Population Analysis of Pair Densities - A Study of Bassis Set Dependence

Based on work reported by several authors, one of us (RP) has introduced a Mulliken-like population analysis of pair densities. This scheme, introduced first at the semi-empirical level, was subsequently generalised both for SCF and for post-SCF ab initio methods. A potential problem is the basis set dependence that can be expected for all kinds of Mulliken-like approaches. The main purpose of the present study is to quantify this basis set dependence for a range of simple molecules, using basis sets ranging from STO-3Gto TZVP quality. It is shown, except for lowquality basis sets, that the »effective«pair populations derived from ab initio SCF calculations are relatively insensitive to the choice of basis set

Population Analysis of Pair Densities - A Study of Bassis Set Dependence

Based on work reported by several authors, one of us (RP) has introduced a Mulliken-like population analysis of pair densities. This scheme, introduced first at the semi-empirical level, was subsequently generalised both for SCF and for post-SCF ab initio methods. A potential problem is the basis set dependence that can be expected for all kinds of Mulliken-like approaches. The main purpose of the present study is to quantify this basis set dependence for a range of simple molecules, using basis sets ranging from STO-3Gto TZVP quality. It is shown, except for lowquality basis sets, that the »effective«pair populations derived from ab initio SCF calculations are relatively insensitive to the choice of basis set

Novel PCB-degrading Rhodococcus strains able to promote plant growth for assisted rhizoremediation of historically polluted soils

Extended soil contamination by polychlorinated biphenyls (PCBs) represents a global environmental issue that can hardly be addressed with the conventional remediation treatments. Rhizoremediation is a sustainable alternative, exploiting plants to stimulate in situ the degradative bacterial communities naturally occurring in historically polluted areas. This approach can be enhanced by the use of bacterial strains that combine PCB degradation potential with the ability to promote plant and root development. With this aim, we established a collection of aerobic bacteria isolated from the soil of the highly PCB-polluted site \u201cSIN Brescia-Caffaro\u201d (Italy) biostimulated by the plant Phalaris arundinacea. The strains, selected on biphenyl and plant secondary metabolites provided as unique carbon source, were largely dominated by Actinobacteria and a significant number showed traits of interest for remediation, harbouring genes homologous to bphA, involved in the PCB oxidation pathway, and displaying 2,3-catechol dioxygenase activity and emulsification properties. Several strains also showed the potential to alleviate plant stress through 1-aminocyclopropane-1-carboxyl-ate deaminase activity. In particular, we identified three Rhodococcus strains able to degrade in vitro several PCB congeners and to promote lateral root emergence in the model plant Arabidopsis thaliana in vivo. In addition, these strains showed the capacity to colonize the root system and to increase the plant biomass in PCB contaminated soil, making them ideal candidates to sustain microbial-assisted PCB rhizoremediation through a bioaugmentation approach

Development and characterization of a high-throughput in vitro cord formation model insensitive to VEGF inhibition

BACKGROUND: Anti-VEGF therapy reduces tumor blood vessels, however, some vessels always remain. These VEGF insensitive vessels may help support continued tumor growth and metastases. Many in vitro assays examining multiple steps of the angiogenic process have been described, but the majority of these assays are sensitive to VEGF inhibition. There has been little focus on the development of high-throughput, in vitro assays to model the vessels that are insensitive to VEGF inhibition. METHODS: Here, we describe a fixed end-point and kinetic, high-throughput stem cell co-culture model of cord formation. RESULTS: In this system, cords develop within 24 hours, at which point they begin to lose sensitivity to VEGF inhibitors, bevacizumab, and ramucirumab. Consistent with the hypothesis that other angiogenic factors maintain VEGF-independent vessels, pharmacologic intervention with a broad spectrum anti-angiogenic antagonist (suramin), a vascular disrupting agent (combretastatin), or a combination of VEGF and Notch pathway inhibitors reduced the established networks. In addition, we used our in vitro approach to develop an in vivo co-implant vasculogenesis model that connects with the endogenous vasculature to form functional blood vessels. Similar to the in vitro system, over time these vessels become insensitive to VEGF inhibition. CONCLUSION: Together, these models may be used to identify novel drugs targeting tumor vessels that are not sensitive to VEGF inhibition

Micro-CT imaging reveals<i> Mekk3 </i>heterozygosity prevents cerebral cavernous malformations in <i>Ccm2</i>-deficient mice

Mutations in CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation in humans. Mouse models of CCM disease have been established by deleting Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. However, the full value of these animal models is limited by the lack of an accurate and quantitative method to assess lesion burden and progression. In the present study we have established a refined and detailed contrast enhanced X-ray micro-CT method to measure CCM lesion burden in mouse brains. As this study utilized a voxel dimension of 9.5μm (leading to a minimum feature size of approximately 25μm), it is therefore sufficient to measure CCM lesion volume and number globally and accurately, and provide high-resolution 3-D mapping of CCM lesions in mouse brains. Using this method, we found loss of Ccm1 or Ccm2 in neonatal endothelium confers CCM lesions in the mouse hindbrain with similar total volume and number. This quantitative approach also demonstrated a rescue of CCM lesions with simultaneous deletion of one allele of Mekk3. This method would enhance the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases

The p38/MK2/Hsp25 Pathway Is Required for BMP-2-Induced Cell Migration

Background: Bone morphogenetic proteins (BMPs) have been shown to participate in the patterning and specification of several tissues and organs during development and to regulate cell growth, differentiation and migration in different cell types. BMP-mediated cell migration requires activation of the small GTPase Cdc42 and LIMK1 activities. In our earlier report we showed that activation of LIMK1 also requires the activation of PAKs through Cdc42 and PI3K. However, the requirement of additional signaling is not clearly known. Methodology/Principal Findings: Activation of p38 MAPK has been shown to be relevant for a number of BMP-2¿s physiological effects. We report here that BMP-2 regulation of cell migration and actin cytoskeleton remodelling are dependent on p38 activity. BMP-2 treatment of mesenchymal cells results in activation of the p38/MK2/Hsp25 signaling pathway downstream from the BMP receptors. Moreover, chemical inhibition of p38 signaling or genetic ablation of either p38¿ or MK2 blocks the ability to activate the downstream effectors of the pathway and abolishes BMP-2-induction of cell migration. These signaling effects on p38/MK2/Hsp25 do not require the activity of either Cdc42 or PAK, whereas p38/MK2 activities do not significantly modify the BMP-2-dependent activation of LIMK1, measured by either kinase activity or with an antibody raised against phospho-threonine 508 at its activation loop. Finally, phosphorylated Hsp25 colocalizes with the BMP receptor complexes in lamellipodia and overexpression of a phosphorylation mutant form of Hsp25 is able to abolish the migration of cells in response to BMP-2. Conclusions: These results indicate that Cdc42/PAK/LIMK1 and p38/MK2/Hsp25 pathways, acting in parallel and modulating specific actin regulatory proteins, play a critical role in integrating responses during BMP-induced actin reorganization and cell migration

The great screen anomaly—a new frontier in product discovery through functional metagenomics

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Badanie modelowe "mock-up" bariery bentonitowej

The mock-up experiment simulates vertical placement of radioactive waste in the underground repository. Relatively small model is basically a stainless steel cylinder (400 mm diameter and 600 mm height), simulating a section of gallery in which the radioactive waste will be disposed. The cylinder is equipped with a 140 mm diameter central tube, in which heating elements - simulating the heat produced by the waste - are placed. Heating temperature is 120 degrees of Celsius. The annular gap between the central tube and the outer lining is backfilled with pre-compacted bentonite blocks. Major part of blocks is made of pure bentonite from Jelsovy Potok deposit and the other from Lieskovec deposit, both milled into < 250 mim/m fraction. Some blocks contain either 5% of pyrite concentrate to simulate pyrite presence in a gallery host rock or 5% of powdered elementary iron to determine iron-bentonite interactions (iron components in gallery). In the host rock the bentonite water saturation occurs naturally. In the experimental mock-up the hydration is ensured from the external water source. Water is distributed using 23 hydration holes placed outside of the backfill block. The water chemistry is based on the composition of the original water present in the most perspective area for geological radioactive waste repository in Slovakia.Eksperyment "mock-up" symuluje pionowe ułożenie odpadów radioaktywnych w podziemnym składowisku. Stosunkowo mały model jest wykonanym ze stali nierdzewnej cylindrem o średnicy 400 mm i wysokości 600 mm. Jest on symulacją części galerii, w której odpady promieniotwórcze zostaną rozmieszczone. W centralnej części cylindra znajduje się rura o średnicy 140 mm, do której wprowadza się elementy grzejne - symulacja ciepła wytworzonego przez odpady. Temperatura grzania wynosiła 120 stopni Celsjusza. Pierścieniową szczelinę pomiędzy rurą i okładzinami zewnętrznymi wypełniono wstępnie zagęszczonymi blokami bentonitowymi. Większa część bloków jest z czystego bentonitu i pochodzi z osadów Jelsovy Potok oraz Lieskovec. Niektóre bloki zawierają albo 5% koncentratu pirytu do symulacji obecności pirytu w skale galerii lub 5% proszku żelaza w celu określenia interakcji żelazo-bentonit. W eksperymencie nawodnienie jest zapewnione z zewnętrznego źródła wody. Woda jest rozprowadzana za pomocą 23 otworów umieszczonych na zewnątrz bloku. Chemia wody bazuje na składzie oryginalnej wody obecnej w większości obszaru składowiska odpadów promieniotwórczych na Słowacji