Francisella tularensis: No Evidence for Transovarial Transmission in the Tularemia Tick Vectors Dermacentor reticulatus and Ixodes ricinus

Background

Tularemia is a zoonosis caused by the Francisella tularensis, a highly infectious Gram-negative coccobacillus. Due to easy dissemination, multiple routes of infection, high environmental contamination and morbidity and mortality rates, Francisella is considered a potential bioterrorism threat and classified as a category A select agent by the CDC. Tick bites are among the most prevalent modes of transmission, and ticks have been indicated as a possible reservoir, although their reservoir competence has yet to be defined. Tick-borne transmission of F. tularensis was recognized in 1923, and transstadial transmission has been demonstrated in several tick species. Studies on transovarial transmission, however, have reported conflicting results.

Objective

The aim of this study was to evaluate the role of ticks as reservoirs for Francisella, assessing the transovarial transmission of F. tularensis subsp. holarctica in ticks, using experimentally-infected females of Dermacentor reticulatus and Ixodes ricinus.

Results

Transmission electron microscopy and fluorescence in situ hybridization showed F. tularensis within oocytes. However, cultures and bioassays of eggs and larvae were negative; in addition, microscopy techniques revealed bacterial degeneration/death in the oocytes.

Conclusions

These results suggest that bacterial death might occur in oocytes, preventing the transovarial transmission of Francisella. We can speculate that Francisella does not have a defined reservoir, but that rather various biological niches (e.g. ticks, rodents), that allow the bacterium to persist in the environment. Our results, suggesting that ticks are not competent for the bacterium vertical transmission, are congruent with this view.     

Emblica officinalis Extract Induces Autophagy and Inhibits Human Ovarian Cancer Cell Proliferation,Angiogenesis, Growth of Mouse Xenograft Tumors

Patients with ovarian cancer (OC) may be treated with surgery, chemotherapyand/or radiation therapy, although none of these strategies are very effective.Several plant-based natural products/dietary supplements, including extractsfrom Emblicaofficinalis (Amla), havedemonstrated potent anti-neoplastic properties. In this study we determined thatAmla extract (AE) has anti-proliferative effects on OC cells under bothin vitro and in vivo conditions. We alsodetermined the anti-proliferative effects one of the components of AE,quercetin, on OC cells under in vitro conditions. AE did notinduce apoptotic cell death, but did significantly increase the expression ofthe autophagic proteins beclin1 and LC3B-II under in vitroconditions. Quercetin also increased the expression of the autophagic proteinsbeclin1 and LC3B-II under in vitro conditions. AE alsosignificantly reduced the expression of several angiogenic genes, includinghypoxia-inducible factor 1α (HIF-1α) in OVCAR3 cells. AE acted synergisticallywith cisplatin to reduce cell proliferation and increase expression of theautophagic proteins beclin1 and LC3B-II under in vitroconditions. AE also had anti-proliferative effects and induced the expression ofthe autophagic proteins beclin1 and LC3B-II in mouse xenograft tumors.Additionally, AE reduced endothelial cell antigen – CD31 positive blood vesselsand HIF-1α expression in mouse xenograft tumors. Together, these studiesindicate that AE inhibits OC cell growth both in vitro andin vivo possibly via inhibition of angiogenesis andactivation of autophagy in OC. Thus AE may prove useful as an alternative oradjunct therapeutic approach in helping to fight OC.     

Porphyromonas gingivalis Evasion of Autophagy and Intracellular Killing by Human Myeloid Dendritic Cells Involves DC-SIGN-TLR2 Crosstalk

Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.     

Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.     

Plasmodium falciparum Erythrocytic Stage Parasites Require the Putative Autophagy Protein PfAtg7 for Normal Growth

Analysis of the Plasmodium falciparum genome reveals a limited number of putative autophagy genes, specifically the four genes involved in ATG8 lipidation, an essential step in formation of autophagosomes. In yeast, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. These four putative P. falciparum ATG (PfATG) genes are transcribed during the parasite’s erythrocytic stages. PfAtg7 has relatively low identity and similarity to yeast Atg7 (14.7% and 32.2%, respectively), due primarily to long insertions typical of P. falciparum. Excluding the insertions the identity and similarity are higher (38.0% and 70.8%, respectively). This and the fact that key residues are conserved, including the catalytic cysteine and ATP binding domain, we hypothesize that PfAtg7 is the activating enzyme of PfAtg8. To assess the role of PfAtg7 we have generated two transgenic parasite lines. In one, the PfATG7 locus was modified to introduce a C-terminal hemagglutinin tag. Western blotting reveals two distinct protein species, one migrating near the predicted 150 kDa and one at approximately 65 kDa. The second transgenic line introduces an inducible degradation domain into the PfATG7 locus, allowing us to rapidly attenuate PfAtg7 protein levels. Corresponding species are also observed in this parasite line at approximately 200 kDa and 100 kDa. Upon PfATG7 attenuation parasites exhibit a slow growth phenotype indicating the essentiality of this putative enzyme for normal growth.     

Host Cell Autophagy Is Induced by Toxoplasma gondii and Contributes to Parasite Growth

Autophagy has been shown to contribute to defense against intracellular bacteria and parasites. In comparison, the ability of such pathogens to manipulate host cell autophagy to their advantage has not been examined. Here we present evidence that infection by Toxoplasma gondii, an intracellular protozoan parasite, induces host cell autophagy in both HeLa cells and primary fibroblasts, via a mechanism dependent on host Atg5 but independent of host mammalian target of rapamycin suppression. Infection led to the conversion of LC3 to the autophagosome-associated form LC3-II, to the accumulation of LC3-containing vesicles near the parasitophorous vacuole, and to the relocalization toward the vacuole of structures labeled by the phosphatidylinositol 3-phosphate indicator YFP-2×FYVE. The autophagy regulator beclin 1 was concentrated in the vicinity of the parasitophorous vacuole in infected cells. Inhibitor studies indicated that parasite-induced autophagy is dependent on calcium signaling and on abscisic acid. At physiologically relevant amino acid levels, parasite growth became defective in Atg5-deficient cells, indicating a role for host cell autophagy in parasite recovery of host cell nutrients. A flow cytometric analysis of cell size as a function of parasite content revealed that autophagy-dependent parasite growth correlates with autophagy-dependent consumption of host cell mass that is dependent on parasite progression. These findings indicate a new role for autophagy as a pathway by which parasites may effectively compete with the host cell for limiting anabolic resources.Macroautophagy (hereafter referred to as autophagy) is a major catabolic process in which cytosolic constituents are sequestered within double-membraned vesicles (autophagosomes) and subsequently delivered to lysosomes for degradation. Current evidence indicates at least two distinct functions for this process. On the one hand, autophagy can be up-regulated under nutrient-limiting conditions to increase nutrient supply via recycling of the products of autophagic degradation, which may be exported from the lysosome (1). The up-regulation of autophagy upon starvation is thought to be mediated by the suppression of signaling through the mTOR pathway (2). On the other hand, autophagy can serve to maintain cellular homeostasis by facilitating the removal of damaged or deleterious elements, such as misfolded protein aggregates (3). An important example of the latter function is the role of autophagy in restricting the growth of intracellular pathogens, including both free bacteria that have escaped into host cytosol, such as group A Streptococcus, and pathogens, such as Mycobacterium tuberculosis, that reside in parasitophorous vacuoles in macrophages (4, 5). In macrophages infected with Toxoplasma gondii, fusion of the parasitophorous vacuole with lysosomes can be induced in an autophagy-dependent manner when host cell anti-parasitic function is activated via CD40 (6). Autophagy as a component of host defense may be up-regulated by inflammatory agents such as lipopolysaccharide (7) and interferon-γ (8).Although the clearance function of autophagy may enhance pathogen killing in host cells that have been activated to generate antimicrobial or antiparasitic function, in permissive host cells, in which the pathogen is less susceptible to sequestration by the autophagosome, autophagy may conceivably play a quite different role. Modulation of the balance between anabolic and catabolic processes may affect the outcome of competition between pathogen and host cell for limiting nutrients. In particular, the nutritive function of autophagy could favor pathogen expansion by providing greater access to host cell biomass. The intracellular apicomplexan parasite, T. gondii, is a suitable agent for the investigation of this hypothesis, because it has been shown to be highly dependent on its host cell for the supply of several nutrients, including amino acids (9), lipids (10), and purines (11). T. gondii replicates within a parasitophorous vacuole that, in permissive host cells, is protected from lysosomal fusion. Recent evidence indicates that in such permissive cells, in which the parasite can differentiate into bradyzoites associated with chronic infection, the pathogen is able to actively sequester host cell lysosome-derived vesicles, thereby potentially gaining access to their contents (12).The ability of intracellular parasites to regulate host cell autophagy has been little examined, and there is also little information with respect to the impact of these pathogens on host cell signals that potentially affect the autophagic pathway. In addition to mTOR, these include calcium ions, which have been implicated in autophagy induced by endoplasmic reticulum stress (13). In this study, we provide evidence that T. gondii induces host cell autophagy by a mechanism dependent on calcium but independent of mTOR and that it exploits the nutritive function of host autophagy to enhance its proliferation.     

FAM5C Contributes to Aggressive Periodontitis

Aggressive periodontitis is characterized by a rapid and severe periodontal destruction in young systemically healthy subjects. A greater prevalence is reported in Africans and African descendent groups than in Caucasians and Hispanics. We first fine mapped the interval 1q24.2 to 1q31.3 suggested as containing an aggressive periodontitis locus. Three hundred and eighty-nine subjects from 55 pedigrees were studied. Saliva samples were collected from all subjects, and DNA was extracted. Twenty-one single nucleotide polymorphisms were selected and analyzed by standard polymerase chain reaction using TaqMan chemistry. Non-parametric linkage and transmission distortion analyses were performed. Although linkage results were negative, statistically significant association between two markers, rs1935881 and rs1342913, in the FAM5C gene and aggressive periodontitis (p = 0.03) was found. Haplotype analysis showed an association between aggressive periodontitis and the haplotype A-G (rs1935881-rs1342913; p = 0.009). Sequence analysis of FAM5C coding regions did not disclose any mutations, but two variants in conserved intronic regions of FAM5C, rs57694932 and rs10494634, were found. However, these two variants are not associated with aggressive periodontitis. Secondly, we investigated the pattern of FAM5C expression in aggressive periodontitis lesions and its possible correlations with inflammatory/immunological factors and pathogens commonly associated with periodontal diseases. FAM5C mRNA expression was significantly higher in diseased versus healthy sites, and was found to be correlated to the IL-1β, IL-17A, IL-4 and RANKL mRNA levels. No correlations were found between FAM5C levels and the presence and load of red complex periodontopathogens or Aggregatibacter actinomycetemcomitans. This study provides evidence that FAM5C contributes to aggressive periodontitis.     

Growth and Yield Responses of Soybean to Aldicarb

A series of greenhouse, phytotron, field, and microplot experiments evaluated factors that influenced plant-growth.stimulation associated with the use of the pesticide aldicarb. A phytotron experiment showed.that aldicarb increased growth, of Ransom soybean at all temperatures but was somewhat phytotoxic to Coker 156 soybean at 30 C. Soybean gave the greatest response to this nematicide at 22 C in a commercially available medium, Metromix 220. Soybean cultivars Ransom and Coker 156. exhibited increased growth in response to aldicarb or, to a lesser extent aldicarb sulfone treatments under greenhouse and microplot conditions. Enhanced soybean growth, however, did not always result in significantly greater soybean seed yield. Soil type affected soybean sensitivity to aldicarb, with. the greatest growth and yield increases generally occurring in fine-textured soils or those with high.organic matter. Plant-growth stimulation by aldicarb occurs in the absence of pests but is dependent upon concentration and edaphic and other environmental factors.     

Drosophila Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation.     

Transgenic Rice Expressing Ictb and FBP/Sbpase Derived from Cyanobacteria Exhibits Enhanced Photosynthesis and Mesophyll Conductance to CO2

To find a way to promote the rate of carbon flux and further improve the photosynthetic rate in rice, two CO₂-transporting and fixing relevant genes, Ictb and FBP/Sbpase, which were derived from cyanobacteria with the 35SCaMV promotor in the respective constructs, were transformed into rice. Three homologous transgenic groups with Ictb, FBP/Sbpase and the two genes combined were constructed in parallel, and the functional effects of these two genes were investigated by physiological, biochemical and leaf anatomy analyses. The results indicated that the mesophyll conductance and net photosynthetic rate were higher at approximately 10.5–36.8% and 13.5–34.6%, respectively, in the three groups but without any changes in leaf anatomy structure compared with wild type. Other physiological and biochemical parameters increased with the same trend in the three groups, which showed that the effect of FBP/SBPase on improving photosynthetic capacity was better than that of ICTB and that there was an additive effect in ICTB+FBP/SBPase. ICTB localized in the cytoplasm, whereas FBP/SBPase was successfully transported to the chloroplast. The two genes might show a synergistic interaction to promote carbon flow and the assimilation rate as a whole. The multigene transformation engineering and its potential utility for improving the photosynthetic capacity and yield in rice were discussed.     

Mycobacterium tuberculosis Is Resistant to Isoniazid at a Slow Growth Rate by Single Nucleotide Polymorphisms in katG Codon Ser315

An important aim for improving TB treatment is to shorten the period of antibiotic therapy without increasing relapse rates or encouraging the development of antibiotic-resistant strains. In any M. tuberculosis population there is a proportion of bacteria that are drug-tolerant; this might be because of pre-existing populations of slow growing/non replicating bacteria that are protected from antibiotic action due to the expression of a phenotype that limits drug activity. We addressed this question by observing populations of either slow growing (constant 69.3h mean generation time) or fast growing bacilli (constant 23.1h mean generation time) in their response to the effects of isoniazid exposure, using controlled and defined growth in chemostats. Phenotypic differences were detected between the populations at the two growth rates including expression of efflux mechanisms and the involvement of antisense RNA/small RNA in the regulation of a drug-tolerant phenotype, which has not been explored previously for M. tuberculosis. Genotypic analyses showed that slow growing bacilli develop resistance to isoniazid through mutations specifically in katG codon Ser³¹⁵ which are present in approximately 50–90% of all isoniazid-resistant clinical isolates. The fast growing bacilli persisted as a mixed population with katG mutations distributed throughout the gene. Mutations in katG codon Ser³¹⁵ appear to have a fitness cost in vitro and particularly in fast growing cultures. Our results suggest a requirement for functional katG-encoded catalase-peroxide in the slow growers but not the fast-growing bacteria, which may explain why katG codon Ser³¹⁵ mutations are favoured in the slow growing cultures.     

Tent5a modulates muscle fiber formation in adolescent idiopathic scoliosis via maintenance of myogenin expression

ObjectiveParavertebral muscle asymmetry may be involved in the pathogenesis of adolescent idiopathic scoliosis (AIS), and the Tent5a protein was recently identified as a novel active noncanonical poly(A) polymerase. We, therefore, explored the function of the AIS susceptibility gene Tent5a in myoblasts.Materials and methodsRNA‐seq of AIS paravertebral muscle was performed, and the molecular differences in paravertebral muscle were investigated. Twenty‐four AIS susceptibility genes were screened, and differential expression of Tent5a in paravertebral muscles was confirmed with qPCR and Western blot. After the knockdown of Tent5a, the functional effects of Tent5a on C2C12 cell proliferation, migration, and apoptosis were detected by Cell Counting Kit‐8 assay, wound‐healing assay, and TUNEL assay, respectively. Myogenic differentiation markers were tested with immunofluorescence and qPCR in vitro, and muscle fiber formation was compared in vivo.ResultsThe AIS susceptibility gene Tent5a was differentially expressed in AIS paravertebral muscles. Tent5a knockdown inhibited the proliferation and migration of C2C12 cells and inhibited the maturation of type I muscle fibers in vitro and in vivo. Mechanistically, the expression of myogenin was decreased along with the suppression of Tent5a.Conclusions Tent5a plays an important role in the proliferation and migration of myoblasts, and it regulates muscle fiber maturation by maintaining the stability of myogenin. Tent5a may be involved in the pathogenesis of AIS by regulating the formation of muscle fiber type I.     

In Vivo Transfer of Intracellular Labels from Locally Implanted Bone Marrow Stromal Cells to Resident Tissue Macrophages

Intracellular labels such as dextran coated superparamagnetic iron oxide nanoparticles (SPION), bromodeoxyuridine (BrdU) or green fluorescent protein (GFP) are frequently used to study the fate of transplanted cells by in vivo magnetic resonance imaging or fluorescent microscopy. Bystander uptake of labeled cells by resident tissue macrophages (TM) can confound the interpretation of the presence of intracellular labels especially during direct implantation of cells, which can result in more than 70% cell death. In this study we determined the percentages of TM that took up SPION, BrdU or GFP from labeled bone marrow stromal cells (BMSCs) that were placed into areas of angiogenesis and inflammation in a mouse model known as Matrigel™ plaque perfusion assay. Cells recovered from digested plaques at various time points were analyzed by fluorescence microscopy and flow cytometry. The analysis of harvested plaques revealed 5% of BrdU⁺, 5–10% of GFP⁺ and 5–15% of dextran⁺ macrophages. The transfer of the label was not dependent on cell dose or viability. Collectively, this study suggests that care should be taken to validate donor origin of cells using an independent marker by histology and to assess transplanted cells for TM markers prior to drawing conclusions about the in vivo behavior of transplanted cells.     

In Situ Transplantation of Alginate Bioencapsulated Adipose Tissues Derived Stem Cells (ADSCs) via Hepatic Injection in a Mouse Model

Objective

Adipose tissue derived stem cells (ADSCs) transplantation has recently gained widespread enthusiasm, particularly in the perspective to use them as potential alternative cell sources for hepatocytes in cell based therapy, mainly because of their capability of hepatogenic differentiation in vitro and in vivo. But some challenges remain to be addressed, including whether ADSCs can be provided effectively to the target organ and whether subsequent proliferation of transplanted cells can be achieved. To date, intrasplenic injection is the conventional method to deliver ADSCs into the liver; however, a number of donor cells retained in the spleen has been reported. In this study, our objective is to evaluate a novel route to transplant ADSCs specifically to the liver. We aimed to test the feasibility of in situ transplantation of ADSCs by injecting bioencapsulated ADSCs into the liver in mouse model.

Methods

The ADSCs isolated from human alpha 1 antitrypsin (M-hAAT) transgenic mice were used to allow delivered ADSCs be readily identified in the liver of recipient mice, and alginate was selected as a cell carrier. We first evaluated whether alginate microspheres are implantable into the liver tissue by injection and whether ADSCs could migrate from alginate microspheres (study one). Once proven, we then examined the in vivo fate of ADSCs loaded microspheres in the liver. Specifically, we evaluated whether transplanted, undifferentiated ASDCs could be induced by the local microenvironment toward hepatogenic differentiation and the distribution of surviving ADSCs in major tissue organs (study two).

Results

Our results indicated ADSCs loaded alginate microspheres were implantable into the liver. Both degraded and residual alginate microspheres were observed in the liver up to three weeks. The viable ADSCs were detectable surrounding degraded and residual alginate microspheres in the liver and other major organs such as bone marrow and the lungs. Importantly, transplanted ADSCs underwent hepatogenic differentiation to become cells expressing albumin in the liver. These findings improve our understanding of the interplay between ADSCs (donor cells), alginate (biomaterial), and local microenvironment in a hepatectomized mouse model, and might improve the strategy of in situ transplantation of ADSCs in treating liver diseases.     

In Vitro Morphogenesis of Arabidopsis to Search for Novel Endophytic Fungi Modulating Plant Growth

Fungal endophytes have shown to affect plant growth and to confer stress tolerance to the host; however, effects of endophytes isolated from water plants have been poorly investigated. In this study, fungi isolated from stems (stem-E) and roots (root-E) of Mentha aquatica L. (water mint) were identified, and their morphogenetic properties analysed on in vitro cultured Arabidopsis (L.) Heynh., 14 and 21 days after inoculation (DAI). Nineteen fungi were analysed and, based on ITS analysis, 17 isolates showed to be genetically distinct. The overall effect of water mint endophytes on Arabidopsis fresh (FW) and dry weight (DW) was neutral and positive, respectively, and the increased DW, mainly occurring 14 DAI, was possibly related to plant defence mechanism. Only three fungi increased both FW and DW of Arabidopsis at 14 and 21 DAI, thus behaving as plant growth promoting (PGP) fungi. E-treatment caused a reduction of root depth and primary root length in most cases and inhibition-to-promotion of root area and lateral root length, from 14 DAI. Only Phoma macrostoma, among the water mint PGP fungi, increased both root area and depth, 21 DAI. Root depth and area 14 DAI were shown to influence DWs, indicating that the extension of the root system, and thus nutrient uptake, was an important determinant of plant dry biomass. Reduction of Arabidopsis root depth occurred to a great extent when plants where treated with stem-E while root area decreased or increased under the effects of stem-E and root-E, respectively, pointing to an influence of the endophyte origin on root extension. M. aquatica and many other perennial hydrophytes have growing worldwide application in water pollution remediation. The present study provided a model for directed screening of endophytes able to modulate plant growth in the perspective of future field applications of these fungi.     

Schizosaccharomyces pombe KAT5 contributes to resection and repair of a DNA double-strand break

Chromatin remodeling is essential for effective repair of a DNA double-strand break (DSB). KAT5 (Schizosaccharomyces pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that coordinates various DNA damage response activities at a DNA DSB, including histone remodeling and activation of the DNA damage checkpoint. In S. pombe, mutations in mst1⁺ causes sensitivity to DNA damaging drugs. Here we show that Mst1 is recruited to DSBs. Mutation of mst1⁺ disrupts recruitment of repair proteins and delays resection. These defects are partially rescued by deletion of pku70, which has been previously shown to antagonize repair by homologous recombination (HR). These phenotypes of mst1 are similar to pht1-4KR, a nonacetylatable form of histone variant H2A.Z, which has been proposed to affect resection. Our data suggest that Mst1 functions to direct repair of DSBs toward HR pathways by modulating resection at the DSB.     

SH2D2A Modulates T Cell Mediated Protection to a B Cell Derived Tumor in Transgenic Mice

Background

T cell specific adapter protein (TSAd), encoded by the SH2D2A gene, modulates signaling downstream of the T cell receptor (TCR). Young, unchallenged SH2D2A-deficient C57BL/6 mice exhibit a relatively normal immune phenotype. To address whether SH2D2A regulates physiologic immune responses, SH2D2A-deficient TCR-transgenic BALB/c mice were generated. The transgenic TCR recognizes a myeloma-derived idiotypic (Id) peptide in the context of the major histocompatibility complex (MHC) class II molecule I-E^d, and confers T cell mediated resistance to transplanted multiple myeloma development in vivo.

Principal Findings

The immune phenotype of SH2D2A-deficient C57BL/6 and BALB/c mice did not reveal major differences compared to the corresponding wild type mice. When challenged with myeloma cells, Id-specific TCR-transgenic BALB/c mice lacking SH2D2A displayed increased resistance towards tumor development. Tumor free TCR-transgenic SH2D2A-deficient mice had higher numbers of Id-specific single positive CD4+ thymocytes compared to TCR-transgenic wild-type mice.

Conclusion

Our results suggest a modulatory role for SH2D2A in T cell mediated immune surveillance of cancer. However, it remains to be established whether its effect is T-cell intrinsic. Further studies are required to determine whether targeting SH2D2A function in T cells may be a potential adjuvant in cancer immunotherapy.