extfieldsofstudy: Medicine Biology

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Risk factors and prevalence of antibodies for Toxoplasma gondii in diaphragmatic fluid in wolverines (Gulo gulo) from the Northwest Territories, Canada

Toxoplasma gondii, a zoonotic food borne parasite that can infect almost all warm-blooded animals including people, and ranks 4th among 24 most significant global foodborne parasites listed by the World Health Organization/United Nations Food and Agriculture Organization (FAO/WHO, 2014). Exposure to T. gondii has been reported in wildlife and people in the Canadian North, despite low densities of feline definitive hosts. The ecology of this host-parasite system could be affected by changing climate and landscape in boreal and sub-Arctic regions, and surveillance data are critically needed. Wolverines are an economically and culturally important species in northern Canada due to their valuable fur. Fluid obtained from diaphragmatic muscle of 127 wolverines (Gulo gulo) were tested for antibodies to T. gondii using an enzyme linked immunosorbent assay (ELISA). A seroprevalence of 62% (Confidence Interval (CI): 53–71%) was observed. This result indicates high levels of exposure, likely either through environmental contamination with T. gondii oocysts shed by infected wild felids, or consumption of carcasses/offal of other intermediate hosts containing tissue cysts with bradyzoites in tissues. We examined factors associated with seropositivity, including age, sex, harvest location, harvest location with respect to treeline, and body condition index. Adult (≥2 years) wolverines had 5.2 times higher odds of being sero-positive than juvenile (<1 years) wolverines. The highest seroprevalence was observed in wolverines from Sahtu and South Slave regions. Proportion of sero-positive wolverines harvested above and below the tree line was not significantly different (60% vs 65%). Age was the only significant predictor of T. gondii exposure in wolverines (using logistic regression analysis); further studies should target larger sample sizes. This study is an example of how fluid from diaphragmatic muscle can be used for screening for T. gondii antibodies in wolverines. The diaphragm, commonly collected for screening for another food borne parasite, Trichinella, in wildlife harvested for human consumption, can be used for screening of T. gondii exposure in wildlife. Due to their predatory and scavenging lifestyle and high trophic level, wolverines could serve as a sentinel species for T. gondii.

Introduction
Toxoplasmosis, an important food borne parasitic zoonosis, has been reported in almost all warm-blooded vertebrates, including livestock, wildlife, birds, marine mammals and humans (Dubey, 2010). Toxoplasma gondii ranks 4th among 24 most significant global foodborne parasites listed by the World Health Organization/United Nations Food and Agriculture Organization (FAO/WHO, 2014). Toxoplasma gondii has three infective stages in its life cycle: sporozoites (in oocyst), tachyzoites and bradyzoites. Felids are the only definitive host, where sexual reproduction of the parasite results in excretion of oocysts in feces. Intermediate hosts become infected by ingesting food or water contaminated with oocysts, which ultimately develop into bradyzoites in tissues. Ingestion of tissue cysts containing bradyzoites can also lead to T. gondii infection. Infection with T. gondii may occur by sexual (via semen) or transplacental routes. In humans, the infection is often asymptomatic, but clinical manifestation can range from mild to life-threatening, particularly among immunocompromised persons and fetuses Gajadhar et al., 2006;Halonen and Weiss, 2013;Hill and Dubey, 2002;Jones et al., 2009).
Exposure to T. gondii is common in people and animals in northern Canada, with seroprevalence as high as 65% in humans (Curtis et al., 1988), 62.5% in dogs (Salb et al., 2008) and 3-40% in wildlife [Caribou/Reindeer (Rangifer tarandus), Muskoxen (Ovibos moschatus) etc.] has been reported in some studies (Johnson et al., 2010;Kutz et al., 2001;Simon et al., 2011). Higher seroprevalence of T. gondii in muskox from the Northwest Territories (NWT), Canada than from Greenland (5% vs. 3%) (Clausen and Hjort, 1986;Kutz et al., 2000) as well as in reindeer from the NWT (40%) (Kutz et al., 2001) than Norway (1%) (Vikoren et al., 2004) and Alaska (6.6%) (Zarnke et al., 2000) has been reported. Wildlife can play an important role in the epidemiology of T. gondii infection, and has been documented or implicated as a source of the parasite in North America including northern Canada. An outbreak of congenital toxoplasmosis in northern Quebec, Canada was statistically linked to consumption of raw dried caribou meat and skinning of animals (McDonald et al., 1990). Toxoplasma gondii infection after consumption of raw venison was also documented in three US hunters (Sacks et al., 1983). An outbreak of toxoplasmosis occurred in British Columbia, Canada due to contamination of municipal drinking water with feces of cougars (Felis concolor vancouverensis) as well as domestic cats (Aramini et al., 1998).
Wolverines (Gulo gulo) are an economically important species in northern Canada because of their valuable fur. Wolverines are classified as vulnerable throughout their circumpolar range by the IUCN; however, the highest population densities in Canada occur in the mountains of the Yukon and Northwest Territories, where they are legally trapped (Naughton, 2012). Due to their high trophic level, wolverines could play the role of "bioaccumulators" of food borne parasites. Important characteristics of a sentinel animal host (Halliday et al., 2007;NRCCAMEH, 1991) may include measurable response (e.g. parasites in tissues, antibodies in blood), sufficient availability (population stability), and high levels of exposure. Their predatory and scavenging lifestyle and large home range predispose wolverines for exposure. Serological tests for the detection of antibodies to T. gondii are commonly used as screening tools for domestic and wildlife species. A variety of serological methods, including agglutination test (MAT), enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT) have been used for detection of antibodies to T. gondii in wildlife species Elmore et al., 2016). The MAT is the most widely used method, it is simple to use and does not require species-specific conjugates.
The first objective of this study was to determine the seroprevalence of T. gondii in wolverines via detecting anti-T. gondii antibodies in fluid collected from diaphragm tissues which are commonly collected in surveys for another foodborne parasite, Trichinella spp. The second objective was to assess the association between exposure to T. gondii and the risk factors such as age, sex, location and body condition index (BCI).

Study area
Bordered by Nunavut to the east and the Yukon to the west, the NWT is the most populous of the three territories in Northern Canada, with an estimated population of 44,718 (Statistics-Canada, 2017). Climate varies from the southern part (subarctic climate) to the northern coast and the islands (polar climate). Communities depend on wildlife for traditional, cultural and economic values. Northwest Territories (NWT) harbors a variety of wildlife species including caribou (Rangifer tarandus), moose (Alces alces), muskox (Ovibos moschatus), mountain goats (Oreamnos americanus), Dall sheep (Ovis dalli), beaver (Castor canadensis), wolves (Canis lupus), foxes (Vulpes vulpes, V. lagopus), lynx (Lynx Canadensis), wolverines (Gulo gulo), and bears (Ursus americanus, U. arctos, U. maritimus).

Collection of samples
Wolverines were collected by trappers as part of the fur harvest during 2005-2012 in NWT (Fig. 1). Trappers submitted carcasses to Environment and Natural Resources, NWT for necropsy. Carcasses were collected from wolverines in four regions, Sahtu, Dehcho, North Slave, South Slave and Inuvik. These regions cover five different ecozones viz. Southern Arctic (14%), Taiga Cordillera (3%), Taiga Plains (65%), Taiga Shields (16%) and Tundra Cordillera (2%). Harvest location was further subdivided into above and below treeline. For wolverine harvested above treeline the straight-line distance between the harvest location and treeline was recorded. Sex and body condition index were determined at necropsy (Robitaille et al., 2012). Age was determined via cementum analysis of the canine tooth at a commercial laboratory (Matson's Laboratory LLC, Milltown, Montana, USA). Wolverines were classified as juveniles (b1 year), yearlings (1-2 years), and adults (≥2 year). Entire diaphragms were collected, frozen at −20°C, and shipped to the Zoonotic Parasite Research Unit at the Western College of Veterinary Medicine, University of Saskatchewan.

Tissue fluid extraction
Diaphragms were placed individually in double plastic bags to avoid cross-contamination between samples and left at 4°C for 24 h to thaw. Approximately, 1 ml of accumulated tissue fluid was removed from the bag and centrifuged at 1000 ×g for 5 min at 4°C. The supernatant was stored at -20°C until used for testing.

Enzyme linked immunosorbent assay (ELISA)
Tissue fluid samples were tested in duplicate for the presence of antibodies against T. gondii using a commercially available multi-species ID Screen Toxoplasmosis Indirect kit as per manufacturer's instructions (ID.vet, Grabels, France). Briefly, 100 μl of tissue fluid (at 1:2 in dilution buffer) were transferred to wells of an antigen coated ELISA plate and incubated for 45 min at room temperature. Wells were then washed 3 times and 100 μl 1× multispecies peroxidase (HRP) conjugate was added. The ELISA plate was incubated for 30 min at room temperature and then washed three times. Substrate solution (100 μl) was added to each well and the plate was incubated in the dark for 15 min at room temperature. After addition of 100 μl stop solution, optical density (O.D) values at 450 nm were recorded using an ELISA automated plate reader (Spectramax, Molecular Devices). Tests were considered valid if the mean value of the positive control OD was N0.350 and if the ratio of the mean OD values of the positive and negative controls was N3.
S/P percentage was calculated using controls provided by the manufacturer as per the following formula.
Samples presenting an S/P ratio ≤ 40% were considered negative. If the S/P ratio was between 40% and 50%, test outcome was considered doubtful and the sample was omitted from statistical analysis. Samples with an S/P ratio ≥ 50% were considered positive.
In addition to positive and negative controls provided in the kit, we included diaphragmatic fluid from four and two wolverines detected positive and negative for T. gondii DNA in heart tissue using magnetic capture PCR Sharma et al., 2018); these tissue-positive and -negative wolverines showed seropositivity and seronegativity, respectively on commercial available ELISA kit.

Statistical analysis
Seroprevalence was calculated from the proportion of sero-positive results of those tested and is presented with 95% confidence intervals. One sample had a doubtful result and was not included in statistical analysis. Possible associations between outcome variable (either positive/negative for antibodies against T. gondii) were evaluated by binary logistic regression. The following predictors were evaluated for inclusion in a final multivariable model using univariable binary logistic regression analysis: Age (juvenile, yearling and adult), sex (female and male), harvest location (Dehcho, Sahtu, Inuvik, North Slave, and South Slave regions), harvest location with respect to tree line (above or below tree line) and body condition (continuous factor). A relaxed level of significance (p ≤ 0.20) was used to identify variables. Stepwise forward multivariable regression analysis was performed to include risk factors and confounders in final model. Goodness of fit of the final model was evaluated by Hosmer Lemeshow test. Variables associated at a significance level of p b 0.05 were retained in the model. To estimate the degree of the association between each predictor and T. gondii sero-status, odds ratios and respective 95% confidence intervals were calculated. All statistical analyses were performed using IBM SPSS (ver. 24; Armonk, New York, USA).

Ethical approval
As animals were harvested for purposes other than research, this was considered Category A and exempt from Animal Research ethics review at the University of Saskatchewan. We worked closely with the Government of the Northwest Territories for wildlife research and export permits.

Association with the risk factors using regression models
On univariable logistic regression, age (p = 0.02) and location (p = 0.139) appeared to be associated with exposure to T. gondii (Table 1). After multivariable regression, only age was significantly associated with sero-positivity. The odds of presence of anti-T. gondii antibodies were five times (odds ratio = 5.2, 95% CI (2.12-13), p = 0.002) higher in adult wolverines than in juvenile wolverines.

Discussion
We successfully detected antibodies against T. gondii in diaphragmatic fluid from wolverines, demonstrating the utility of this approach in wildlife (especially in remote regions) from which sera are not routinely available. We reported high seroprevalence of T. gondii in wolverines from the NWT in comparison to seroprevalence reported in other wildlife species in NWT [5% in Muskoxen, 3%-40% in Caribou/Reindeer, 22% in Harbour Seal (Phoca vitulina), 10% in Ringed Seal (Phoca hispida), and 10% in Bearded Seal (Erignathus barbatus)] (Johnson et al., 2010;Kutz et al., 2000;Kutz et al., 2001;Simon et al., 2011), although results were on par with those observed in dogs in a community in the southern NWT (Salb et al., 2008). The 62% of the sero-positive wolverines from NWT, which is higher than the seroprevalence reported in two previous reports of T. gondii exposure in wolverines in Canada: 42% of 41 wolverines in Nunavut (Reichard et al., 2008) and none of 20 in BC (Philippa et al., 2004). Variation in seroprevalence among wolverines from these three different provinces/territories could be due to differences in serological methods [ELISA vs MAT in, current study and (Philippa et al., 2004), MAT (Reichard et al., 2008)], specimens used [serum in (Philippa et al., 2004), spleen exudates in (Reichard et al., 2008)], diaphragmatic fluid in current study and dietary habits of wolverines from different locations. We chose an ELISA, which requires less time, and results are more objective in comparison to MAT. As species-specific conjugates are not available for wolverine, we used a commercially available ELISA kit (ID.vet, Grabels, France) with A/G conjugates, which has been previously used in other wildlife species (Racka et al., 2015;Roqueplo et al., 2011). This ELISA had sensitivity and specificity equal or superior to two serological tests (MAT, and IFAT) using magnetic capture qPCR as a reference test. We used a 1:2 dilution as a cut off as recommended by the manufacturer and confirmed in wolverines using different dilutions of meat juice (Sharma et al., 2018). As sensitivity and specificity of ELISA using diaphragmatic fluid to detect anti-T. gondii antibodies is unknown, we reported apparent seroprevalence.
There are many reports of prevalence of antibodies to T. gondii in blood collected from immobilized or live-trapped wildlife by biologists and researchers (Elmore et al., 2016;Johnson et al., 2010); however, sera are rarely available from trapped and hunted wildlife, which are most important from the perspective of human exposure. We adapted this test for use on tissue fluid as sera were not available, and previous studies reported a high degree of agreement between MAT and ELISA tests performed on sera and tissue fluid Halos et al., 2010). As tissue fluid has fewer antibodies than serum, we used a lower dilution (1:2) as compared to serum (1:10) (Nockler et al., 2005;Racka et al., 2015). This study suggests that diaphragmatic fluid is a suitable substrate for tests for antibody to T. gondii. Diaphragm is frequently collected in wildlife being screened for another foodborne parasite, Trichinella spp. Therefore, diaphragm and its fluid can be a convenient sample for testing Trichinella spp. infection, and T. gondii exposure, respectively. Our in-house controls (tissue fluid obtained from diaphragms of wolverines of known T. gondii status) performed well (tissue-positives and -negatives were sero-positives and -negatives, respectively) with a commercial ELISA kit, and the use of wildlife species-specific controls whenever possible is recommended. Further studies are required on validation of serological tests for detection of T. gondii antibodies in tissue fluid/sera of wolverines. Our finding that significantly higher proportion of seropositive adult than juvenile wolverines is not surprising due to the longevity of antibodies (potentially lifelong) and more cumulative exposure to food or water contaminated with oocysts and/or consumption of meat having tissue cysts. However, the dynamics of anti-T. gondii antibodies are not known in wolverines. Higher prevalence in adult wolverines is in agreement with surveys conducted in other wildlife species from different geographical areas, e.g. wolves (Canis lupus) from Alaska, (Zarnke et al., 2000); maned wolves (Chrysocyon brachyurus) from Brazil, (Vitaliano et al., 2004); Eurasian otter (Lutra lutra) from England and Wales, (Chadwick et al., 2013)]. On the other hand, our findings are not consistent with those reported in wolverines from Nunavut (Reichard et al., 2008), in which apparently smaller percentage of adult wolverines (37%, 11/30) were positive for antibodies to T. gondii than juveniles (55%, 6/11). No significant association between age and T. gondii exposure in wolverines from Nunavut was reported (Reichard et al., 2008), nor in feral cats from Prince Edward Island, Canada (Stojanovic and Foley, 2011). A slight difference of seroprevalence among males and females was not significant (despite a male bias in harvest), similar to that reported in other wildlife surveys (feral cats, (Stojanovic and Foley, 2011), e.g. Eurasian otter, (Chadwick et al., 2013); grizzly and black bears, (Chomel et al., 1995); caribou, (Kutz et al., 2001). The body condition of wolverines in our study did not differ significantly with exposure to T. gondii, similar to findings in free roaming cats and dogs from Mexico (Ortega-Pacheco et al., 2017) as well as in wild and domestic cats from France (Afonso et al., 2013). Exposure to T. gondii in wolverines across all sites (region-specific seroprevalence ranged from 44 to 75%) indicates widespread transmission of the parasite across NWT, and geographical differences were not significant, likely as a result of small sample size.
Lynx (Lynx canadensis) are the only wild felid present in northern Canada and they are rarely seen above the treeline; thus, we hypothesized decreased opportunity for exposure to oocysts above the treeline (i.e. some parts of Inuvik and North Slave). Among 15 wolverines harvested above the tree line (13 from Inuvik and 2 from North Slave), mean straight-line distance from tree line was 22 km (range 4-99 km). Both the wolverines [IDs: W2010-086 (71 km) and NS06-92 (99 km)] from North Slave had highest straight line distance and were positive and negative for anti-T. gondii antibodies, respectively. Wolverine (ID: TU11/02) from Inuvik harvested at highest straightline distance was 33 km above the tree line and was sero-positive. Presence of sero-positive wolverines above the tree line indicates either environmental contamination with oocysts or presence of potential intermediate hosts (caribou and muskoxen). Also, as home range of wolverines varies from 100 to 800 km 2 , possibility of migration of sero-positive wolverines cannot be ignored. An association between the presence of antibodies to T. gondii and proximity to the treeline has been reported in wolverines from Nunavut (Reichard et al., 2008) and in caribou and muskoxen (Kutz et al., 2000;Kutz et al., 2001). Future studies could evaluate the spatio-temporal occurrence of T. gondii infection in wild carnivores and potential intermediate hosts in NWT relative to treeline, explore new serological methods that can distinguish exposure to oocysts vs tissue cysts, and compare genotypes of T. gondii circulating in wolverines and Canadian lynx. Further studies are needed on T. gondii in domestic and wild felids in NWT as well as their relative contribution to environmental oocyst contamination.
Finally, little is known about the effects of T. gondii in free ranging wildlife. Clinical toxoplasmosis has been reported in wild carnivores e.g. foxes (Dubey et al., 1990;Dubey and Pas, 2008), wild boars (Calero-Bernal et al., 2013), and raccoons, Procyon lotor (Dubey et al., 1992). An outbreak reported in mink (close relative of wolverines) resulted in abortions and neonatal mortalites occurring in 26% of the female mink (n = 7800) (Frank, 2001). Fatal toxoplasmosis has been reported in sea otters (Enhydra lutris), brown hare (Lepus europaeus P.), Eurasian red squirrels (Sciurus vulgaris), Pallas' cats (Otocolobus manul) (Kreuder et al., 2003, Brown et al., 2005, Gustafsson and Uggla, 1994, Jokelainen and Nylund, 2012. Further studies are required to see if T. gondii is responsible for pathology and decreased reproductive success in wolverines. Detection of antibodies to T. gondii in animals indicates prior exposed to the parasites but does not always indicate if it is infected (Kornacka et al., 2016). Therefore, our study can only speak to exposure, and bioassays (cat and mouse) or molecular methods are needed to determine tissue status and therefore any hazard to trappers/hunters handling fresh tissues of wolverines. However, as association between skinning and human exposure to T. gondii has been reported (McDonald et al., 1990), hunters/ trappers should avoid unintentional ingestion of tissues or fluids and follow good sanitary practices while processing wolverine carcasses (Hill et al., 2014). It is not recommended to leave carcasses and offal where they can be scavenged, perpetuating the life cycle of the parasite, especially near human habitations. In addition to wolverines, other wild animals, for example, moose (Alces alces) (Remes et al., 2018) and wild boar (Sus scrofa) (Ranucci et al., 2013) could act as sentinels for the exposure to T. gondii, however, moose, which are browsing herbivores, serve as indicators of oocyst transmission, while more omnivorous species such as boar and wolverine may also serve as indicators of overall transmission, including consumption of tissue cysts via carnivory.

Conclusion
Our results indicate that T. gondii circulates in wolverines throughout NWT. Due to their apex position in the food chain, dietary habits, and reported high seroprevalence, wolverines can serve as useful sentinels of risks of transmission to other wildlife and people from their shared environment in the Canadian north. We report the successful application of a commercial serological test to a sample substrate (diaphragmatic fluid) commonly available in harvested wildlife, which may have food safety applications. Finally, we found, as expected, that older animals were more likely to be exposed to T. gondii, while other risk factors for seropositivity should be explored with a larger sample size.


extfieldsofstudy: Biology Medicine

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Plant Hsp90 is a novel adjuvant that elicits a strong humoral and cellular immune response against B- and T-cell epitopes of a Toxoplasma gondii SAG1 peptide

Background The 90-kDa heat-shock protein (Hsp90) from Nicotiana benthamiana (NbHsp90.3) is a promising adjuvant, especially for those vaccines that require a T cell-mediated immune response. Toxoplasma gondii SAG1 is considered one of the most important antigens for the development of effective subunit vaccines. Some epitopes located in the SAG1 C-terminus region have showed a strong humoral and cellular immune response. In the present study, we aimed to assess the efficacy of NbHsp90.3 as carrier/adjuvant of SAG1-derived peptide (SAG1HC) in a T. gondii infection murine model. Methods In the present study, C57BL/6 mice were intraperitoneal immunized with the NbHsp90.3-SAG1HC fusion protein (NbHsp90.3-SAG1HC group), mature SAG1 (SAG1m group), NbHsp90.3 (NbHsp90.3 group) or PBS buffer 1× (PBS group). The levels of IgG antibodies and the cytokine profile were determined by ELISA. Two weeks after the last immunization, all mice were orally challenged with 20 cysts of T. gondii Me49 strain and the number of brain cysts was determined. In addition, both humoral and cellular immune responses were also evaluated during the acute and chronic phase of T. gondii infection by ELISA. Results The characterization of the immune response generated after vaccination with NbHsp90.3 as an adjuvant showed that NbHsp90.3-SAG1HC-immunized mice produced antibodies that were able to recognize not only rSAG1m but also the native SAG1 present in the total lysate antigen extract (SAG1TLA) from T. gondii tachyzoites, while control groups did not. Furthermore, anti-rSAG1m IgG2a/2b antibodies were significantly induced. In addition, only the spleen cell cultures from NbHsp90.3-SAG1HC-immunized mice showed a significantly increased production of IFN-γ. During the chronic phase of T. gondii infection, the antibodies generated by the infection were unable to detect the recombinant protein, but they did react with TLA extract. In addition, splenocytes from all groups showed a high production of IFN-γ when stimulated with rGRA4, but only those from NbHsp90.3-SAG1HC group stimulated with rSAG1m showed high production of IFN-γ. Finally, NbHsp90.3-SAG1HC-immunized mice exhibited a significant reduction in the cyst load (56%) against T. gondii infection. Conclusions We demonstrated that NbHsp90.3 enhances the humoral and cell-mediated immune response through a Th1 type cytokine production. Mice vaccinated with NbHsp90.3-SAG1HC exhibited a partial protection against T. gondii infection and it was correlated with the induction of memory immune response. We developed and validated a vaccine formulation which, to our knowledge, for the first time includes the NbHsp90.3 protein covalently fused to a peptide from T. gondii SAG1 protein that contains T- and B-cell epitopes. Electronic supplementary material The online version of this article (10.1186/s13071-019-3362-6) contains supplementary material, which is available to authorized users.

Background
SAG1 is present in tachyzoites of T. gondii parasites. This protein is a stage-specific protein, which presents low polymorphism between different T. gondii strains and elicits a strong specific antibody response [1,2]. SAG1 is able to stimulate the production of IFN-γ of T cells in seropositive individuals [3]. For this reason, this antigen has been suggested as an excellent candidate for vaccine development in the prevention of toxoplasmosis.
In several studies, SAG1 has been used as a nativepurified protein, recombinant protein or DNA expression vector in the development of vaccines against T. gondii [4,5]. Likewise, various peptides of SAG1 have demonstrated to stimulate host humoral and cellular immunity and to provide partial protection against T. gondii [6]. The selection of proper epitopes becomes necessary to eliminate deleterious components and to retain the advantageous elements in the design of an efficient and safe vaccine [7,8]. Therefore, many studies have identified different short peptides present in SAG1 that would have promising immunogenic properties and could be used in the development of acellular vaccines [9][10][11][12]. Wang et al. [11] showed that peptides of SAG1 containing B-cell epitopes are highly immunogenic. In addition, Godard et al. [9] observed that the C-terminus of SAG1 is the dominant antigenic and immunogenic region and, in particular, the SAG1 238-256 peptide is an important T-cell epitope. Likewise, Siachoque et al. [10] scanned the B-cell epitopes present in SAG1 and revealed that they are primarily placed near the C-terminus. Furthermore, Cardona et al. [11] found that B-cell epitopes of SAG1 enclosed in the SAG1 301-320 position show the highest reactivity against human sera from T. gondii-infected patients.
Another important aspect in the development of acellular vaccines is the selection of the appropriated adjuvant, since it plays an important role in the efficacy of the immunizations. It is generally accepted that a T helper 1 (Th1) response associated with IFN-γ producing cells, is the main mediator of the immunity against T. gondii infection [13,14]. Therefore, Hsp90s and Hsp70s have been used as antigen/carriers in order to induce B-and T-cell mediated immune responses [15][16][17][18][19]. Our group showed that plant Hsp90s (pHsp90s) are B cell mitogens and that the presence of toll-like receptor 4 (TLR4) is necessary for a suitable response [18]. In addition, our results showed that pHsp90s could be incorporated as adjuvants in a vaccine formulation that needs a Th1mediated response along with the stimulation of cytotoxic CD8 + cells to confer immunity [19].
Previously, we expressed SAG1 fused to Leishmania infantum 83-kDa heat-shock protein (LiHsp83-SAG1) to improve the stability of the recombinant protein when expressed in plants [20]. In addition, oral immunization with plant-made LiHsp83-SAG1 produced a significant reduction of the cyst load and this correlated with an increment of specific antibodies anti-rSAG1 [20]. A further advantage of employing Hsp90 as adjuvants/carriers is the possibility of using peptides containing B-and T-cell epitopes, instead of the full-length antigen or large polypeptides that may enclose toxic or unstable regions that can affect their correct expression in a heterologous system. While LiHsp83 is a pathogenic-associated adjuvant, pHsp90s offers the advantage of being a nonpathogenic adjuvant. Bearing this in mind, we characterized the role of pHsp90 in the modulation of the immune response. Thus, we evaluated the efficacy of Nicotiana benthamiana Hsp90.3 (NbHsp90.3) as a carrier/adjuvant of a T-and B-cell epitopes SAG1 peptide in a T. gondii infection model.

Plasmid construction
A peptide of Toxoplasma gondii SAG1 (SAG1 HC ) (from residue 221 to residue 319 of the protein) that encodes both T-and B-cell epitopes [10] was amplified by PCR using the plasmid pRSET-A-SAG1 77-322 as a template [21]. The forward primer sequence was 5′-ggt acc ATA AAG TTC CTC AAG ACA AC-3′ and the reverse primer sequence was 5′-aag ctt CTA AAT GGA AAC GTG ACT GGC-3′ flanked by KpnI and HindIII restriction sites (lowercase), respectively. The N. benthamiana Hsp90.3 full length sequence (NbHsp90.3) was amplified by PCR using the plasmid pRSET-A-NbHsp90.3 as a template [18]. The forward degenerated primer sequence was 5′-gga tcc ATG GCG GAS GCA GAR ACS TTT GCW TTY CAA GC-3′ and the reverse primer sequence was 5′-ctc gag GTC TAC TTC CTC CAT CTT TTC AGC ATC ATC AGC-3′ flanked by BamHI and XhoI restriction sites (lowercase), respectively. The PCR products were first cloned separately into pGEM-T easy vectors (Promega, Fitchburg, WI, USA). After sequencing, the isolated segments were sequentially cloned into pRSET-A validated a vaccine formulation which, to our knowledge, for the first time includes the NbHsp90.3 protein covalently fused to a peptide from T. gondii SAG1 protein that contains T-and B-cell epitopes.

Preparation of the total lysate antigens (TLA) from Toxoplasma gondii tachyzoites
TLA were obtained from a suspension of T. gondii RH tachyzoites. RH tachyzoites were sonicated (10 s pulse ON with 30 s intervals OFF, for 1 min) using a 130 W ultrasonic processor (Vibra Cell ™ ; Sonic, Newtown, CT, USA) and centrifuged at 8000× g for 5 min at 4 °C. Then, the supernatant was quantified by Bradford protein assay and stored at -80 °C until use.

Mice and vaccination
C57BL/6 (H-2b) mice specific-pathogen-free were obtained from the central bioterium of Facultad de Ciencias Exactas y Naturales of Universidad de Buenos Aires. Six-to eight-week-old female mice were bred and housed following the institutional guidelines of the Universidad de General San Martín (C.I.C.U.A.E., IIB-UNSAM, 09/2016). Mice had access to food and water ad libitum and were maintained at 21-22 °C, 12:12 h light-dark photocycle. Five to eight mice per group were intraperitoneally immunized with equimolar amount of SAG1m (34-kDa, 2.5 μg), NbHsp90.3 (84.5-kDa, 7.5 μg) and NbHsp90.3-SAG1 HC fusion protein (96-kDa, 9 μg). Each group received 4 doses (every 15 days) of each protein or PBS control. One day before immunizing, mice were bled from the tail vein and sera were stored at -20 °C until analysis.

Cytokine analysis
The cytokine production was evaluated in independent experiments, either 15 days after the last immunization (pre-challenge) or 30 days after challenging with 20 Me49 tissue cysts (post-challenge). Spleens from 5 immunized mice from each group were removed and the splenocytes (1.25 × 10 6 cells/well) were stimulated with either 10 µg/ ml of rSAG1m, rNbHsp90.3 or rGRA4. After 72 h, the production of IL-4, IL-10 and IFN-γ in harvested cellfree supernatants was assessed as previously described by Corigliano et al. [19].

Challenge infection
Two weeks after the last immunization, mice were orally challenged with 20 tissue cysts of T. gondii Me49 strain as previously described [21]. Mice were observed daily for mortality determination. One month after the challenge, animals were sacrificed, and their brains were removed for tissue cyst quantification. Each brain was homogenized in 2 ml of PBS by eight passages through a 21-gage needle [22]. The mean number of cysts per brain was determined by observation with an optical microscope, by counting four samples of 20 μl aliquots of each homogenized brain [21].

Statistical analysis
Statistical analysis was carried out with Prism 5.0 (Graph-Pad, San Diego, CA, USA) using one-and two-way analysis of variance (ANOVA). Statistical analysis was carried out using Tukey's multiple comparisons test to compare means among groups. Values of P < 0.05 were considered significantly different.

Expression of recombinant proteins
We expressed peptide of Toxoplasma gondii SAG1 from aa 221 to aa 319 (SAG1 HC ). This peptide encloses B-and T-cell epitopes [10]. The peptide was cloned in frame with Nicotiana benthamiana Hsp90.3 (NbHsp90.3) [18] and was expressed and purified from E. coli.
The efficacy of vaccination in immunized mice was determined by serological analysis. First, we analyzed by Western blot whether the sera from mice immunized with the different formulations were able to recognize SAG1 antigen present in TLA (SAG1 TLA ). The sera pools from mice immunized with NbHsp90.3-SAG1 HC fusion protein or SAG1m were able to immunodetect SAG1 TLA (Additional file 1: Figure S1). Therefore, we used SAG1 TLA and recombinant SAG1m (rSAG1m) in all serological analysis. Production of IgG antibodies against rSAG1m and SAG1 TLA was evaluated by ELISA 60 days after the first immunization. The levels of anti-rSAG1m and anti-SAG1 TLA total IgG antibodies were strongly increased in NbHsp90.3-SAG1 HC -immunized mice (Fig. 1). In fact, significant differences were observed between the NbHsp90.3-SAG1 HC group and control groups (SAG1m-NbHsp90.3-and PBS-immunized mice) (Fig. 1). Therefore, NbHsp90.3-SAG1 HC formulation Fig. 1 Levels of anti-SAG1 total IgG antibodies in the sera of C57BL/6 immunized mice. Two weeks after the last immunization, serum samples (8 mice per group) were collected to analyze IgGt by ELISA with rSAG1m (a) and SAG1 TLA (b) as the bound target. Sera were diluted 1:1000 or 1:100 for detecting rSAG1m or SAG1 TLA , respectively. Each bar represents the group mean ± SEM. Results represent one of three similar experiments. Pre-immune sera were used as a negative control. a ****P < 0.0001, NbHsp90.3 HC vs PBS, NbHsp90.3 and SAG1m groups. b *P < 0.05, NbHsp90.3 HC vs SAG1m group; ***P < 0.001, NbHsp90.3 HC vs PBS and NbHsp90.3 groups. Statistical analysis was performed by one-way analysis of variance (ANOVA) using Tukey's multiple comparisons test elicits a strong humoral response in comparison with SAG1m group, suggesting that NbHsp90.3 works as an efficient adjuvant to stimulate B-cell epitopes when associated to the antigen of interest. In addition, no anti-rSAG1m or anti-SAG TLA IgGt was detected in the PBS or NbHsp90.3 groups (Fig. 1).
To investigate the isotype profile induced after immunization, we evaluated the presence of IgG1, IgG2a and IgG2b antibodies in serum samples on day 60, by using rSAG1m or SAG1 TLA as the bound targets (Fig. 2). All NbHsp90.3-SAG1 HC -immunized mice produced IgG1 and IgG2a antibodies that were able to recognize rSAG1m but not SAG1 TLA (Fig. 2). In addition, NbHsp90.3-SAG1 HC -immunized mice produced high levels of anti-SAG1 TLA IgG2b antibody isotype, whereas SAG1m-immunized mice showed a predominant specific IgG1 response against SAG1 TLA . Furthermore, higher levels of anti-rSAG1m IgG2b antibodies were observed in NbHsp90.3 and SAG1m groups respect to PBS group, which were not observed when the SAG1 TLA was used as the bound target (Fig. 2). This could be due to the shared N-terminus sequences for both recombinant proteins.

Cellular immune response generated by NbHsp90.3-SAG1 HC fusion protein
To evaluate the cytokine response profile, splenocytes from vaccinated mice were stimulated with either rSAG1m or rNbHsp90.3 and the amounts of cytokine production (IFN-γ, IL-4 and IL-10) in different groups were measured. As shown in Fig. 3a, NbHsp90.3-SAG1 HC -immunized mice generated a significantly higher level of IFN-γ compared with control groups (SAG1m-, NbHsp90.3-and PBS-immunized mice).

Parasite load and humoral and cellular response after T. gondii infection
To assess the effect on parasite load against T. gondii infection, two weeks after the vaccination schedule was completed, immunized mice were orally challenged and the number of T. gondii brain cysts was analyzed one month later. All mice survived for 30 days. The behavior of NbHsp90.3-SAG1 HC -immunized mice was apparently normal, while SAG1m-, NbHsp90.3-and PBS-immunized mice exhibited lack of movement and a general aspect indicating illness. While the brain cyst loads in SAG1-and NbHsp90.3-immunized mice were not significantly lower compared to PBS-immunized mice, the NbHsp90.3-SAG1 HC -immunized mice presented a significant reduction in the cyst load (56%) when compared to control groups (SAG1m-, NbHsp90.3-and PBSimmunized mice) (Fig. 4a). In addition, we evaluated the humoral immune response in the chronic phase of T. gondii infection (Fig. 4b, c). Thirty days after the challenge, the levels of anti-TLA IgGt were significantly increased in all groups (Fig. 4b), especially in the NbHsp90.3-SAG1 HC -immunized mice (Fig. 4b). By contrast, high levels of anti-rSAG1m IgGt antibodies were detected in NbHsp90.3-SAG1 HC -and SAG1m-immunized mice, while no response was observed in NbHsp90.3 and PBS groups (Fig. 4c). Furthermore, we evaluated the cytokine profile in sera from immunized and challenged mice during the acute and chronic phases of T. gondii infection (10 Fig. 2 Analysis of IgG isotype antibodies against SAG1 in the sera of C57BL/6 immunized mice. Two weeks after the immunization schedule was completed, serum samples (8 mice per group) were collected and diluted 1:100 to analyze IgG1, IgG2a and IgG2b sub-classes with rSAG1m (a) or SAG1 TLA (b) as the bound target. Each bar represents the group mean ± SEM. Results represent one of three similar experiments. Pre-immune sera were used as a negative control. *P < 0.05; ***P < 0.001; ****P < 0.0001. Statistical analysis was performed by two-way analysis of variance (ANOVA) using Tukey's multiple comparisons test and 30 days after the challenge, respectively). The levels of IFN-γ were induced in the acute phase but they were significantly diminished in the sera from all mice with chronic infection. Actually, no significant differences were observed among groups (Fig. 5a). On the other hand, IL-10 and IL-4 were undetectable in sera from all mice with acute and chronic infection (data not shown). In addition, the cytokine profile was analyzed in supernatant of spleen cell cultures. IL-10, IL-4 and IFN-γ production was measured in the supernatants from spleen  immunized mice. Two weeks after the last immunization, mice were orally challenged with 20 tissue cysts of ME49 T. gondii strain (sub-lethal dose). Thirty days after the infection, mice were sacrificed, and their brains were removed for cyst load determination. Each bar represents the group mean ± SEM. Results represent one of three similar experiments. **P < 0.01, NbHsp90.3-SAG1 HC vs NbHsp90.3 group; ****P < 0.0001, NbHsp90.3-SAG1 HC vs PBS and SAG1m groups. b, c Antigen-specific antibodies in sera from C57BL/6 immunized mice with chronic T. gondii infection. Thirty days after mice were orally challenged with 20 tissue cysts of ME49 T. gondii strain (sub-lethal dose), serum samples (8 mice per group) were obtained and IgGt was determined by ELISA with TLA (b) or rSAG1m (c) as the bound targets (sera were diluted 1:16,000 and 1:1000, respectively). Each bar represents the group mean ± SEM. Results represent one of two similar experiments. b ***P < 0.001, NbHsp90.3-SAG1 HC vs PBS, NbHsp90.3 and SAG1m groups. c **P < 0.01, NbHsp90.3-SAG1 HC vs SAG1m group; ***P < 0.001, NbHsp90.3-SAG1 HC vs PBS and NbHsp90.3 groups. Pre-immune sera were used as a negative control. Statistical analysis was performed by one-way analysis of variance (ANOVA) using Tukey's multiple comparisons test cells culture of immunized mice in the chronic phase of T. gondii infection. Splenocytes were stimulated in vitro with rSAG1m and recombinant GRA4 (rGRA4) (Fig. 5b). The stimulation with rGRA4 was carried out in order to distinguish the cellular response induced by the T. gondii challenge from the cellular response induced by the vaccination and the challenge. As expected, after the challenge, IFN-γ levels were strongly induced in all groups when spleen cells were stimulated with rGRA4 (Fig. 5b), while IL-10 was not secreted (Fig. 5c). On the other hand, the spleen cells from SAG1m-and NbHsp90.3-SAG1 HC -immunized mice stimulated with rSAG1m showed different cytokine profile production (Fig. 5b,  c). While, the levels of IFN-γ were significantly induced in the NbHsp90.3-SAG1 HC -immunized mice (Fig. 5b), IL-10 was only secreted by splenocytes from SAG1mimmunized mice (Fig. 5c). Notably, splenocytes from NbHsp90.3-and PBS-immunized mice stimulated with rSAG1m did not show an increase of any of the analyzed cytokines. Finally, IL-4 was not secreted by spleen cells of any of the groups stimulated with either rGRA4 or rSAG1m (data not shown).

Discussion
Several Hsp90s from different organisms, in particular Gp96, are being studied as promising adjuvants to enhance innate and adaptive immune responses, especially as potent T cell activators for designing vaccines against infectious diseases [19,[23][24][25][26][27][28]. According to these properties, Hsp90s have been explored as adjuvants for enhancing vaccine potency [28][29][30]. The most useful, simple and feasible strategy employed is to covalently link the desired antigen to Hsp90. In previous studies, it was demonstrated that immunization with Hsp90 from Leishmania infantum (LiHsp83) fused to a reporter antigen (MBP-LiHsp83) or a T. gondii Rop2 antigen (Rop2-LiHsp83), generated mainly IgG2a isotype antibodies inferring the involvement of an immune response mediated by Th1 cells [23,24,31]. Like other Hsp90s, the Hsp90.3 isoform from Nicotiana benthamiana (NbHsp90.3) is also considered a promising adjuvant with the advantage that NbHsp90.3 comes from a non-pathogenic source and it can be used for any pathogen-associated antigens, whereas LiHsp83 could interfere with antigens from other pathogens such as trypanosomatids.
In agreement with this, we previously demonstrated that BALB/c mice immunized with MBP-NbHsp90.3 were able to elicit a Th1 immune response against MBP [19]. Since analysis with generic antigens will not necessarily reproduce the immune response against microbialor parasitic-derived antigens, we aimed to evaluate the potential of NbHsp90.3 as a carrier/adjuvant of a peptide from T. gondii SAG1 protein.
SAG1 is considered an important antigen for the development of effective diagnostic tests or subunit vaccines [6]. Significant progress has been made towards characterization of its immunogenicity and a significant amount of information is available to date on the different antigenic regions related to T-or B-cell responses [10,12,[32][33][34]. In the present study, we analyzed both humoral and cellular immune responses induced by NbHsp90.3 covalently linked to SAG1 HC (a peptide that contains a T-and B-cell epitopes) [10]. NbHsp90.3, SAG1m or PBS were defined as control groups and the experiments were carried out in the murine model.
Humoral response analysis showed that NbHsp90.3-SAG1 HC -immunized mice induced a high production of specific-antibodies that were able to recognize rSAG1m and also SAG1 TLA . Sera from NbHsp90.3-SAG1 HCimmunized mice showed a significant increase of anti-rSAG1m IgG2a and IgG2b antibody isotypes and only anti-SAG1 TLA IgG2b antibodies. This finding suggests that the B-cell epitope present in the SAG1 HC peptide would generate antibodies which are able to distinguish rSAG1m from SAG1 TLA . Another interesting result is that the SAG1-immunized mice showed a significative increase of the anti-SAG1 TLA IgG1 levels, suggesting that humoral immune response induced by NbHsp90.3-SAG1 HC formulation is addressed towards a Th1 profile when the antigenic peptide is carried by NbHsp90.3. These outcomes are consistent with those found by Corigliano et al. [19]. However, it must be noted that while in the present study SAG1 HC is fused to the C-terminus of NbHsp90.3, the carried protein was fused to the N-terminus in Corigliano et al. [19]. Therefore, the adjuvant properties present in NbHsp90.3 will be maintained independently where the carrier protein is situated. It is also worthy of mention that in order to trigger a specific humoral immune response against the carried antigen, it is not necessary to fuse the complete protein of interest to NbHsp90.3, but only those epitopes with antigenic capacity.
As cytokines play an important role in the protection against T. gondii infection, we assessed their production. Our previous studies showed that NbHsp90.3 elicited a high production of IFN-γ when used as adjuvant [19]. It is important to note that IFN-γ is a key mediator in the mechanisms of T. gondii resistance and plays an important role in protecting hosts during both acute and chronic phases of toxoplasmosis [35][36][37]. In this study, we observed that splenocytes from NbHsp90.3-SAG1 HC -immunized mice produced the highest amounts of IFN-γ when stimulated with rSAG1m. These results confirm that the presence of the selected T-and B-cell epitopes of SAG1 is enough to elicit a Th1 immune response when they are fused to NbHsp90.3. On the other hand, it is generally assumed that IL-10 production correlates with a Th2 T-cell response [38]. In our study, high levels of IL-10 were elicited by spleen cells from mice of control groups (SAG1m and NbHsp90.3 groups). Actually, the high levels of IL-10 could explain the absence of IFN-γ in the SAG1m-and NbHsp90.30-immunized mice. According to these results, the immunization with SAG1m alone would induce a Th2 immune response with a production of IgG1 antibodies and IL-10 secretion. In fact, several lineal and conformational B-cell epitopes have been identified in SAG1 protein, most of them localized in the N-terminus region [39]; thus, it is expected that immunization with SAG1m alone would induce a Th2 immune response.
As the main goal of vaccination is the stimulation of a protective immune response against a defined pathogen, immunized mice were infected with T. gondii. In order to assess the protective immunity against T. gondii infection, the humoral and cellular immune responses were analyzed after the challenge. Interestingly, while antibodies from NbHsp90.3-SAG1 HC -immunized mice were able to respond to rSAG1m and SAG1 TLA , the antibodies generated after the infection were unable to react with rSAG1m, but they did with the TLA extract. This could be explained by the posttranslational modification that takes place to the native SAG1 into the parasite [32] which could contribute in modifying the conformational profile of epitopes of both proteins. Similar results were observed by Sánchez et al. [40]. The authors showed that sera from mice infected with T. gondii were unable to recognize the recombinant TgPI-1 protein [40]. Taken together, these results suggest that rSAG1m y SAG1 TLA carried dissimilar epitopes that make it possible to differentiate between vaccinated and infected mice.
On the other hand, the analysis of cytokines after the challenge in the chronic phase of T. gondii infection showed that rGRA4-stimulated spleen cells enhanced a high production of IFN-γ without secretion of either IL-10 or IL-4, indicating the T. gondii infection elicited a strong adaptative Th1 T cell immune response in mice from all groups. Only rSAG1m-stimulated spleen cells from NbHsp90.3-SAG1 HC group also showed high production of IFN-γ in agreement with what was observed before the challenge. In addition, SAG1m group triggered a Th2 adaptative immune response against rSAG1m indicating that T. gondii infection did not modify the cytokine profile against the SAG1m antigen elicited by the immunization. Taken together these results indicate that the NbHsp90.3 protein not only enhances cell memory response against the carried antigen but also modulates the response towards a Th1 profile in the immunized mice, which could be responsible for the conferred immune protection against T. gondii infection. More importantly, NbHsp90.3-SAG1 HC -immunized mice showed a significant reduction in the parasitic load (56%) with respect to the other groups concurrently in agreement with the cellular and humoral response observed in this group. Therefore, the capability of NbHsp90.3-adjuvanted vaccines to induce memory T cells can respond more rapidly to challenge and provide a long-term protective anti-T. gondii immunity. Although the percentage of cyst load reduction is comparable to others immunized with recombinant proteins [40][41][42][43][44], we emphasize that in the NbHsp90.3-SAG1 HC group no exogenous adjuvant was added. In summary, these results indicate that the cellular and humoral immune responses induced by NbHsp90.3-SAG1 HC formulation are addressed towards a Th1 immune profile with a production of IFN-γ, IgG2a and IgG2b antibodies against the carried antigen. We demonstrated the ability of NbHsp90.3 as an excellent adjuvant that modulates the immune response against T. gondii antigens in an appropriate manner.

Conclusions
In the present study we validated the use of NbHsp90.3 as an adjuvant in a murine model. We developed and validated a vaccine formulation which, to our knowledge, for the first time includes the NbHsp90.3 protein covalently fused to a peptide from T. gondii SAG1 protein that contains T-and B-cell epitopes. We also demonstrated that NbHsp90.3 enhances a humoral and cell-mediated immune response along with Th1 cytokine production. After the challenge, mice vaccinated with NbHsp90.3-SAG1 HC exhibited a partial protection against the T. gondii infection which correlated with the induction of the memory immune response. In conclusion, NbHsp90.3 is an attractive adjuvant to be incorporated in vaccine formulations that require the generation of a Th1 profile along with a T-cell mediated response to confer immunity.


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Long non-coding RNA RNCR3 promotes glioma progression involving the Akt/GSK-3β pathway

Increasing evidence has confirmed that long non-coding RNAs (lncRNAs) serve critical roles in the development of a large number of human malignancies, including glioma. Several previously published studies have reported that the lncRNA retinal non-coding RNA3 (RNCR3; also termed LINC00599) exerts important roles in certain human malignancies; however, the precise biological role and underlying molecular mechanisms of RNCR3 in the development of glioma are yet to be fully elucidated. In the present study, it was revealed that the expression of RNCR3 was increased in glioma tissues compared with in corresponding adjacent normal tissues. Furthermore, increased levels of RNCR3 expression were associated with tumor progression and poor survival rates of patients with glioma. In addition, the U87 and U251 cell lines were selected to investigate the biological function and potential mechanisms of RNCR3 in glioma, and it was observed that RNCR3 knockdown led to an impairment of the proliferative and invasive abilities of cells; furthermore, G1 phase arrest was induced in glioma cells in vitro. Finally, the results of western blot analyses revealed that knockdown of RNCR3 led to a decrease in the expression levels of phosphorylated Akt and glycogen synthase kinase-3β (GSK-3β), without any clear effect on the expression levels of total Akt and GSK-3β. Collectively, these results suggested that RNCR3 is able to regulate cell proliferation, the cell cycle and cell invasion in glioma, potentially via the Akt/GSK-3β signaling pathway.

Introduction
Glioma is the most common and most aggressive primary malignant tumor arising in the central nervous system of adults globally (1). It is also one of the main causes of cancerassociated mortality (2). Although certain progress has been made regarding effective early diagnosis of the disease, the majority of patients with glioma have progressed to the advanced stages by the time of diagnosis (3). Intensive research has been conducted to examine the biological mechanisms underlying the progression of glioma; however, the prognosis of patients in this category remains rather poor (4,5).
With the rapid development of genome and transcriptome sequencing technologies, numerous novel non-protein-coding RNAs have been identified (6). Whereas >80% of the genome is transcribed into mRNA transcripts, only ~2% of the genome codes for proteins (6,7). Long non-coding RNAs (lncRNAs) comprise a class of non-protein-coding RNAs that are >200 nucleotides in length (8). LncRNAs serve key regulatory roles in a variety of biological and pathological processes, including tumorigenesis, transcription regulation and epigenetic post-transcription regulation (9)(10)(11).
Retinal noncoding RNA3 (RNCR3, also termed LINC00599) is a lncRNA that is transcribed from the intergenic regions of the genome, and is highly conserved in mammals (12). A number of studies have reported that RNCR3 exerts important regulatory functions in cell proliferation and differentiation, and the process of atherosclerosis (13,14). Regarding human cancers, Tian et al (15) reported that RNCR promoted prostate cancer development by regulating microRNA (miR)-185-5p. However, the biological functions and molecular mechanisms of RNCR3 in glioma are yet to be fully elucidated.
In the present study, RNCR3 expression levels in glioma tissues and in paired normal tissues were evaluated, following which the biological function of RNCR3 in glioma cell lines, and the underlying mechanisms of RNCR3 in glioma, were further investigated. To the best of our knowledge, the present study is the first to demonstrate that RNCR3 functions as an oncogene in glioma development.

Materials and methods
Clinical samples. The present study was approved by the Ethics and Research Committees of the Sunshine Union Hospital of Shandong Province (Weifang, China), and performed in accordance with the principles of the Declaration of Helsinki. A total of 54 pairs of glioma tissue samples and paired adjacent normal tissues were obtained from patients undergoing resection at the Department of Neurosurgery of Sunshine Union Hospital between January 2005 and December 2010. The patients included 30 males and 24 females with a mean age of 53.9 years (range, 41-74 years). All the patients were pathologically confirmed, and none of the patients had received preoperative chemotherapy or radiation therapy. The tissues were collected during surgery and stored immediately in liquid nitrogen (-196˚C). Written informed consent was collected from all subjects. The clinical characteristics of all the patients are summarized in Table I.
Cell culture. A human astroglia cell line, HA, was acquired from BeNa Culture Collection. Two human glioma cell lines, SHG-44 and U251, and a glioblastoma of unknown origin, U87 (cat. no. HTB14) were purchased from the American Type Culture Collection. Cell lines were authenticated via short tandem repeat cell authentication profiling. All the cell lines were maintained in Gibco™ DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA), and 50 U/ml penicillin and 0.1 mg/ml streptomycin (Biowest). All the cell cultures were incubated at 37˚C with 5% CO 2 .
RNA extraction and reverse transcription-quantitative PCR (RT-qPCR). Following the manufacturer's protocol, TRIzol ® reagent (Thermo Fisher Scientific, Inc.) was used to isolate the RNA of tissues or cells, and the isolated RNA was subsequently reverse-transcribed using an Invitrogen™ PrimeScript RT Reagent kit (Thermo Fisher Scientific, Inc.). SYBR Premix Ex Taq™ reagent (Takara Biotechnology Co., Ltd.) was used for the qPCR assay, and qPCR was performed using an Applied Biosystems™ ABI PRISM 7500 PCR system (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. PCRs were performed in a total volume of 20 µl with 10 µl 2x SYBR premix ex-taq, 5 µl cDNA, 0.8 µl primers (2.5 µM) and 4.2 µl ddH 2 O). The PCR thermocycling conditions were; denaturation at 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec, 55˚C for 15 sec and 72˚C for 10 sec with a final extension step at 60˚C for 1 min. Melting curves of the amplified products were analyzed at the end of each PCR to confirm that only one product was amplified and detected. Expression was calculated via the relative quantification cycle (Cq) value and was normalized to the expression of the internal control gene GAPDH. Relative expression of RNCR3 was calculated using the 2 -ΔΔCq method (16). The primer sequences for RNCR3 and GAPDH are presented in Table II. The mean value was selected as the cut-off between high and low RNCR3 expression in patients with glioma. The relative expression of control groups was normalized to 1.
RNCR3 short hairpin RNA (shRNA). RNCR3 shRNA (sh-RNCR3) was purchased from Shanghai GenePharma Co. Ltd. And transfected into cells at a final concentration of 50 nM. An Invitrogen™ Lipofectamine ® 3000 kit (Thermo Fisher Scientific, Inc.) was used to perform the transfections, according to the manufacturer's protocol. RT-qPCR was subsequently used to evaluate the knockdown efficiency. For all experiments involving transfected cells, cells were transfected for 48 h before subsequent experiments were performed.
Cell Counting Kit-8 (CCK-8) assay. Cell proliferation was examined every 24 h, according to the manufacturer's protocol. Cells were placed into the 96-well plates at a density of ~3,000 cells/well. Subsequently, 10 µl CCK-8 (Dojindo Molecular technologies, Inc.) was added, and the cells were incubated at 37˚C for a further 2 h. Finally, the absorbance at 450 nm was determined using a spectrometer.
Cell cycle assay. Cells were collected, washed 3 times with cold PBS, and then fixed in 70% ethanol at 4˚C overnight. Subsequently, the cells were washed, re-suspended, and incubated in a solution of 10 µg/ml RNase and 1 mg/ml propidium iodide (Sigma-Aldrich; Merck KGaA) at 37˚C for 30 min in the dark. Finally, a FACSCalibur™ Cell Analyzer with ModFit version 5.0 (both BD Biosciences) was used to analyze the cells and the resultant flow cytometry data.
Cell invasion assay. The invasive ability of the cells was determined using BD 24-well Transwell ® chambers (Costar; Corning Inc.) pre-coated with Matrigel™ coating, according to the manufacturer's protocol. Briefly, 1x10 5 cells suspended in 200 µl serum-free medium were seeded into the upper chamber, and 800 µl DMEM supplemented with 10% FBS was placed into the lower chamber. After incubating the cells for 18 h at 37˚C, cells on the lower chamber membranes were fixed with 4% formaldehyde 15 min at room temperature and stained with 1% crystal violet at room temperature for 10 min. Cells in five randomly selected fields of the membrane were counted under a light microscope (magnification, x40).
Western blot analysis. RIPA buffer (Beyotime Institute of Biotechnology) was used to extract proteins from the cells. Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit. The proteins (30 µg per lane) were resolved on a 10% SDS-PAGE and then electrophoretically transferred to polyvinylidene fluoride membranes (Bio-Rad   ) was used to detect the intensities of the immunoblots, which were visualized following exposure to X-ray film. Protein expression levels were quantified relative to β-actin levels using ImageJ (version 1.8.0 National Institutes of Health).

Statistical analysis.
Experimental data from at least three independent experiments are presented as the mean ± standard deviation. SPSS 18.0 software (SPSS, Inc.) was used to perform the statistical analyses. The associations between the levels of RNCR3 and clinicopathological factors was assessed by χ 2 test. Differences between multiple groups were evaluated by one-way analysis of variance followed by Tukey's post hoc test. Differences between two groups were evaluated using an unpaired or paired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Expression of RNCR3 is increased in glioma tissues and cell lines.
RT-qPCR analysis was performed to measure the relative expression of RNCR3 in 54 pairs of glioma tissue and adjacent normal tissue, we have used the normal tissues as  control for normalization. The results obtained suggested that RNCR3 expression was markedly higher in glioma tissues compared with the corresponding adjacent normal tissues (Fig. 1A). RNCR3 expression was further examined in cell lines: A human astroglia cell line (HA), two human glioma cell lines (SHG-44 and U251), and a glioblastoma of unknown origin (U87). These experiments revealed that the expression of RNCR3 was higher in the glioma cell lines compared with that in the HA cell line (Fig. 1B).

Upregulated expression of RNCR3 is associated with progression of the disease, and poor prognosis of patients with glioma.
Subsequently, the correlation between RNCR3 expression levels and clinicopathological factors in 66 glioma individuals was investigated (Table I). This analysis revealed that an elevated expression of RNCR3 was associated with tumor size and clinical stage, although no significant correlations were identified between RNCR3 expression and age and gender. In addition, KaplanMeier survival curves suggested that glioma patients who had higher expression levels of RNCR3 in their glioma tissues had significantly poorer survival rates compared with glioma patients who had correspondingly lower expression levels of RNCR3 in their glioma tissues (Fig. 1C).
Knockdown of RNCR3 inhibits glioma cell proliferation. The results of the RT-qPCR assay confirmed that the RNCR3 expression levels of U87 and U251 cells transfected with sh-RNCR3 were lower compared with that of the control untransfected cells, or those U87 and U251 cells that were transfected with NC shRNA ( Fig. 2A and B). RNCR3 knockdown led to a repression of cell proliferation in the U87 (Fig. 2C) and U251 (Fig. 2D) glioma cell lines.

Knockdown of RNCR3 induces G 1 phase arrest of glioma cells.
The results of the cell cycle assay experiments revealed that knocked-down RNCR3 induced the G 1 phase arrest of U87 and U251 cells (Fig. 3). Fig. 4, the cell invasion assays confirmed that RNCR3 knockdown repressed cell invasion in the U87 and U251 cells.

Knockdown of RNCR3 inhibits cell invasion in the glioma cell lines. As shown in
Knockdown of RNCR3 inhibits the Akt/GSK-3β pathway in glioma cells. As has been well established, the Akt/GSK-3β signaling pathway is constitutively active in a large number of different types of human cancer. To further investigate whether RNCR3 regulates glioma development through Akt/GSK-3β pathway activation, western blot assays were performed to evaluate the levels of total and phosphorylated Akt and GSK-3β in the glioma cells. As shown in Fig. 5, silencing of RNCR3 did not exert a clear influence on the total level of Akt and GSK-3β, but the levels of phosphorylated Akt and GSK-3β were significantly reduced. These results indicated that overexpression of RNCR3 activated the Akt/GSK-3β pathway in human glioma cells.

Discussion
In view of the increasing number of studies that are being published focusing on the functional attributes of lncRNAs, emerging evidence has indicated that lncRNAs have important roles in various physiological and pathological processes, including cell proliferation, apoptosis and differentiation, and the development of different types of cancer (9,17,18). LncRNAs fulfill a range of different roles, including the regulation of gene transcription in basal transcription machinery, post-transcriptional regulation of RNA splicing and epigenetic regulation (19). For example, the lncRNA small ubiquitin-like modifier 1 pseudogene 3 promoted breast cancer progression by negatively regulating miR-320a (9). LncRNA highly upregulated in liver cancer   triggers autophagy by stabilizing sirtuin-1, and impairs the chemosensitivity of hepatocellular carcinoma cells (18). LncRNA HOxA transcript at the distal tip enhances tumorigenesis and metastasis in esophageal squamous carcinoma cells through the transcriptional and post-transcriptional regulation of homeobox protein A13 (19).
In the present study it was revealed for the first time, to the best of our knowledge, that the expression of lncRNA RNCR3 was upregulated in glioma tissues and cell lines. Additionally, the increased expression levels of RNCR3 in glioma tissues were associated with tumor progression and poor survival rates in patients with glioma. Furthermore, RNCR3 knockdown inhibited the proliferative and invasive abilities, and induced the G 1 phase arrest of glioma cells. Finally, the present study suggested that the effects of RNCR3 on cell proliferation, the cell cycle and cellular invasion may involve regulation of the Akt/GSK-3β signaling pathway.
A number of studies have suggested that aberrant signaling of the Akt pathway contributes to cell proliferation and invasion in various human malignancies (20). The results of the present study suggested that RNCR3 knockdown led to an inhibition of the phosphorylation of Akt, although there was no clear influence on the total levels of Akt. Akt phosphorylation has previously been reported to inhibit the activity of GSK-3β, which is an important downstream target protein of Akt (20). These results are consistent with previous studies, which reported on the function of Akt/GSK-3β in glioma (21,22). In addition, a large number of lncRNAs have been reported to regulate human cancers via actions on the Akt/GSK-3β pathway. For example, the lncRNA lung cancer-associated transcript 1 was revealed to enhance proliferation and invasion in clear cell renal cell carcinoma via the Akt/GSK-3β pathway (23). The lncRNA urothelial carcinoma-associated 1 (UCA1) was identified to promote tumorigenesis by regulating the AKT/GSK-3β pathway in cholangiocarcinoma (24). LncRNA UCA1, induced by SP1 transcription factor, was also shown to enhance cell proliferation by recruiting enhancer of zeste 2 polycomb repressive complex 2 subunit, the catalytic subunit of the polycomb repressive complex 2, and activating the Akt signaling pathway in gastric cancer (25).
In conclusion, the present study demonstrated that the expression level of RNCR3 was increased in glioma tissues and in cell lines, and that RNCR3 may therefore serve as a novel prognostic indicator for patients with glioma. Silencing RNCR3 in glioma cells led to a suppression of the proliferative and invasive abilities of the cells, and induced cell cycle arrest involving the Akt/GSK3β pathway.


ISOLATION OF ENDOPHYTES FROM CASTOR PLANT AND MEASURING ITS ACTIVITY AGAINST HEAVY METALS AND POLLUTANTS

Ms. Supriya Yadav. Department of microbiology, K. J. Somaiya College of Science and Commerce, Mumbai, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 12 February 2019 Final Accepted: 14 March 2019 Published: April 2019

The ever-increasing pollution of the environment has been one of the greatest concerns for science and the general public in the last fifty years. The rapid industrialization of agriculture, expansion of the chemical industry, and the need to generate cheap forms of energy has caused the continuous release of man-made organic chemicals into natural ecosystems. The benefits of combining endophytic bacteria with plants for increased remediation of pollutants have been successfully tried for toxic metal removal from contaminated soils. Endophytic bacteria reside within plant hosts without causing disease symptoms. Several methods have been devised for the treatment and removal of heavy metal in contaminated soils. Biological approach has the great potential that contributed for the bioaccumulation of heavy metals by endophytic bacterial assisted phytoremediation. Three endophytic bacteria were isolated from leaf, stem and root of Ricinus communis . All endophytic bacteria were able to grow at concentration of 5% Pb and 5% Zn and could be considered resistant to these metals. The plants which were under heavy metal stress and inoculated with endophytes showed the better growth as compared to control plants. This means that heavy metals are used by the organism for its own metabolism which converts the heavy metals from complex organic compounds into simple byproduct that promotes the growth of the plant. Endophytic association with host moong plants significantly increased the shoot length and biomass as compared to non inoculated control plants. The efficiency of all the three isolate was good in bioremediation of heavy metals. So if these isolates are used in consortium the yield will be more in bioremediation of heavy metals in highly contaminated soil. These isolates can be used as growth promoters in agriculture and also in the soil contaminated with heavy metals and other pollutants.

Introduction:-
The ever-increasing pollution of the environment has been one of the greatest concerns for science and the general public in the last fifty years. The rapid industrialization of agriculture, expansion of the chemical industry, and the need to generate cheap forms of energy has caused the continuous release of man-made organic chemicals into natural ecosystems. Consequently, the atmosphere, bodies of water, and many soil environments have become ISSN: 2320-5407 Int. J. Adv. Res. 7(4), 861-868 862 polluted by a large variety of toxic compounds. Metals like cadmium (Cd) and copper (Cu) are added in the agricultural soil during irrigation. These metals have toxic effect on living cells if these metals are present in high concentration. Cadmium can inhibit the growth and yield of plants as a result of chlorosis, instability of lipid membrane, oxidative damage and can react with biochemical functions. Copper is naturally present in the soil which play a vital role in synthesizing enzyme and proteins which are used in various metabolic processes by plants. (Mukti G. et al.,2014). However, if these metals are present in higher concentration can prove toxic for crops because it interferes with numerous physiological process.
This industrial water contain very high amount of heavy metals. These heavy metals and other pollutants have to be removed from the soil through low cost technology before it is applied in agriculture or other purpose. Many methods have been developed to remove the heavy metals like physico-chemicals which include reverse osmosis, solvent extraction, lime coagulation, ion exchange and chemical precipitation which is very expensive. Bioremediation is one such technique which is very efficiently used these days for treatment of heavy metals and other pollutants. Bioremediation is the technique that use microbial metabolism to remove pollutants.
The use of microbe have been reported a good biological absorbents to remove heavy metals. During last decades, a specific techniques such as bioremediation have been evolved which includes phytoremediation enhanced by endophytic microorganisms, rhizoremediation. Endophytes are microorganisms that reside within the inner parts of plants without causing any disease symptoms.Cultivable endophytic communities can be isolated after surface sterilization of the plant material. Endophytes exist in a range of tissue types within a broad range of plants, colonizing the plant systemically with bacterial colonies and biofilms, residing latently in intercellular spaces, inside the vascular tissue or within cells. It has been reported that many isolates provide beneficial effects to their hosts like preventing disease development by synthesizing novel compounds and antifungal metabolites. Investigations of biodiversity of endophyte strains for novel metabolites may identify new drugs for the treatment of human, plant and animal diseases.

Sampling:
Mature and healthy plants will be collected; samples should be processed within 8 hours. Endophytic organisms will be isolated from roots and rhizomes of castor plant. Plant tissue such as leaves stem and roots will be broadly screened for presence of endophytic microbes from castor plant.

Processing of the sample for isolation of the endophytes:
Sampling different parts of the selected castor plant, samples will be taken from roots, stems and leaves.

Disinfecting leaf samples:
Samples will be immersed in 70% ethanol for 1-3 min and 4% aqueous solution of sodium hypochlorite 15min, 1min with 70% ethanol again and finally rinsed 4-5 times with sterile distilled water.

Disinfecting stem samples:
With some modification samples will be treated with 70% ethanol for 3-4min and 4% aqueous solution of sodium hypochlorite 2 min, 0.1% mercury chloride for 1.5 min and rinsed with sterile distilled water.

Disinfecting root samples:
Root tissues will be immersed in 70% ethanol for 1-3min and 5% aqueous solution of sodium hypochlorite 1 min, 2 min with 70% ethanol, and 1 min with 0.15 mercury chloride and rinsed with sterile distilled water.

Enrichment of the sample:
Small disinfected pieces of root, leaves and stem sample was inoculated into 100mL sterile LB medium each. All the three flasks were incubated on shaker conditions at room temperature for 1 week.

Isolation of endophytic bacteria:
863 Endophytes were isolated from each of enriched broth onto sterile LB agar plates.Incubated at R.T for 24-48 hours.Pure endophytic cultures were isolated from crowded plates and maintained on fresh nutrient agar medium.

Resistance to heavy metal:
Minimum Inhibitory Concentration of both the heavy metals i.e Pb and Zn in the form of Pb(NO 3 ) and ZnSO 4 were checked for all the three isolates. 5% stock solution of heavy metals Pb(NO 3 ) ,ZnSO 4 was prepared in sterile LB medium. Sterile LB medium without heavy metals were used as a diluents. MIC of all the three isolates i.e R, S and L were carried out with increasing concentration of both the heavy metals along with positive and negative control. All the tubes were incubated at R.T for 24-48 hours and were checked for the inhibition.
Plant growth promotion: (Ruby J., 2013) On the basis of result obtained isolates i.e R, S and L was selected and was used for the pot assay. In pot assay different combination of isolates and heavy metals were carried out along with individual isolates, heavy metals and control.
The bioefficacy was determined based on root, shoot length and biomass of the plant. Moong seeds and soil was used as carrier for formulation. The enough amount of healthy monotous seeds of moong were separated by hand and superficial disinfection done with 95% ethanol within 30 sec and 3 min in HgCl 2 (0.2%) and finally were washed with DW at least 4-5min.. Sterile LB media was inoculated with all three endophytic isolates and incubated for 24-72 hrs at R.T. Sterile seeds were coated with inoculated broth and saw with equal distance in the pots. Pots were irrigated with water every day. After interval of 30 days seedlings were removed and shoot length, root length and biomass of the plant were measured.

Result:-
The three different bacterial species were isolated from different parts of the castor plant and the biochemical tests were performed was done to identify the organisms. Among them one were identified as Bacillus subtilis species and other were identified as Micrococcus luteus and Bacillus species by performing biochemicals.
The isolated endophytic bacteria exhibited tolerance to different heavy metals i.e Pb, Zn. All endophytic bacteria were able to grow at concentration of 5% Pb and 5% Zn and could be considered resistant to these metals. Since the average lead and zinc content of the soil are 0.003% and 0.02% respectively, the 5% of lead and zinc is very high so even if the organisms are not able tolerate this concentration of heavy metals it is not a problem.
To further assess the role of endophyte in improving plant physiology and relieving the adverse effects of Pb and Zn stress several parameters like root length , shoot length , leave length and biomass of the plants were determined The bioefficacy was determined based on root, shoot length and biomass of the plant. Moong seeds and soil was used as carrier for formulation. After interval of 30 days all the 12 seedlings from each pot were removed and shoot length, root length and biomass of the plant were measured. During Pb and Zn stress shoot length, root length, leave length were considerably reduced in endophyte free as compared to endophyte inoculated plants. Increased curling, drying and reduced leaf area was observed in the pots which were not inoculated with endophytes and were under heavy metal stress. This showed that plant was not able to tolerate heavy metals and it had effect on growth and physiology of the plant. The reduced growth of shoot and root was significant in non inoculated pots than in inoculated pots with heavy metals.The plants which were under heavy metal stress and inoculated with endophytes showed the better growth as compared to control plants. This means that heavy metals are used by the organism for its own metabolism which converts the heavy metals from complex organic compounds into simple byproduct that promotes the growth of the plant.Endophytic association with host moong plants significantly increased the shoot length and biomass as compared to non inoculated control plants. The plants which were inoculated with endophytes and were not under heavy metal stress showed the best growth. The leaf area was larger than the control plants. The root and shoot length was significantly larger than the control plants. The biomass of the endophyte inoculated plants was much higher than control plants.The pot which was inoculated with isolate R gave the best shoot length whereas one which was inoculated with isolate S gave the best root length of the plants as compared to control and other two isolates. In the pot inoculated with isolate L, the biomass of the plant was higher as compared to other two isolates. Also the leave length was more than the results obtained from other two isolates (  In the present study three bacterial endophytes were isolated from root, shoot and leaf interior of the Ricinus communis based on their morphological differences. Tolerance of the isolate towards heavy metal was the first parameter evaluated and all the isolates were able to tolerate these metals upto 5%. These three isolates showed the resistance to very high concentration of Pb and Zn. In many other studies Pb and Zn resistant bacteria were also isolated; however, they exhibited resistance to comparatively lower concentration of these metals. Similar study showed that the application of the heavy metal-resistant endophytic bacterial strains effectively increased the bioavailability of Pb in the rhizosphere soils and promoted the growth of rape plants, consequently increasing the total Pb uptake of the rape plants even under nonsterile conditions. In this study, comparatively less seed germination and root and shoot development was observed with plants cultivated in the heavy metal contaminated soil as compared to the plants vegetated in control soil ( Table No.

1).
The presence of Pb and Zn and other heavy metals in soil reduces seed germination and plant growth. Despite the toxic effects of heavy metals present in the contaminated soil, inoculation of Pb and Zn-resistant plant growthpromoting bacteria increased seed germination and root and shoot development. Particularly, three individual strains improved plant growth and development to a greater extent. Similarly, it was found that bacterial inoculation reduced Pb and Zn toxicity and improved seed germination and plant growth. In this study,the ability of the inoculated endophytic bacteria to improve plant growth in the heavy metal contaminated soil might be due to the combined effect of heavy metal tolerance ability as well as plant growth-promoting activities.
In this study, moong was found to be able to tolerate Pb and Zn contaminated soil. The application of Pb and Znresistant plant growth-promoting endophytic bacteria to moong further enhanced the tolerance of Pb and Zn from the soil. Similarly, earlier studies reported that the inoculation of Pb and Zn-resistant plant growth-promoting bacteria enhanced the heavy metals uptake by plants. (Mani Rajkumar et al, 2007). The present study suggests that the use of Pb and Zn-resistant plant growth-promoting bacteria protects the plant against the inhibitory effects of heavy metals present in the heavy metal contaminated soil and facilitates the transportation of heavy metals from soil in to above ground plant biomass.
In this study the plants which were under heavy metal stress and inoculated with endophytes showed the best growth of plants. There was significance increase in root, shoot, leave length and biomass of the plant as compared to control plants . the plants which were inoculated with only endophytes separately showed the significant increase in root ,shoot,leave length and biomass as compared to control plants. There wre decrease in root,shoot, leave length and biomass of the plant which were inoculated with only heavy metals separately. This means that heavy metals affect the growh and physiology of the plant. The growth of plants hich were under heavy metal stress and inoculated with only endophytes sepately wre much good as compared to plants which were under heavy metal stress.
The endophytic bacteria colonizing the plant tissue interior might protect plant against the inhibitory effects of high concentration of Pb and Zn which promotes the plant growth. Several earlier study also reported that this plant can accumulate high concentration of different metals in its roots, shoots and leaves. This might be one of the reasons that Ricinus commuinis hosts several bacteria in its tissue which can tolerate high concentration of heavy metals.
(Mani Rajkumar et al, 2007) The findings of the present study suggests that endophytes such as Bacillus circulans, Bacillus subtilis and Microccocus leutus cannot only improve the plant biomass but also resist the toxic effects of metal concentration. Such beneficial impacts may be due to secretion of bioactive compounds which might help in converting metal into stable complexes. However it is also essential to understand the in vivo mechanism by which the bioactive compounds producing endophytes reduce the metal toxicity.

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Plant-bacteria partnerships can be exploited to enhance phytoremediation efficiency of soil and water contaminated with organic and inorganic pollutants. The beneficial effects of heavy metal-resistant and plant growth-promoting bacteria include reduced heavy metals toxicity and accelerated root development, resulting in better access to nutrients and water and thus faster initial growth, leading to enhanced remediation of contaminated soil environmentally and economically sustainable plant biomass production. Improved yields on contaminated land might also reduce the need to clear and use additional areas of land for food, feed fiber and biofuel feedstock production for a growing world population, consequentially saving native ecosystems and biodiversity. Overall, the combined use of plants and bacteria can act as decontaminators by improving phytoremediation or protecting the food chain by decreasing the levels of pesticide residues in crops. (Khan et al., 2015) In present study, it was found that Ricinus commuinis hosted 3 culturable Pb and Zn -resistant bacteria in its interior tissue and their inoculation to moong improved plant growth and the remediation of heavy metal contaminated soil, suggesting their potential use in the remediation of heavy metal contaminated soil. The stimulatory effects of Pb and Zn-resistant plant growth-promoting endophytic bacteria on moong growth might be due to the additive effects of different plant growth-promoting properties of the isolated endophytic bacteria. Further plant-inoculation experiments in different combinations are needed to better understand the stimulatory effects of the combined inoculation strategy. The very high level of metal tolerance of the isolated bacteria of Ricinus commuinis makes them interesting candidates for further studies on the genes involved in this tolerance. Plants growing on heavy metal contaminated sites could be excellent ecosystems to isolate bacterial genes involved in metal resistance and/or plant growth promotion.

Conclusion:-
This study investigate the effects of inoculation with the endophytic bacterium, R,S,and L, on plant growth, root morphology, and biomass, a study that might facilitate the understanding of mechanisms of microbe-assisted phytoremediation. Through pot assay, all three bacterial isolate inoculations significantly enhanced the efficiency of zinc and lead phytoextraction by increasing R. communis biomass, promoting zinc and lead absorption, improving root, shoot and leave morphology. The application of endophytes to phytoremediation not only strengthens the ability of phyto-accumulators to acclimate to the contaminated environment, but also expands the application range of phytoremediation.
Thus, these promising endophytes may be commercially formulated effective biocontrol agents for the management of heavy metal contamination of the soil. These isolates were further selected for further studies on their effect on plant growth parametes, diseases incidences.