Brachypodium distachyon as a model plant toward improved biofuel crops: Search for secreted proteins involved in biogenesis and disassembly of cell wall polymers

Polysaccharides make up about 75% of plant cell walls and can be broken down to produce sugar substrates (saccharification) from which a whole range of products can be obtained, including bioethanol. Cell walls also contain 5–10% of proteins, which could be used to tailor them for agroindustrial uses. Here we present cell wall proteomics data of Brachypodium distachyon, a model plant for temperate grasses. Leaves and culms were analyzed during active growth and at mature stage. Altogether, 559 proteins were identified by LC‐MS/MS and bioinformatics, among which 314 have predicted signal peptides. Sixty‐three proteins were shared by two organs at two developmental stages where they could play housekeeping functions. Differences were observed between organs and stages of development, especially at the level of glycoside hydrolases and oxidoreductases. Differences were also found between the known cell wall proteomes of B. distachyon, Oryza sativa, and the Arabidopsis thaliana dicot. Three glycoside hydrolases could be immunolocalized in cell walls using polyclonal antibodies against proteotypic peptides. Organ‐specific expression consistent with proteomics results could be observed as well as cell‐specific localization. Moreover, the high number of proteins of unknown function in B. distachyon cell wall proteomes opens new fields of research for monocot cell walls.     

Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs

Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs.     

Anti-Tumor Effects of Ganoderma lucidum (Reishi) in Inflammatory Breast Cancer in In Vivo and In Vitro Models

The medicinal mushroom Ganoderma lucidum (Reishi) was tested as a potential therapeutic for Inflammatory Breast Cancer (IBC) using in vivo and in vitro IBC models. IBC is a lethal and aggressive form of breast cancer that manifests itself without a typical tumor mass. Studies show that IBC tissue biopsies overexpress E-cadherin and the eukaryotic initiation factor 4GI (eIF4GI), two proteins that are partially responsible for the unique pathological properties of this disease. IBC is treated with a multimodal approach that includes non-targeted systemic chemotherapy, surgery, and radiation. Because of its non-toxic and selective anti-cancer activity, medicinal mushroom extracts have received attention for their use in cancer therapy. Our previous studies demonstrate these selective anti-cancer effects of Reishi, where IBC cell viability and invasion, as well as the expression of key IBC molecules, including eIF4G is compromised. Thus, herein we define the mechanistic effects of Reishi focusing on the phosphoinositide-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, a regulator of cell survival and growth. The present study demonstrates that Reishi treated IBC SUM-149 cells have reduced expression of mTOR downstream effectors at early treatment times, as we observe reduced eIF4G levels coupled with increased levels of eIF4E bound to 4E-BP, with consequential protein synthesis reduction. Severe combined immunodeficient mice injected with IBC cells treated with Reishi for 13 weeks show reduced tumor growth and weight by ∼50%, and Reishi treated tumors showed reduced expression of E-cadherin, mTOR, eIF4G, and p70S6K, and activity of extracellular regulated kinase (ERK1/2). Our results provide evidence that Reishi suppresses protein synthesis and tumor growth by affecting survival and proliferative signaling pathways that act on translation, suggesting that Reishi is a potential natural therapeutic for breast and other cancers.     

External Validation of the Derived Neutrophil to Lymphocyte Ratio as a Prognostic Marker on a Large Cohort of Pancreatic Cancer Patients

Background

With growing evidence on the role of inflammation in cancer biology, the presence of a systemic inflammatory response has been postulated as having prognostic significance in a wide range of cancer types. The derived neutrophil to lymphocyte ratio (dNLR), which represents an easily determinable potential prognostic marker in daily practise and clinical trials, has never been externally validated in pancreatic cancer (PC) patients.

Methods

Data from 474 consecutive PC patients, treated between 2004 and 2012 at a single centre, were evaluated retrospectively. Cancer-specific survival (CSS) was assessed using the Kaplan-Meier method. To evaluate the prognostic relevance of dNLR, univariate and multivariate Cox regression models were applied.

Results

We calculated by ROC analysis a cut-off value of 2.3 for the dNLR to be ideal to discriminate between patients’ survival in the whole cohort. Kaplan-Meier curve reveals a dNLR≥2.3 as a factor for decreased CSS in PC patients (p<0.001, log-rank test). An independent significant association between high dNLR≥2.3 and poor clinical outcome in multivariate analysis (HR = 1.24, CI95% = 1.01–1.51, p = 0.041) was identified.

Conclusion

In the present study we confirmed elevated pre-treatment dNLR as an independent prognostic factor for clinical outcome in PC patients. Our data encourage independent replication in other series and settings of this easily available parameter as well as stratified analysis according to tumor resectability.     

Complement C3 and C5 Deficiency Affects Fracture Healing

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3^-/- and C5^-/- as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3^-/-: -25%, p=0.02; C5^-/-: -20% p=0.052) and newly formed bone (C3^-/-: -38%, p=0.01; C5^-/-: -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3^-/- mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.     

Triploid Atlantic salmon Salmo salar have a higher dietary phosphorus requirement for bone mineralization during early development

The effect of a dietary phosphorus regime in freshwater on vertebra bone mineralization was assessed in diploid and triploid Atlantic salmon, Salmo salar. Fish were fed either a low phosphorus (LP) diet containing 10.5 g kg⁻¹ total phosphorus or a normal phosphorus (NP) diet containing 17.4 g kg⁻¹ total phosphorus from ∼3 to ∼65 g (day 126) in body weight. Two further groups were fed the NP diet from ∼3 g in body weight, but were then switched to the LP diet after 38 (∼10 g in body weight) or 77 (∼30 g in body weight) days. Growth, vertebral ash content (% ash) and radiologically detectable vertebra pathologies were assessed. Triploids were initially smaller than diploids, and again on day 77, but there was no ploidy effect on days 38 or 126. Vertebral ash content increased with increasing body size and those fish fed the NP diet had higher vertebral ash content than those groups fed the LP diet during the intervening time period, but this diet effect became less apparent as fish grew, with all groups having relatively equal vertebral ash content at termination. In general, triploids had lower vertebral ash content than diploids on day 38 and this was most evident in the group fed the LP diet. On day 77, those triploids fed the LP diet during the intervening time period had lower vertebral ash content than diploids. At termination on day 126, the triploids had the same vertebral ash content as diploids, irrespective of diet. There was a ploidy × diet interaction on vertebral deformities, with triploids having higher prevalences of fish with ≥1 deformed vertebra in all dietary groups except continuous NP. In conclusion, between days 0 and 77 (3–30 g body size), triploids required more dietary phosphorus than diploids in order to maintain similar vertebral ash content. A possible link between phosphorus feeding history and phosphorus demand is also discussed.     

Trait-dependency of trophic interactions in zooplankton food webs

Anthropogenic change in the abundance or identity of dominant top predators may induce reorganizations in whole food webs. Predicting these reorganizations requires identifying the biological rules that govern trophic niches. However, we still lack a detailed understanding of the respective contributions of body size, behaviour (e.g. match between predator hunting mode and prey antipredator strategy), phylogeny and/or ontogeny in determining both the presence and strength of trophic interactions. Here, we address this question by measuring zooplankton numerical response to fish predators in lake enclosures. We compared the fit to zooplankton count data of models grouping zooplankters based either on 1) body sizes, 2) antipredator behaviour, 3) body size combined with antipredator behaviour or on 4) phylogeny combined with ontogeny (i.e. different life stages of copepods). Body size was a better predictor of zooplankton numerical response to fish than antipredator behaviour, but combining body size and behaviour provided even better predictions. Models based on phylogeny combined with ontogeny clearly outperformed those based on other zooplankton grouping rules, except when phylogeny was poorly resolved. Removing ontogenetic information plagued the predictive power of the highly-resolved (genus-level) phylogenetic grouping but not of medium-resolved or poorly-resolved phylogenetic grouping. Our results support the recent use of phylogeny as a superior surrogate for traits controlling trophic niches, and further highlight the added value of combining phylogeny with ontogenetic traits. Further improvements in our mechanistic understanding of how trophic networks are shaped are bound to uncovering the trophic traits captured by phylogeny and ontogeny, but that currently remain hidden to us.