Genome profiles of pathologist-defined cell clusters by multiregional LCM and G&T-seq in one triple-negative breast cancer patient.

Pathological examination is the gold standard for cancer diagnosis, and breast tumor cells are often found in clusters. We report a case study on one triple-negative breast cancer (TNBC) patient, analyzing tumor development, metastasis, and prognosis with simultaneous DNA and RNA sequencing of pathologist-defined cell clusters from multiregional frozen sections. The cell clusters are isolated by laser capture microdissection (LCM) from primary tumor tissue, lymphatic vessels, and axillary lymph nodes.

Zika Virus Causes Persistent Infection in Porcine Conceptuses and may Impair Health in Offspring.

Outcomes of Zika virus (ZIKV) infection in pregnant women vary from the birth of asymptomatic offspring to abnormal development and severe brain lesions in fetuses and infants. There are concerns that offspring affected in utero and born without apparent symptoms may develop mental illnesses. Therefore, animal models are important to test interventions against in utero infection and health sequelae in symptomatic and likely more widespread asymptomatic offspring.

Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin.

Background: Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein.

Milk composition studies in transgenic goats expressing recombinant human butyrylcholinesterase in the mammary gland.

The use of the mammary gland of transgenic goats as a bioreactor is a well established platform for the efficient production of recombinant proteins, especially for molecules that cannot be adequately produced in traditional systems using genetically engineered microorganisms and cells. However, the extraordinary demand placed on the secretory epithelium by the expression of large amounts of the recombinant protein, may result in a compromised mammary physiology. In this study, milk composition was compared between control and transgenic goats expressing high levels (1-5 g/l) of recombinant human butyrylcholinesterase in the milk.

IL-15 transpresentation augments CD8+ T cell activation and is required for optimal recall responses by central memory CD8+ T cells.

Although the adaptive immune system has a remarkable ability to mount rapid recall responses to previously encountered pathogens, the cellular and molecular signals necessary for memory CD8(+) T cell reactivation are poorly defined. IL-15 plays a critical role in memory CD8(+) T cell survival; however, whether IL-15 is also involved in memory CD8(+) T cell reactivation is presently unclear. Using artificial Ag-presenting surfaces prepared on cell-sized microspheres, we specifically addressed the role of IL-15 transpresentation on mouse CD8(+) T cell activation in the complete absence of additional stimulatory signals.

Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk.

The production of recombinant proteins in the milk of transgenic animals has attracted significant interest in the last decade, as a valuable alternative for the production of recombinant proteins that cannot be or are inefficiently produced using conventional systems based on microorganisms or animal cells. Several recombinant proteins of pharmaceutical and biomedical interest have been successfully expressed in high quantities (g/l) in the milk of transgenic animals. However, this productivity may be associated with a compromised mammary physiology resulting, among other things, from the extraordinary demand placed on the mammary secretory cells.

Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning.

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated.

Laparoscopic ovum pick-up followed by in vitro embryo production for the reproductive rescue of aged goats of high genetic value.

The efficiency of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production (IVEP) in the propagation of aged goats with poor reproductive performance was evaluated in the present study. Follicular development was stimulated in donor goats with 80 mg follicle-stimulating hormone and 300 IU equine chorionic gonadotrophin administered 36 h before LOPU. In addition, goats were heat synchronised with intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days and a luteolytic injection of 125 microg cloprostenol 36 h before sponge removal and LOPU.

Assessment of mumps virus growth on various continuous cell lines by virological, immunological, molecular and morphological investigations.

Vero cells have been used as a convenient laboratory substrate for the isolation of mumps virus but may not be very sensitive and may select for particular adapted variants from clinical specimens. Continuous cell lines were evaluated for their ability to support the replication of mumps virus. Criteria included the production of infectious virus, detection of intracellular mumps proteins by immunofluorescence and electron microscopy and detection of specific nucleic acid by RT-PCR.

Detection and quantitation of human immunodeficiency virus type-1 particles by confocal microscopy.

A method is described to visualise directly human immunodeficiency virus type-1 (HIV-1) particles. HIV-1 containing samples were adsorbed onto a plastic surface and doubly labeled with antibodies specific for viral proteins and sensitive nucleic acids dyes. Laser scanning confocal microscopy detected co-localization of viral proteins and nucleic acids, thus allowing specific identification of HIV.

In vivo uptake of an experimental microencapsulated diphtheria vaccine following sub-cutaneous immunisation.

Previous studies demonstrated in vitro phagocytosis of poly(lactide-co-glycolide) (PLGA) microspheres (MS) by macrophages and dendritic cells and the biodistribution of fluorescent PLGA particles following oral or intranasal administration. In this study, we report the uptake and biodistribution of sub-cutaneously administered fluorescent labelled PLGA MS loaded with diphtheria toxoid (DT). The cell type and percentage of fluorescent positive cells were determined by flow cytometry and confirmed by fluorescent microscopy.

Optimization of nb-4 and hl-60 differentiation for use in opsonophagocytosis assays.

Production of effective vaccine formulations is dependent on the availability of assays for the measurement of protective immune responses. The development and standardization of in vitro human cell-based assays for functional opsonophagocytic antibodies require critical evaluation and optimization of the preparation of cells for the assay. We report evaluation of a number of protocols with two continuous cell lines (NB-4 and HL-60) for the provision of differentiated cells for use in functional assays.

Multiphase transfer processes in waste rock piles producing acid mine drainage 2. Applications of numerical simulation.

Acid mine drainage (AMD) results from the oxidation of sulfides, mainly pyrite, present in mine wastes, either mill tailings or waste rock. This is the second of two papers describing the coupled physical processes taking place in waste rock piles undergoing AMD production. Since the oxidation of pyrite involves the consumption of oxygen and the production of heat, the oxidation process initiates coupled processes of gas transfer by diffusion and convection as well as heat transfer.

Multiphase transfer processes in waste rock piles producing acid mine drainage 1: Conceptual model and system characterization.

Acid mine drainage (AMD) results from the oxidation of sulfides, mainly pyrite, present in mine wastes, either mill tailings or waste rock. This is the first of two papers describing the coupled physical processes taking place in waste rock piles undergoing AMD production. Since the oxidation of pyrite involves the consumption of oxygen and the production of heat, the oxidation process initiates coupled processes of gas transfer by diffusion and convection as well as heat transfer.

In situ hybridization and immunolabelling study of the early replication of simian immunodeficiency virus (SIVmacJ5) in vivo.

The distribution of virus-infected cells in cynomolgus macaques was determined at 4, 7, 14 and 28 days following intravenous challenge with 1000 TCID(50) of the wild-type simian immunodeficiency virus SIVmacJ5 (stock J5C). At each time-point, pairs of macaques were killed humanely and the presence of SIV was determined and quantified in blood, spleen, peripheral and mesenteric lymph nodes, thymus, lung and ileum by virus co-cultivation with C8166 cells, by quantitative DNA PCR or by in situ hybridization (ISH). At day 4 post-infection (p.

Ultrastructural localization of the RNA of immunodeficiency viruses using electron microscopy in situ hybridization and in vitroinfected lymphocytes.

Cells infected in vitro with immunodeficiency viruses have been examined by electron microscopy in situ hybridization (EM ISH) methods for localization of viral RNA. Techniques used for preparation of specimens and probes are described. Unambiguous positive results were obtained using a mixture of two or three single negative strand DNA oligonucleotides complementary to regions of the gag, env and nef genes, each 200-300 bases and labelled with dig-11-UTP.

Comparison of in vitro and in vivo methods to study stability of PLGA microencapsulated tetanus toxoid vaccines.

The purpose of this study was to investigate the utility of various in vitro and in vivo methods to assess the stability of experimental vaccines containing tetanus toxoid (TT) within PLGA microspheres. In vitro, the breakdown of the encapsulating polymers into their acid components led to changes in the structure of TT, as determined by the physico-chemical methods, rendering it undetectable by capture ELISA and altering its structural integrity. The changes in TT were directly related to increasing acidity of the vaccine supernate.

Development of a continuous IL-7-dependent murine pre-B cell line PB-1 suitable for the biological characterisation and assay of human IL-7.

Human interleukin-7 (IL-7) is a cytokine that appears to be critical for early T- and B-cell development and although IL-7 is currently under investigation as a therapeutic agent in a variety of hematolymphopoietic disorders, there have been few instances of the detection or investigation of this cytokine using a biological assay. This has been due, in the main, to the lack of a widely available, stable, easy to maintain and use, IL-7 responsive cell line. We have developed a pre-B-cell line, PB-1, from murine bone marrow, that is dependent on IL-7 for growth and has been maintained continually for up to 1 year without loss of responsiveness.

Evaluation of transmission electron microscopy for examination of components of acellular pertussis and combination vaccines.

New acellular pertussis and combination vaccines vary in the concentration and presence or absence of specific components and in extent of adsorption to adjuvants. There is a pressing need to develop new control methods for acellular pertussis vaccines. Negative staining electron microscopy has been evaluated as a method for assessing the purity of individual vaccine components and the amount of adsorption to aluminium gels.

Approaches to the control of acellular pertussis vaccines.

The quality control of acellular pertussis vaccines presents particular problems related to the differences in composition and method of detoxification used in the various type of preparation. These vaccines are not amenable to potency assay by the active mouse protection test used for whole-cell pertussis vaccines and assurance of protective activity is problematic. In contrast, monitoring of these vaccines for safety is relatively straightforward and is centred on assays for the lipooligosaccharide endotoxin, active pertussis toxin and absence of reversion to toxicity of detoxified product.

Roles of matrix, p2, and N-terminal myristoylation in human immunodeficiency virus type 1 Gag assembly.

Human immunodeficiency virus type 1 Gag protein is cotranslationally myristoylated at the N terminus and targeted to the plasma membrane, where virus particle assembly occurs. Particle assembly requires the ordered multimerization of Gag proteins, yet there is little direct evidence of intermediates of the reaction or of the domains that lead to each stage of the oligomerization process. In this study, following the expression in insect cells of C-terminally truncated Gag proteins and their purification, both the multimeric nature of each Gag protein and the ability to form Gag virus-like particles (VLP) were analyzed.

Further evidence for hexagonal organization of HIV gag protein in prebudding assemblies and immature virus-like particles.

The fullerene-like model for the organization of HIV gag encoded precursor pr55gag was based on the study of prebudding assemblies at the plasma membrane of cells infected with a recombinant baculovirus expressing HIV-1 gag protein. The objective of the present study was to support the model by image processing of virus-like particles (VLP). In this work we used VLP purified by density gradient centrifugation, which caused partial or occasionally complete loss of the lipid bilayer in some VLP without the use of detergent.

Detection of a trimeric human immunodeficiency virus type 1 Gag intermediate is dependent on sequences in the matrix protein, p17.

Previous studies have shown that single amino acid changes in the amino-terminal matrix (MA) domain, p17, of the human immunodeficiency virus type 1 Gag precursor Pr55, can abrogate virion particle assembly. In the three-dimensional structure of MA such mutations lie in a single helix spanning residues 54 to 68, suggesting a key role for this helix in the assembly process. The fundamental nature of this involvement, however, remains poorly understood.