Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections. Giardia duodenalis  is a non-invasive protozoan parasite of the upper small intestines of mammals, including humans. This parasite is a common cause of waterborne diarrhoeal disease worldwide, and is estimated to infect over 280 million individuals annually with 20,000 cases reported per year in the US alone [G. duodenalis infections can also result in the development of extraintestinal or post-infectious complications [G. duodenalis-related illness in the developing world, its impairment on development and socioeconomic improvements, and its close association with poverty, this parasite has been included on the World Health Organization’s (WHO) Neglected Diseases Initiative since 2006 [G. duodenalis has on society warrants a better understanding of the disease pathophysiology. To date, it has been established that G. duodenalis causes increased rates of enterocyte apoptosis, intestinal epithelial barrier dysfunction, shortening of small intestinal brush border microvilli in a CD8+ lymphocyte dependent manner, anion hypersecretion, and increased rates of intestinal transit . Furtheo humans , 12. The debated , 14, and debated , are assG. duodenalis virulence factors have been identified. Of these, the best characterized are ventral adhesive disc proteins and surface lectins that ensure attachment, the four pairs of flagella that confer re-colonization and movement, and variant surface proteins (VSPs) that evade host IgA-directed clearance [G. duodenalis also produces an arginine deiminase that prevents intestinal epithelial cells (IECs) from utilizing arginine, thereby impairing intestinal epithelial proliferation and nitric oxide production [G. duodenalis-mediated pathogenesis remain largely unknown. Cathepsin cysteine proteases contain an active site cysteine and histidine residue, and are categorized as clan CA cysteine proteases; in addition, these proteases are further subdivided into cathepsin B (catB) or cathepsin L (catL) superfamilies , leading to attenuated CXCL8-induced neutrophil chemotaxis [G. duodenalis pathogenesis, and help identify specific parasitic proteases that may become therapeutic targets [Only a small number of learance , 15, 16.oduction –20. Althiewed in ). Cathepiewed in , 23), intolytica , 25. Theroteases , 27 and cultures –31. Howecultures , 33. Moremotaxis . However targets .G. duodenalis trophozoites are non-invasive, the intestinal epithelium represents a primary point of a contact between host and parasite. This layer is comprised of a single layer of IECs that separate the external environment of the intestinal lumen from underlying host tissues . As suchin IECs –40; howeGiardia is known to shorten epithelial brush border microvilli, thereby contributing to malabsorptive diarrhea [in vitro, and its deletion in vivo enhances susceptibility to dextran sodium sulfate (DSS)-induced colitis due to increased rates of IEC apoptosis [Giardia has been found to cause epithelial apoptosis [G. duodenalis. Indeed, previous research has demonstrated that G. duodenalis trophozoites disrupt intestinal epithelial F-actin [G. duodenalis infection in vivo via CD4+ and CD8+ immune responses [G. duodenalis parasite products directly target villin. The objectives of this study were to characterize cysteine protease activity in G. duodenalis trophozoites during co-incubations with IECs, and to determine whether there is a role for these proteases in intestinal epithelial cytoskeletal villin breakdown and disruption.Villin is a unique cytoskeletal protein expressed in gastrointestinal, renal, and urogenital epithelial cells. Within the gut, villin is primarily expressed in the microvilli of IEC where it provides an essential function in regulating the organization of epithelial brush border microfilaments during periods of physiological stress . This prdiarrhea , 43, 44.poptosis . In its poptosis , 46. In poptosis , 47–50.  F-actin . Moreoveesponses . Howevertrans-Epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E64d) [Formononetin, the broad-spectrum clan CA cysteine protease inhibitor -r (E64d) , and ther (E64d) , the catr (E64d) , and ther (E64d)  were purin vitro [2, and 96% humidity. Cells were used between passages 22 and 34.Previous research has validated the human adenocarcinoma Caco-2 cell line (ATCC HTB-37) as a reliable model for studying giardiasis in vitro , 56, 57.G. duodenalis NF trophozoites (Assemblage A) were originally obtained following an epidemic of human giardiasis in Newfoundland, Canada [G. duodenalis S2 isolate parasites (Assemblage A) were obtained from a sheep [G. duodenalis GS/M isolate (Assemblage B) was obtained from the American Type Culture Collection (ATCC 50581) [, Canada , 39, G.  a sheep , 58, andC 50581) . TrophozC 50581) , 61 suppG. duodenalis trophozoites were collected by cold shock on ice for 30 min. Following this, 15ml tubes were pooled into 50 ml polypropylene tubes, centrifuged at 500g for 10 min at 4°C, and resulting supernatants were aspirated. The pellets were resuspended in 10ml of chilled sterile 1X phosphate buffered saline (PBS) (Sigma-Aldrich) and centrifuged at 500g for 10 min at 4°C. The resulting pellet was resuspended in Caco-2 growth media, the trophozoites enumerated using a hemocytometer, and their concentration was adjusted to a multiplicity of infection (MOI) of 10 parasites per 1 host cell (MOI of 10:1).Confluent tubes of g for 5 min. The supernatants were decanted and the pellets were washed twice more by repeating the centrifugation. Trophozoites were suspended in a final volume of 1 ml PBS. Total DNA was extracted from the trophozoites using the DNeasy Tissue Kit , using a modified protocol. Two hundred microliters of the suspended trophozoites were transferred to 1.5 ml microcentrifuge tubes, and lysed overnight at 56°C using 180 μl of lysis buffer and 20 μl of proteinase K (20 mg/ml) supplied with the DNeasy Tissue Kit. The manufacturer’s instructions were then followed to purify the DNA. Nucleic acid was eluted with 100 μl of elution buffer. Positive control, G. duodenalis cysts , and negative control, DNase-free water , were also extracted in parallel.In preparation for DNA extraction, trophozoites were harvested by placing culture tubes on ice for 10 min. Tubes were then gently inverted to mix the contents, and 2 ml of each suspension were pipetted into 15 ml centrifuge tubes , and PBS pH 7.4 was added to a final volume of 10 ml. Tubes were centrifuged at 1,000Giardia 16S rRNA gene as described [Giardia, and a restriction fragment length polymorphism (RFLP) assay for genotyping, was performed as described [A nested-PCR was performed to amplify fragments of the escribed . PCR ampescribed .G. duodenalis Assemblages and trimmed to identical lengths of 189 bp or 367 bp for 16S rRNA and gdh genes, respectively, using Bioedit v7.1.3.0 [http://www.megasoftware.net/) with a Kimura two parameter model neighbour-joining analysis, with 100 bootstraps and pair-wise deletion.DNA sequencing of the products of both 16S rRNA and gdh PCR was performed at the McGill University and Genome Quebec Innovation Centre in Montreal, QC, using a 3730xl DNA Analyser . PCR products were purified and bi-directional sequencing was performed using the same primers as the original amplifications. DNA sequences were assembled, edited and aligned using SeqScape v2.5 (ABI). Resulting consensus sequences were then aligned with representative sequence data from v7.1.3.0 . ConsensG. duodenalis trophozoites was used to assess parasite viability after 2- or 24-hour co-incubation. Subsequently, Caco-2 cell supernatants were collected, vortexed, and 10 μL of cell supernatant was analyzed on a hemocytometer. The ratio of motile, or swimming, trophozoites to total trophozoite counts was assessed as a marker of viability [Motility of iability .G. duodenalis trophozoites  were co-incubated with confluent Caco-2 monolayers at an MOI of 10:1 for 2 or 24 hours. Identical experiments were performed, except trophozoites were initially pre-treated with E-64d (10 μM), Ca-074Me (10 μM), or vehicle control  3 hours prior (see below) to co-incubation with in vitro Caco-2 monolayers. Similarly, Caco-2 monolayers were pre-treated with one of E-64d (10 μM), Ca-074Me (10 μM), ML-9 (40 μM), Z-DEVD-FMK (50 μM), or vehicle control (DMSO) prior to co-incubation with G. duodenalis trophozoites for 2 or 24 hours. In complimentary experiments, G. duodenalis NF trophozoites were co-incubated with Caco-2 monolayers grown to confluence on the bottom of 12-well plates; in this instance, G. duodenalis trophozoites were seeded into the top compartment of 0.4 μm transwells to prevent direct contact between in vitro monolayers and parasites. Incubation conditions were maintained at 37°C, 5% CO2, and 96% humidity for experimental duration. At the end of the incubation period, cell supernatants were collected and processed for cathepsin activity assays (see below), while monolayers were washed once with PBS and subsequently processed for Western blotting analysis (see below).G. duodenalis NF trophozoite sonicates for 2 hours. Briefly, culture media from confluent Caco-2 monolayers was removed, and cells were washed once with PBS. Following this, the PBS was aspirated, the Caco-2 monolayers were collected in 200μL protease inhibitor-free radioimmunoprecipitation assay (RIPA) buffer  in microcentrifuge tubes, and sonicated at level 3 for 5 seconds . Caco-2 lysates protein concentrations were adjusted to 3mg/ml using the Bradford assay method . At the same time, G. duodenalis NF trophozoites were adjusted to a concentration of 1x107 trophozoites/ml and sonicated three times at level 4 for 30 seconds on ice . The resulting Caco-2 lysates and G. duodenalis trophozoite sonicates were co-incubated for 2 hours in the presence of 10 mM DTT and in the presence or absence of E-64 (200 μM) or Ca-074Me (200 μM). After 2 hours, samples were processed for Western blotting analysis (see below).In parallel experiments, Caco-2 monolayer cellular lysates were co-incubated with g for 10 min at 4°C. The resulting supernatant was collected and protein concentrations determined using the Bradford assay method [At the end of the co-incubation for 2 or 24 hours, trophozoites were removed from the co-culture via three ice-cold PBS washes. Caco-2 monolayers were subsequently collected in RIPA buffer supplemented with a protease inhibitor cocktail tablet . After a 30 min incubation at 4°C, cellular lysates were sonicated at level 3 for 5 seconds and centrifuged at 10,000y method . Proteinhttp://rsbweb.nih.gov/ij/). The membranes were stripped using 0.5M acetic acid (45 min incubation) and 0.2 M sodium hydroxide (5 min incubation), and re-probed with a GAPDH antibody to ensure equal loading of the gels. In all instances, GAPDH was used as the loading control.Protein samples were separated via SDS-PAGE (7–12%) and transferred to nitrocellulose membranes  over 1 hour at 100V. The membranes were blocked using 5% fat-free milk solution in 1X Tris-buffered saline + 0.1% Tween (TBS-T) for 1 hour. Primary antibodies were diluted in the same 5% milk solution and incubated with the membranes overnight at 4°C. After three 15-min washes with TBS-T, HRP-conjugated secondary antibodies, also diluted in 5% milk solution, were added to the membranes for 1 hour at room temperature. The membranes were washed three times with TBS-T for 15 min each and then visualized using ECL-plus chemifluorescence detection system . ECL-plus was added the membranes for 5 min. The membranes were visualized on ECL film . The films were scanned for densitometric analysis using the software ImageJ  for 2 or 24 hours. Following this, samples were collected and processed for cathepsin activity assays and Western blotting analysis. In separate experiments, confluent tubes of G. duodenalis trophozoites were treated with E64d (10μM), Ca-074Me (10μM), or vehicle control (DMSO) for 3 hours. Following this, G. duodenalis trophozoites were harvested by cold shock on ice for 30 min and then co-incubated with Caco-2 monolayers for 2 or 24 hours. Samples were again collected for cathepsin activity assays and Western blotting analysis.Previous research has demonstrated that concentrations of 10μM E-64d or Ca-074Me do not affect activity , 67. TheG. duodenalis trophozoites with Caco-2 monolayers (as described above), supernatants, parasites, and IECs were collected and processed for cathepsin activity assays. Cell supernatants were collected, centrifuged at 500g, 4°C for 10 min and stored for further use. The supernatant pellet, representing G. duodenalis trophozoites, was resuspended in ice-cold PBS, centrifuged at 500g, 4°C for 10 min, and the trophozoite pellet re-suspended in RIPA buffer; this fraction was then sonicated at level 4 for 30 seconds on ice and centrifuged at 10,000g, 4°C for 10 min. A Bradford assay was performed and samples normalized to 1.0 mg/ml. Adherent G. duodenalis trophozoites were removed from Caco-2 monolayers by modifying a previously described protocol [2 for 60 min. Following this, the formononetin solution was aspirated, and monolayers were washed with ice-cold PBS three times. Monolayers were then collected and lysed in RIPA buffer not containing a protease inhibitor table, sonicated on level 3 for 5 seconds, and then centrifuged for 10 min at 10,000g at 4°C. A Bradford assay was performed on collected Caco-2 lysates and samples were then normalized to 3.0 mg/ml. All samples were stored at-70°C until further use.After co-incubation of protocol . In shore, Molecular Devices, Sunnyvale, CA) at 37°C with excitation and emission wavelengths of 354nm and 445nm, respectively. Measurements were recorded every 30 seconds for 5 min. For all experiments, cathepsin assay buffer was adjusted to a pH of 7.2 to mimic the luminal pH of the upper small intestine.Assessment of cathepsin cysteine protease activity was performed via recording the liberation of 7-aminomehtylcoumarin (AMC) from fluorogenic substrates, whereby cathepsin protease activity correlates to an increase in detectable relative light units (RFUs) over time , 54. To 7G. duodenalis NF trophozoites pretreated for 3 hours with E64d (10uM) or vehicle control  at 37°C, 5% CO2. In another set of experiments, Caco-2 monolayers were pre-treated with ML-9 (40μM) for 30 min prior to co-incubation with G. duodenalis NF trophozoites for 2 or 24 hours. At the end of the co-incubation, the infection medium was aspirated off out of each chamber, followed by two ice-cold PBS washes. To fix/permeabilize the cells, ice-cold methanol was then added to each well and the chamber slides were subsequently incubated at 4°C for 30 min. After two more PBS washes, the cells were blocked with heat inactivated-fetal bovine serum  for 15 min at room temperature. Primary antibodies were prepared in a solution of 2% FBS-PBS at appropriate concentrations that was subsequently administered to cells for 1 hour at 37°C. Following two PBS washes, the cells were incubated with fluorescent secondary antibodies  for 1 hour at 37°C. The cells were subsequently washed twice with PBS. Nuclei were counterstained with 1μM Hoechst fluorescent staining 33258 (Invitrogen Life Technologies) for 30 min at 37°C. The cells were washed once with PBS and the slides were mounted with Aqua Poly/Mount . Micrographs were obtained using a Leica DMR Microscope, with a Retiga 2000R  at 400x. All images were collected with the same gain and exposure time lengths. Micrographs presented in the Results section are representative images of 2 replicate monolayers from three separate experiments.Caco-2 monolayers grown to confluence in Lab-Tek chamber slides  (8–10 days) were co-incubated for 2 or 24 hours with 1.0x10All data are representative of at least three separate experiments and expressed as means ± SEM, where applicable. All statistical analyses were performed using the software, Graphpad Prism 6, which ensures normality of data prior to analysis. Comparisons between groups were made using a Student t-test or one-way ANOVA, followed by Tukey’s test for multiple comparison analysis. Statistical significance was established at p<0.05.G. dudoenalis isolates, matching the expected banding pattern for G. duodenalis Assemblage A  gene revealed identical banding patterns for both NF and S2 mblage A . DNA seqG. duodenalis trophozoites contain, as well as release, cathepsin cysteine proteases into cell supernatants [G. duodenalis trophozoites, IECs, or cell supernatants during co-incubation of parasites and IECs remain unknown. Therefore, cathepsin cysteine protease activity levels were assessed within parasites, IECs, and cell supernatants, following co-incubation. Cathepsin cysteine protease activity was determined by calculating the slope from the RFU versus time, as illustrated in G. duodenalis NF trophozoite sonicates alone  were co-incubated in Caco-2 growth media in the presence or absence of Caco-2 monolayers. Hydrolysis of the catB/L substrate ZFR-AMC  and values were not statistically significant from each other  following their exposure to Caco-2 monolayers, while this further increase could not be detected when cells were co-incubated with the Assemblage B GS/M.Slope values were determined when  ZFR-AMC  and catB ZFR-AMC  was obsech other . A signich other  and ZRR-ch other  hydrolys-2 cells . This tr-2 cells . Hydroly-2 cells  and ZRR--2 cells  was not G. duodenalis NF trophozoites were used for the rest of the study to assess whether and how parasite catB/L proteases may affect host IECs. Initial experiments sought to determine whether catB/L activity could be inhibited within G. duodenalis trophozoites via the broad-spectrum clan CA cysteine protease inhibitor E64d (10 μM) or the catB-specific inhibitor Ca-074Me (10 μM). Parasite viability was not affected by 3-hour treatment with E-64d or Ca-074Me; compared against control G. duodenalis trophozoites, no significant difference in the proportion of motile to non-motile trophozoites in groups treated with E64d, Ca-074Me, or vehicle control (DMSO) was observed -dependent manner [G. duodenalis infection in vivo [G. duodenalis parasite products are capable of directly targeting intestinal epithelial cytoskeletal proteins such as villin. As previous findings have shown that Entamoeba histolytica proteolytically degrades villin using cysteine proteases [G. duodenalis cathepsin-like cysteine proteases was able to degrade and disrupt villin.Recent research has focused on the ability of proteins , 40, 69.t manner . Moreove in vivo . It is nroteases , we hypoG. duodenalis NF trophozoites with Caco-2 monolayers for 2 hours yielded a ~ 45kDa cleavage product as determined by Western blotting  [G. duodenalis clan CA cysteine proteases affect ZO-1 in a manner similar to villin. As determined via Western blotting and corresponding densitometry, co-incubation of G. duodenalis trophozoites with Caco-2 monolayers resulted in loss of full-length ZO-1 protein at 2  –39, 69. ein at 2  and 24 , weanalysis . Interes24 hours . No signes for 2  or 24 hoes for 2 . CollectG. duodenalis infections, and implicate them as potential virulence factors. CatB/L activity was observed within G. duodenalis trophozoites and in parasite supernatants following exposure to host IECs. This activity was inhibited when parasites were pre-treated with the broad-spectrum clan CA, cysteine protease inhibitor E-64d [G. duodenalis trophozoites from any isolate, ruling out any translocation of the cysteine proteases into the cells from the supernatant. G. duodenalis trophozoites degraded and redistributed the intestinal epithelial cytoskeletal protein villin; this required direct contact between the parasite and IEC, and was prevented when parasites were pre-treated with E-64d prior to incubation with intestinal monolayers. In addition, this study elucidated that degradation and redistribution of the tight junction protein ZO-1 was not dependent on factors sensitive to inhibition with E-64d, thereby suggesting that G. duodenalis may induce multiple pathophysiological responses within host IECs, some mediated by parasite clan CA cysteine proteases, some not. Finally, this study demonstrates that degradation and disruption of villin is partially dependent upon MLCK activation. At early time points, MLCK inhibition in intestinal monolayers exposed to G. duodenalis trophozoites did not prevent villin degradation; however, MLCK inhibition prevented G. duodenalis-induced villin degradation at later time points. Collectively, these results suggest that G. duodenalis surface clan CA cysteine proteases induce villin degradation and redistribution within IECs and that this effect, at least in part, is mediated by MLCK activation within IECs.Results from this study reveal a previously unrecognized role for parasite surface clan CA cysteine proteases in the pathophysiology of or E-64d  or the cor E-64d . Co-incuG. duodenalis infections, we used a well-established in vitro cell culture model [G. duodenalis [G. duodenalis trophozoites promote the disruption of intestinal epithelial barrier proteins in vitro [in vivo [To elucidate the role of parasite cathepsin cysteine proteases in the pathophysiology of re model , 71, 72.re model , 55, 73,odenalis , 67. Resin vitro , 44, 69,[in vivo , and dis[in vivo . These sG. duodenalis lipid rafts prevents actin cytoskeletal remodelling [G. duodenalis trophozoites were unable to induce villin degradation and redistribution within IECs when the two were separated by transwells. Collectively, these results suggest that intestinal epithelial actin cytoskeletal remodelling during giardiasis is dependent at least in part on contact between G. duodenalis trophozoites and host IECs. It remains to be elucidated whether actin cytoskeletal remodelling via villin activation is necessary for establishment of G. duodenalis infection.Within the gastrointestinal tract, the cytoskeletal protein villin is exclusively expressed by IECs, and its primary function is to maintain microvillous brush border integrity during periods of stress via its ability to promote the assembly and disassembly of the actin cytoskeleton (reviewed in ). Pathogodelling . ConsistG. duodenalis infection in vivo, and is dependent on CD4+ and CD8+ lymphocytes [G. duodenalis uses more than one mechanism to disrupt villin and the intestinal epithelial brush border. A variety of invasive gastrointestinal pathogens are reliant on the intestinal epithelial expression of villin and its ability to remodel the actin cytoskeleton to facilitate entry and dissemination through host tissues [Salmonella sp. and enteropathogenic Escherichia coli induce villin redistribution, this occurs in the absence of villin proteolysis [G. duodenalis-mediated disruption of villin may significantly affect the ability of other gastrointestinal pathogens to colonize and cause disease. Moreover, G. duodenalis infections have been reported to occur simultaneously with other pro-inflammatory gastrointestinal pathogens [G. duodenalis had reduced incidence of diarrhoeal disease and fever, and lower serum inflammatory scores when compared to individuals not infected with G. duodenalis [G. duodenalis infections may prevent other pathogens from establishing and/or inducing disease within their host.Disruption of villin and the related protein ezrin occurs during the late stages of phocytes . Data fr tissues –81. Furtteolysis , 83. Theathogens –87. A reodenalis . More reG. duodenalis genome contains genes for multiple cathepsin B and L cysteine proteases, but their functions have only begun to be described [G. duodenalis cathepsin cysteine proteases are involved in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin, in keeping with the observation that Entamoeba histolytica cysteine proteases promote villin breakdown [G. duodenalis cathepsin cysteine proteases had been known to play a role in trophozoite encystation and excystation [G. duodenalis-mediated attenuation of CXCL8 did not require direct contact between parasites and host IEC monolayers [G. duodenalis trophozoites possess multiple types of cathepsin cysteine proteases, and each may have a different role in disease pathogenesis. Indeed, single-celled parasites can possess both surface-associated and secreted cathepsin cysteine proteases which may have unique roles in pathogenesis [Several parasites contain and produce cathepsin cysteine proteases . Thescribed . Resultsreakdown . The rolystation , and degystation . Importanolayers . These rogenesis –92.G. duodenalis cathepsin cysteine proteases in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin. These results support previous observations that disruption of the intestinal epithelial cytoskeleton requires intimate contact between parasites and intestinal epithelial cells. The present findings also suggest that Giardia cysteine proteases disrupt villin at least in part by engaging epithelial MLCK. Our findings are the first to indicate that G. duodenalis cathepsin cysteine proteases represent potential parasite virulence factors capable of inducing pathophysiological responses within host intestinal epithelial cells.In conclusion, our data reveal a novel role for


The polymorphism of IL28B was described as important in the pathogenesis of infections caused by some viruses. The aim of this research was to evaluate whether IL28B gene polymorphisms (SNP rs8099917 and SNP rs12979860) were associated with HAM/TSP. The study included 144 subjects were classified according to their neurological status in two groups: Group I (60 asymptomatic HTLV-1 carriers) and Group II (84 HAM/TSP patients). Blood samples were collected, and PBMC separated by Ficoll density gradient centrifugation. DNA was extracted using a commercial kit, and the DNA was stored at -80°C for later analysis. After this, the proviral load was quantified, and the rs8099917 and rs12979860 SNPs in the region of IL28B-gene were analyzed by StepOnePlus Real-time PCR System. A multivariate model analysis, including gender, age, and HTLV-1 DNA proviral load, showed that IL28B polymorphism SNP rs12979860 was not independently associated with HAM/TSP outcome. In contrast, the SNP rs8099917 allele GG was independently associated with HAM/TSP outcome . Persons with SNP rs8099917 genotype GG may present a distinct immune response against HTLV-1 infection. So, it seems reasonable to suggest that a search for IL28B polymorphisms should be performed for all HTLV-1-infected subjects in order to monitor their risk for disease development; however, since this is the first description of this finding in the literature, we should first replicate this study with more HTLV-1-infected persons to strengthen the evidence already provided by our results.


Although the etiologic difference in pathogenic properties of HTLV-1 and HTLV-2 still remains unclear, it has been suggested that it could be attributed to the differential structure and activities of their transactivating Tax proteins. Tax-1 and Tax-2, although having 85% amino acid (aa) similarity, present phenotypic differences consistent with a more robust transformation capacity of Tax-1. Interestingly, the HTLV-2C Brazilian variant present in Amerindians and in IDU from urban areas is genotypically close to HTLV-2A but Tax-2C possesses an additional 25aa in the C-terminal region similar to that of Tax-2B. We have already demonstrated that Tax-1 and Tax-2B have several common domains, but present differential cellular distribution. To add some information concerning the structure and site domains present in Tax-2C we conducted the present study. We have obtained the Tax-2C sequence from 25 different HTLV-2C subjects and analyzed the aminoacid homology between Tax-2B and Tax-2C variants. We found that they differ for amino acidic substitutions in eleven different positions that may affect cellular localization or post-translational modification. Studies on phenotypic properties and cellular localization of Tax-2C are in progress. Support: MCT/CNPq # 303545/2012-7, CAPES, IAL # 49D/2010), Brazil.


Heart rate variability (HRV) can reflect the changes in the autonomic nervous system (ANS) that are affected by apnea or hypopnea events among patients with obstructive sleep apnea hypopnea syndrome (OSAHS). To evaluate the possibility of using HRV to screen for OSAHS, we investigated the relationship between HRV and polysomnography (PSG) diagnostic indices using electrocardiography (ECG) and PSG data from 25 patients with OSAHS and 27 healthy participants. We evaluated the relationship between various PSG diagnostic indices  and heart rate variability (HRV) parameters using Spearman’s correlation analysis. Moreover, we used multiple linear regression analyses to construct linear models for the AHI, MI, and ODI. In our analysis, the AHI was significantly associated with relative powers of very low frequency (VLF [%]) , relative powers of high frequency (HF [%]) , ratio between low frequency and high frequency powers (LF/HF) , normalized powers of low frequency (LF [n.u.]) , and normalized powers of high frequency (HF [n.u.])  among patients with OSAHS. The MI was significantly related to standard deviation of RR intervals (SDNN) , VLF [%] , HF [%] , LF/HF , LF [n.u.] , HF [n.u.] , and absolute powers of very low frequency (VLF [abs])  among patients with OSAHS. The ODI was significantly correlated with VLF [%] , HF [%] , LF [n.u.] , and HF [n.u.]  among patients with OSAHS. The linear models for the PSG diagnostic indices were AHI = -38.357+1.318VLF [%], MI = -13.389+11.297LF/HF+0.266SDNN, and ODI = -55.588+1.715VLF [%]. However, the PSG diagnostic indices were not related to the HRV parameters among healthy participants. Our analysis suggests that HRV parameters are powerful tools to screen for OSAHS patients in place of PSG monitoring. Obstructive sleep apnea hypopnea syndrome (OSAHS) has a prevalence of 2% to 4% among middle-aged adults and is an independent risk factor for many systemic disorders . AdverseFull-night polysomnography (PSG) is recommended as a routine procedure in the diagnosis of OSAHS . During Heart rate variability (HRV) reflects the status of the autonomic nervous system (ANS) in patients with physiological and pathological conditions , providiOur study explored the associations between various PSG diagnostic indices  and HRV parameters; moreover, we constructed linear models to describe them. We also analyzed the changes in autonomic nervous activity among patients with OSAHS during nocturnal sleep. We hope that this study provides a theoretical basis for the use of HRV as a diagnostic index to replace PSG among patients with OSAHS.We retrospectively studied 25 patients with normal sinus rhythm (23 men and two women) who had OSAHS-characteristic symptoms and 27 healthy participants (24 men and three women) without OSAHS at Xili People’s Hospital of Shenzhen. All participants were enrolled from November 2013 to November 2014. We measured their heights and weights to calculate the body mass indices (BMIs) for all patients. Nine, four, and twelve patients had mild, moderate and severe OSAHS, respectively, in our study according to the PSG diagnostic sleeping report that two experienced doctors independently created. The exclusion criteria for this study were as follows: potential cardiovascular disease, diabetes mellitus, pulmonary disease, thyroid disease, automatic nervous system disorders, other diseases causing daytime sleepiness , a clinical history of treatment for OSAHS or a history of medication affecting the HRV analysis results. The ethics committee of Xili People’s Hospital of Shenzhen approved our study. Written consent forms were obtained from all participants.Each participant underwent eight-hour nocturnal monitoring using PSG in bed. We informed all participants not to ingest alcohol or caffeine, nap or engage in long or strenuous exercises on the day of monitoring. During the PSG monitoring, we collected physiological data including EEG, EMG, EOG, ECG, oxygen saturation, body movement, and nasal airflow. Apnea was defined as the absence of respiration for more than 10 seconds. Hypopnea was defined as the reduction of at least 50% ventilation resulting in a decrease in arterial saturation of 4% or more. OSAHS was defined as apnea or hypopnea occurring no fewer than five times per hour, lasting for at least 10 seconds. The AHI records the number of apnea-plus-hypopnea events per hour during sleep. The MI was calculated using the average value of the micro-arousals related to hourly respiratory events during sleep. The ODI assesses the average number of oxygen desaturation events per hour during sleep.Raw ECG data were acquired using a PSG machine. This machine collects ECG data by lead II. The sampling rate was 512 Hz. Because 8-hour ECG recordings are too large to analyze, we divided these signals into 96 segments to more easily and accurately distinguish R-peaks. Every five minutes was counted as one segment. The average HRV parameters of all segments were calculated as the monitored HRV parameters. We used the freely available software package ARTiiFACT 2.0  to detect R-peaks and extract inter-beat intervals. After using ARTiiFACT to detect the RR intervals, one experienced doctor marked the missing R-peaks and deselected incorrect R-peaks. Next, the software computed all of the basic HRV parameters, including the time and frequency domain parameters, using the HRV analysis module. The time domain parameters were MEANRR, MEDIANRR, MEANHR, SDNN, RMSSD, NN50, and pNN50. Frequency bands were divided into very low frequency (0.003 to 0.04 Hz), low frequency (0.04 to 0.15 Hz), and high frequency (0.15 to 0.4 Hz) categories. The frequency domain parameters were VLF [%], LF [%], HF [%], LF/HF, LF [n.u.], HF [n.u.], VLF [abs], LF [abs], and HF [abs]. Data are expressed as the mean±SD. All data were analyzed using the statistical software SPSS 17.0 . We used a two-tailed Spearman’s correlation analysis to explore the linear relationship between the OSAHS diagnostic indices and the HRV parameters. We used multiple linear regression analyses to construct linear models for AHI, MI, and ODI. These models were adjusted for baseline characteristics including age, gender, BMI, and neck circumference. We performed a power analysis for statistics. Power>0.75 was considered as reliable. P<0.05 was considered as significant.2, and 41.18±3.46 (range = 28.1–45.3) cm, respectively. These values were 38.85±12.14 (range = 6–55) years old, 166.12±13.50 (range = 135–179) cm, 75.88±13.23 (range = 25–103) kg, 27.01±2.44 (range = 15.9–32.8) kg/m2, and 37.18±4.53 (range = 30.3–46.0) cm for the control group, respectively.The mean age, height, weight, BMI, and neck circumference of the OSAHS group were 36.96±11.09 (range = 4–53) years old, 164.48±12.53 (range = 115–178) cm, 79.88±15.23 (range = 21.5–105) kg, 29.01±3.35 (range = 16.3–33.1) kg/mThe means±SDs of MEANRR, MEDIANRR, MEANHR, SDNN, RMSSD, NN50 and pNN50 of the time domain variables are presented for all patients in The AHI, MI and ODI were significantly correlated with different frequency domain variables with regard to the OSAHS group. The mean AHIs were 34.03±24.09 (range = 5.5–82.0) and 2.11±2.08 (range = 2.1–4.9) for the OSAHS and control groups, respectively. As The mean MIs were 27.52±18.62 (range = 8.3–60.5) and 6.10±5.24 (range = 4.2–13.0) for the OSAHS group and control group, respectively. The mean ODIs were 38.58±31.92 (range = 5.7–110.7) and 1.40±2.01 (range = 1.2–4.2) with regard to the OSAHS and control groups, respectively. Based on Spearman’s correlation analysis between the PSG diagnostic indices and the HRV parameters for the OSAHS group, multiple regression analyses were performed using stepwise selection to construct linear models. OSAHS is receiving increased attention because of growing concern regarding its effect on quality of life; thus, its diagnosis is particularly important. We attempted to make this complicated, traditional examination easier by analyzing the correlation between PSG indices and HRV parameters. PSG reflects respiratory and oxygenation patterns . TherefoTime domain parameters are frequently used to investigate OSAHS. Roche et al. found that the SDNN index and RMSSD remained significant predictors of OSAHS . In our Currently, spectral analyses of HRV are a frequently used to assess the autonomic modulation of heart rate. Spectral analyses provide precise information on sympathetic and parasympathetic function . In addiA previous study showed that cyclical changes in heart rate are associated with respiration . RespiraParasympathetic activity predominated in supine and quiet positions, whereas the predominance of sympathetic activity was lesser . Once paShiomi et al. found augmentation in VLF and detected a VLF peak in patients with OSAHS . In thatIn general, we found that the AHI, MI, and ODI showed significant linear relationships with certain frequency domain parameters for the OSAHS group. We also built linear models for the AHI, MI, and ODI. The models were AHI = -38.357+1.318VLF [%], MI = -13.389+11.297LF/HF + 0.266SDNN, and ODI = -55.588+1.715VLF [%]. We found many single correlations between the PSG diagnostic indices and the HRV parameters among patients with OSAHS. We should use these three models to accurately estimate the AHI, MI, and ODI. Therefore, we speculate that VLF [%], LF/HF, and SDNN are meaningful HRV parameters for clinical use. The data obtained using non-invasive methods might be more accurate than those using invasive methods because of changes in the ANS caused by the latter . Based oFinally, the present study has several limitations. First, it is obvious that PSG diagnostic indices associated with more than one HRV parameters in our study. It is a major limitation for us not to make multiple comparisons. In the subsequent research, we will improve the multiple comparisons. Second, the size of the sample was relatively small. Third, we performed a single institutional study. Forth, no subgroups were created based on severity. We are looking forward to performing large-sample and multi-center research. At last, we did not account for the changes in sleep stage that might also influence the frequency domain parameters . ChangesOSAHS is a complex medical condition that results in high morbidity and mortality rates across both developed and developing regions. The current study found significant associations between PSG diagnostic indices and HRV parameters as well as constructed linear models for the AHI, MI, and ODI with regard to OSAHS participants. The recurrence of apnea, hypopnea, and micro-arousal could result in the changes in the ANSs of OSAHS participants. HRV indicators represent the sympathetic, parasympathetic, and sympathetic-parasympathetic regulation of heart rate. PSG examination is relatively expensive and time-consuming. However, ECG examination is efficient and cheap. The strong significant correlation and our established models show that HRV examination can replace cumbersome sleep monitoring as a powerful tool to diagnose and assess the status of OSAHS.S1 Dataset(XLSX)Click here for additional data file.


The laser bandwidth grows with increasing order, but it is almost independent of power in the 1–10 W range, amounting to ~1, ~2 and ~3 nm for orders 1–3, respectively. So, the random Raman laser exhibits no degradation of output characteristics with increasing Stokes order. A theory adequately describing the unique laser features has been developed. Thus, a full picture of the cascaded random Raman lasing in fibers is shown.Random Raman lasers attract now a great deal of attention as they operate in non-active turbid or transparent scattering media. In the last case, single mode fibers with feedback via Rayleigh backscattering generate a high-quality unidirectional laser beam. However, such fiber lasers have rather poor spectral and polarization properties, worsening with increasing power and Stokes order. Here we demonstrate a linearly-polarized cascaded random Raman lasing in a polarization-maintaining fiber. The quantum efficiency of converting the pump (1.05 μm) into the output radiation is almost independent of the Stokes order, amounting to 79%, 83%, and 77% for the 1 Random lasers represent now a rapidly growing class of light sources, in which a conventional optical cavity is substituted by a multiple-scattering feedback in a disordered gain medium, such as laser-crystal or semiconductor powders, see182 molecules in a glass lattice, whereas the feedback is provided by elastic Rayleigh scattering of the SRS-induced Stokes wave on sub-micron irregularities of the glass structure, with a small part (~10−3) of scattered light coming back into the fiber. Though the feedback is very weak, it is sufficient for lasing in a kilometers-long passive fiber given that the integral Raman gain is proportional to the fiber length and pump power.For the development of new light sources, a competitive device performance becomes quite a challenge. In this sense, fiber-based random lasers4151614As shown recentlyHere we report on the first demonstration of high-order random Raman lasing in a polarization-maintaining (PM) fiber enabling linearly-polarized output radiation of extreme stability, efficiency and narrow bandwidth at a power level up to ~10 W. As the generation thresholds of the Stokes components are two times lower than those in depolarized RRFLs, we obtained up to 4th order in only 1-km long fiber. The cascaded generation of the RRFL in the all-fiber PM configuration exhibits no degradation of output characteristics with increasing Stokes order. The unique features of the cascaded RRFL are analyzed within the framework of the developed analytical model. In the studied scheme, all waves have the same linear polarization, which simplifies simulation of power conversion and spectral broadening processes. The obtained formulae predict with a high accuracy the output power and bandwidth as a function of the pump power and Stokes order, which is useful both in fundamental research and practical applications of random fiber lasers.RRFLs employing conventional single-mode fibers under depolarized pumping generate depolarized (or randomly polarized) light14151617181920212223242526R amounts to 91% at 1.11 μm (1st Stokes), 66% at 1.17 μm (2nd Stokes), 36% at 1.23 μm (3rd Stokes), and as low as 12% at 1.3 μm (4th Stokes) in correspondence with coupling ratio of the PM fiber coupler at these wavelengths.The experimental setup is schematically shown in As an output fiber end is cleaved with an angle of >10° to eliminate the Fresnel reflection, the feedback in this scheme is provided by random Rayleigh backscattering distributed along the PM fiber and by localized reflection from the FLM. When the pump-induced Raman gain becomes higher than the round trip losses in such half-open cavity, the RRFL starts to lase. The output laser power and spectra are measured by a power meter and an optical spectrum analyzer (OSA) Yokogawa AQ6370, respectively. The polarization properties of the generated radiation are investigated with the measurement scheme based on the Glan-Thompson polarizer and polarimeter, see Methods section for more details. As we use linearly polarized pumping whose axis coincides with a chosen (slow) axis of the PM fiber, the Raman gain for another polarization component is strongly discriminated and generation of one (slow) linearly polarized component is expected, similar to conventional Raman fiber lasers with polarized pumping31st (1.11 μm) to higher Stokes orders, which appear consecutively in the output spectrum , 2nd Stokes (circles) and 3rd Stokes (stars) orders at the output as functions of the input pump power. Only the residual pump wave slightly (by ~15%) attenuated in the 1-km passive fiber is present at the fiber output below the lasing threshold. Its power grows proportionally to the input pump power up to the 1st Stokes threshold (2.6 W). Then the 1st Stokes power starts to grow and the output pump power is depleted as long as almost all the pump power is converted into the Raman lasing. The 1st Stokes power grows up to the 2nd Stokes threshold (5.9 W) and then starts to deplete, and so on for the higher Stokes orders. The maximum input pump power (Pin = 13.6 W) almost corresponds to the 4th Stokes wave threshold revealing a rather low output power (~0.02 W). The absolute optical efficiency of pump-to-Stokes conversion calculated as a ratio of the corresponding j-order Stokes power PSj  to the input pump power Pin exceeds 75% for the first and second Stokes waves and approaches 70% for the third Stokes line. Those values are close to the corresponding quantum limits  and set record values for the 2nd and the 3rd Raman Stokes waves in random fiber lasers, which are only slightly lower than the maximum efficiency demonstrated for the 1st order (around 88%)19j = 1,2,3, respectively. Note that the output power in RRFLs with a half-open cavity is only weakly dependent on the FLM reflectivityhtly by ~% attenua20−1 relative to the pumpst Stokes output spectrum consists of two generation lines at 1106 and 1111 nm, accordingly. The distribution between the lines is appreciably changed with an increase in the generated power141728The linear polarization state of the components principally changes the power evolution of the generated spectra as compared to that for depolarized radiation−1 Raman peak varies in the range of 1.1–1.5 nm, 1.4–2.5 nm and 2.3–3.4 nm for the 1st, 2nd and 3rd Stokes components, correspondingly, whereas the 490-cm−1 peak for the 1st Stokes wave is considerably narrower (0.5–1.2 nm). The evolution of the spectral linewidth comprising rather large constant value at the threshold and small power-variable part is principally different from theoryThe linewidth of the first peak is plotted for all Stokes orders as a function of the power in the corresponding line . In addiTo explain these rather unique features of the cascaded PM random fiber laser, we developed an analytical theory, which is described in the next section.P0 into the j-th order Stokes electromagnetic wave at a wavelength λj with a power Pj within the framework of the balance model described in the Methods section. Under assumptions of equal attenuation for all waves and special relations for their Raman gain coefficients, we succeeded in deriving analytical solutions for their power distributions Pj(x). The output power Pj(L) of the generated j-th order Stokes wave is expressed asLet us consider the Stimulated Raman Scattering (SRS) process for inelastic scattering of the pump electromagnetic wave with a power λ0 and Pin are the wavelength and input power of the pump wave, respectively, α is the average attenuation coefficient, L and gR is the Raman gain coefficient of the 1st Stokes wave which is set for higher orders too, and j-th Stokes wave j = 1) or of the previous Stokes component playing the role of a pump at j > 1. Herewith, the power of the pump wave (or previous Stokes component) starts to decrease exponentially with increasing input pump power above the threshold:Here st, 2nd, and 3th orders. The experimental values for the threshold powers and parameters L = 1 km, α = 0.15 km−1, and gR = 2 W−1*km−1 are used in the calculations.The power curves (solid lines) calculated from rd-order generation whose wavelength is longer by 17% than that for the input pump. The model parameters start deviating noticeably from the experimental ones. If we replace the model gain coefficient gR in the formula for the 3rd-order output power by the real value ( gR3 = 1.3 W−1*km−1), the agreement between the theory and experiment becomes appreciably better. Moreover, this parameter affects only the intermediate power domain whereas the maximum generated power remains unchanged. It is clear that the power growth above the threshold and the corresponding depletion of previous component are adequately described by the exponential functions predicted by the theory for all orders.A comparison shows that the derived formulae agree quite well with the experimental data for the first and second orders of the cascaded Raman generation. The difference becomes noticeable for the 3Pin = 6 and 9.7 W, which nearly correspond to the maximum output of the 1st and 2nd Stokes waves. It is seen that the transition regions in the power distribution are described by the steep hyperbolic tangent function predicted by the theory, and different waves are almost fully separated in space. The pump distribution is concentrated near the left end (x = 0), whereas the output Stokes order is at the right end (x = L), and intermediate ones are in between. The output order number is defined only by the available pump power and fiber length.The longitudinal power distributions calculated from j-th Stokes order the kinetic model was modified accordingly taking into account that the experiment is characterized by a relatively large group velocity dispersion as compared with the integral nonlinearity and Raman gain, see Methods section for details. The resulting FWHM linewidth is expressed as a cubic-root function of the output power of the j-th Stokes wave Pjout:To obtain the spectral characteristics of the cascaded Raman generation with the Rayleigh feedback, one should treat kinetic equations20st Stokes wave generation in RFLs20which is supplemented by the pump-induced cross-phase modulation (XPM) effect. The XPM contribution can be estimated at the corresponding threshold, similar to the case of the 1γSPM and γXPM are the Kerr nonlinearity coefficients for the SPM and XPM processes, βj and Δβj are the second-order dispersion and dispersion walk-off coefficients, respectively, Pmax(j−1) is the maximum power of the (j − 1)-th order Stokes wave (or pump wave if j = 1) corresponding to the j-th Stokes generation threshold, and Δg(j) is the HWHM width of the Raman gain spectral peak. The solid curves in FWHM = ∆SPM + ∆XPM calculated with the nonlinear coefficients γSPM = γXPM/2 = 6 W−1*km−1 and experimental values for the power and Raman widths Δ j)g defines the minimum bandwidth of the generated spectrum amounting to 0.17–0.3, 0.48, 0.72 and 1.03 for the 1st, 2nd, 3rd and 4th Stokes waves, respectively, growing nearly proportionally to the pump power at corresponding threshold. At the same time, the SPM linewidth is a slowly-growing cubic root function of the generated power for all orders. This is quite different from the results obtained for the 1Another interesting spectral effect observed in the studied laser is the reduction of the linewidth for transmitted pump radiation in the regime of its strong depletion by the cascaded Raman generation see . Taking st, 2nd and 3rd Stokes waves, correspondingly. Moreover, 4th order at 1.3 μm appears at 13.6 W pumping. Therefore, conversion of the 1.055-μm pump wave to the 7th Stokes order beyond 1.55 μm with nearly the same efficiency seems feasible in our scheme at the input power of about 26 W. The pump power level will be reduced proportionally for longer fibers at the expense of decreasing transmission/efficiency, but some efficiency compensation is expected from sufficient reduction of the Rayleigh losses reaching the minimum value of ~0.2 dB/km at 1.55 μm, similar to the effect of increasing number of transmitted photons in non-polarized RRFL with a large Raman shiftThe observed power/efficiency behavior of the cascaded random RFL is also quite different from that for conventional RFLs, either linearly polarized3136Thus, we demonstrated a linearly-polarized cascaded random Raman lasing in a 1-km-long piece of a PM fiber terminated by a fiber loop mirror at one side. The quantum efficiency of converting the pump radiation (1.05 μm) to the output Stokes wave is around 80%, independent of the Stokes order, which is close to the transmission coefficient of the 1-km fiber used. The output power and efficiency of the Stokes components in the cascaded RRFLs with a half-open cavity is almost independent of the reflectivity of the terminating mirror which is different for different Stokes orders. Herewith, the laser exhibits high power stability, and its PER values exceed 22 dB for all generated orders without any polarization controller.The proposed approach enables high-efficiency generation of high-quality linearly-polarized laser radiation at almost any wavelength (including the telecom range around 1.55 μm) by using existing high-power pump sources near 1 μm. A complete description of the cascaded Raman lasing in fibers realized here (both in theory and experiment) enables development on this base of high-performance devices, which offer wide opportunities for advanced applications, especially in telecommunications and sensing based on fiber optics links, in which all-fiber PM RRFLs can be easily integrated. Linear polarization and a high power at a relatively narrow bandwidth  with depolarized output radiation and two polarization maintaining Yb-doped fiber amplifiers (YDFAs). Fiber polarization beam splitter placed behind the YDFL extracts the linearly polarized component of radiation, which is then launched into the YDFAs. As a result, the pump laser generates linearly polarized radiation at 1054.6 nm with the output power up to 15 W in the fundamental transverse mode.Pmax/Pmin), where Pmin and Pmax correspond to the minimum and maximum powers transmitted by the polarizer during the rotation of its optical axis. The transmitted power is measured by the polarimeter which provides a large dynamic range (from −60 to 10 dBm) in 1000–1350 nm spectral regions. We do not use any spectrally selective elements in the measurement setup; therefore, the PER value of an individual spectral component is measured when its output power dominates over the others . The lens and attenuator are used to focus and attenuate the output radiation in front of the polarizer. The polarization extinction ratio is defined as PER = 10log into the j-th order Stokes waves x, are written as followsIn the SRS process, every pump quantum with a frequency νj, αj and gR(j) are the frequency, attenuation and Raman gain coefficient for j-th order Stokes wave, respectively; and α0 is pump attenuation coefficient. A rather simple analytical solution for the system of differential P0(0) = Pin. In addition, the reflecting mirror is also placed at x = 0, thus ε) of the co-propagating wave (Pj+) into the counter-propagating wave (Pj−) can be substituted by a local reflector with a reflection coefficient Reff(j) ≪ 10−4 placed at the point x = LRSj where Pj+(x) reaches its maximum value:Here Pj− can be neglected in the right side of α0 = αj = α and Raman gain coefficient has the model frequency dependence:The Rayleigh scattering of the gR1 = gR:Using this relation, we can express the power threshold for the all Stokes orders obtained from the gain and loss balance in the iteration form with single gain coefficient where Pin exceeds the generation threshold for the j-th Stokes wave, the Stokes power distribution along the fiber becomes inhomogeneous:If the input pump power P0(x) and Pj(x) are the longitudinal distributions involving a specific hyperbolic tangent function for the pump (0) and j-th Stokes (j) waves at the k-th cascade (j = 1…k); and the coordinateHere Pjout in x = L under conditions is the point, where RRFL see , their dWe assume that the effective dispersion length in the studied RRFL is much larger than the gain length, which allows us to neglect the dispersion effect in the kinetic equations20ω is detuning from the line center, and solving equations (A2–A4) fromwhere γSPM is the Kerr nonlinear coefficient for the SPM processes, β is the second-order dispersion coefficient, and ΔRMS is the spectral half width. The left side describes spectral filtration over the round trip  leading to the Schawlow-Townes spectral narrowing, whereas the right side describes SPM-induced broadening. The solution of this equation iswhere j-th Stokes component in the case of gRPin ≪ βΔRMS2 is similar to that for the 1st component under the same conditionand the corresponding spectral density of the This formula converted to the −3 dB width is given together with the estimated XPM linewidth in How to cite this article: Babin, S. A. et al. High-order random Raman lasing in a PM fiber with ultimate efficiency and narrow bandwidth. Sci. Rep.6, 22625; doi: 10.1038/srep22625 (2016).


The Gag polyprotein is the main retroviral structural protein and is essential for the assembly and release of virus particles. In this study, we have analyzed the morphology and Gag stoichiometry of human T-cell leukemia virus type 1 (HTLV-1)-like particles and authentic, mature HTLV-1 particles by using cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission electron microscopy (STEM). HTLV-1-like particles mimicked the morphology of immature authentic HTLV-1 virions. Importantly, we have observed for the first time that the morphology of these virus-like particles (VLPs) has the unique local feature of a flat Gag lattice that does not follow the curvature of the viral membrane, resulting in an enlarged distance between the Gag lattice and the viral membrane. Other morphological features that have been previously observed with other retroviruses include: (1) a Gag lattice with multiple discontinuities; (2) membrane regions associated with the Gag lattice that exhibited a string of bead-like densities at the inner leaflet; and (3) an arrangement of the Gag lattice resembling a railroad track. Measurement of the average size and mass of VLPs and authentic HTLV-1 particles suggested a consistent range of size and Gag copy numbers in these two groups of particles. The unique local flat Gag lattice morphological feature observed suggests that HTLV-1 Gag could be arranged in a lattice structure that is distinct from that of other retroviruses characterized to date. Approximately 10–20 million people are infected with human T-cell leukemia virus type 1 (HTLV-1) worldwide ,4. Theseet al. [Like other retroviruses, the assembly and budding of HTLV-1 particles is directed by the viral Gag polyprotein . Brieflet al. . A previet al. . InfectiThe diameters of retrovirus particles are typically variable and commonly appear to form a normal distribution ,14,15,16In this study, a comparative analysis of HTLV-1-like particles and authentic, mature HTLV-1 particles was performed by cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission electron microscopy (STEM). These findings provide the first demonstration of the morphology of these virus-like particles (VLPs) having the unique feature of local flat Gag lattice regions that did not follow the curvature of the viral membrane and had an enlarged distance toward the membrane. Morphological features similar to that observed with other retroviruses  include gag gene expression construct  (6 human embryonic kidney 293T cells in 6 mL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Clone III were co-transfected with the pN3-HTLV-1 Gag expression construct along with an HTLV-1 envelope protein expression construct (ratio of 10:1) using GeneJet  following the manufacturer’s instructions. Twenty-four hours post-transfection, 2 mL of fresh media were added to each plate and incubated for additional 24 h at 37 °C in 5% CO2. To harvest VLPs, cell culture supernatants from transfected cells were centrifuged at 3000× g for 5 min to remove large cellular debris and then filtered through a 0.2 µm filter. The samples were then concentrated and purified in the same manner as with authentic particles.A codon-optimized HTLV-1 erlined) A. In ordg for 5 min to remove large cellular debris and then filtered through a 0.2 µm filter.Authentic HTLV-1 particles were produced from MT-2 cells, a T-cell line chronically infected with HTLV-1, which was obtained from Dr. Douglas Richman through the NIH AIDS Reagent Program, Division of Acquired Immune Deficiency Syndrome (AIDS), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) ,23. MT-2i.e., authentic virus particles or VLPs) were then ultracentrifuged through an 8% OptiPrep  cushion at 109,000× g for 1.5 h in a 50.1 Ti rotor  at 4 °C. The particle pellet was resuspended in 0.5 mL of 1× STE buffer , and overlaid onto a 4 mL 10%–40% OptiPrep gradient and centrifuged to equilibrium in a SW55 Ti rotor (Beckman) at 250,000× g for 3 h at 4 °C. The virus- or VLP-containing fraction, at about 20% OptiPrep, was removed from the gradient using a hypodermic needle. The collected virus particles were diluted 10 fold in 1 × STE and pelleted at 195,000× g for 1 h in a SW55 Ti rotor at 4 °C. Following centrifugation, the pellet was re-suspended in ~15 µL of 1 × STE at 4 °C overnight and then analyzed by cryo-TEM or STEM. The compound 2, 2′-dithiodipyridine  was used to inactivate authentic HTLV-1 infectivity prior to cryo-TEM or STEM analysis as previously described [The concentrated particles (escribed .2) and 1 to 5 µm under focus conditions using a Gatan 4 k by 4 k CCD camera .Virus and VLP samples were prepared for cryo-TEM as previously described . BrieflyCryo-TEM images were analyzed by using ImageJ software . For each virus particle or VLP analyzed, two perpendicular diameters were used to calculate the average diameter . Histogr2) was used to help to reduce mass loss (less than 1%) caused by electron radiation as well as to eliminate contamination from mobile hydrocarbons. Each point in the STEM image corresponds to an area of 0.625 nm2 over the specimen. The whole image corresponds to 512 by 512 nm in the specimen with the center of the points separated by 1 nm. A large- and a small-angle annular dark-field detector were used to digitally record the number of scattered electrons in each scanning point. The number of scattered electrons at any scanning point is proportional to the sample mass in that local region.The mass of virus particles or VLPs was determined by quantitative dark-field STEM, which was developed at the Brookhaven National Laboratory  . This mei.e., a sphere) in order to mimic the virus density profile. The diameter of the sphere was based on the dimension of the measured particle. The mass of each virus particle was then calculated using the sum of the electron densities within the mask and a scale factor, which was determined using the image of TMV and its mass per unit length  [STEM images were analyzed by using the PCMass software developed by the BNL STEM facility . Each virus particle or VLP in the STEM micrograph was first masked by a density profile model (1 kDa/Å) ,27. The vs. 8 nm)  and Rous sarcoma virus (RSV)) [HTLV-1-like particles produced using the HTLV-1 Gag-only expression construct A were obs. 8 nm) A,B. Abous (RSV)) ,28,29.i.e., in ~10% of particles analyzed) lining along the inner leaflet of the viral membrane  . The extracted viral RNA was quantified by determining the ultraviolet (UV) absorption at 260 nm and a conversion factor of 40 µg/mL × A260 optical density unit x dilution factor using a Beckman DU-65 spectrophotometer . The Thermo Scientific Pierce BCA Protein Assay Kit  was used to estimate the protein content of the same sample used to determine the VLPs’ RNA content. The VLPs’ Gag/RNA mass ratio was determined to be 14.4:1, equivalent to about 4% of the averaged molecular mass of the VLPs measured by STEM . Based uarticle) .Assuming that the mass of the Gag protein in HTLV-1-like particles is similar to that of other retroviruses, approximately 70%–90% of the total protein mass ,35, the in vitro translation of viral RNA [The same methodology used to calculate the Gag copy number in HTLV-1-like particles was used to estimate the Gag stoichiometry in authentic immature HTLV-1 particles by calculating the Gag copy number in authentic mature HTLV-1 particles. Based upon the average size of the authentic mature HTLV-1 particles, which is 113 nm, we estimated the lipid mass to be approximately 80 MDa . The viriral RNA , it was iral RNA ,38.Although HTLV-1 was the first human retrovirus to be discovered ,40, the i.e., average diameter of 110 nm without tag versus 75 nm with tag) as well as the Gag-Gag interactions, given the distinct morphological differences in the presence and absence of the fluorophore tag [The electron density of the HTLV-1 Gag lattice appears more compact than what has been previously observed for Gag lattices from HIV-1, MPMV or RSV ,28,45. Thore tag . This ishore tag . Taken tSTEM analysis led to the observation that the HTLV-1 Gag copy number distribution per particle spanned a wide range for both HTLV-1-like particles and HTLV-1 authentic particles B, which The observations made by this study emphasize both unique and common morphological features of the HTLV-1-like particles in comparison to other retrovirus immature VLPs. Future studies will include a detailed determination of the immature Gag lattice, which should provide important new insights into the unique aspects of HTLV-1 particle assembly, in particular, and new insights into retroviral assembly, in general.


Cities are a key system in anthropogenic phosphorus (P) cycling because they concentrate both P demand and waste production. Urban agriculture (UA) has been proposed as a means to improve P management by recycling cities’ P-rich waste back into local food production. However, we have a limited understanding of the role UA currently plays in the P cycle of cities or its potential to recycle local P waste. Using existing data combined with surveys of local UA practitioners, we quantified the role of UA in the P cycle of Montreal, Canada to explore the potential for UA to recycle local P waste. We also used existing data to complete a substance flow analysis of P flows in the overall food system of Montreal. In 2012, Montreal imported 3.5 Gg of P in food, of which 2.63 Gg ultimately accumulated in landfills, 0.36 Gg were discharged to local waters, and only 0.09 Gg were recycled through composting. We found that UA is only a small sub-system in the overall P cycle of the city, contributing just 0.44% of the P consumed as food in the city. However, within the UA system, the rate of recycling is high: 73% of inputs applied to soil were from recycled sources. While a Quebec mandate to recycle 100% of all organic waste by 2020 might increase the role of UA in P recycling, the area of land in UA is too small to accommodate all P waste produced on the island. UA may, however, be a valuable pathway to improve urban P sustainability by acting as an activity that changes residents’ relationship to, and understanding of, the food system and increases their acceptance of composting. People have significantly altered the P biogeochemical cycle, changing P flows between ecosystems , modifyiAnthropogenic changes to the P cycle pose a two-sided problem. On the one hand, we face scarcity of non-renewable mined-P resources ,8, with Understanding urban P cycling is a key component in understanding anthropogenic P cycling at regional and global scales . Cities In order to transform cities from centers, or hotspots, of P cycling to ecosystems that contribute to sustainable P management, we also require better information about the real potential and feasibility of proposed solutions in specific cities. One proposed solution is the use of urban and peri-urban agriculture to recycle urban P-waste back into food production at the local scale.  acknowleUnderstanding the role of UA in P recycling is particularly important in cities where new urban planning and management documents are changing to include sustainability goals. Montreal  has adopted a new organic waste management policy and is experiencing growing public, government, and private support for UA, which could impact P cycling in the city. The Quebec provincial government has mandated that 100% of all organic waste  be recycled by 2020. Assuming that compost products are applied to agricultural land, this increase in organic waste recycling would translate into P recycling. At the same time, there has been increasing public support for UA, culminating in 29,000 signatures on a petition asking the municipality for a formal public consultation process on UA in Montreal in 2011 . In respHowever, we do not know how much P is currently cycling (or being recycled) through the food and waste system of Montreal, or through the UA system on the island. Such benchmark information is essential to understand how policies and practices in Montreal may change P cycling in the future. Here, we aim to better understand the current and potential role of UA in urban P cycling and recycling by examining two key systems in urban P cycling on the island of Montreal, Canada . We quan2, population 1.98 million in 2012) is located in the Saint-Lawrence River [We used substance flow analysis  to quance River . BecauseTo calculate P flows through the Montreal food system we quantified P in food imports to the island (1), food consumed on the island (2), human urine and feces produced on the island (3), sewage waste going to the wastewater treatment plant (4), sewage treatment plant losses to the Saint-Lawrence river (5), biosolids sent to landfill (6), septic storage (7), food and green waste produced on island (8), food and green waste produced recycled through compost (9), and food and green waste produced sent to landfill , we quantified the following, where the letters refer to the symbology used in To obtain quantitative data on P flows and information on general nutrient management practices, we conducted in-person surveys with commercial farmers , private and community gardeners (83), and organizations managing collective, institutional, and work-place gardens (50) between April and November 2013. We scaled these survey results by the estimated area under UA production to calculate the overall P budget for the UA flows on the island of Montreal. McGill University Research Ethical Board approved the protocol for administering the survey, survey questions, and data management and storage protocols (REB File # 995-0213). Written consent was obtained from participants whenever possible through signature, although oral consent was also approved, and was documented by the researcher checking the consent box on the survey form  farms, which included for-profit enterprises and large-scale university farms, 2) collective, institutional, and business gardens, which included gardens where many individuals may participate in the gardening, but decisions about fertilization, management, and harvest are made collectively or centrally by an organization or agronomic advisor, and 3) community and private citizen gardens, where each individual gardener makes decisions about his/her plot of cultivated land. We used different sampling strategies for these three categories. For farms and collective gardens, we developed an initial list of UA practitioners to survey  and usedSome conversions and assumptions were necessary to transform survey answers into P flows at the garden scale and to calculate P flows for the island as a whole. We estimated the total area under UA production on the island of Montreal to scale our survey results, and thus P flows, to the whole Montreal UA system .In all scenarios we assume a sustainable situation in which P budgets are balanced  were imported to the island of Montreal in food, 0.36 Gg P (flow 5) was exported in wastewater to the Saint-Lawrence river, and 2.63 Gg P (flow 6+10) were exported to landfills . The maj-1 (flows a+c-b). That is, UA practitioners harvested less P in food and feed (0.012 GgP yr-1 flow b) than was applied to soils in 2012  are only 2.58% of the P imported to Montreal in the larger food system (comparing flow 1 to flow a). P in crops, milk, and eggs produced on the island through UA represent only 0.43% of the P in food distributed to people through grocery stores and restaurants (comparing flow 2 to flow f).Two ways to increase P recycling on the island are to increase the percentage of P inputs to UA from on-island recycled sources and to expand the area under cultivation. Increasing the percentage of recycled P inputs to UA is possible but is unlikely to play a large role in augmenting overall P recycling in Montreal, as UA practitioners already use recycled P for 73% of their fertilizer applications. However, a phone survey by the city of Montreal on UA practices indicated that only 23% of Montreal UA practitioners composted . P recyc2 of UA  to utilize all P waste currently produced  of data reported in  and the (DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


A direct antigen-sandwich ELISA using an enzyme-labeled detector antibody has been used as an easy, quick and quantitative assay for many antigens. Generation of such a quantitative and easy ELISA for various HTLV-1 specific antigens and HTLV-1-related host cell antigens will be helpful in various HTLV-1 studies. Thus, we screened our monoclonal antibody (mAb) library to find appropriate combinations of capture and HRP-labeled detector mAbs that enabled quantitation of target antigens. Using our mouse and rat mAbs, we have established direct ELISA kits specific for HTLV-1 core p24, gp46 and Tax, and Tax-induced host antigens including OX40 (CD134), OX40L (CD252) and IL-2R alpha (CD25). These kits were applicable for quantitation of antigens in not only culture supernatants and cell lysates but also human serum/plasma. Preliminary experiments using these kits showed that HTLV-1 p24 and gp46 together with OX40 and CD25, but not Tax or OX40L, were released into culture supernatants of HTLV-1-infected T cells. Interestingly, examination of plasma from various HTLV-1 infected donors showed that soluble OX40 as well as CD25 levels were significantly higher in ATL patients than the others. Soluble OX40L, p24, gp46 and Tax antigens were negative in any plasma tested. We hope that these ELISA kits will be helpful for many fields of studies on HTLV-1 and related diseases.


Giardia duodenalis trophozoites have included comparisons of the electrophoretic mobility of enzymes and of chromosomes, and sequencing of genes encoding β-giardin, triose phosphate isomerase, the small subunit of ribosomal RNA and glutamate dehydrogenase. To date, G. duodenalis organisms have been sub-classified into eight genetic assemblages (designated A–H). Genotyping of G. duodenalis organisms isolated from various hosts has shown that assemblages A and B infect the largest range of host species, and appear to be the main (or possibly only) G. duodenalis assemblages that undeniably infect human subjects. In at least some cases of assemblage A or B infection in wild mammals, there is suggestive evidence that the infection had resulted from environmental contamination by G. duodenalis cysts of human origin.Techniques for sub-classifying morphologically identical  Giardia organisms, designated Giardia duodenalis  [G. duodenalis is the only Giardia species that causes human infection; other currently recognised species in this genus include the following (hosts are mentioned in parentheses): Giardia muris (rodents) [G. microti  [G. psittaci (budgerigars) [G. ardeae (great blue herons) [G. agilis (amphibians) [Morphologically similar or identical lamblia) , can infrodents) , G. micruskrats) , G. psiterigars) , G. arde herons)  and G. ahibians) .G. duodenalis organisms. Early work of this type involved examination of the electrophoretic mobility of G. duodenalis enzymes [G. duodenalis, designated assemblages A and B [G. duodenalis emerged from study of the electrophoretic mobility of Giardia chromosomes [G. duodenalis DNA, and restriction fragment length polymorphism (RFLP) analysis and sequencing of the resulting PCR products, added further insight into the heterogeneity of the organism, confirming the existence of assemblages A and B, and – in conjunction with data from enzyme electrophoresis – delineating six additional assemblages (C–H) [From the 1980s onwards, increasingly precise methods have been developed to sub-classify morphologically identical  enzymes . In the  A and B . Additioomosomes . Polymeres (C–H) , 34–37.Giardia duodenalis genes (genetic loci) used for genotyping the organisms include genes encoding β-giardin (bg), triose phosphate isomerase (tpi), the small subunit of ribosomal RNA (ssu) and glutamate dehydrogenase (gdh) [Giardia duodenalis assemblages have been shown to be either relatively specific to certain hosts (assemblages C–H) or essentially unrestricted in terms of the species of host that they can infect  [G. duodenalis exclusively to one or other assemblage. This point is pertinent to G. duodenalis, and non-human hosts for the respective assemblages [se (gdh) . Giardiablage A) , a situaemblages .G. duodenalis cysts excreted by animals), is limited. One study from the United Kingdom suggested that contact with farm animals  and with pets  was a risk factor for giardiasis in human subjects [G. duodenalis can be transmitted between dogs and human subjects was obtained from a study in a tea-growing community in northeast India [G. duodenalis infection in human subjects and in dog(s) occupying the same household. For one such household, genetic identity between G. duodenalis in a dog and in human subject(s) was reported [G. duodenalis might have been either, or both, dog-to-human or human-to-dog. One caveat that applies to genetic studies of G. duodenalis that rely on faecal cysts as the starting material for molecular analysis is whether the presence of such cysts necessarily reflects infection, rather than resulting merely from coprophagy of faecal material containing cysts, and passage of these cysts through an animal’s gastrointestinal tract without causing infection [Unambiguous direct evidence that human giardiasis can be an example of a zoonosis, i.e. a human infection acquired from non-human hosts under “natural” conditions  [Giardia infection can occur under experimental conditions. It is, however, unclear whether the result of the experiment just described constitutes evidence for the zoonotic transmissibility of G. duodenalis, under “normal field conditions”.Dogs have been infected with organism . There ihed rat) . This woG. duodenalis organisms obtained from human subjects with Giardia infection has shown that assemblages A and B appear to be the only ones that undeniably cause human infections [G. duodenalis organisms that have genetic markers characteristic of non-A, non-B, assemblages [G. duodenalis concurrently have been described [G. duodenalis.Genotyping of fections , althougemblages , 49. Mixescribed , 18, 28.G. duodenalis organisms can show sequence differences between different copies of the same gene  [Individual ygosity) , 7.G. duodenalis has focussed on actual or presumed animal-to-human transmission, there is increasing evidence that Giardia cysts of human origin can contaminate the environment and infect wild mammals  [G. duodenalis genetic assemblages has provided a level of precision and specificity that was lacking when essentially the only tool was morphological examination of trophozoites. For example, excretion of assemblage B cysts by Australian sea lions, and relative proximity of colonies of these animals to human settlements at coastal sites, speaks to the probability of initial infection of the animals by cysts of human origin [G. duodenalis infection in freely ranging gorillas may reflect human-to-gorilla transmission of the parasite [Although much of the literature on inter-species transmission of ubjects) . The abin origin . Similarparasite , 21.G. duodenalis infection in Canadian communities during the 1990s, one ceased and the other diminished after, in each case, a beaver excreting G. duodenalis cysts was removed from the water source supplying the respective community [G. duodenalis cysts excreted by the beavers. The respective studies predated current knowledge of G. duodenalis assemblages. Archival material (Giardia organisms) from these outbreaks was, however, available for study by modern molecular techniques some two decades later [G. duodenalis, whereas the water contained G. duodenalis of assemblage B, and assemblages A and B were isolated from the infected human subjects. In the other outbreak, the beaver was found to be infected with assemblage B, whereas the infected human subjects included one with assemblage A infection [Giardia infection in beavers and in human subjects merely involves beaver-to-human transmission of the parasite, have yielded to a more nuanced appreciation of environmental contamination by human-derived G. duodenalis cysts that may infect beavers [Of two outbreaks of human ommunity . These oes later . Using snfection . Consequ beavers .


Soil OC and TSN did not differ with grazing exclusion at heavily encroached sites, but were twice as high inside enclosure compared to open grazing soils at low encroached sites. Mean SOC and TSN in soils of 0–20 cm depth were up to 120% higher than that of the 21–40 cm soil layer. Soil OC was positively related to TSN, cation exchange capacity (CEC), but negatively related to sand content. Our results show that soil OC and TSN stocks are affected by grazing, but the magnitude is largely influenced by woody encroachment and soil texture. We suggest that improving the herbaceous layer cover through a reduction in grazing and woody encroachment restriction are the key strategies for reducing SOC and TSN losses and, hence, for climate change mitigation in semi-arid rangelands.High grazing intensity and wide-spread woody encroachment may strongly alter soil carbon (C) and nitrogen (N) pools. However, the direction and quantity of these changes have rarely been quantified in East African savanna ecosystem. As shifts in soil C and N pools might further potentially influence climate change mitigation, we quantified and compared soil organic carbon (SOC) and total soil nitrogen (TSN) content in enclosures and communal grazing lands across varying woody cover i.e. woody encroachment levels. Estimated mean SOC and TSN stocks at 0–40 cm depth varied across grazing regimes and among woody encroachment levels. The open grazing land at the heavily encroached site on sandy loam soil contained the least SOC (30 ± 2.1 Mg ha Given t2 fixation in photosynthetic tissue and reduction in belowground C inputs through lower root production and higher root litter turnover [Previous studies have shown mixed results of grazing effects on soil organic carbon (SOC) and soil organic nitrogen (SON), with studies showing positive , neutralturnover , 10. Furturnover . Ruminanturnover . Changesturnover  or increturnover . Grazingturnover . The effturnover –14. Henc2 concentration and climate change [Over the last century, African savannas have been encroached by woody species despite differing climate and management practices such as variable domestic herbivore stocking rates and fire regimes . Woody ee change . Similare change . GrazingLarge areas of southern Ethiopian rangelands have become encroached by woody plant species, resulting in a substantial reduction of the herbaceous layer and, as a result, of pastoral productivity . While wA recent assessment of aboveground vegetation biomass and cover data has indicated an increasing potential for aboveground C stocks by encroaching woody plant species in a semi-arid Ethiopian rangeland . HoweverThe main aim of this study was to investigate whether soil C and N stocks have increased with woody encroachment and how this dynamics interacts with grazing practices. We expect that severely woody encroached sites will contain the most SOC and total soil nitrogen (TSN) stocks. We further would expect that long-term grazing relaxation (rangeland enclosure) will increase SOC and TSN stocks. Thirdly, we hypothesize that woody encroachment and grazing exclusion will interact and that, as a result, severely encroached sites from which grazing has been excluded for a long time would have greater SOC and TSN than sites that are less encroached and grazed.The permit for soil and plant sampling was obtained from the Yabello District Agricultural Office and Village level pastoral community leaders, which are responsible for the management of the communal rangelands of Borana pastoral community.Study sites were located in a semi-arid pastoral system within approximately 10–70 km apart in the Yabello and Dire Districts, Borana, southern Ethiopia. This semi-arid rangeland is used predominantly for livestock  production . The sitFifty years (1957–2012) of climate data  indicated a long-term mean annual precipitation of 550 mm in the region, with a 66% coefficient of variation across years. Rainfall has a bimodal distribution, with 55% of the annual precipitation occurring in March—May, followed by 30% in September—November. Mean annual air temperature is 20°C, with a mean monthly maximum of 21°C in February and a mean monthly minimum of 18.5°C in July.Historically, the land use system in Borana was largely characterized by sustainable exploitation of rangeland resources based on herd mobility associated with flexible stocking densities . Movemen-2(1TLU = 250 kg live weight)[-2[−2 during and after the 1999/2000 drought year, respectively [The livestock density in 1982, measured by aerial observation, was 14.3 and 11.9Tropical Livestock Unit (TLU) kme weight) domestice weight). By 2000eight)[-2. Similarectively . Though ectively ,33.More exclusive forms of land ownership have been introduced since the mid-1960s by the establishment of traditional rangeland enclosures and government ranches . The forBy 1980s, with the expansion of ponds, boreholes and shallow wells and government settlement programs, crop cultivation has drastically expanded into wetter and more valuable grazing areas . By 2000The study was conducted along a gradient of woody plant encroachment, representing four levels of woody encroachment in southern Ethiopian pastoral rangelands. The levels were based on the stage of woody encroachment determined through personal interviews with local people and district agricultural office managers, and also supported by ground quantification of the woody plant canopy cover and density . Woody vEach woody encroachment level was divided into two grazing regimes namely, limited grazing (hereafter named ‘enclosure’) and communal grazing land (hereafter named ‘open’) .The open2 were effectively used for sampling shrub dominated vegetation [2) were randomly nested within the larger 100 m2 plots used for woody species.We randomly selected three replicates of both grazing regimes within the same age group and 10–25 ha in size and 2–5 km apart  and weighed using a 0.1 g scale.Vegetation sampling was done at the end of the long and short rainy seasons , soil samples were collected at the end of the long rainy season. Herbaceous aboveground biomass was destructively quantified ; grass a®, Model A10) [-3) was calculated as the mass of the fine soil (<2 mm) fraction divided by the volume of the entire core to avoid overestimating the mass of the soil when stones and gravels were present [Four soil core samples at 0–20 and 21–40 cm depth were collected from each corner of the subplots using an intact soil core sampler of 6.5 cm diameter and using the intact soil core sampling method. Samplesdel A10) . A fractdel A10) . Standardel A10)  and TSN del A10) . Ammoniudel A10) , pH and del A10) . Bulk de present . Percent present :SOC(kghd = depth (cm), C = organic carbon concentration, N = total nitrogen concentration and cf is the conversion factor = (kg cm-3) ×  × .Where The influences of grazing, woody encroachment and soil depth and their interactions on SOC % and SOC stock, herbaceous root OC content, TSN % and total TSN stock, SOC:TSN ratio, and soil bulk density were evaluated using SAS version 9.1 mixed model procedures (Proc MIXED). Differences in all response variables were evaluated by treating woody encroachment level as main effect, grazing management regime and soil depth was nested within woody encroachment level, age of enclosures was considered as random effect. Mean comparisons were made using Tukey’s test (p < 0.05). All values reported are means (± SE). Linear regressions were used to determine the relationship between SOC and TSN concentration, soil texture, CEC, pH, and soil bulk density.Significant differences between herbaceous aboveground C (Hag OC) and root stocks (HR OC) were found between open and enclosure plots with the exception of the LE site which had lower stocks in a comparison to the other sites . However-1respectively, in the open grazing soils at the HE site to 81.0 ± 8.04 and 9.2 ± 1.1.32 Mg ha-1 respectively, in the enclosure soils at the LE site  to about 70% higher values in the enclosure and open grazing land of SE site (-3 (in LE enclosure) to 1.80 ± 0.05 g m-3 (in SE enclosure).Significant differences in soil bulk density were not found between open and enclosure area with the exception of the LE site which had lower soil bulk density in the enclosures compared to the open area . The bul SE site . The deeSoil OC fractions were linked to the TSN fractions (0–20 cm soil depth) for all study areas . Soil OCOur results showed that the response of herbaceous above- and belowground biomass C stocks to grazing was strongly influenced by woody encroachment levels. The pattern of the herbaceous above-and belowground biomass C stock response to grazing in most of the woody encroachment sites  is in agreement with a herbaceous biomass decrease observed in other semi-arid environments , 38. AngOur result demonstrated that the response of SOC and TSN to grazing interacted significantly with woody encroachment levels suggesting that the increasing level of woody encroachment and other related site variation  may affect the response of SOC and TSN stocks to grazing. Soil OC and TSN stocks were significantly higher in the enclosures than in the open grazed in low woody encroachment levels on sandy clay soils, but the difference in SOC and TSN was statistically the same in moderate to highest encroachment levels on sandy loam soils. The increase maybe related to increased vegetation (woody and herbaceous) production, litter quality and nutrient cycling , and theHigh SOC and TSN (SOM) in the enclosures can also potentially improve soil physical properties such as soil structure and total porosity, which in turn may increase water infiltration rates into the soil , 58. In -1fall within the range reported by earlier studies for tropical woodland and savanna ecosystems, i.e.,20–80 Mg ha-1[On average, our SOC stocks of about 44Mg ha0 Mg ha-1, 61.Our 0 Mg ha-1. The fin0 Mg ha-1. Jackson0 Mg ha-1. Hudak e0 Mg ha-1. Woody s0 Mg ha-1, which m0 Mg ha-1.Prosopis can establish in a southern Texas savanna rangeland [However, several studies have shown that many other biotic and abiotic factors can determine SOC stocks . Soil prangeland . Similarangeland . Similarangeland . Soils wangeland .The histangeland . Hence, angeland . Furtherangeland . TherefoLack of sufficient replications in our experimental design limits us to isolate the role of woody encroachment for SOC and TSN storage decline at heavily encroached sites in the presence of uncontrolled many potential factors  that could determine the SOC stocks in these semiarid rangelands. On the basis of our data presented here SOC and TSN stocks tended to decrease as a result of the expansion of woody encroachment into semiarid savanna ecosystem. However, as savanna soils, vegetation structure and climate are highly variable and the SOC and TSN storage can be determine by these factors further field studies will be needed to evaluate the large scale net effects of woody plants encroachment and site characteristics on SOC storage in the Borana rangeland ecosystem.The results show that SOC and TSN stocks were partly affected by woody encroachment and grazing management but the magnitude of their effect depended on soil sand content. High woody cover did not increase or maintained SOC and TSN stocks at our sites on sandy loam soils. Hence improving the herbaceous layer cover through a reduction in livestock grazing and woody encroachment restriction are the key strategies to maintain SOC and TSN stocks or reduce their losses and, thereby, for climate change mitigation in semi-arid rangelands.


Several investigations in Latin America have monitored health problems in urban spaces using what is called “sentinel areas” On the basis of this strategy, we detected an overall HTLV-1 prevalence of 1.7% in a representative sample of general population in Salvador, Northeast Brazil. In order to evaluate the sexual transmission of HTLV-1 in the general population of the city of Salvador, Northeast Brazil, the study analyzed a collection of 3,451 serum samples obtained by a simple random sampling procedure without replacement. Samples were in 30 “sentinel areas” f the city of Salvador, Bahia, Northeast Brazil collected from May 1998 to July 2000. HTLV-1 Syphilis and HIV infection were tested as a sexual transmission marker. Overall prevalence of HTLV-1 was 1.48% . Sixty-two percent of the seropositive individuals were women and the majority (65.3%) earned two minimum wages or less. Overall prevalence of syphilis and HIV was 26.67%  and 0.6% , respectively. Syphilis was present in 12 out of 51 (23.53%) individuals infected by HTLV-1 , while only one person was infected with HIV (4.76%). HTLV-1 infection was associated with family income , age  and syphilis . In the logistic regression analysis stratified by sex, HTLV-1 infection remained associated with age and syphilis diagnosis in males and with age, income and syphilis diagnosis in females. These data strongly suggest a predominance of heterosexual transmission of HTLV-1 in Salvador, Bahia. Furthermore, the majority of infected individuals were poor, had low levels of education and lived in worse living conditions, confirming our previous results.