The Shadow Moiré method using the phase shifting technique and digital image processing: computational implementation and application to the 3D-reconstruction of a buckled plate

The Shadow Moiré fringe patterns are level lines of equal depth generated by interference between a master grid and its shadow projected on the surface. In simplistic approach, the minimum error is about the order of the master grid pitch, that is, always larger than 0,1 mm, resulting in an experimental technique of low precision. The use of a phase shift increases the accuracy of the Shadow Moiré technique. The current work uses the phase shifting method to determine the surfaces three-dimensional shape using isothamic fringe patterns and digital image processing. The current study presents the method and applies it to images obtained by simulation for error evaluation, as well as to a buckled plate, obtaining excellent results. The method hands itself particularly useful to decrease the errors in the interpretation of the Moiré fringes that can adversely affect the calculations of displacements in pieces containing many concave and convex regions in relatively small areas.

Introducing the concept of critical Fo in batch heat processing

The determination of the sterilization value for low acid foods in retorts includes a critical evaluation of the factory's facilities and utilities, validation of the heat processing equipment (by heat distribution assays), and finally heat penetration assays with the product. The intensity of the heat process applied to the food can be expressed by the Fo value (sterilization value, in minutes, at a reference temperature of 121.1 °C, and a thermal index, z, of 10 °C, for Clostridium botulinum spores). For safety reasons, the lowest value for Fo is frequently adopted, being obtained in heat penetration assays as indicative of the minimum process intensity applied. This lowest Fo value should always be higher than the minimum Fo recommended for the food in question. However, the use of the Fo value for the coldest can fail to statistically explain all the practical occurrences in food heat treatment processes. Thus, as a result of intense experimental work, we aimed to develop a new focus to determine the lowest Fo value, which we renamed the critical Fo. The critical Fo is based on a statistical model for the interpretation of the results of heat penetration assays in packages, and it depends not only on the Fo values found at the coldest point of the package and the coldest point of the equipment, but also on the size of the batch of packages processed in the retort, the total processing time in the retort, and the time between CIPs of the retort. In the present study, we tried to explore the results of physical measurements used in the validation of food heat processes. Three examples of calculations were prepared to illustrate the methodology developed and to introduce the concept of critical Fo for the processing of canned food.

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples

Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

BIOFILM FORMATION OF Vibrio cholerae ON STAINLESS STEEL USED IN FOOD PROCESSING

Vibrio cholerae represents a significant threat to human health in developing countries. This pathogen forms biofilms which favors its attachment to surfaces and its survival and transmission by water or food. This work evaluated the in vitro biofilm formation of V. cholerae isolated from clinical and environmental sources on stainless steel of the type used in food processing by using the environmental scanning electron microscopy (ESEM). Results showed no cell adhesion at 4 h and scarce surface colonization at 24 h. Biofilms from the environmental strain were observed at 48 h with high cellular aggregations embedded in Vibrio exopolysaccharide (VPS), while less confluence and VPS production with microcolonies of elongated cells were observed in biofilms produced by the clinical strain. At 96 h the biofilms of the environmental strain were released from the surface leaving coccoid cells and residual structures, whereas biofilms of the clinical strain formed highly organized structures such as channels, mushroom-like and pillars. This is the first study that has shown the in vitro ability of V. cholerae to colonize and form biofilms on stainless steel used in food processing.

Minimum detection limit of an in-house nested-PCR assay for herpes simplex virus and varicella zoster virus

Introduction Herpes simplex virus (HSV) and varicella zoster virus (VZV) are responsible for a variety of human diseases, including central nervous system diseases. The use of polymerase chain reaction (PCR) techniques on cerebrospinal fluid samples has allowed the detection of viral DNA with high sensitivity and specificity. Methods Serial dilutions of quantified commercial controls of each virus were subjected to an in-house nested-PCR technique. Results The minimum detection limits for HSV and VZV were 5 and 10 copies/µL, respectively. Conclusions The detection limit of nested-PCR for HSV and VZV in this study was similar to the limits found in previous studies.

A malaria merozoite surface protein (MSP1)-structure, processing and function

Merozoite surface protein-1 (MSP-1, also referred to as P195, PMMSA or MSA 1) is one of the most studied of all malaria proteins. The proteins. The protein is found in all malaria species investigated and structural studies on the gene indicate that parts of the molecule are well-conserved. Studies on Plasmodium falciparum have shown that the protein is in a processed form on the merozoite surface, a result of proteolytic cleavage of the large percursor molecule. Recent studies have identified some of these cleavage sites. During invasion of the new red cell most of the MSP1 molecule is shed from the parasite surface except for a small C-terminal fragment which can be detected in ring stages. Analysis of the structure of this fragment suggests that it contains two growth factor-like domains that may have a functional role.

Proteolytic processing of the Cyt1Ab1 toxin produced by Bacillus thuringiensis subsp. medellin

Bacillus thuringiensis produces d-endotoxins that require proteolytic processing to become active. The activation of the B. thuringiensis subsp. medellin 28 kDa (Cyt1Ab1) cytolytic toxin by trypsin, chymotrypsin and gut extract from Culex quinquefasciatus larvae was analyzed. The Cyt1Ab1 toxin of B. thuringiensis subsp. medellin was processed by all proteases tested to fragments between 23 and 25 kDa, while processing of the Cyt1Aa1 toxin produce fragments between 22.5 and 24.5 kDa. The Cyt1Ab1 toxin was preferentially processed at the alkaline pH of 12. The in vitro proteolytic processing of the Cyt1Ab1 toxin by C. quinquefasciatus larvae midgut extract showed a 25 kDa fragment; a similar result was observed when the activation was performed in the in vivo experiments. The solubilized Cyt1Ab1 toxin and the protease resistant cores generated by in vitro processing showed hemolytic activity but not mosquitocidal activity. Amino terminal sequence of the C. quinquefasciatus gut extract resistant fragment indicated that the cutting site was located between Lys31 and Asp32, with a sequence DDPNEKNNHNS; while for the trypsin-resistant fragment the cutting site was determined between Leu29 and Arg30, and for the chymotrypsin-resistant fragment between Arg30 and Lys31.

Inhibition of the polymerase chain reaction by sputum samples from tuberculosis patients after processing using a silica-guanidiniumthiocyanate DNA isolation procedure

With the objective to evaluate PCR-mediated detection of Mycobacterium tuberculosis DNA as a diagnostic procedure for diagnosis of tuberculosis in individuals attending ambulatory services in Primary Health Units of the City Tuberculosis Program in Rio de Janeiro, Brazil, their sputum samples were collected and treated with a DNA extraction procedure using silica-guanidiniumthiocyanate. This procedure has been described to be highly efficient for extraction of different kind of nucleic acids from bacteria and clinical samples. Upon comparing PCR results with the number of acid-fast bacilli, no direct relation was observed between the number of bacilli present in the sample and PCR positivity. Part of the processed samples was therefore spiked with pure DNA of M. tuberculosis and inhibition of the PCR reaction was verified in 22 out of 36 (61%) of the samples, demonstrating that the extraction procedure as originally described should not be used for PCR analysis of sputum samples.

Trypanosoma cruzi: establishment of permeable cells for RNA processing analysis with drugs

Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independent transcripts. For the first time, we present the standardization of Trypanosoma cruzi permeable cells (Y strain) as a model for trans-splicing study of mRNAs in trypanosomes, following by RNase protection reaction, which localizes the SL exon and intron. This trans-splicing reaction in vitro was also used to analyze the influence of NFOH-121, a nitrofurazone-derivative, on this mechanism. The results suggested that the prodrug affects the RNA processing in these parasites, but the trans-splicing reaction still occurred.

Slime production and proteinase activity of Candida species isolated from blood samples and the comparison of these activities with minimum inhibitory concentration values of antifungal agents

Slime and proteinase activity of 54 strains consisting of 19 Candida parapsilosis and 35 C. albicans strains isolated from blood samples were investigated in this study. Ketoconazole, amphothericin B, and fluconazole susceptibility of Candida species were compared with slime production and proteinase activity of these species. For both Candida species, no correlation was detected between the slime activity and minimum inhibitory concentration (MIC) values of the three antifungal agents. For both Candida species no correlation was detected between the proteinase activity and the MIC values of amphothericin B, and fluconazole however, statistically significant difference, was determined between the proteinase activity and MIC values of ketoconazole (p = 0.007). Slime production was determined by using modified Christensen macrotube method and proteinase activity was measured by the method of Staib. Antifungal susceptibility was determined through the guidelines of National Committee for Laboratory Standards (NCCLS M27-A).

Attaining the minimum target of resolution WHA 54.19 for schistosomiasis control in the Rainforest Zone of the state of Pernambuco, Northeastern Brazil

Resolution 19 of the 54th World Health Assembly (WHA-54.19) urged member nations to promote preventive measures, ensure treatment and mobilize resources for control of schistosomiasis and soil-transmitted helminthiases (STH). The minimum target is to attend 75% of all school-age children at risk by year 2010. The Brazilian Ministry of Health (MoH) recommends biennial surveys of whole communities and treatment of the positives through the Schistosomiasis Control Program within the Unified Health System (PCE-SUS). However, by 2004 the PCE-SUS had covered only 8.4% of the 1.2 million residents in the Rainforest Zone of Pernambuco (ZMP). Six of the 43 municipalities still remained unattended. Only three of the municipalities already surveyed reached coverage of 25% or more. At least 154 thousand children in the 7-14 years old range have to be examined (and treated if positive) within the next five years to attend the minimum target of the WHA 54.19 for the ZMP. To make this target feasible, it is suggested that from 2006 to 2010 the PCE-SUS actions should be complemented with school-based diagnosis and treatment, involving health and educational organs as well as community associations to include both children in schools and non-enrolled school-age children.

The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.