An assessment of the ultrasonic probe-based enhancement of protein cleavage with immobilized trypsin

The use of ultrasonic probe, in conjunction with immobilized trypsin, has been explored in this work for potential enhancement of protein digestion. Several solid supports commonly used to immobilize trypsin were subjected to different ultrasonication amplitudes and time in order to investigate their mechanical resistance to ultrasonic energy when provided by the ultrasonic probe. Glass beads and magnetic particles were found to remain intact in most conditions studied. It was found that immobilized trypsin cannot be reused after ultrasonication since the enzymatic activity was greatly diminished. For comparative purposes, vortex shaking was also explored for protein cleavage. Four standard proteins--bovine serum albumin, α-lactalbumin, carbonic anhydrase and ovalbumin--were successfully identified using peptide mass fingerprint, or peptide fragment fingerprint. In addition, the performance of the classical protein cleavage (overnight, 12 h) and the ultrasonic methods was found to be similar when the digestion of a complex proteome, human plasma, was assessed through 18-O quantification. The digestion yields found were 90-117% for the ultrasonic and 5-21% for the vortex when those methods were compared with the classical overnight digestion.     

The role of the parenchyma sheath and PCD during the development of oil cavities in Pterodon pubescens (Leguminosae-Papilionoideae)

Pterodon pubescens cavities are constituted by lumen and uniseriated epithelium surrounded by multiseriate parenchyma sheath. We studied the development of secretory cavities, including the role of parenchyma sheath, using light and transmission electron microscopy. A Tunel assay was performed to verify whether programmed cell death (PCD) occurs during the process. The lumen is formed by schizogeny and lysigeny occur in later developmental stages of the secretory cavities. Ultrastructurally, epithelial cells in later developmental stages become dark and with sinuous walls; the protoplast becomes retracted and the cytoplasm shows low organelle definition. Degenerated cells are released toward the lumen. Our results showed that PCD occurs during later developmental stages of cavities and plays a critical role in functioning of these glands. New cells originated from the parenchyma sheath differentiate into secretory cells and replace those degenerated ones. This fact associated to PCD guarantees epithelium renovation during the secretory cycle and the maintenance of secretory activity of cavities.     

Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains

Background

Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity.

Methodology and Findings

We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer.

Conclusions

These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers {"type":"entrez-nucleotide","attrs":{"text":"CP001809","term_id":"340539261","term_text":"CP001809"}}CP001809 and {"type":"entrez-nucleotide","attrs":{"text":"CP001829","term_id":"302205157","term_text":"CP001829"}}CP001829.     

Migration and ramification of microglia in quail embryo retina organotypic cultures

Organotypic cultures of retina explants preserve the complex cellular microenvironment of the retina and have been used as a tool to assess the biological functions of some cell types. However, studies to date have shown that microglial cells activate quickly in response to the retina explantation. In this study, microglial cells migrated and ramified in quail embryo retina organotypic cultures (QEROCs) according to chronological patterns bearing a resemblance to those in the retina in situ, despite some differences in cell density and ramification degree. Retinal explants from quail embryos at 9 days of incubation (E9) proved to be the best in vitro system for reproducing a physiological-like behavior of microglial cells when cultured in Eagle's basal medium supplemented with horse serum. During the first week in vitro, microglial cells migrated tangentially in the vitreal part of QEROCs, and some began to migrate radially from 3 days in vitro (div) onward, ramifying in the inner and outer plexiform layers, thus mimicking microglia development in the retina in situ, although reaching a lower degree of ramification after 7 div. From 8 div onward, microglial cells rounded throughout the explant thickness simultaneously with the nonphysiological appearance of dead photoreceptors and round microglia in the outernuclear layer. Therefore, E9 QEROCs can be used during the first week in vitro as a model system for experimental studies of molecules putatively involved in microglial migration and ramification.     

Genetic differentiation, structure, and a transition zone among populations of the pitcher plant moth Exyra semicrocea: implications for conservation

Pitcher plant bogs, or carnivorous plant wetlands, have experienced extensive habitat loss and fragmentation throughout the southeastern United States Coastal Plain, resulting in an estimated reduction to <3% of their former range. This situation has lead to increased management attention of these habitats and their carnivorous plant species. However, conservation priorities focus primarily on the plants since little information currently exists on other community members, such as their endemic arthropod biota. Here, we investigated the population structure of one of these, the obligate pitcher plant moth Exyra semicrocea (Lepidoptera: Noctuidae), using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. Examination of 221 individuals from 11 populations across eight southeastern US states identified 51 unique haplotypes. These haplotypes belonged to one of two divergent (～1.9-3.0%) lineages separated by the Mississippi alluvial plain. Populations of the West Gulf Coastal Plain exhibited significant genetic structure, contrasting with similarly distanced populations east of the Mississippi alluvial plain. In the eastern portion of the Coastal Plain, an apparent transition zone exists between two regionally distinct population groups, with a well-established genetic discontinuity for other organisms coinciding with this zone. The structure of E. semicrocea appears to have been influenced by patchy pitcher plant bog habitats in the West Gulf Coastal Plain as well as impacts of Pleistocene interglacials on the Apalachicola-Chattahoochee-Flint River Basin. These findings, along with potential extirpation of E. semicrocea at four visited, but isolated, sites highlight the need to consider other endemic or associated community members when managing and restoring pitcher plant bog habitats.     

Dynamics of viral evolution and CTL responses in HIV-1 infection

Improved understanding of the dynamics of host immune responses and viral evolution is critical for effective HIV-1 vaccine design. We comprehensively analyzed Cytotoxic T-lymphocyte (CTL)-viral epitope dynamics in an antiretroviral therapy-naïve subject over the first four years of HIV-1 infection. We found that CTL responses developed sequentially and required constant antigenic stimulation for maintenance. CTL responses exerting strong selective pressure emerged early and led to rapid escape, proliferated rapidly and were predominant during acute/early infection. Although CTL responses to a few persistent epitopes developed over the first two months of infection, they proliferated slowly. As CTL epitopes were replaced by mutational variants, the corresponding responses immediately declined, most rapidly in the cases of strongly selected epitopes. CTL recognition of epitope variants, via cross-reactivity and de novo responses, was common throughout the period of study. Our data demonstrate that HIV-specific CTL responses, especially in the critical acute/early stage, were focused on regions that are prone to escape. Failure of CTL responses to strongly target functional or structurally critical regions of the virus, as well as the sequential cascade of CTL responses, followed closely by viral escape and decline of the corresponding responses, likely contribute to a lack of sustainable viral suppression. Focusing early and rapidly proliferating CTL on persistent epitopes may be essential for durable viral control in HIV-1 infection.     

A new approach to dengue fatal cases diagnosis: NS1 antigen capture in tissues

Abstract/Background

Dengue is the most important arthropod borne viral disease worldwide in terms of morbidity and mortality and is caused by any of the four serotypes of dengue virus (DENV-1 to 4). Brazil is responsible for approximately 80% of dengue cases in the Americas, and since the introduction of dengue in 1986, a total of 5,944,270 cases have been reported including 21,596 dengue hemorrhagic fever and 874 fatal cases. DENV can infect many cell types and cause diverse clinical and pathological effects. The goal of the study was to investigate the usefulness of NS1 capture tests as an alternative tool to detect DENV in tissue specimens from previously confirmed dengue fatal cases (n = 23) that occurred in 2002 in Brazil.

Methodology/Principal Findings

A total of 74 tissue specimens were available: liver (n = 23), lung (n = 14), kidney (n = 04), brain (n = 10), heart (n = 02), skin (n = 01), spleen (n = 15), thymus (n = 03) and lymph nodes (n = 02). We evaluated three tests for NS1 antigen capture: first generation Dengue Early ELISA (PanBio Diagnostics), Platelia NS1 (BioRad Laboratories) and the rapid test NS1 Ag Strip (BioRad Laboratories). The overall dengue fatal case diagnosis based on the tissues analyzed by Dengue Early ELISA, Platelia NS1 and the NS1 Ag Strip was 34.7% (08/23), 60.8% (14/23) and 91.3% (21/23), respectively. The Dengue Early ELISA detected NS1 in 22.9% (17/74) of the specimens analyzed and the Platelia NS1 in 45.9% (34/74). The highest sensitivity (78.3%; 58/74) was achieved by the NS1 Ag Strip, and the differences in the sensitivities were statistically significant (p<0.05). The NS1 Ag Strip was the most sensitive in liver (91.3%; 21/23), lung (71.4%; 10/14), kidney (100%; 4/4), brain (80%; 8/10), spleen (66.6%, 10/15) and thymus (100%, 3/3) when compared to the other two ELISA assays.

Conclusions/Significance

This study shows the DENV NS1 capture assay as a rapid and valuable approach to postmortem dengue confirmation. With an increasing number of DHF and fatal cases, the availability of new approaches useful for cases confirmation plays an important tool for the disease surveillance.     

Extracellular purine and pyrimidine catabolism in cell culture

The presence of purines and pyrimidines bases, nucleosides, and nucleotides in the culture medium has shown to differently affect the growth of a Chinese hamster ovary (CHO) cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP; Carvalhal et al., Biotech Prog. 2003;19:69-83). CHO, BHK, as well as Sf9 cell growth was clearly reduced in the presence of purines but was not affected by pyrimidines at the concentrations tested. The knowledge about the mechanisms by which nucleotides exert their effect when present outside the cells remains very incomplete. The catabolism of both extracellular purines and pyrimidines was followed during the culture of CHO cells. Purines/pyrimidines nucleotides added at a concentration of 1 mM to the culture medium decreased to negligible concentrations in the first 2 days. Purine and pyrimidine catabolism originated only purinic and pyrimidic end-products, respectively. The comparison between AMP catabolism in serum-free cultures (CHO cells expressing Factor VII and Sf9 cells) and in cultures containing serum (CHO cells expressing SEAP and BHK cells expressing Factor VII) showed that AMP extracellular catabolism is mediated by both cells and enzymes present in the serum. This work shows that the quantification of purines and pyrimidines in the culture medium is essential in animal cell culture optimization. When using AMP addition as a chemical cell growth strategy for recombinant protein production improvement, AMP extracellular concentration monitoring allows the optimization of the multiple AMP addition strategy for a prolonged cell culture duration with high specific productivity.