Labeling and enrichment of Arabidopsis thaliana matrix metalloproteases using an active-site directed, marimastat-based photoreactive probe

Matrix metalloproteases (MMPs) are secreted or membrane-bound zinc-containing proteases that play diverse roles in development and immunity in plants and in tissue remodeling in animals. We developed a photoreactive probe based on the MMP inhibitor marimastat, conjugated to a 4-azidotetrafluorobenzoyl moiety as photoreactive group and biotin as detection or sorting function. The probe labels At2-MMP, At4-MMP, At5-MMP, and likely other plant MMPs in leaf extracts, as shown by transient At-MMP expression in Nicotiana benthamiana, protein blot, and LC-MS/MS analysis. This MMP probe is a valuable tool to study the post-translational status of MMPs during plant immunity and other MMP-regulated processes.     

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2'',7''-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.     

Generation and characterization of a functional human adipose-derived multipotent mesenchymal stromal cell line

Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the non-immortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications.     

Opportunistic predation on birds trapped in mist nets in two areas in the Atlantic Forest of southeastern Brazil

Mist netting is the most popular method for capturing birds, but it can increase the predation rates of individuals trapped in the nets. From 2008 to 2017, we recorded eight instances of opportunistic bird predation from mist nets (MNs) in a matrix mixing restored forest and fragments of semideciduous seasonal forest in southeastern Brazil, three times (37.5%) by exotic primates and five times (62.5%) by birds of prey. Overall predation rates (1.17–1.20%) at these two sites were considered high but were lower than in other Brazilian studies. Placing MNs near the edges of forest fragments may have allowed attacks by either forest predators or marmosets, which are exotic edge species. Some previously described precautions may decrease the predation rates of birds in MNs, such as shorter observation intervals, greater attention to given site selection and maintaining a safe distance between the MNs and the ground.     

Dichloro(6-aminoethylaminopurine)platinum(II) and its hydroxy analogues: Synthesis and preliminary evaluation

The synthesis, chemical characterization and functional evaluation are reported for dichloro(6-aminoethylaminopurine)platinum(II) and dichloro(6-hydroxyethylaminopurine)platinum(II) and dichloro(6-hydroxyethylamethylaminopurine)platinum(II) (i.e. Pt(6-AEAP), Pt(6-HEAP) and Pt(6-MHEAP) new complexes of platinum(II). Certain reaction conditions favored the formation of the tripurine platinum complex, but the monopurine complex could be obtained either by hydrolysis of the tripurine or by reacting at reduced temperature and concentration. Although neither compound was as effective as cis-diamminedichloroplatinum(II) (i.e. DDP) at reducing tumor cell viability or proliferation, both were associated with much less renal toxicity than DDP in the mouse kidney (i.e. Pt(6-AEAP):～20 × less; Pt(6-MHEAP): ～100 × less).     

Pathology background of targeted therapy; quality control in pathology

The administration of targeted therapy of gastric carcinoma is a very important recent improvement of its treatment and prognosis. The basis of the successful treatment is the excellent quality of pathology, now including HER2 testing: the use of validated methods and strict criteria. This is especially important if we consider that many gastric cancers are diagnosed in small biopsy material, in which HER2 testing is challenging. This requires standardized, validated methods and experienced pathologists. Being of diagnostic and predictive significance, high quality of both the technique and the interpretation of the test is mandatory. In order to achieve general high quality in this field, technical and interpretation external quality control of HER2 testing is necessary. Hungarian pathologists with the help of Roche Hungary Ltd. completed an external quality control round which showed that most of the participating laboratories are able. Kulka J, Szirtes I, Szász AM, Kupcsulik P, Kenessey I, Lotz G, Tímár J. Pathology background of targeted therapy; quality control in pathology.     

Vault nanocapsules as adjuvants favor cell-mediated over antibody-mediated immune responses following immunization of mice

Background

Modifications of adjuvants that induce cell-mediated over antibody-mediated immunity is desired for development of vaccines. Nanocapsules have been found to be viable adjuvants and are amenable to engineering for desired immune responses. We previously showed that natural nanocapsules called vaults can be genetically engineered to elicit Th1 immunity and protection from a mucosal bacterial infection. The purpose of our study was to characterize immunity produced in response to OVA within vault nanoparticles and compare it to another nanocarrier.

Methodology and Principal Findings

We characterized immunity resulting from immunization with the model antigen, ovalbumin (OVA) encased in vault nanocapsules and liposomes. We measured OVA responsive CD8⁺ and CD4⁺ memory T cell responses, cytokine production and antibody titers in vitro and in vivo. We found that immunization with OVA contain in vaults induced a greater number of anti-OVA CD8⁺ memory T cells and production of IFNγ plus CD4⁺ memory T cells. Also, modification of the vault body could change the immune response compared to OVA encased in liposomes.

Conclusions/Significance

These experiments show that vault nanocapsules induced strong anti-OVA CD8⁺ and CD4⁺ T cell memory responses and modest antibody production, which markedly differed from the immune response induced by liposomes. We also found that the vault nanocapsule could be modified to change antibody isotypes in vivo. Thus it is possible to create a vault nanocapsule vaccine that can result in the unique combination of immunogen-responsive CD8⁺ and CD4⁺ T cell immunity coupled with an IgG1 response for future development of vault nanocapsule-based vaccines against antigens for human pathogens and cancer.     

Programmed cell death in the embryonic central nervous system of Drosophila melanogaster

Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence remain largely uninvestigated. We provide here a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord (VNC). We traced the spatio-temporal pattern of PCD and compared the appearance of, and total cell numbers in, thoracic and abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos. Furthermore, we have examined the clonal origin and fate of superfluous cells in H99 mutants by DiI labeling almost all neuroblasts, with special attention to segment-specific differences within the individually identified neuroblast lineages. Our data reveal that although PCD-deficient mutants appear morphologically well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva. Using cell-specific markers we were able to precisely identify some of the progeny cells, including the GW neuron, the U motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning in the embryonic CNS.