heterologous cell factories in which the identified genes are transformed. These enzymes could be the base for detoxification processes of products and juices derived from pome fruits.This is the first study where a next generation sequence (NGS) technique has been applied to depict the complex molecular effects of the mycotoxin PAT on a non-conventional basidiomycete yeast that is able to degrade it and has biocontrol activity against the PAT-producing fungus Sporobolomyces sp. strain IAM 13481 was used in this study. The strain was routinely cultivated on yeast peptone dextrose medium . Sporobolomyces sp. was incubated at concentration of 1×107 CFU/ml in Lilly Barnett medium  [595 in a microplate reader. Transcriptomic analysis was performed for Sporobolomyces sp. incubated in the presence of 5 μg/ml and 50 μg/ml of PAT. With 5 μg/ml of PAT, two biological replicates were used; Sporobolomyces sp. cells were added to 200 ml of LiBa medium and incubated on an orbital shaker at 24 °C until the growth reached a OD595 of ~ 0.08 (~3 h). The untreated cultures were Sporobolomyces sp. incubated in LiBa without PAT at the same growth level. The cultures were centrifuged to collect cells for RNA extraction, and an aliquot of each supernatant obtained was analyzed through HPLC as previously described [Sporobolomyces sp. cultures of 50 ml were incubated on an orbital shaker at 24 °C and cells for RNA extraction were collected by centrifugation at OD595 of ~ 0.08 from one culture, and at OD595 of ~ 0.25 from the other; untreated conditions were LiBa medium without PAT inoculated with Sporobolomyces sp. IAM 13481 reaching the same growth levels. In this case the supernatants obtained were used to assess through TLC analysis [Sporobolomyces growth and PAT degradation for several days, since it was pivotal to verify that the mechanisms behind PAT resistance and degradation were active.r Liter)  supplemeescribed  to monitanalysis  the degrSporobolomyces sp. RNA was extracted also from cells collected on YPD agar after two days of growth, and from fired ballistospores collected from a mirror YPD plate [Sporobolomyces sp. reference transcriptome . Quality and concentration of total RNA was checked by gel electrophoresis and a spectrophotometer, respectively. Libraries construction and Illumina sequencing were performed at the DNA Core Facility of the University of Missouri-Columbia. For RNA samples obtained from Sporobolomyces sp. cells incubated in the presence of 50 μg/ml of PAT and respective controls, TruSeq non-strand specific libraries were prepared. For RNA samples extracted from Sporobolomyces sp. cells incubated in the presence of 5 μg/ml of PAT and from a mirror plate, including their respective controls, TruSeq strand-specific libraries were prepared since this method allows a more accurate generation of a reference transcriptome  and annotation (.gtf format) were downloaded from the US Department of Energy’s Joint Genome Institute website (http://genome.jgi-psf.org/Sporo1/Sporo1.home.html). Cleaned reads generated from all six conditions were individually mapped to the Sporobolomyces sp. genome sequence using Tophat. Generated .bam files were used to run Cufflinks with the RABT (Reference Annotation Based Transcript) assembly, an option that it is able to integrate novel transcripts in the existing genome annotation. Cuffmerge was then used to merge all the .gtf files generated by Cufflinks into the reference transcriptome used in downstream differential analysis. Novel Sporobolomyces sp. genes were those that did not find a corresponding match in the JGI Sporobolomyces sp. genome annotation. Gene names were assigned according to the Saccharomyces genome database (SGD) following BLASTx analysis; matches with E-value higher than 1E-3 were not considered because they indicated different orthologs having only low similarity. Cuffdiff, the statistical software for gene expression analysis, was run to compare the Tophat outputs deriving from samples of Sporobolomyces sp. incubated with and without 5 μg/ml of PAT  annotation and InterProScan [Bioinformatic analyses were performed using the Tuxedo tools . First, rProScan .Sporobolomyces sp. incubated with or without 50 μg/ml of PAT at two time points were analyzed using Tophat-Cufflinks as described above; the newly generated strand-specific based reference transcriptome was used. Because samples were as single biological replicates, DEGs were selected based on the Log2FC expression values, with those ≥ 2 being upregulated and those ≤ -2 downregulated. DEGs selected were compared with those obtained for Sporobolomyces sp. incubated with 5 μg/ml of PAT, as indicative of the reliability of our analysis since these datasets originated from three different and independent conditions. Furthermore, in order to evaluate the genetic effects of the PAT concentration and the incubation time, the Log2FC expression values of the main upregulated DEGs of these three datasets were compared.Non-strand sequences generated from samples of All the supporting data are included as Additional file.


Vitamin D, a secosteroid hormone, has roles in the optimal functioning of many organ systems and illnesses . Recent 2), pregnancy. Statistical analysis was carried out by linear regression and t test with SPSS 13. This study was approved by the Internal Review Board. Informed consent was obtained.In a 10-bed general ICU at the emergency department of a tertiary teaching hospital in Florence , 71 medical patients with severe sepsis or septic shock  admitted to the ICU between January 2013 and September 2013 were studied. Vitamin D levels were measured by radioimmunoassay at admission, as well as demographic data (Table P = 0.300) or length of stay in hospital (P = 0.154). Also our data do not demonstrate a statistical significance difference at t test between the value of vitamin D in the dead group and the alive group.Most patients showed vitamin D levels below 20 ng/ml, and we have not demonstrated a statistical significance correlation in the univariate regression between vitamin D level and both SAPS (Several groups have reported an inverse association between vitamin D levels in critically ill patients, severity of disease, outcome length of ICU stay and mortality ,4. In ou


We initiated a litter manipulation experiment to test the response of litter decomposition to litter removal/addition in three successional forests in southern China, namely masson pine forest (MPF), mixed coniferous and broadleaved forest (MF) and monsoon evergreen broadleaved forest (MEBF). Results showed that litter removal decreased litter decomposition rates by 27%, 10% and 8% and litter addition increased litter decomposition rates by 55%, 36% and 14% in MEBF, MF and MPF, respectively. The magnitudes of changes in litter decomposition were more significant in MEBF forest and less significant in MF, but not significant in MPF. Our results suggest that change in litter quantity can affect litter decomposition, and this impact may become stronger with forest succession in tropical forest ecosystem.Global changes such as increasing CO The Dinghushan Biosphere Reserve (DHSBR) consists of three typical forest types in southern China at different forest successional stages in terms of age and exposure to human disturbances; hence it provides an excellent opportunity to study the response of litter decomposition to litter manipulation along a forest successional gradient. The three forest types are a pioneer masson pine forest (MPF), a transitional coniferous and broad-leave mixed forest (MF) and a climax monsoon evergreen broad-leaved forest (MFBF) No specific permits were required for the described field studies. This research station (DHSBR) belongs to South China Botanical Garden, Chinese Academy of Sciences, which also supported the study. We confirmed that the location is not privately owned. We also confirmed that the field studies did not involve endangered or protected species. Data will be made available upon request.o33’ E and 23o10’ N). Average annual precipitation is 1927 mm, with 75% occurring from March to August and only 6% from December to February The study was conducted in Dinghushan Biosphere Reserve (DHSBR), which is in the middle part of Guangdong province, southern China  Hemsl., Machilus chinensis (Champ. ex Benth.) Hemsl., Syzygium rehderianum Merr. & Perry in the canopy layer  Copel in the understory layer. The texture of top soil in MEBF is light gravel-heavy loam. The water retention capacity of top soil is the highest with field moisture capacity and wilting coefficient of 34.6% and 11.4% gravimetric water content, respectively There are three main forest types in the reserve; a pioneer masson pine forest (hereafter named as MPF), a coniferous and broad-leave mixed forest (hereafter named as MF) and a monsoon evergreen broad-leaved forest (hereafter named as MEBF) s in MPF , which wna Hance . The texpy layer  and CalaIn each forest, we used a randomized complete block design with five blocks . In each block, we set up three 1 m×1 m plots to be used as a control (CT), a litter removal (L−), and a litter addition (L+) plots making up a total of 15 plots in each forest. There was at least 3 m buffer zone between two adjacent plots to avoid overlapping effects of different treatments. The litter decomposition experiment started in February, 2007, by placing litterbags on the surface of litter layer in the plots. We removed all litter every month from the L− plots and added it to the corresponding L+ plots to cover the litterbags. Litter removal from the L− plots caused low average litter input but did not cause entire absence of litter input to the plots because the plots was receiving litter in the following days of each mouth. The controls received the normal input of aboveground litter.Schima superba Chardn. & Champ. (SS) and Castanopsis chinensis Hanc (CC) was collected in MEBF and MF. Leaf litter of SS, CC and Pinus. massoniana Lamb (PM) was collected in MPF. These species were chosen in this study because they are the dominant tree species and contribute 40–91% the total leaf litter fall in these forests Leaf litter was collected using litter traps and nylon mesh placed on the forest floor under the trees in the study sites in May and June 2006, during which litterfall is peak Litter decomposition was determined by placing fresh litter in mesh bags in the plots. A total of 2625 litter bags were prepared from 25×25 cm polyvinyl screen with 0.5×0.5 mm mesh in the bottom and 2×2 mm in the top. Leaf litter of each species was mixed before filling in the mesh bags. Each bag was filled with 10 g air-dried mass of litter. Only one litter type was put in each bag and the litter bags were evenly distributed among each plot. Litter bags were retrieved at about 3, 6, 9, 12, and 18 months  after the start of the study. Five litter bags of each species  were randomly selected and collected from each plot at each sample date. The average of the 5 litterbags of each species per plot at each sampling date was used in the statistical calculations. After removing roots, soil and other extraneous materials, the leaf residue in each litter bag was oven dried at 45°C for 48 h and weighed. Litter from the last sampling date was measured for N and P concentrations using the method as described above.Soil sampling was conducted in August 2008 (1.5 years after litter manipulation). From each plot, 5 soil cores with 2.5 cm inner diameter were collected at random points from a 0–10cm soil depth and combined to create one composite sample per plot. Changes in soil microbial biomass was studied only in MEBF using phospholipid fatty acid (PLFA) analysis as described in Bossio and Scow kt), where y is the fraction of mass remaining at a specific time t (years), “e” the base of natural logarithm, k the decomposition coefficient (year−1). To determine changes in decomposition due to treatment effect, k value changes were given by (ktrt–conk)/kcon, where ktrt is the decomposition coefficient in treatments (L− or L+), conk the decomposition coefficient in control. Nutrient content was calculated by multiplying the nutrient concentration by the mass remaining. Nutrient content was then reported as a proportion of the initial leaf content To determine litter decomposition rates we used the following decomposition model k) among forests or litter species in controls, and to test the difference in PLFAs among treatments in MEBF. Two-way ANOVA was used to determine the interaction effect of litter manipulation treatments and litter types on litter decomposition rate and nutrient remaining in each forest. Repeated measure ANOVA was used to determine the difference in litter mass remaining among forests, litter manipulation treatments and litter species. In all ANOVAs, the block was included as random factors. In addition, data was log transformed to fulfill the requirements of normality and homogeneity of variance. All analyses were conducted using SAS software . Statistically significant differences were set with P values <0.05 unless otherwise stated. Mean values ± standard errors were reported in the text.One-way Analysis of Variance (ANOVA) was used to test the difference in decomposition rate  was significantly higher in MEBF than in MF and MPF for all possible species comparison. However, no significant difference in litter decomposition rate was found between MF and MPF for all species. Average litter decomposition rates (k) were 1.97, 0.92, and 0.73 for MEBF, MF, and MPF, respectively. When compared within forest type, litter decomposition rate was significantly lower in CC leaf litter than in SS in the MEBF and MF . However, there was no significant difference among the selected three species in the MPF (P = 0.261).The leaf litter decomposition rates in control plots varied among forest types and tree species , Table 2P = 0.045 and 0.030, respectively). Repeated measure ANOVA with Turkey’ HSD test showed that mass remaining was significantly higher in L− than in control plots in the second, third, fourth and fifth sampling dates for SS leaf litter. For CC leaf litter, significant difference in litter decomposition between L− and control plots was observed only after the last sampling date . However, such a response of leaf litter decomposition rates to litter addition showed temporal variation between the two species. For SS, significant difference between control and L+ plots was found in the second, third, and fourth sampling dates, whereas the reported response for CC was observed in the third and fourth sampling dates . However, the effect of litter removal on litter decomposition varied between the two species (P = 0.033), but not for CC (P = 0.842). Repeated measure ANOVA with Turkey’ HSD test indicated that litter addition significantly increased decomposition of SS leaf litter in the third, fourth and fifth sampling dates, and significantly increased decomposition of CC leaf litter in the second and third sampling dates .In MPF, decomposition rate increased slightly with litter addition and decreased slightly with litter removal for all species , but onek values in response to the treatments decreased in the order: MEBF>MF>MPF (P<0.001). On the other hand, litter addition increased leaf litter decomposition by 55% in the MEBF, which is significantly larger than the average percent increase in leaf litter decomposition rates in MF (36%) and MPF (14%) (P<0.001).The relative change in F>MF>MPF . AverageP>0.05). In MPF, litter removal tended to increase N and P remaining and litter addition tended to decrease N and P remaining in all species, but the magnitude of these changes varied among the treatments and species. For example, litter removal significantly increased N remaining in PM (P = 0.005), P remaining in SS and CC .Nutrient (N and P) remaining in all leaf litter types was measured after the last sampling date and it varied depending on forests and species . In MEBFP = 0.074). The average total PLFAs were 55.40 (6.91), 57.94 (7.9), and 45.34 (6.1) nmol g−1 in control, L−, and L+ plots, respectively. Similarly, there was no significant difference among litter manipulation treatments for the Bac PLFAs, Fun PLFAs, and F:B  of leaf litter observed in the controls (ranging from 0.68 to 2.19) were similar to those found in subtropical forests of south China k values in our study were higher than those reported from temperate forests In the present study, decomposition rates  in tropical forest and consequently affect nutrient cycle, and for any sudden events in the forest such as hurricane and tree diseases which also changes quantity of litter input to the forest floor. However, recent mechanisms are still far from clear, thus we also suggest similar studies in temperate and other tropical forest ecosystems to further strengthen the findings.Table S1Indices of the tree structure in the three tropical forest types. The survey was conducted in February 2007.(DOC)Click here for additional data file.Table S2Initial chemical characteristics of three studied leaf litter.(DOC)Click here for additional data file.