The immune response of allophenic mice to the synthetic polymer GLΦ

The immune response of allophenic mice of type C57BL/6(A × SJL) F₁ to GL administered in complete Freund's adjuvant was tested. Control mice of the three strains C57BL/6, A, and SJL are all nonresponders to this antigen. However, the F₁ generations of C57BL/6 × A, C57BL/6 × SJL, and A × SJL were all responders to the antigen, so that the complementarity of at least two genes is confirmed. The allophenic mice showed no further complementation beyond the F₁ generation, a result which may argue against the possibility that more than two genes control the response to GL in these mouse strains. Characterization of the allophenic mice over several months showed that they exhibit chimeric drift, both in their coat color and in peripheral white blood cell population. There is no apparent correlation of coat color to the lymphocyte composition of the mice at any one time. The mice are true chimeras, since killing of the two populations of white blood cells with two different anti-H-2 sera produced a 100 percent killing. The immune response of individual allophenic mice to GL showed a good correlation to the number of A × SJL lympho-cytes in the animal.Abbreviations used in this paper are GL an amino acid polymer of 57 %l-glutamic acid, 38%l-lysine, and 5%l-phenylalanine - GLT¹⁵ an amino acid polymer ofl-glutamic acid,l-lysine, and 15 %l-tyrosine - (T,G)-A-L an amino acid polymer having a polylysine backbone with side chains of polyd-l-alanine, terminating in short sequences of tyrosine and glutamic acid - GAT₁₀ an amino acid polymer of 60%l-glutamic acid, 30%l-alanine, and 10%l-tyrosine - GLA₅ an amino acid polymer of 57%l-glutamic acid, 38%l-lysine, and 5%l-alanine - DNP 2,4 dinitrophenyl - BGG bovine gamma globulin - FCS fetal calf serum - PWBC peripheral white blood cell - SWBC spleen white blood cell - T cell thymus-derived lymphocyte - B cell bone marrow-derived lymphocyte     

Intraoperative electron radiation therapy after salvage surgery in gynecological cancers and retroperitoneal sarcomas: outcomes and adverse effects

BackgroundSalvage surgery is considered an option for isolated recurrences of retroperitoneal and pelvic tumors, in patients who have undergone previous radiotherapy. In order to increase local control intra operative electron radiation therapy (IOERT) can be used in these patients to administer additional radiation dose. We evaluated the outcomes and adverse effects in patients with retroperitoneal sarcoma and gynecologic tumors after salvage surgery and IOERT.Materials and methodsTwenty patients were retrospectively analyzed. Twenty-three IOERT treatments were performed after surgery. Six (30%) were sarcoma and 14 (70%) were gynecological carcinoma. Administered dose depended on previous dose received with external beam radiotherapy (EBRT) and proximity to critical structures. The toxicities were scored using the Common Terminology Criteria for Adverse Events version 4.0.ResultsThe median age of the patients was 51 years (range 34–70). After a median follow-up of 32 months (range 1–68), in the sarcoma group the local control rate was 66.6%; while in the gynecological group the local control rate was 64.3%. In relation to late toxicity, one patient had a Grade 2 vesicovaginal fistula, and one patient presented Grade 4 enterocolitis and enteric intestinal fistula.ConclusionsIOERT could have a role in the treatment of retroperitoneal sarcomas in primary tumors after EBRT, as it may suggest a benefit in local control or recurrences after surgical resection in those at high risk of microscopic residual disease. The addition of IOERT to salvage resection for isolated recurrence of gynecologic cancers suggest favorable local control in cases with concern for residual microscopic disease.     

Drought‐induced changes in flow regimes lead to long‐term losses in mussel‐provided ecosystem services

Extreme hydro‐meteorological events such as droughts are becoming more frequent, intense, and persistent. This is particularly true in the south central USA, where rapidly growing urban areas are running out of water and human‐engineered water storage and management are leading to broad‐scale changes in flow regimes. The Kiamichi River in southeastern Oklahoma, USA, has high fish and freshwater mussel biodiversity. However, water from this rural river is desired by multiple urban areas and other entities. Freshwater mussels are large, long‐lived filter feeders that provide important ecosystem services. We ask how observed changes in mussel biomass and community composition resulting from drought‐induced changes in flow regimes might lead to changes in river ecosystem services. We sampled mussel communities in this river over a 20‐year period that included two severe droughts. We then used laboratory‐derived physiological rates and river‐wide estimates of species‐specific mussel biomass to estimate three aggregate ecosystem services provided by mussels over this time period: biofiltration, nutrient recycling (nitrogen and phosphorus), and nutrient storage (nitrogen, phosphorus, and carbon). Mussel populations declined over 60%, and declines were directly linked to drought‐induced changes in flow regimes. All ecosystem services declined over time and mirrored biomass losses. Mussel declines were exacerbated by human water management, which has increased the magnitude and frequency of hydrologic drought in downstream reaches of the river. Freshwater mussels are globally imperiled and declining around the world. Summed across multiple streams and rivers, mussel losses similar to those we document here could have considerable consequences for downstream water quality although lost biofiltration and nutrient retention. While we cannot control the frequency and severity of climatological droughts, water releases from reservoirs could be used to augment stream flows and prevent compounded anthropogenic stressors.     

Reduced plasma angiotensin II levels are reversed by hydroxyurea treatment in mice with sickle cell disease

Aims

Sickle cell disease (SCD) pathogenesis leads to recurrent vaso-occlusive and hemolytic processes, causing numerous clinical complications including renal damage. As vasoconstrictive mechanisms may be enhanced in SCD, due to endothelial dysfunction and vasoactive protein production, we aimed to determine whether the expression of proteins of the renin–angiotensin system (RAS) may be altered in an animal model of SCD.

Main methods

Plasma angiotensin II (Ang II) was measured in C57BL/6 (WT) mice and mice with SCD by ELISA, while quantitative PCR was used to compare the expressions of the genes encoding the angiotensin-II-receptors 1 and 2 (AT1R and AT2R) and the angiotensin-converting enzymes (ACE1 and ACE2) in the kidneys, hearts, livers and brains of mice. The effects of hydroxyurea (HU; 50–75 mg/kg/day, 4 weeks) treatment on these parameters were also determined.

Key findings

Plasma Ang II was significantly diminished in SCD mice, compared with WT mice, in association with decreased AT1R and ACE1 expressions in SCD mice kidneys. Treatment of SCD mice with HU reduced leukocyte and platelet counts and increased plasma Ang II to levels similar to those of WT mice. HU also increased AT1R and ACE2 gene expression in the kidney and heart.

Significance

Results indicate an imbalanced RAS in an SCD mouse model; HU therapy may be able to restore some RAS parameters in these mice. Further investigations regarding Ang II production and the RAS in human SCD may be warranted, as such changes may reflect or contribute to renal damage and alterations in blood pressure.     

Alpha-synuclein cell-to-cell transfer and seeding in grafted dopaminergic neurons in vivo

Several people with Parkinson's disease have been treated with intrastriatal grafts of fetal dopaminergic neurons. Following autopsy, 10-22 years after surgery, some of the grafted neurons contained Lewy bodies similar to those observed in the host brain. Numerous studies have attempted to explain these findings in cell and animal models. In cell culture, α-synuclein has been found to transfer from one cell to another, via mechanisms that include exosomal transport and endocytosis, and in certain cases seed aggregation in the recipient cell. In animal models, transfer of α-synuclein from host brain cells to grafted neurons has been shown, but the reported frequency of the event has been relatively low and little is known about the underlying mechanisms as well as the fate of the transferred α-synuclein. We now demonstrate frequent transfer of α-synuclein from a rat brain engineered to overexpress human α-synuclein to grafted dopaminergic neurons. Further, we show that this model can be used to explore mechanisms underlying cell-to-cell transfer of α-synuclein. Thus, we present evidence both for the involvement of endocytosis in α-synuclein uptake in vivo, and for seeding of aggregation of endogenous α-synuclein in the recipient neuron by the transferred α-synuclein. Finally, we show that, at least in a subset of the studied cells, the transmitted α-synuclein is sensitive to proteinase K. Our new model system could be used to test compounds that inhibit cell-to-cell transfer of α-synuclein and therefore might retard progression of Parkinson neuropathology.     

Enhanced heterologous protein production in Pichia pastoris under increased air pressure

Pichia pastoris is a widely used host for the production of heterologous proteins. In this case, high cell densities are needed and oxygen is a major limiting factor. The increased air pressure could be used to improve the oxygen solubility in the medium and to reach the high oxygen demand of methanol metabolism. In this study, two P. pastoris strains producing two different recombinant proteins, one intracellular (β‐galactosidase) and other extracellular (frutalin), were used to investigate the effect of increased air pressure on yeast growth in glycerol and heterologous protein production, using the methanol AOX1‐inducible system. Experiments were carried out in a stainless steel bioreactor under total air pressure of 1 bar and 5 bar. The use of an air pressure raise of up to 5 bar proved to be applicable for P. pastoris cultivation. Moreover, no effects on the kinetic growth parameters and methanol utilization (Mut) phenotype of strains were found, while an increase in recombinant β‐galactosidase‐specific activity (ninefold) and recombinant frutalin production was observed. Furthermore, the air pressure raise led to a reduction in the secreted protease specific activity. This work shows for the first time that the application of an air pressure of 5 bar may be used as a strategy to decrease protease secretion and improve recombinant protein production in P. pastoris. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1040–1047, 2014     

Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins

Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses.     

Global gene expression in rice blast pathogen Magnaporthe oryzae treated with a natural rice soil isolate

The rhizospheric microbiome is comprised of many microbes, some of which reduce the virulence of their phytopathogenic neighbors; however, the mechanisms underlying these interactions are largely unknown. Rice soil isolate Pseudomonas chlororaphis EA105 strongly inhibits Magnaporthe oryzae’s in vitro growth by restricting fungal diameter as well as inhibiting the formation of the appressorium, required for penetration. We were interested in elucidating M. oryzae’s response to EA105 treatment, and utilized a microarray approach to obtain a global perspective of EA105 elicited changes in this pathogen. Based on this analysis, three genes of interest were knocked out in M. oryzae 70-15, and their sensitivity to EA105 treatment as well as their ability to infect rice was determined. Priming rice plants with EA105 prior to M. oryzae infection decreased lesion size, and the mutants were tested to see if this effect was retained. A null 70-15 mutant in a trichothecene biosynthesis gene showed less susceptibility to bacterial treatment, forming more appressoria than the parental type 70-15. A similar pattern was seen in a null mutant for a stress-inducible protein, MGG_03098. In addition, when this mutant was inoculated onto the leaves of EA105-primed rice plants, lesions were reduced to a greater extent than in 70-15, implicating the lack of this gene with an increased ISR response in rice. Understanding the global effect of biocontrol bacteria on phytopathogens is a key for developing successful and lasting solutions to crop loss caused by plant diseases and has the potential to greatly increase food supply.     

Airborne pollen in Padua (NE-Italy): A comparison between two pollen samplers

During recent years a gradual decrease inallergenic airborne pollen concentration hasbeen observed in the monitoring station ofPadua (Italy). Because technical checks of thesampler were not able to explain this trend,the results obtained from two twinpollen-samplers (Lanzoni VPPS 2000), placed twometres apart, were compared.An eight-week sampling was carried out duringthe year 2000 from July to September.Subsequent analysis revealed no statisticallysignificant difference between the dataobtained with the two instruments. On the otherhand, both samplers captured high levels offungal spores. We conclude that the observednegative trend in pollen count is real and notrelated to technical biases.     

Molecular phylogeny and biogeography of the eastern Tapaculos (Aves: Rhinocryptidae: Scytalopus, Eleoscytalopus): Cryptic diversification in Brazilian Atlantic Forest

Scytalopus and the recently erected Eleoscytalopus are among the Neotropical groups of birds whose taxonomy is most difficult to resolve given their very conservative morphology. We investigated the phylogeny and species limits of Eleoscytalopus and the eastern Scytalopus using two mitochondrial genes and two nuclear introns of multiple individuals from all species of these groups. The eastern Scytalopus are separated in three well defined clades also supported by morphological or vocal characteristics, although the relationships between these clades could not be resolved. We found several allopatric and very divergent lineages in these genera whose characteristics are consistent with species-level divergence, especially in S. speluncae. The great divergence between E. psychopompus and its sister species supports the former as a valid species. Our results corroborate the importance of the Bahia refuge as an avian center of endemism.