Phase I/II study of immunotherapy with T-cell peptide epitopes in patients with stage IV melanoma

Previous studies in small groups of patients suggested that immunization of melanoma patients with peptide epitopes recognized by T cells could induce regression of melanoma. This approach was tested in 36 patients with stage IV melanoma. The (MHC class I–restricted) peptides were from gp100, MART-1, tyrosinase, and MAGE-3. The gp100 and MART-1 peptides had been modified to increase their immunogenicity. In half the patients (groups 3 and 4) the peptides were given in the adjuvant Montanide-ISA-720, and half the patients in both groups were given GM-CSF s.c. for 4 days following each injection. Treatment was well tolerated except for two severe erythematous responses to Montanide-ISA-720 and marked inflammatory responses at sites of GM-CSF administration in three patients. There were no objective clinical responses but stabilization of disease for periods from 3 to 12 months were seen in seven patients. Five of these were patients given the peptides in Montanide-ISA-720. Delayed-type hypersensitivity (DTH) skin test responses were also seen mainly in the patients given the peptides in Montanide-ISA-720. GM-CSF did not increase DTH responses in patients in the latter group but may have increased DTH responses in those not given peptides in Montanide-ISA-720. Inflammatory responses around s.c. metastases or regional lymph nodes were observed in two patients. These results suggest that the peptides are more effective when given in the adjuvant Montanide-ISA-720. Nevertheless, results from this study, together with those from a number of comparable studies, indicate that peptide vaccines are currently of minimal benefit to patients and support the need for ongoing development of new strategies in treatment of this disease.     

Whole genome amplification from filamentous fungi using Phi29-mediated multiple displacement amplification

The availability of genomic DNA of sufficient quality and quantity is fundamental to molecular genetic analysis. Many filamentous fungi are slow growing or even unculturable and current DNA isolation methods are often unsatisfactory. We have used multiple displacement amplification (MDA) to amplify whole genomes for two fungal species, Penicillium paxilli and the slow growing endophyte of grasses Epichloe festucae. Up to 10 microg of high molecular weight DNA was routinely amplified from less than 10 ng of template DNA obtained using glass bead-mediated disruption of fungal spores or alkaline lysis of mycelium. PCR was possible from MDA-generated DNA and amplicons up to 10 kb were successfully amplified. RFLP analysis was successful, with bands of up to 5 kb routinely detected. Hybridization of MDA-amplified DNA to a cosmid library illustrated that the MDA product amplified from E. festucae is representative of the genome. MDA is a reliable method that could be applied to applications ranging from high-throughput screening of deletion mutants to genomic library construction.     

The N-Terminal Domain of the Tomato Immune Protein Prf Contains Multiple Homotypic and Pto Kinase Interaction Sites

Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats).     

Microbial Ecology of the Hive and Pollination Landscape: Bacterial Associates from Floral Nectar,the Alimentary Tract and Stored Food of Honey Bees (Apis mellifera)

Nearly all eukaryotes are host to beneficial or benign bacteria in their gut lumen, either vertically inherited, or acquired from the environment. While bacteria core to the honey bee gut are becoming evident, the influence of the hive and pollination environment on honey bee microbial health is largely unexplored. Here we compare bacteria from floral nectar in the immediate pollination environment, different segments of the honey bee (Apis mellifera) alimentary tract, and food stored in the hive (honey and packed pollen or “beebread”). We used cultivation and sequencing to explore bacterial communities in all sample types, coupled with culture-independent analysis of beebread. We compare our results from the alimentary tract with both culture-dependent and culture-independent analyses from previous studies. Culturing the foregut (crop), midgut and hindgut with standard media produced many identical or highly similar 16S rDNA sequences found with 16S rDNA clone libraries and next generation sequencing of 16S rDNA amplicons. Despite extensive culturing with identical media, our results do not support the core crop bacterial community hypothesized by recent studies. We cultured a wide variety of bacterial strains from 6 of 7 phylogenetic groups considered core to the honey bee hindgut. Our results reveal that many bacteria prevalent in beebread and the crop are also found in floral nectar, suggesting frequent horizontal transmission. From beebread we uncovered a variety of bacterial phylotypes, including many possible pathogens and food spoilage organisms, and potentially beneficial bacteria including Lactobacillus kunkeei, Acetobacteraceae and many different groups of Actinobacteria. Contributions of these bacteria to colony health may include general hygiene, fungal and pathogen inhibition and beebread preservation. Our results are important for understanding the contribution to pollinator health of both environmentally vectored and core microbiota, and the identification of factors that may affect bacterial detection and transmission, colony food storage and disease susceptibility.     

The proliferative effects of retinoic acid on primary cultures of adult rat type II pneumocytes depend upon cell density

Retinoic acid (RA) is important for maintaining integrity of alveolar epithelial cells, but the mechanism has not been defined. We cultured type II pneumocytes at confluent, high cell density (10⁴ cells/mm²) and found that RA (10⁻⁶ M) inhibited thymidine incorporation to 60% of control, despite a dose-dependent increase in epidermal growth factor receptor (EGFR) levels. However, at lower, subconfluent density (10² cells/mm²), RA stimulated thymidine incorporation to 280% of control. EGF increased thymidine incorporation at concentrations as low as 0.1 ng/mL, but no further increase was observed at higher concentrations up to 100 ng/mL. In subconfluent cells co-treated with EGF (100 ng/mL) and increasing concentrations of RA (10⁻⁸ M–10⁻⁵ M RA), thymidine incorporation was significantly greater at all concentrations than RA alone, with greatest increases observed at 10⁻⁷ (422% of control) and 10⁻⁶ (470% of control) M RA. In summary, the effects of RA on thymidine incorporation are sensitive to changes in cell density. RA inhibits thymidine incorporation at high cell density and stimulates thymidine incorporation at low density. RA increases EGFRs in cultured type II pneumocytes, and EGF stimulates thymidine incorporation independent of the cultured cell density. These data may help to explain how RA mediates lung repair in vivo.     

Phylogeography of Pogonomyrmex barbatus and P. rugosus harvester ants with genetic and environmental caste determination

We present a phylogeographic study of at least six reproductively isolated lineages of new world harvester ants within the Pogonomyrmex barbatus and P. rugosus species group. The genetic and geographic relationships within this clade are complex: Four of the identified lineages show genetic caste determination (GCD) and are divided into two pairs. Each pair has evolved under a mutualistic system that necessitates sympatry. These paired lineages are dependent upon one another because their GCD requires interlineage matings for the production of F1 hybrid workers, and intralineage matings are required to produce queens. This GCD system maintains genetic isolation among these interdependent lineages, while simultaneously requiring co-expansion and emigration as their distributions have changed over time. It has also been demonstrated that three of these four GCD lineages have undergone historical hybridization, but the narrower sampling range of previous studies has left questions on the hybrid parentage, breadth, and age of these groups. Thus, reconstructing the phylogenetic and geographic history of this group allows us to evaluate past insights and hypotheses and to plan future inquiries in a more complete historical biogeographic context. Using mitochondrial DNA sequences sampled across most of the morphospecies’ ranges in the U.S.A. and Mexico, we conducted a detailed phylogeographic study. Remarkably, our results indicate that one of the GCD lineage pairs has experienced a dramatic range expansion, despite the genetic load and fitness costs of the GCD system. Our analyses also reveal a complex pattern of vicariance and dispersal in Pogonomyrmex harvester ants that is largely concordant with models of late Miocene, Pliocene, and Pleistocene range shifts among various arid-adapted taxa in North America.     

Key Edaphic Properties Largely Explain Temporal and Geographic Variation in Soil Microbial Communities across Four Biomes

Soil microbial communities play a critical role in nutrient transformation and storage in all ecosystems. Quantifying the seasonal and long-term temporal extent of genetic and functional variation of soil microorganisms in response to biotic and abiotic changes within and across ecosystems will inform our understanding of the effect of climate change on these processes. We examined spatial and seasonal variation in microbial communities based on 16S rRNA gene sequencing and phospholipid fatty acid (PLFA) composition across four biomes: a tropical broadleaf forest (Hawaii), taiga (Alaska), semiarid grassland-shrubland (Utah), and a subtropical coniferous forest (Florida). In this study, we used a team-based instructional approach leveraging the iPlant Collaborative to examine publicly available National Ecological Observatory Network (NEON) 16S gene and PLFA measurements that quantify microbial diversity, composition, and growth. Both profiling techniques revealed that microbial communities grouped strongly by ecosystem and were predominately influenced by three edaphic factors: pH, soil water content, and cation exchange capacity. Temporal variability of microbial communities differed by profiling technique; 16S-based community measurements showed significant temporal variability only in the subtropical coniferous forest communities, specifically through changes within subgroups of Acidobacteria. Conversely, PLFA-based community measurements showed seasonal shifts in taiga and tropical broadleaf forest systems. These differences may be due to the premise that 16S-based measurements are predominantly influenced by large shifts in the abiotic soil environment, while PLFA-based analyses reflect the metabolically active fraction of the microbial community, which is more sensitive to local disturbances and biotic interactions. To address the technical issue of the response of soil microbial communities to sample storage temperature, we compared 16S-based community structure in soils stored at -80°C and -20°C and found no significant differences in community composition based on storage temperature. Free, open access datasets and data sharing platforms are powerful tools for integrating research and teaching in undergraduate and graduate student classrooms. They are a valuable resource for fostering interdisciplinary collaborations, testing ecological theory, model development and validation, and generating novel hypotheses. Training in data analysis and interpretation of large datasets in university classrooms through project-based learning improves the learning experience for students and enables their use of these significant resources throughout their careers.     

Shading effect of photovoltaic panels on horticulture crops production: a mini review

Reviews in Environmental Science and Bio/Technology - Agrivoltaics (APV) combine crops with solar photovoltaics (PV) on the same land area to provide sustainability benefits across land, energy and...